Characterisation of Proteins from Grevillea robusta · Characterisation of Proteins from Grevillea...

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Characterisation of Proteins from Grevillea robusta

and NMR studies of the Serine Protease Inhibitor

Sarah Jane Kruger

B.BioMedSci (Hons)

School of Science

Faculty of Science

Griffith University

Submitted in fulfilment of the requirements

Of the Degree of Doctor of Philosophy

April 2004

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Abstract _____________________________________________________________________________________

Abstract

Proteins that recognise the sugar surface structures on cells have an enormous potential

to be used as tools in the characterisation of these structures. A group of proteins, called

lectins, have been identified that can bind to carbohydrate complexes on the receptors of

cells. The crude extract from Grevillea robusta seeds was found to contain lectin-like

proteins that were different from most other lectins, as they would specifically target the

receptors of white blood cells and not those found on red blood cells. Therefore, the

lectin isolated from G.robusta could be used as a tool to identify the specific surface

structures on white blood cells.

The lectin was isolated using affinity chromatography where a complex

(oligosaccharide) matrix was used. Agglutination, binding and sugar inhibition assays

confirmed the isolated protein was a lectin. The lectin was found in low amounts (up to

5% of the total protein content) within the seeds of G.robusta. As a result of this low

yield, the identification of the lectin by PAGE was difficult because the levels of protein

were beyond the detection limit of the commercial staining reagents. The lectin was

called the GR2 protein and was characterised as a monocot mannose binding lectin

based on its sugar specificity for only mannose.

A serine protease inhibitor was isolated from the seeds of G.robusta using two different

chromatography methods, reverse phase HPLC (GR1.HPLC) and gel filtration

chromatography (GR1.GF). Ion exchange chromatography was used to initially

separate the proteins in the crude extract and the fraction containing the GR1 protein

was further purified using reverse phase HPLC (GR1.HPLC). N-terminal sequencing

results of the GR1.HPLC protein, showed evidence of proteolytic cleavage during the

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Abstract _____________________________________________________________________________________

extraction process, which lead to the second purification method being established.

Protease inhibitors were added to the buffers prior to being purified by gel filtration

chromatography, which resulted in the GR1 protein being isolated from the crude

extract without the presence of the contaminating protein.

Mass spectroscopy identified the molecular weight of the GR1 protein to be 6669Da

and the full amino acid sequence was derived by cDNA techniques. Sequence

alignment studies of the GR1 protein showed significant similarities with the Bowman-

Birk inhibitor. The positioning of the cysteine residues were conserved throughout the

Bowman-Birk superfamily, however these residues were not conserved within the GR1

protein. Competitive inhibition assays on the GR1 protein revealed the protein could

inhibit both trypsin and chymotrypsin at similar levels to that seen for the Bowman-Birk

inhibitor. Therefore, the GR1 protein was characterised as a member of the Bowman-

Birk superfamily of serine protease inhibitors.

The three-dimensional structure of the GR1 protein was determined using two-

dimensional NMR spectroscopy. Computer programs such as XEASY, DYANA and

SYBYL® were used to tabulate the information taken from the 2D experiments,

generate structures and minimise these structures respectively. The solution structure of

the GR1 protein was found to contain a region of antiparallel β-sheet structure that

corresponded to the trypsin binding site and the remainder of the protein consisted of

loops and turns that were held together by disulfide bridges (the chymotrypsin-binding

region). Structural similarities between the GR1 protein and the Bowman-Birk inhibitor

existed only in the trypsin-binding site of the Bowman-Birk inhibitor. The GR1 protein

is the first member of the Proteaceae family to be characterised as a Bowman-Birk

inhibitor.

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Abstract _____________________________________________________________________________________

This thesis outlines the isolation and biochemical characterisation of the two proteins

found within Grevillea robusta and also describes the steps involved and results

obtained in determining the three-dimensional structure of the GR1 protein.

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Table of Contents _____________________________________________________________________________________

Table of Contents

ABSTRACT............................................................................................................................................... II

TABLE OF CONTENTS.......................................................................................................................... V

LIST OF FIGURES ..............................................................................................................................VIII

LIST OF TABLES ...................................................................................................................................XI

ACKNOWLEDGMENTS ..................................................................................................................... XII

STATEMENT OF ORIGINALITY.....................................................................................................XIII

ABBREVIATIONS ...............................................................................................................................XIV

CHAPTER 1 INTRODUCTION.............................................................................................................. 2

1.1 CARBOHYDRATES AND CELL RECOGNITION................................................................................... 2 1.2 LEUKOCYTE FUNCTION .................................................................................................................. 3 1.3 NEUTROPHIL ANTIGENS ................................................................................................................. 7 1.4 CHARACTERISATION OF NEUTROPHIL GLYCOPROTEINS ............................................................... 10 1.5 CARBOHYDRATE SPECIFICITY OF THE LECTIN ............................................................................... 12 1.6 ANIMAL LECTINS .......................................................................................................................... 15

1.6.1 C-type lectins...................................................................................................................... 15 1.6.2 I-type lectins ....................................................................................................................... 18 1.6.3 Galectins ............................................................................................................................ 19 1.6.4 Pentraxins........................................................................................................................... 21 1.6.5 P-type lectins ...................................................................................................................... 22

1.7 PLANT LECTINS............................................................................................................................. 24 1.7.1 Legume lectins.................................................................................................................... 24 1.7.2 Monocot Mannose binding lectins...................................................................................... 27 1.7.3 Chitin-binding lectins ......................................................................................................... 28 1.7.4 Type II ribosome inactivating protein (RIP) ...................................................................... 29 1.7.5 The Jacalin family .............................................................................................................. 30

1.8 ROLES OF LECTINS IN PLANTS ....................................................................................................... 32 1.9 APPLICATIONS OF PLANT LECTINS ................................................................................................ 33 1.10 SERINE PROTEASE INHIBITORS................................................................................................. 34

1.10.1 Functional Role of Serine Protease Inhibitors ................................................................... 39 1.10.2 Applications of Serine Protease inhibitors......................................................................... 40

1.11 INITIAL RESEARCH................................................................................................................... 41 1.12 AIMS AND EXPECTED OUTCOMES ............................................................................................ 43

CHAPTER 2 EXTRACTION OF PROTEINS FROM GREVILLEA ROBUSTA.............................. 45

2.1 INTRODUCTION ............................................................................................................................. 45 2.2 AMMONIUM SULFATE PRECIPITATION OF CRUDE PROTEINS. ......................................................... 47 2.3 N-TERMINAL SEQUENCING OF THE CRUDE EXTRACT..................................................................... 49 2.4 BIOASSAYS ................................................................................................................................... 50

2.4.1 Agglutination...................................................................................................................... 51 2.4.2 Granulocyte Agglutination test (GAT) ............................................................................... 51 2.4.3 Granulocyte immunofluorescence Test (GIFT) .................................................................. 52 2.4.4 Sugar blocking granulocyte immunofluorescence test ....................................................... 56 2.4.5 Bioassay results for the crude extract of G.robusta ........................................................... 59

2.5 CONCLUSION ................................................................................................................................ 61

CHAPTER 3 PURIFICATION AND CHARACTERISATION OF A LECTIN ISOLATED FROM THE SEEDS OF GREVILLEA ROBUSTA. ........................................................................................... 63

3.1 INTRODUCTION ............................................................................................................................. 63 3.2 PURIFICATION OF THE LECTIN FROM G.ROBUSTA........................................................................... 64 3.3 BIOASSAYS ................................................................................................................................... 67

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3.4 N-TERMINAL SEQUENCING OF THE LECTIN.................................................................................... 70 3.5 CONCLUSION ................................................................................................................................ 72

CHAPTER 4 PURIFICATION OF THE GR1.HPLC PROTEIN ...................................................... 74

4.1 INTRODUCTION ............................................................................................................................. 74 4.1 INITIAL PURIFICATION OF THE PROTEINS FROM THE CRUDE EXTRACT OF G.ROBUSTA .................. 75

4.1.1 Gel Filtration (GF) Chromatography ................................................................................ 75 4.1.2 Ion Exchange (IEX) Chromatography................................................................................ 77

4.2 LARGE SCALE PREPARATION OF THE CRUDE EXTRACT FROM G.ROBUSTA...................................... 83 4.3 FURTHER PURIFICATION OF G.ROBUSTA PROTEINS........................................................................ 90

4.3.1 Further purification of the GR1.Qseph and SO4/5.Qseph proteins. .................................. 91 4.3.2 Further purification of the GR1.HPLC protein.................................................................. 92 4.3.3 Bioassays............................................................................................................................ 95 4.3.4 Purification of the GR1.HighQ protein .............................................................................. 96

4.4 N-TERMINAL SEQUENCING ........................................................................................................... 98 4.5 DETERMINATION OF THE FULL AMINO ACID SEQUENCE AND SEQUENCE ALIGNMENT STUDIES OF THE GR1.HPLC PROTEIN..................................................................................................................... 100 4.6 MASS SPECTROSCOPY (MS) OF THE GR1.HPLC PROTEIN ......................................................... 105 4.7 CONCLUSION .............................................................................................................................. 108

CHAPTER 5 PURIFICATION & CHARACTERISATION OF THE GR1.GF PROTEIN .......... 110

5.1 INTRODUCTION ........................................................................................................................... 110 5.2 EXTRACTION OF THE PROTEINS FROM THE SEEDS OF G.ROBUSTA. ............................................... 111 5.3 PURIFICATION OF THE GR1.GF PROTEIN FROM THE CRUDE EXTRACT ........................................ 112 5.4 BIOASSAYS ................................................................................................................................. 115 5.5 N-TERMINAL SEQUENCING OF THE GR1.GF PROTEIN ................................................................. 117 5.6 MASS SPECTROSCOPY OF THE GR1.GF PROTEIN........................................................................ 118 5.7 SERINE PROTEASE INHIBITION ASSAYS........................................................................................ 119 5.8 CONCLUSION .............................................................................................................................. 121

CHAPTER 6 NMR ASSIGNMENT OF THE GR1 PROTEIN FROM GREVILLEA ROBUSTA. 123

6.1 INTRODUCTION ........................................................................................................................... 123 6.2 NMR SPECTROSCOPY................................................................................................................. 125

6.2.1 One dimensional NMR experiments ................................................................................. 125 6.2.2 Two dimensional NMR experiments ................................................................................. 127

6.2.2.1 Correlated spectroscopy (COSY) and Double quantum filtered COSY (DQF-COSY) ..............129 6.2.2.2 Total correlation spectroscopy (TOCSY)....................................................................................130 6.2.2.3 Nuclear Overhauser Enhancement spectroscopy (NOESY)........................................................131

6.3 THE 1H NMR ASSIGNMENT OF THE GR1 PROTEIN ..................................................................... 133 6.3.1 Solvent suppression .......................................................................................................... 133 6.3.2 Spin system identification................................................................................................. 135 6.3.3 Sequential Assignment of the GR1 protein ....................................................................... 139

6.3.3.1 Sequential assignment of Residues 1-29 .....................................................................................141 6.3.3.2 Sequential assignment of Residues 30-46 ...................................................................................145 6.3.3.3 Sequential assignment of Residues 47-61 ...................................................................................148

6.4 CONCLUSION .............................................................................................................................. 153

CHAPTER 7 STRUCTURAL STUDIES OF THE GR1 PROTEIN................................................. 155

7.1 INTRODUCTION ........................................................................................................................... 155 7.2 SECONDARY STRUCTURE OF THE GR1 PROTEIN ......................................................................... 155

7.2.1 Prediction of secondary structure .................................................................................... 155 7.2.2 Secondary structure assignment....................................................................................... 159

7.3 POSITIONING OF THE DISULFIDE BRIDGES IN THE GR1 PROTEIN ................................................. 161 7.4 THREE DIMENSIONAL STRUCTURE OF THE GR1 PROTEIN............................................................ 164

7.4.1 Structural Restraints......................................................................................................... 164 7.4.2 Structural Calculations .................................................................................................... 165

7.4.2.1 Initial GR1 structures ..................................................................................................................166 7.5 FURTHER REFINEMENT OF THE GR1 PROTEIN............................................................................. 168 7.6 THE OPTIMISED 3D STRUCTURE OF THE GR1 PROTEIN. .............................................................. 174

7.6.1 The structure of section one of the GR1 protein............................................................... 175 7.6.2 The structure of section two of the GR1 protein............................................................... 179 7.6.3 The structure of section three for the GR1 protein........................................................... 183

7.7 CONCLUSION .............................................................................................................................. 186

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CHAPTER 8 CONCLUSIONS............................................................................................................. 190

CHAPTER 9 EXPERIMENTAL ......................................................................................................... 196

9.1.1 Ammonium sulfate precipitation of proteins. ................................................................... 196 9.1.2 Ammonium sulfate precipitation of proteins using protease inhibitors............................ 197

9.2 POLYACRYLAMIDE GEL ELECTROPHORESIS ................................................................................ 197 9.2.1 Sodium Dodecyl Sulfate (SDS) PAGE.............................................................................. 197 9.2.2 Native PAGE .................................................................................................................... 198

9.3 PROTEIN CONCENTRATION ESTIMATION ..................................................................................... 198 9.4 PURIFICATION OF THE GR1 PROTEIN - PART 1............................................................................ 199

9.4.1 Ion exchange chromatography (IEX) ............................................................................... 199 9.4.2 Reverse phase-HPLC ....................................................................................................... 200 9.4.3 High Q chromatography .................................................................................................. 200

9.5 PURIFICATION OF THE GR1 PROTEIN – PART 2 ........................................................................... 200 9.5.1 Gel filtration chromatography ......................................................................................... 200

9.6 PURIFICATION OF A LECTIN FROM G.ROBUSTA............................................................................. 201 9.7 N-TERMINAL SEQUENCING OF PROTEINS ISOLATED FROM G.ROBUSTA ........................................ 201

9.7.1 Deglycosylation of proteins.............................................................................................. 201 9.7.2 Native PAGE and Electroblotting of proteins .................................................................. 202

9.8 BIOASSAYS ................................................................................................................................. 202 9.8.1 Biotinylation of proteins................................................................................................... 202 9.8.2 Granulocyte harvest ......................................................................................................... 203 9.8.3 Granulocyte Agglutination Test (GAT) ............................................................................ 204 9.8.4 Granulocyte Immunofluorescence Test (GIFT)................................................................ 204 9.8.5 Sugar blocking GIFT........................................................................................................ 205

9.9 MASS SPECTROSCOPY................................................................................................................. 206 9.10 NMR SPECTOSCOPY .............................................................................................................. 206

9.10.1 NMR measurements.......................................................................................................... 206 9.10.2 NMR distance restraints................................................................................................... 207 9.10.3 Structure calculations....................................................................................................... 208 9.10.4 Further refinement of the generated structures................................................................ 209

APPENDIX A METHODOLOGY USED TO DETERMINE THE FULL AMINO ACID SEQUENCE OF THE GR1 PROTEIN FROM GREVILLEA ROBUSTA...................................... 211

A-1 RNA EXTRACTION...................................................................................................................... 211 A-2 3’RACE ..................................................................................................................................... 212 A-3 5’RACE ..................................................................................................................................... 214 A-4 SEQUENCING OF THE CDNA ....................................................................................................... 215

APPENDIX B ENZYMATIC INHIBITORY STUDIES OF THE GR1 PROTEIN ISOLATED FROM THE SEEDS OF G.ROBUSTA................................................................................................. 217

B-1 TRYPSIN AND CHYMOTRYPSIN INHIBITION ASSAYS .................................................................... 217

APPENDIX C .............................................................. EXPERIMENTAL RANDOM COIL VALUES 218

APPENDIX D ..................................................................................... STRUCTURE CALCULATIONS 219

D-1 CALIBA AND ANNEAL MACROS USED TO GENERATE THE DYANA STRUCTURES .................. 219 D-2 STEREOCHEMICAL QUALITY OF THE DYANA STRUCTURES ...................................................... 220 D-3 STEREOCHEMICAL QUALITY OF THE SYBYL® MINIMISED STRUCTURES .................................... 221

REFERENCES....................................................................................................................................... 224

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List of Figures _____________________________________________________________________________________

List of Figures

FIGURE 1.1: MAMMALIAN CIRCULATORY CELLS........................................................................................... 4 FIGURE 1.2: (A) P-SELECTIN COMPLEXED WITH SIALYL LEX AND (B) THE SIALYL LEX IN THE BINDING SITE

OF THE SELECTIN. ................................................................................................................................ 5 FIGURE 1.3: SCHEMATIC REPRESENTATION OF LEUKOCYTE TRAFFICKING. ................................................... 6 FIGURE 1.4: THE STRUCTURE OF THE FCγRIIIB. ........................................................................................... 9 FIGURE 1.5: MONOSACCHARIDE STRUCTURE.............................................................................................. 13 FIGURE 1.6: THE ARRANGEMENT OF THE HYDROXYL GROUPS AROUND THE PYRANOSE RING..................... 14 FIGURE 1.7: A SCHEMATIC REPRESENTATION OF THE COLLECTIN PROTEINS. .............................................. 16 FIGURE 1.8: THE STRUCTURE OF THE MBP-A TAKEN FROM THE SIDE (A) AND THE TOP (B). ..................... 17 FIGURE 1.9: STRUCTURAL SIMILARITIES OF THE CRD BETWEEN THE GALECTIN-7 AND LEGUME LECTINS. 21 FIGURE 1.10: THE STRUCTURE OF THE C-REACTIVE PROTEIN (CRP) AND THE SERUM AMYLOID COMPONENT

(SAP). ............................................................................................................................................... 22 FIGURE 1.11: THE STRUCTURES OF LEGUME LECTINS. ................................................................................ 26 FIGURE 1.12: THE STRUCTURE OF THE SNOWDROP LECTIN COMPLEXED WITH METHYL α-D-MANNOSIDE (A)

AND A CLOSE UP OF THE CARBOHYDRATE BINDING SITE (B).............................................................. 27 FIGURE 1.13: THE CRYSTAL STRUCTURE OF THE RICIN A-CHAIN. ............................................................... 30 FIGURE 1.14: THE STRUCTURE OF THE JACALIN LECTIN.............................................................................. 31 FIGURE 1.15: SEQUENTIAL ALIGNMENT OF THE MEMBERS OF THE BOWMAN-BIRK INHIBITOR FAMILY. ..... 36 FIGURE 1.16: THE STRUCTURE OF THE BOWMAN-BIRK INHIBITOR. ............................................................ 39 FIGURE 2.1: GREVILLEA ROBUSTA OR SILKY OAK TREE. .............................................................................. 46 FIGURE 2.2: SDS (A) AND NATIVE (B) PAGE OF THE CRUDE EXTRACT FROM G.ROBUSTA. ....................... 47 FIGURE 2.3: THE BIOTINYLATION OF PROTEINS........................................................................................... 53 FIGURE 2.4: THE SCHEMATIC REPRESENTATION OF THE GIFT BIOASSAY. .................................................. 55 FIGURE 2.5: SCHEMATIC REPRESENTATION OF THE SUGAR BLOCKING GIFT BIOASSAY. ............................ 58 FIGURE 3.1: AFFINITY CHROMATOGRAPHY OF THE CRUDE EXTRACT FROM G.ROBUSTA.............................. 66 FIGURE 3.2: NATIVE PAGE OF THE ELUTED FRACTIONS............................................................................. 67 FIGURE 3.3: N-TERMINAL SEQUENCING RESULTS OF THE LECTIN................................................................ 70 FIGURE 4.1: ELUTED PROTEINS FROM G.ROBUSTA USING GEL FILTRATION CHROMATOGRAPHY.................. 76 FIGURE 4.2: SDS PAGE (A) AND NATIVE PAGE (B) OF ELUTED GF.S200.PK2 FROM GEL FILTRATION

CHROMATOGRAPHY........................................................................................................................... 77 FIGURE 4.3: ANION EXCHANGE CHROMATOGRAPHY OF ELUTED PROTEINS FROM G.ROBUSTA..................... 78 FIGURE 4.4: THE ELUTION PROFILE OF THE CRUDE EXTRACT AFTER MODIFICATIONS TO THE ELUTION

BUFFER AND GRADIENT. .................................................................................................................... 79 FIGURE 4.5: MODIFICATIONS TO THE SALT CONCENTRATION, FLOW RATE AND GRADIENT CONDITIONS..... 80 FIGURE 4.6: NATIVE PAGE OF THE ELUTED PEAKS FROM IEX CHROMATOGRAPHY................................... 81 FIGURE 4.7: ADJUSTMENT OF THE GRADIENT CONDITIONS USING A 10ML Q -SEPHAROSE FF COLUMN. ..... 82 FIGURE 4.8: LARGE SCALE Q-SEPHAROSE COLUMN AT PH 8.0.................................................................... 84 FIGURE 4.9: LARGE SCALE Q-SEPHAROSE COLUMN AT PH 8.5.................................................................... 88 FIGURE 4.10: HYDROXYAPATITE CHROMATOGRAPHY OF THE PROTEINS SO1/2 AND SO4/5 DERIVED FROM

THE Q SEPHAROSE METHOD............................................................................................................... 92 FIGURE 4.11: RP-HPLC OF GR1/4 PROTEINS USING THE GRADIENT 25-32% ACETONITRILE/0.1% TFA. .. 93 FIGURE 4.12: RP-HPLC USING AN ISOCRATIC FLOW RATE AT 29% ACETONITRILE/TFA 0.1%. ................. 94 FIGURE 4.13: THE SEPARATION OF GR1/4.QSEPH PROTEINS USING A HIGH Q COLUMN. ............................ 97 FIGURE 4.14: N-TERMINAL SEQUENCES OF PROTEINS SEEN ON NATIVE PAGE. .......................................... 98 FIGURE 4.15: COMPARISON BETWEEN THE FULL AMINO ACID SEQUENCE AND THE N-TERMINAL

SEQUENCING RESULTS FOR THE GR1 PROTEIN................................................................................. 100 FIGURE 4.16: SEQUENCE ALIGNMENT OF THE GR1 PROTEIN WITH A NUMBER OF PROTEASE INHIBITORS

FROM THE BOWMAN-BIRK PROTEASE INHIBITOR FAMILY................................................................ 102 FIGURE 4.17: POSITION OF THE DISULFIDE BRIDGES OF THE BOWMAN-BIRK INHIBITOR AND SEQUENCE

ALIGNMENT WITH THE GR1 PROTEIN. ............................................................................................. 103 FIGURE 4.18: POSITIVE ELECTROSPRAY OF THE GR1.HPLC PROTEIN. ..................................................... 106 FIGURE 5.1: NATIVE PAGE OF THE CRUDE EXTRACTS PROCESSED WITHOUT AND WITH PROTEASE

INHIBITORS. ..................................................................................................................................... 112 FIGURE 5.2: GEL FILTRATION CHROMATOGRAPHY OF THE CRUDE EXTRACT (CONTAINING PROTEASE

INHIBITORS)..................................................................................................................................... 114 FIGURE 5.3: NATIVE PAGE OF ELUTED FRACTIONS FROM GEL FILTRATION CHROMATOGRAPHY. ............ 114 FIGURE 5.4: N-TERMINAL SEQUENCING HOMOLOGY OF THE ELUTED GR1 PROTEINS. .............................. 117 FIGURE 5.5: MASS SPECTRUM OBTAINED FOR THE GR1.GF PROTEIN. ...................................................... 119 FIGURE 6.1: 1D SPECTRUM OF THE AMIDE REGION FOR THE GR1 PROTEIN............................................... 126

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List of Figures _____________________________________________________________________________________

FIGURE 6.2: THE 1D NMR SPECTRA FOR THE AMIDE REGION OF THE (A) GR1.HPLC AND (B) GR1.GF PROTEINS......................................................................................................................................... 127

FIGURE 6.3: THE SCHEMATIC REPRESENTATION OF THE NOESY EXPERIMENT......................................... 128 FIGURE 6.4: PULSE SEQUENCE FOR THE (A) COSY AND (B) DQF-COSY EXPERIMENTS......................... 129 FIGURE 6.5: THE HN-Hα REGION OF THE DQF-COSY EXPERIMENT FOR THE GR1 PROTEIN. .................. 130 FIGURE 6.6: THE PULSE SEQUENCE FOR THE TOCSY EXPERIMENT. ......................................................... 131 FIGURE 6.7: THE (A) SCHEMATIC PULSE SEQUENCE FOR THE NOESY EXPERIMENT AND (B) THE HN-HN

REGION OF THE NOESY SPECTRA FOR THE GR1 PROTEIN............................................................... 132 FIGURE 6.8: SCHEMATIC REPRESENTATION OF THE WATER SUPPRESSION SEQUENCES FOR (A)

PRESATURATION AND (B) WATERGATE. ..................................................................................... 134 FIGURE 6.9: AN EXAMPLE OF A 2D SPECTRUM.......................................................................................... 136 FIGURE 6.10: A SUMMARY OF THE IDENTIFIED SPIN SYSTEMS IN THE TOCSY SPECTRUM FOR THE GR1

PROTEIN........................................................................................................................................... 138 FIGURE 6.11: SEQUENTIAL ASSIGNMENT OF THE PROTEIN. ....................................................................... 139 FIGURE 6.12: THE TOCSY SPECTRA FOR THE GR1 PROTEIN THAT SHOWS THE REGIONS OF OVERLAPPING

PEAKS. ............................................................................................................................................. 140 FIGURE 6.13: THE FINGERPRINT REGION OF NOESY SPECTRA OF THE GR1 PROTEIN AT 303 K IN 18%

CD3CN/ H2O. ................................................................................................................................ 143 FIGURE 6.14: THE HN-HN REGION OF THE NOESY SPECTRA FOR THE GR1 PROTEIN. ............................ 144 FIGURE 6.15: THE (A) HNI –HNI+1 AND (B) HαI-HNI+1 CONNECTIVITIES FOR RESIDUES 32-46 WITHIN THE

GR1 PROTEIN. ................................................................................................................................. 147 FIGURE 6.16: THE (A) HN-HN AND (B) HαI-HNI+1 CONNECTIVITIES FOR THE RESIDUES 48-61 SEEN IN THE

NOESY SPECTRA............................................................................................................................ 149 FIGURE 7.1: SECONDARY STRUCTURE PREDICTION OF THE GR1 PROTEIN. ............................................... 156 FIGURE 7.2: SECONDARY STRUCTURE PREDICTION COMPARISON BETWEEN THE GR1 PROTEIN (THIS WORK)

AND BOWMAN-BIRK INHIBITOR (WERNER & WEMMER, 1991) FOR THE TRYPSIN-BINDING REGION......................................................................................................................................................... 158

FIGURE 7.3: SECONDARY STRUCTURE PREDICTION COMPARISON BETWEEN THE BOWMAN-BIRK INHIBITOR (WERNER & WEMMER, 1991) AND THE GR1 PROTEIN (THIS WORK) FOR THE CHYMOTRYPSIN-BINDING REGION.............................................................................................................................. 159

FIGURE 7.4: SCHEMATIC REPRESENTATION OF THE HYDROGEN BONDS BETWEEN THE RESIDUES 10-15 AND 19-24............................................................................................................................................... 160

FIGURE 7.5: THE CONSERVATION OF CYSTEINE RESIDUES WITHIN A NUMBER OF BOWMAN-BIRK INHIBITORS AND THE GR1 PROTEIN (THIS WORK). ............................................................................................. 162

FIGURE 7.6: THE NUMBER OF NOE UPPER DISTANCE LIMITS PER RESIDUE IN THE AMINO ACID SEQUENCE OF THE GR1 PROTEIN. .......................................................................................................................... 167

FIGURE 7.7: CONVERGED STRUCTURES OF THE RESIDUES 8-25 OF THE GR1 PROTEIN (A) BEFORE AND (B) AFTER MINIMISATION USING SYBYL®. ........................................................................................... 171

FIGURE 7.8: CONVERGED STRUCTURES OF THE RESIDUES 29-48 OF THE GR1 PROTEIN (A) BEFORE AND (B) AFTER MINIMISATION USING SYBYL®. ........................................................................................... 172

FIGURE 7.9: CONVERGED STRUCTURES FOR THE RESIDUES 50-61 OF THE GR1 PROTEIN (A) BEFORE AND (B) AFTER MINIMISATION USING SYBYL®...................................................................................... 173

FIGURE 7.10: THE SOLUTION STRUCTURE OF THE GR1 PROTEIN............................................................... 174 FIGURE 7.11: CONVERGED STRUCTURES OF THE TRYPSIN-BINDING REGION OF THE GR1 PROTEIN........... 176 FIGURE 7.12: THE SEQUENTIAL ALIGNMENT OF THE TRYPSIN-BINDING REGION OF THE BOWMAN-BIRK

INHIBITOR WITH THE GR1 PROTEIN. ................................................................................................ 177 FIGURE 7.13: STRUCTURAL SIMILARITIES BETWEEN THE (A) GR1 AFTER MINIMISATION AND THE (B)

BOWMAN-BIRK INHIBITOR. ............................................................................................................. 178 FIGURE 7.14: TOP 10 REFINED STRUCTURES FOR RESIDUES 28-48 IN THE GR1 PROTEIN. ......................... 180 FIGURE 7.15: THE SEQUENTIAL SIMILARITIES BETWEEN THE CHYMOTRYPSIN-BINDING REGION OF THE

BOWMAN-BIRK INHIBITOR AND THE GR1 PROTEIN. ........................................................................ 180 FIGURE 7.16: THE CHYMOTRYPSIN-BINDING REGION OF THE (A) BOWMAN-BIRK INHIBITOR AND (B) THE

MINIMISED GR1 PROTEIN. ............................................................................................................... 182 FIGURE 7.17: SEQUENCE ALIGNMENT OF RESIDUES 50-61 FROM THE GR1 PROTEIN WITH THE

CORRESPONDING REGION IN THE BOWMAN-BIRK INHIBITOR. .......................................................... 183 FIGURE 7.18: THE SOLUTION STRUCTURE OF THE FINAL SECTION OF THE GR1 PROTEIN. ......................... 184 FIGURE 7.19: STRUCTURAL DIFFERENCES BETWEEN THE (A) BOWMAN-BIRK INHIBITOR (WERNER &

WEMMER, 1992) AND (B) THE GR1 PROTEIN (THIS WORK)............................................................. 185 FIGURE 7.20: THE SOLUTION STRUCTURE OF THE GR1 PROTEIN............................................................... 187 FIGURE 8.1: SEQUENCE ALIGNMENT OF THE GR1 PROTEIN AND THE BOWMAN-BIRK INHIBITOR. ............ 192 FIGURE A-1: A LIST OF THE DEGENERATIVE PRIMERS DESIGNED FROM THE N-TERMINAL SEQUENCING

RESULTS. ......................................................................................................................................... 213 FIGURE A-2: THE SPECIFIC PRIMERS DEVELOPED FROM THE 3’RACE RESULTS. ...................................... 214

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List of Figures _____________________________________________________________________________________

FIGURE A-3: THE COMPLETE AMINO ACID SEQUENCE OF THE GR1 PROTEIN FROM THE SEEDS OF G.ROBUSTA......................................................................................................................................................... 216

FIGURE B-1: THE INHIBITION CURVES FOR THE GR1 PROTEIN. ................................................................. 217 FIGURE D-1: PROCHECK RESULTS OF THE DYANA GENERATED STRUCTURES..................................... 220 FIGURE D-2: PROCHECK RESULTS OF THE SYBYL® MINIMISED STRUCTURES AFTER (A) 2000 STEPS AND

(B) 4000 STEPS................................................................................................................................ 221 FIGURE D-3: PROCHECK RESULTS AFTER 8000 STEPS OF MINIMISATION USING SYBYL®. ................... 222

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List of Tables _____________________________________________________________________________________

List of Tables

TABLE 1.1: SUMMARY OF THE REVISED NOMENCLATURE OF GRANULOCYTE ALLOANTIGENS.#.................... 8 TABLE 1.2: THE MEMBERS OF THE I-TYPE LECTIN FAMILY.......................................................................... 19 TABLE 1.3: THE MEMBERS OF THE GALECTIN FAMILY.#.............................................................................. 20 TABLE 1.4: MEMBERS OF THE LEGUME LECTIN FAMILY.............................................................................. 25 TABLE 1.5: FAMILIES OF PLANT PROTEIN PROTEASE INHIBITORS. ............................................................... 34 TABLE 1.6: PRELIMINARY BIOLOGICAL CHARACTERISTICS OF THE 8 SPECIES OF PLANTS. .......................... 42 TABLE 2.1: N-TERMINAL SEQUENCES OF THE CRUDE EXTRACT FROM G.ROBUSTA.# .................................... 49 TABLE 2.2: THE GAT AND GIFT RESULTS OF THE CRUDE EXTRACT FROM G.ROBUSTA.# ............................ 59 TABLE 2.3: SUGAR-BLOCKING GIFT RESULTS OF THE CRUDE EXTRACT FROM G.ROBUSTA.# ...................... 60 TABLE 3.1: GAT AND GIFT BIOASSAY RESULTS OF THE PROTEINS ELUTED FROM THE MANNAN-AGAROSE

COLUMN. # ......................................................................................................................................... 68 TABLE 3.2: CONFIRMATION OF THE SUGAR SPECIFICITY OF THE LECTIN.† ................................................... 69 TABLE 4.1: NATIVE PAGE AND GAT BIOASSAY RESULTS OF ELUTED FRACTION FROM THE LARGE-SCALE

PURIFICATION OF PROTEINS FROM G.ROBUSTA SEEDS. # ..................................................................... 85 TABLE 4.2: SATURATION POINT ON GRANULOCYTES USING THE QSEPH.8.5.PK2 FRACTION. # .................... 86 TABLE 4.3: SUGAR-BLOCKING GIFT RESULTS OF QSEPH.8.PK2. †.............................................................. 87 TABLE 4.4: SUMMARY OF NATIVE PAGE AND BIOASSAY RESULTS OF THE PROTEINS ELUTED FROM THE

LARGE-SCALE Q-SEPHAROSE COLUMN AT PH 8.5. #........................................................................... 89 TABLE 4.5: GAT BIOASSAY AND THE BIOLOGICAL EFFECTS OF THE SOLVENTS ON THE CRUDE EXTRACT. # 95 TABLE 4.6: THE GAT AND GIFT BIOASSAY RESULTS OF THE ELUTED FRACTIONS USING HIGH-Q RESIN. # 98 TABLE 5.1: GAT AND GIFT BIOASSAY RESULTS. ..................................................................................... 116 TABLE 6.1: THE SUMMARY OF THE ASSIGNMENT OF THE GR1 PROTEIN.................................................... 150 TABLE 6.2: CHEMICAL SHIFT ASSIGNMENT OF THE GR1 PROTEIN IN 18% CD3CN/ H2O PH 3.5 AT 303 K.

........................................................................................................................................................ 151 TABLE 7.1: SUMMARY OF NMR RESTRAINTS AND STRUCTURAL STATISTICS FROM DYANA FOR ALL 20

STRUCTURES.................................................................................................................................... 168 TABLE 7.2: THE SUMMARY OF THE 20 ENERGY-MINIMIZED NMR STRUCTURES OF THE GR1 PROTEIN

BEFORE AND AFTER SYBYL® MINIMISATION. ................................................................................. 169

___________________________________________________________________ xi

Acknowledgments ____________________________________________________________________________________

Acknowledgments

Firstly, I would like to thank all of my supervisors; Dr Robyn Minchinton, Dr Greg

Pierens and Professor Ron Quinn for giving me the opportunity to undertake this project

and for providing unfailing support and advice when it was greatly needed.

I would like to thank the Australian Postgraduate Award (industry) and Natural

Products Discovery (formally AstraZeneca) for funding me throughout the duration of

the project.

A very special thanks to all of the staff at the Australian Red Cross Blood Service

(ARCBS) and Natural Products Discovery for their continual help, guidance and

support throughout the years. I would like to thank Helen Clague for her contribution to

the project, as without her sequencing results, the NMR assignment would have been

very challenging.

To my family, thank you very much for the support and encouragement through all the

good and the difficult times. I would also like to thank Frank Stevenson and Lyle

McMillen, for reviewing the thesis.

To all my friends, in particular, Jay, Elizabeth, Heather, Matthew, Sylvia, and Rama,

thank you for your support and listening to me over the years. Thank you for not

saying, “Have you finished yet?”

Finally I would like to thank Shane who has put up with me during my self-doubting

stages, and also providing support throughout.

_________________________________________________________________ xii

Statement of Originality ____________________________________________________________________________________

Statement of Originality

This work has not previously been submitted for a degree or diploma in any university.

To the best of my knowledge and belief, the thesis contains no material previously

published or written by another person except where due reference is made in the thesis

itself.

………………………………

Sarah Jane Kruger

_________________________________________________________________ xiii

Abbreviations ____________________________________________________________________________________

Abbreviations

Ala alanine Arg arginine Asn asparagine Asp aspartic acid BBI Bowman-Birk inhibitor BLAST basic local alignment search tool cDNA copy DNA CD3CN acetonitrile CHT hydroxyapatite COSY correlated spectroscopy CRD carbohydrate recognition domain Cys cysteine DMSO dimethyl-sulfoxide DSS sodium 3-(trimethylsilyl)-1-propanesulfonic acid DQF-COSY double quantum filtered correlated spectroscopy DYANA dynamic algorithm for NMR applications EDTA ethylenediaminetetra acetic acid FID Fourier Induced Decay FITC fluorescein isothiocyanate FT Fourier Transform fuc fucose gal galactose GalNAc N-acetylgalactosamine GAT granulocyte agglutination test GIFT granulocyte immunofluorescence test glc glucose GlcNAc N-acetylglucosamine Gln glutamine Glu glutamic acid Gly glycine His histidine HNA human neutrophil antigen Ile isoleucine lac lactose Leu leucine LMW low molecular weight Lys lysine malt maltose man mannose MBP mannose binding protein MCF mean channel fluorescence Met methionine MWCO molecular weight cut off NaCl sodium chloride NeuNAc N-acetylneuraminic acid NMR nuclear magnetic resonance

_________________________________________________________________ xiv

Abbreviations ____________________________________________________________________________________

NOESY Nuclear Overhauser Enhancement spectroscopy PAGE polyacrylamide gel electrophoresis PBS phosphate buffered saline PDB protein databank Phe phenylalanine PIC protease inhibitor cocktail PMSF phenylmethylsulfonyl fluoride Pro proline PVDF polyvinylidene difluride rf radio frequency RMSD root mean square deviation SDS sodium dodecyl sulfate Ser serine SiaLex sialyl Lewis x isomer SiaLea sialyl Lewis a isomer TBS Tris buffered saline Thr threonine TOCSY total correlation spectroscopy Trp tryptophan Tyr tyrosine Val valine

_________________________________________________________________ xv

Chapter 1 Introduction _________________________________________________________________________________

Chapter 1 Introduction

_________________________________________________________________ 1


Chapter 1 Introduction

1.1 Carbohydrates and Cell Recognition

Cell recognition plays an important role in a number of biological events including

processes such as fertilisation, embryogenesis, cell migration, organ formation, immune

defence and microbial infection (Sharon & Lis, 1989). In order for these processes to

occur, specific interactions between molecules are essential. Emil Fisher in 1897

pioneered the concept that specific molecules interact with each other in a similar way

that a key fits specifically into a lock (Sharon & Lis, 1993). This “ lock and key”

concept is still used today to explain the specific interactions between enzymes and

substrates.

In the 1970’s it was established that almost all cells have carbohydrates on their surfaces

in the form of glycoproteins, glycolipids and polysaccharides. However at this time, the

possibility of carbohydrates on the cell surfaces playing a role in cell recognition was a

very farfetched concept due to the complexity of the surface structures. It was this

complexity which prompted the notion that carbohydrates could encode large amounts

of biological information on their monomer units.

Theoretically, peptides and oligonucleotides could exchange information based on the

number of monomeric units they contain and also their sequence. Carbohydrates on the

other hand, could encode more information due to the position and the configuration (α

or β) of the glycosidic units (Sharon and Lis, 1989). If two molecules of a single amino

acid or nucleotide were taken for example, they could form one dipeptide or one

dinucleotide while 2 molecules of a monosaccharide could form 11 different

_________________________________________________________________ 2


disaccharides. Therefore, if 4 different amino acids were taken, only 24 different

tetrapeptides can be formed while 4 different monosaccharides can form a staggering

35560 different tetrasaccharides. It could be proposed that the carbohydrate may be

involved in transfer of biological information from cell to cell via its receptor.

Therefore, the probability of carbohydrates playing a role in the transfer of biological

information was possible.

1.2 Leukocyte Function

The circulatory system of animals is made up of 2 types of cells: red blood cells and

white blood cells. Both of these cells are both derived from the pluripotent stem cell in

the bone marrow. Red blood cells or erythrocytes function by transporting oxygen and

carbon dioxide (bound to haemoglobin) in the blood around the body. The white blood

cells or leukocytes function as the defence system against infection from foreign

material and are able to migrate across the blood vessel walls to the site of infection

(Alberts et. al., 1994).

A number of different types of cells are defined as leukocytes and they include

polymorphonuclear granulocytes, monocytes and lymphocytes (Figure 1.11).

Granulocytes are made up of three different cell types called neutrophils, basophils and

eosinophils where the neutrophils are the most common of the granulocytes and

function in conjunction with the macrophages, to destroy and phagocytise small

microorganisms. Basophils secrete histamine to help in inflammatory reactions and

eosinophils destroy parasites and regulate allergic reaction responses and monocytes

mature into macrophages, which aids in the destruction of foreign matter. Lymphocytes

_________________________________________________________________ 3


are divided into either T- or B-lymphocytes, which are commonly referred to as T- and

B-cells (Alberts et. al, 1994).

l

Polymorphonuclear ulocytes Gran

Figure 1.1: Mammalian circulato

The recruitment of the leukocytes to

a group of structurally related lect

lectin family, are defined as C

transmembrane proteins and are lo

1993). These proteins are made up

N-terminus (outside of the cell) a

_____________________________

Pluripotent stem cel

ry c

th

ins

a2+

cate

of

nd

___

Leukocytes
Erythrocytes
s Lymphocytes

B- & T- lymphocytes

ells.

e site of injury resul

called selectins. S

dependent protein

d on the surface o

a carbohydrate reco

an epidermal grow

_______________

Monocyte

s
neutrophils basophils eosinphil
tin

el

s

f

g

th

__

macrophage

g in inflammation involves

ectins belong to the C-type

are classified as type I

cells (Drickamer & Taylor,

nition domain (CRD) at the

factor (EGF)-like domain.

________________ 4


This is followed by a series of short complement binding proteins like units, a

membrane spanning region and finally the C-terminus located in the cytoplasm (Weis et

l, 1998, Lis & Sharon, 1998).

1,3-linked fucose to be important for binding to all

., 1999).

mplexed with Sialyl Lex and (B) the Sialyl Lex in the

was taken from the RSCB PDB database

DB ID: 1G1R: Somers et. al., 2000).

a

These selectins are specific for sialyl-Lex and its isomer sialyl-Lea oligosaccharide

structures and this interaction is shown in Figure 1.2. Sialyl Lewisx is a sialyated,

fucocylated tetrasaccharide and is defined by the following structure

NeuNAcα2→3Galβ1→4(Fucα1→3) GlcNAc-. Mutational studies on this

tetrasaccharide have shown the α

selectins (Fukuda et. al

A

B

Figure 1.2: (A) P-selectin co

binding site of the selectin.

The Sialyl Lex is shown in yellow. The structure

(P

_________________________________________________________________ 5


Selectins mediate the adhesion of the circulating leukocytes to the endothelial cells,

which leads to the removal and relocation of cells to the site of infection. The

mechanism behind the leukocyte removal involves the specific binding of the selectin

(located on either the leukocyte (L-selectin) or on the endothelium (E- or P- selectin))

to their corresponding receptor (SiaLex and SiaLea). Prior to this binding, the leukocyte

“rolls” along the endothelium, making and breaking bonds as it rolls, resulting in the

slowing down and the eventual binding of the circulating cells to the endothelial

ceptors (Figure 1.3). Once bound, the leukocytes are removed from circulation and

relocated to the site of infection.

re

F

(

o

S

_

A

igure 1.3: Schematic representation of leuk

) Adhesion and migration of cells through the enA

f (B) selectins to lymphocytes and (C) neutrop

pringer 1990.

_____________________________________

B

ocyte trafficking.

dothelial wall. Mechanism for the adhesion

hils to the endothelial cells. Taken from

___________________________ 6


P-selectins are found on both the platelets and endothelial cells and are expressed within

a short period of time (minutes) by molecules such as thrombin, histamine, substance-P

and peroxide. L-selectins found on lymphocytes such as T-cells are involved in the

circulation of lymphocytes through the peripheral lymph nodes and their expression

decreases with the introduction of inflammatory mediators such as cytokines. However,

an increase in the level of cytokines can result in the activation of the E-selectins, which

re found on the surface of the endothelium. E-selectins bind to monocytes and

infection (chemotaxis)) and phagocytosis. Degranulation of

reign material using peroxides formed from the neutrophil and the intracellular

destruction of the foreign material are also functions of the neutrophil (De Haas et. al,

1995; Zhou et. al. 1993).

he allele, are also shown in this table (Clay and

a

neutrophils and they are expressed within hours rather than minutes like the P-selectins

(Lasky, 1992).

Glycoproteins and glycolipids found on the surface of neutrophils are involved in a

number of documented neutrophil functions. These functions include the initial

identification of foreign material (which results in the recruitment and migration of

neutrophils to the site of

fo

1.3 Neutrophil Antigens

Like red blood cells, neutrophil surface glycoproteins also contain blood group antigens.

The Granulocyte Antigen and the Australian Society for Blood Transfusion (ASBT)

working parties on platelet and granulocyte serology (1999) have reviewed the

nomenclature of these antigens as shown in Table 1.1. Previous nomenclature of the

neutrophil antigens (NA1, NA2 etc.), the membrane location of the antigen on the

glycoprotein and the location on t

_________________________________________________________________ 7


Stroncek, 1994). Some of the previously assigned neutrophil antigens (NB2, 5a, 9a, &

they

have not been fully characterised.

Table 1.1: Summary of the revised nome #

System nym

9b) are not listed in the table, however they do exist but are not included because

nclature of Granulocyte alloantigens.

Antigen Antigen Location Acro Alleles

HNA-1 HNA-1

HNA-1a FcgRIIIb

HNA-2 HNA-2a gp 50-64 NB1 not defined HNA-3 HNA-3a gp 70-95 5b not defined

NA1 FCGR3B*1b FcgRIIIb NA2 FCGR3B*2

HNA-1c FcgRIIIb SH FCGR3B*3

HNA-4 HNA-4a CD11b MART CD11B*1 HNA-5 HNA-5a CD11a OND CD11A*1

# HNA = Human neutrophil antigen, gp = glycoprotein.

The HNA-1a (NA1) and HNA-1b (NA2) antigens seen in Table 1.1 are located on the

FcγRIIIb receptor. The FcγRIIIb is one of two FcγRIII receptors (or CD16), that have a

low affinity for IgG and is found on the surface of neutrophils where it is anchored via a

glycan-phosphatidylinositol moiety (Vossebeld et. al., 1997). The differences between

the HNA-1a and HNA-1b allotypes are the result of 5 nucleotides that correspond to the

tal number of glycosylation sites within the antigen. The HNA-1b antigen contains 6

in calcium influx, the release of

xygen and the phagocytosis of opsonised bacteria or viruses (Moldovan et al., 1999).

The expression of the receptor is lost when the neutrophil is exposed to inflammatory

9).

to

glycosylation sites compared to 4 for HNA-1a (Deo et. al., 1997). The structure of the

FcγRIIIb determined by X-ray crystallography is shown in Figure 1.4.

Neutrophil activation of the FcγRIIIb often results

o

signals such as cytokines (Moldovan et. al., 199

_________________________________________________________________ 8


Figure 1.4: The structure of the FCγRIIIb.

The FcγRIIIb is made up of two regions of antiparallel β-sheet structures that contain 2 α-

helices. Structures were taken from the RSCB PDB database (PDB ID: 1E4J, Sondrmann et.

al., 2000).

A number of severe disorders are the result of neutrophil antigens including neonatal

alloimmune neutropenia, immune neutropenia after bone marrow transplantation

(decrease in the circulating white blood cells) (Stroncek et. al, 1994), and severe

transfusion reactions resulting in acute lung injury (Bux, 1996; Popovsky et. al., 1992).

The characterisation of these neutrophil antigens (and the receptors they target) would

be advantageous for the diagnosis and treatment of these disorders.

_________________________________________________________________ 9


1.4 Characterisation of Neutrophil Glycoproteins

Protein-carbohydrate interactions are common in nature. Previously, the biochemical

characterisation of neutrophil glycoproteins was completed using murine monoclonal

antibodies. This proved to be very expensive and alternative methods were required.

The discovery of a group of sugar binding proteins (called lectins), which were found to

bind to the neutrophil surface glycoproteins, provided an alternate and more cost

effective method for the study of these surface structures (Minchinton, 1995;

Minchinton et. al., 1997a; Stroncek et. al., 1994; Zhou et. al., 1993).

Lectins were first identified over a century ago in plants where an extract from caster

beans (ricin) was found to agglutinate red blood cells. William Boyd first saw the

discovery of the effects of lectins on different blood groups. He found the extract from

lima beans (Phaseolus limensis) caused agglutination in some individuals but not all

and this lectin extract was found to be specific for blood type A and it also reacted

weakly with blood type B cells (only when concentrated). Powerful agglutinins were

identified and derived from the tufted vetch (Vicca cracca) and were found to have a

stronger specificity for blood type A than for the blood type B or O cells. Boyd

concluded that these proteins were selective in their interactions with the carbohydrates

and cells and that the sugars were immunodeterminates for the specificity of the blood

types (Sharon & Lis, 1995). This “selective” agglutination of red blood cells resulted in

the ABO blood typing system used today.

Since then, lectins have been found in animals, bacteria, viruses and fungi. They are

defined as non-immune proteins that bind reversibly and with high specificity to the

carbohydrate surface structures of the cell (Goldstein et. al, 1980). Each lectin molecule

_________________________________________________________________ 10


contains two or more carbohydrate binding sites, which allows the protein to form

cross-links between the cells resulting in agglutination. The exception to this is the

toxin ricin, which only has one binding site. The majority of these lectins were isolated

using affinity chromatography where specific immobilised carbohydrate resins were

used (Rüdiger, 1998).

_________________________________________________________________ 11


1.5 Carbohydrate specificity of the lectin

Lectins are defined by their ability to bind reversibly to simple or complex

carbohydrates. They can be specific for a range of different carbohydrates and some

will bind with a higher affinity than others will. Different lectins can be specific for the

same carbohydrate and this specificity is usually for foreign glycans (Peumans & Van

Damme, 1998). The multivalency and the spatial relationship with the binding site of

the lectin and its oligosaccharide mediate the lectin function (Rini, 1995; Weis &

Drickamer, 1996).

Lectins are subdivided into groups that are based on their specificity for

monosaccharides. These monosaccharide groups are the mannose/glucose specific,

galactose/GalNAc specific, GlcNAc specific, fucose specific, sialic acid specific

(Goldstein & Poretz, 1986), monocot mannose binding lectins (Van Damme et. al.,

1995) and the maltose/mannose binding lectins (Peumans et. al., 1997). The structures

of these monosaccharides are shown in Figure 1.5. Lectins that do not fall into any of

these groups are referred as “complex carbohydrates” due to their specificity for

complex carbohydrates (Van Damme et. al., 1998).

_________________________________________________________________ 12


)

Fig

All

Van

carb

The

betw

is c

with

___

Mannose (Man)

N-ac

ure 1.5: Monosaccharide stru

sugars are in the D-conformation u

der Waals interactions and h

ohydrate ring has been foun

refore, the positioning of the hy

een the lectin and carbohydrat

hosen by the lectin, the size o

in this site are critical (Drickam

_________________________

Glucose (Glc

etylgalactosamine (GalNAc)

cture.

nless otherwise indicated.

ydrogen bonding with the

d to stabilise the lectin-

droxyl groups, hydrogen bo

e are important. To ensure

f the lectin binding site an

er, 1997).

_______________________

N-acetylglucosamine(GlcNAc)

Galactose (Gal)
N-acetylneuraminic acid
(Neu-Nac)
L-fucose (Fuc)
oxygen atom within the

carbohydrate interaction.

nds and steric exclusions

the specific carbohydrate

d the number of contacts

______________ 13


Structural studies on lectin-carbohydrate interactions have shown the positioning of the

3- and 4- hydroxyl groups within the pyranose ring to be essential for specificity. The

arrangement of the hydroxyl groups for the Man/Glc and GlcNAc lectins were found to

be equatorial at positions 3 & 4 and in the axial conformation at position 4 in the

Gal/GalNAc lectins (Figure 1.6). Therefore, lectins defined as mannose specific will

not bind to the monosaccharide galactose due to the orientation of the hydroxyl group

(Drickamer, 1997).

Figure 1.6: The arrangement of the hydroxyl groups around the pyranose ring.

Lectins that are specific for mannose are usually specific for glucose but are not specific for

galactose due to the positioning of the 4-hydroxyl group (shown in red).

_________________________________________________________________ 14


1.6 Animal lectins

A number of lectins have been identified in animals. These lectins are divided into five

groups that are dependent on their structural homologies rather than their carbohydrate

specificities. These groups are the C-type, I-type, galectins (S-type), pentraxins and P-

type families.

1.6.1 C-type lectins

The C-type or Ca2+-dependent lectin family are made up of a number of proteins that

contain a homologous carbohydrate recognition domain (CRD). The CRD for this

family of proteins are made up of 115-130 amino acids where 18 of these are highly

conserved and 14 are identical. A number of different domains are attached to the

CRD, which forms the bulk of the protein. There are three subfamilies of C-type

lectins: endocytic lectins, collectins and selectins.

Endocytic lectins are type II transmembrane proteins that contain a short N-terminal

cytoplasmic domain, a hydrophobic membrane spanning domain and a neck region

where the C-terminal CRD is linked (Lis and Sharon, 1998). The mammalian and rat

hepatic asialoglycoprotein receptors are two examples of endocytic lectins (Lis &

Sharon, 1998). The macrophage mannose binding lectin is another example of an

endocytic lectin. This lectin differs from other endocytic lectins as it is a type I

transmembrane protein ie. the C-terminus is in the cytoplasm. The extracellular matrix

of the mannose binding lectin contains three domains including a cysteine rich domain,

_________________________________________________________________ 15


fibronectin repeats (that are similar to type II proteins) and a string of 8 CRD’s close to

the membrane (Taylor et al 1992; Taylor & Drickamer, 1993).

Collectins are soluble proteins that contain a cysteine-rich amino terminus, a region of

collagen-like repeats and a α-helical neck region that contains the CRD’s at the C-

terminus. Examples of these proteins include the serum mannose binding protein

(MBP) A & C (Drickamer et al, 1986, Childs et al, 1990), the pulmonary surfactant

proteins, SP-A and SP-D (Haagsman et al 1987, Childs et al, 1992, Persson et al 1990),

and the CL-43 from bovine serum and bovine conglutinin (Haagsman et al 1987).

Collectins can be distinguished from other lectins by the length of their collagenous

domain. The smaller number of Gly-X-Y repeats have been found to form a bouquet

like structure (MBP-A, MBP-C & SP-A) and larger Gly-X-Y repeats form cruciform

like structures (SP-D) as seen in Figure 1.7 (Weis et. al., 1998).

Figure 1.

(A) The c

A) and (C

________

A

7: A schematic representation of th

ollectin monomer is made up of four reg

) the cruciform (SP-D) of collectins.

______________________________

B

e

io

_

C

collectin proteins.

ns. (B) The bouquet form (MBP-A & SP-

__________________________ 16


The mannose binding proteins are made up of a trimer of subunits, formed by the triple

helix of collagenous regions of the protein (Figure 1.8). It is approximately 32 kDa in

size and it is found in the serum for MBP-A (where is circulates as a hexamer of

trimeric units) and in the liver for MBP-C (Rini and Lobsanov, 1999).

A B

Figure 1.8: The structure of the MBP-A taken from the side (A) and the top (B).

The MBP-A is made up of three domains each containing a α-helical region that is followed by

the CRD. Structures were taken from the RSCB PDB database (PDB ID: 1AFA; Kolatkar &

Weis, 1996).

The mannose binding protein functions by directly attaching to pathogens and initiating

the complement cascade, which activates the expression of the MBP-associated

proteases (MASP-1 and MASP-2). The MBP-A recognises the bacteria, Escherichia

coli and Salmonella montevideo and also the fungi in the Candida and Cryptococcus

groups (Weis et. al., 1998; Epstein et. al., 1996).

The surfactant proteins have been found to be the first line of defence against airborne

pathogens that may attach to the fluid lining of the lungs. The SP-A binds to the

Haemophilus influenzae and Streptococcus pneumoniae and the SP-D has been found to

_________________________________________________________________ 17


bind to Klebiella pneumoniae and Escherichia coli. Both of these proteins require other

mechanisms to remove the bound bacteria, as they do not have the ability to fix

complement like the MBP’s (Weis et. al., 1998; Epstein et. al., 1996).

Selectins are made up of three proteins that are involved in the migration of

lymphocytes from the lymph node endothelium and the removal of circulating

neutrophils and monocytes to the site of inflammation and infection (Lis & Sharon,

1998). There are three members of the selectin family and they are the L-selectin (found

on leukocytes) P-selectin (platelets) and E-selectin (endothelium) (Weis et. al., 1998;

Lis & Sharon, 1998). These proteins have been discussed earlier in this chapter

(Section 1.2) and will not be discussed in detail here.

1.6.2 I-type lectins

I-type lectins are members of the immunoglobulin superfamily as their extracellular

domain is made up of immunoglobulin-like structures. A number of proteins are

classified as I-type lectins and they are listed in Table 1.2. These proteins are

characterised as type I transmembrane proteins in which the amino terminal in the

extracellular domain is found to be similar to the variable region (V-type domain) of the

immunoglobulin IgG (Gabius, 1997; Lis & Sharon, 1998).

_________________________________________________________________ 18


Table 1.2: The members of the I-type lectin family. Name Occurrence Carbohydrate ligand ICAM-1 (CD54) endothelial cells, many activated cell types hyaluronic acid PECAM-1 (CD31) platelets, endothelial cells, myeloid and B lymphoid lineage cells Heparin N-CAM central and peripheral nervous system oligomannoside (and complex ?) glycans Heparin Po glycoprotein peripheral nervous system HNK-1 epitope Myelin-associated glycoprotein peripheral nervous system Neu5Acα2-3Gal Sialoadhesin macrophages in hemopoietic and secondary Neu5Acα2-3Gal lymphoid tissues CD22 mature B cells Neu5Acα2-3Gal CD33 myeloid progenitor cells, monocytes Neu5Acα2-3Gal

1.6.3 Galectins

Galectins are a family of β-galactosidase binding lectins found in mammals and they

consist of 1 or 2 highly conserved carbohydrate recognition domains that are 135 amino

acids long (Hughes, 1999). Galectins are thought to be involved in intra- and

extracellular functions such as modulating cell-cell and cell-matrix interactions (Rini,

1995). These lectins have specificity for galactose, however some galectins are have

been found to be specific for lactose and N-acetyllactosamine. There are four different

structural arrangements including monomers and dimers, and large polypeptides (that

contain 1 or 2 copies of the CRD in association with the repeating linker domains).

Table 1.3 lists the galectins that have been identified.

_________________________________________________________________ 19


Table 1.3: The members of the Galectin family.# Name Occurrence Structural Features Galectin -1 (galaptin, L-14) many cell types homodimer; one CRD/subunit (12-16kDa; prototype Galectin-2 lower small intestine; clone from homodimer; one CRD/subunit (14kDa); prototype human hepatoma Galectin-3 (CBP35, Mac-2, IgE- many cell types monomer with one CRD; Pro-,Tyr-, Gly rich repeats binding protein, L-29, L-34 in N-terminal section (29-37kDa); chimera type Galectin-4 colon, small intestine, stomach monomer with 2 partially homologous but distinct CRD oral epithelium, oesophagus connected by a linker region (36kDa); tandom repeat Galectin-5 blood cells monomer with one CRD (17kDa); prototype Galectin-6 small intestine, colon tandom repeat arrangement of two CRDs (33kDa) Galectin-7 keratinocytes one CRD (12.7kDa); prototype Galectin-8 several tissues homologous to galectin-4 & -6 (tandom repeat of two CRDs with unique link peptide: 34kDa) #Taken from Gabius, 1997.

Galectins are divided into three groups based on the differences seen within their

binding specificity in the CRD (the proto-type, the chimera-type and the tandem repeat

groups). The proto-type galectins are small proteins of 15kDa that contain one CRD

and Galectins -1, -2, -5 & -7 & are all members of this group.

The chimera-type galectins are found in mammals only and have an approximate

molecular weight of 30-35 kDa. The C-terminus contains one CRD and a region of

sequence that is rich in proline, tyrosine, glycine and glutamine amino acid residues.

The N-terminus of chimera galectins is not homologous with any other galectins but

there are some regions of similarity with the heteronuclear ribonucleoprotein complex

(hnRNP) to which Galectin-3 is a member of. The last group of lectins in the galectin

family is the tandem repeat group. These lectins Galectin –4, -6, -8, contain two CRD

domains within the single polypeptide chain (Kasai & Hirabayashi, 1996; Hughes,

1999).

_________________________________________________________________ 20


The three dimensional structures of galectin 1 & 2 have been found to be very similar to

the legume lectin family however, there is very little sequence homology between the

two families. The structures of galectin-7 and concanavilin A are shown in Figure 1.9

where the positioning of the combination site was different between the lectins but were

located on the same face of the CRD.

F

l

(

c

a

1

P

i

l

t

a

a

b

_

A

igure 1.9: Structural similarities of t

ectins.

A) Galectin-7 monomer unit (PDB ID: 1B

oncanavilin A (PDB ID: 1CN1; Shoham et

ntiparallel β-sheet structures.

.6.4 Pentraxins

entraxins are a group of lectins that ar

e, the subunits are arranged in a pentam

ectins require Ca2+ to bind to their carb

his family and they are the C-reactive

nd the serum amyloid P component (SA

re thought to act in the early stages of

een found to precipitate the pneumoco

________________________________

B

he CRD between the Galectin-7 and legume

KZ; Leonidas et. al., 1998) and (B) demetalised

. al., 1979). Both proteins contain 5 or 6 stranded

e defined by the arrangement of their subunits,

er formation as seen in Figure 1.10 and these

ohydrates. Two proteins have been defined in

protein (CRP) (Kilpatrick & Volanakis 1991)

P) (Steel & Whitehead 1994). These proteins

the defence of the host cell where the CRP has

ccal somatic C-polysaccharide (Gabius, 1997).

________________________________ 21


A B

Figure 1.10: The structure of the C-reactive protein (CRP) and the serum amyloid

component (SAP).

(A) The C-reactive protein (PDB ID: 1GNH; Shrive et. al., 1996) and (B) the serum amyloid

component (PDB ID: 1SAC; White et. al., 1994). Note the pentamer arrangement of monomer

units that characterise this group of lectins. These structures were taken from the RSCB PDB

databank.

1.6.5 P-type lectins

There are only two lectins that contain the CRD that defines the P-type lectins. These

lectins are the cation dependent mannose 6-phosphate receptors (CD-MPR) and the

insulin growth factor II/ cation independent mannose-6-phosphate receptor (IGF-II/CI-

MPR). They are type-I transmembrane glycoproteins that contain a single

transmembrane domain, a cytoplasmic C terminus and an extracytoplasmic N-terminus

containing the CRD. The CD-MPR contains a single 159 amino acid domain in the N-

terminus and requires cations to bind with the Man-6-P (with high affinity). The IGF-

II/ CI-MPR is larger than the CD-MPR and it is made up of 15 adjacent but homologous

repeating units with two carbohydrate-binding domains (domain 3 & 9) that will bind

Man-6-P in the absence of cations (Rini and Lobsanov, 1999; Kornfeld, 1992; Lis &

Sharon, 1998).

_________________________________________________________________ 22


The structure of the CD-MPR has been determined by X-ray crystallography in the

presence of manganese and Man-6-P. The receptor is a dimer, which has been found to

contain a relatively large interface. Binding studies between the CD-MPR and the Man-

6-P have shown the C2 hydroxyl group, the 6-phosphate group and the manganese ion

to be important for binding. The clustering of residues around the C2 hydroxyl group of

the carbohydrate is conserved in the domains 3 and 9 of the IGF-II/CD-MPR, which

suggests the recognition of the mannose, is important in the functioning of these

proteins.

_________________________________________________________________ 23


1.7 Plant lectins

Since their discovery in 1880’s, a large number of plant lectins have been identified and

isolated and are found predominantly in the storage organs of the plant, such as the

seeds (Rüdiger, 1998). In legume lectins, the lectin is localised in the cotyledons, for

the caster bean, the lectin is found in the endosperm and in wheat, the lectin is found

confined to the primary axis of the seed (Van Damme et. al., 1998). Lectins can

constitute up to 10% of the total protein content however, it is more likely to be 0.1-5%

(Sharon & Lis, 1990).

Plant lectins are further divided into subfamilies based on their sequence homology and

their molecular weight and it is common to have a large number of lectins with different

carbohydrate specificity in the same family. Plant lectins are divided into the following

families: legume lectins, monocot mannose binding lectins, chitin binding lectins, type

2 ribosome inhibiting proteins, and the jacalin family.

1.7.1 Legume lectins

A large number of legume lectins have been characterised and the majority of these

lectins are located in the seeds where they make up an astonishing 10% of the total

protein content (Sharon & Lis, 1990). Legume lectins are also found in leaves, stems,

bark and roots but in low amounts. Their carbohydrate specificity varies, however the

majority of these lectins are specific for Man/Glc and Gal/GalNAc. Table 1.4 lists a

number of legume lectins identified, the species that they have been derived from and

their carbohydrate specificity.

_________________________________________________________________ 24


Table 1.4: Members of the legume lectin family. Origin Species Carbohydrate specificity

Caesalpinaceae Griffonia simplicifolia Gal/GalNAc, GlcNAc, oligosaccharides Papilionaceae - Abreae Abrus precatorius Gal/GalNAc Papilionaceae - Carageae Caragana arborescens (Pea tree) Man(Glc) Diocleae Canavalia ensiformis (ConA) Man(Glc) Phaseoleae Glycine max (soybean) Gal/GalNAc Phaseoleae Phaseolus lunatus limensis (lima Bean) Gal/GalNAc Phaseoleae Phaseolus vulgaris (kidney bean) oligosaccharides Vicieae Lens culinaris (lentil) Man(Glc) Vicieae Pisum sativum (garden pea) Man(Glc) Vicieae Vicia faba (fava bean) Man(Glc) Vicieae Vicia cracca (common vetch) Man(Glc), Gal/GalNAc Loteae Lotus tetragonalobus (asparagus pea) L-fucose

Legume lectins consist of two or four identical or nearly identical subunits of about 30

kDa, which is composed of 6- or 7-stranded antiparallel β-sheets. Each subunit contains

a single carbohydrate-binding domain that has the same carbohydrate specificity. Most

legume lectins form what is commonly known as the canonical legume monomer as

seen in Figure 1.11. The dimeric form is defined as a large 12 stranded β-sheet that

results from the association of two 6 stranded β sheets as seen in

Figure 1.11B.

_________________________________________________________________ 25


___________________

Figure 1.11: The stru

(A) The canonical mono

Banerjee et. al., 1996). S

The formation of the ca

presence of two diva

(PDB ID: 1CN1; Shoham

teracting with the m

lectins also have hydro

that binds to non-pol

pockets often result in

(Sharon & Lis, 1990; V

carbohydrate-binding s

impaired (Sharon & L

in

C

A

__________________

ctures of legume lecti

mer (PDB ID: 2ENR;

tructures were taken from

rbohydrate-binding si

lent cations (Mn

et. al., 1979) and (C) t

etal ions are highly

phobic binding sites

ar compounds such a

a 10-50-fold increas

an Damme et. al., 199

2+

ite is not formed pro

is, 1990). The ami

B

____________________________ 26

ns.

Bouckaert et. al., 1996), (B) concanavalin A

the RCSB PDB database.

te of the legume lectins is dependent on the

he peanut agglutinin tetramer (PDB ID: 2PEL;

id residues that are responsible for

conserved among these lectins. Legume

located near the carbohydrate-binding site

s indoleacetic acid and adenine. These

e in binding of hydrophobic glycosidases

7).

and Ca2+). Without these cations, the

perly and the functioning of the lectin is

no ac


1.7.2 Monocot Mannose binding lectins

Monocot mannose binding lectins are a group of proteins that are specific for mannose

nly. They were first discovered in the snowdrop bulbs, and other lectins have been

und in a number of different families including Amaryllidaceae, Alliaceae, Araceae,

rchidaceae and Liliaceae (Van Damme et. al. 1987). The structure of the Snowdrop

ctin complexed with its sugar is shown in Figure 1.12.

igur

mann

from

A

o

fo

O

le

F

(A) E

mann

key re

Mono

have

Hom

the c

of th

prepr

____

e 1.12: The structure of the Snowdro

oside (A) and a close up of the carbohy

the RSCB PDB database (PDB ID: 1MSA; He

rotein into the mature polypeptide. The

ach subunit is made up of primarily β-

osides per subunit bind (shown in yellow). (

sidues involved in forming hydrogen bonds

cot mannose-binding lectins are approxim

been three different types identified (hom

omers contain 2 or 4 identical subunits of

leavage of the signal peptide during trans

e peptide during the post-translational s

op

__________________________________

B

p lectin complexed with methyl α-D-

drate binding site (B).

ster et. al., 1995).

heterodimer was formed using the same

sheet structures where three methyl-α-D-

B) The carbohydrate is shown in yellow and

are shown in red. The structure was taken

ately 12 kDa in size, and to date, there

omer, heterodimer and heterotetramer).

approximately 12 kDa and are formed by

lation and the removal of the C-terminus

tage, resulting in the conversion of the

___________________________ 27


methods as the homomer and these polypeptides contain 2 different but very similar

contains two mannose-binding domains. The

eterodimer in the A.sativum lectin (ASA-I) possess two tandomly arrayed highly

homologous domains. The mature heterodimer is formed by the removal of the

glycosylated linker found between the two domains and the non-glycosylated C-

terminus (Van Damme et. al. 1992).

Urticaceae, Solanaceae, Papaveraceae,

uphorbiaceae, Phytolaccaceae and Viscaceae. This domain is a small chitin binding

subunits of approximately 12 kDa that were derived from different preproproteins. The

lectin isolated from Allium ursinum is an example of the heterodimer (Smeets et. al.,

1994).

The second type of monocot mannose binding lectin includes the heterodimer and

heterotetramer forms. They are the result of two different subunits produced from the

same precursor where each subunit

h

1.7.3 Chitin-binding lectins

Chitin binding lectins contain a hevein domain and have been found in a number of

plant families such as Graminceae,

E

protein of 43 residues that was first found in the latex rubber tree Hevea brasiliensis.

An interesting characteristic of this protein is the primary sequence contains a large

number of cysteine and glycine residues.

The Graminceae lectins have been found in Triticum aestivum, T.durum, Secale cereale,

Hordeum valgare and Oryza sativa. All of these lectins have two subunits of 18 kDa

(Peumans & Stinissen, 1983). The barley, rye and rice lectins contain only one isoform

and therefore only one subunit while the wheat germ agglutinin (WGA) is made up of

_________________________________________________________________ 28


three isoforms (or three different subunits). Each subunit of the WGA is approximately

17 kDa and contains four homologous subdomains of 43 amino acids that are held

together by 32 disulfide bridges. Each subdomain is arranged in a pseudo four fold

screw-related fashion (Rini, 1995) where the monomers are paired head to tail which

sults in the formation of the dimer (Lis and Sharon, 1998). Proteins within this family

contain a number of different carbohydrate specificities as a result of the variability

between the sequences within each subdomain. For example, WGA is specific for N-

acetylglucosamine and N-acetylneuraminic acid.

ere the lectins specificity is for Neu

Acα(2,6) Gal/GalNAc (Van Damme et. al., 1996; 1997). Lectins that belong to this

family include ricin (Euphorbiaceae), abrus (Abrus precatorius - Fabaceae) and SNA-I

& SNA-V (Sambucus nigra – Sambucaceae) (Peumans and Van Damme, 1998). The

crystal structure of ricin is shown in Figure 1.13.

re

1.7.4 Type II ribosome inactivating protein (RIP)

Type 2 ribosome inactivating protein (RIP) contains two chains (α and β) separated by

a disulfide bond where each chain is made up of 1, 2 or 4 subunits. The α-chain

contains the N-glycosidase activity that results in the cleavage of the rRNA and the β-

chain contains the carbohydrate-binding domain. There is substantial homology

between the sequences for both chains when compared to all type 2 RIPs. The

carbohydrate specificity for type 2 RIP is mainly Gal or GalNAc, however, two cases

have been identified in the Sambucus species wh

5

_________________________________________________________________ 29


Figure 1.13: The crystal structure of the ricin A-chain.

Figure was taken from the RSCB PDB database (PDB ID: 1APG; Katzin et. al., 1991).

1.7.5 The Jacalin family

Jacalin is a galactose specific, tetrameric lectin with a molecular weight of 66 kDa,

derived from the Artocarpus integrifolia or the jackfruit. Each subunit is made up of a

heavy (133 residues) and light (20 residues) chain that form up to four-stranded

antiparallel β-sheets (Figure 1.14). These subunits are stabilised by a number of non-

covalent bonds such as hydrogen bonds. This lectin is derived from one precursor that

is modified into the mature form by a series of complex post-translational modifications

(Lis and Sharon, 1998; Van Damme et. al., 1997; Peumans et. al., 1998).

_________________________________________________________________ 30

Chapter 1 Introduction _________________________________________________________________________________ Chapter 1 Introduction _________________________________________________________________________________

___

Fig

(A)

loca

(PD

The

bee

β1,

(4 p

A

___
________________________________
ure 1.14: The structure of the Jacalin

The full structure of the jacalin lectin and

ted between the two heavy chains. The str

B ID: 1JAC; Sankaranarayanan et. al., 1996

Jacalin lectin from Artocarpus integri

n found to interact with high specificity

3-GalNAc-α) (Kabir & Daar, 1994). Th

er tetramer) that is located at one end of

________________________________

B

______________________________ 31

lectin.

(B) the light chain is shown in red, which is

ucture was taken from the RSCB PDB database

).

folia is a galactose specific lectin that has

to the α-linked T-antigen disaccharide (Gal-

ere is one galactose-binding site per subunit

the β fold.

______________________________ 31


1.8 Roles of lectins in plants

Plant lectins are thought to function either directly within the plant or as a defence

system against pathogen invasion. Within the plant, the lectin may be involved in the

transport of sugars; it can be stored as a source of nitrogen, cell-cell interactions and the

regulation of cell division. Externally, most plant lectins are involved in the defence of

the plant by interacting with surface glycoproteins on the digestive tract of organisms

(Peumans & Van Damme, 1998; Van Damme et. al., 1997).

Several plant lectins have been found to affect the growth and development of insects

when the lectin has been orally ingested. The lectin binds to the carbohydrates on the

surface of the digestive tract resulting in the harmful effects to the insect and these

effects have been studied. It was found that the lectin purified from Phaseolus vulgaris

agglutinin (PHA), binds to the brush border cells of the intestine of insects resulting in

hyperplasia and hypertrophy of the small intestine (Pusztai and Bardocz, 1996). In

addition to this, the ingestion of raw beans of PHA by the insects resulted in nausea,

vomiting and diarrhoea (Peumans & Van Damme, 1998). Therefore, lectins in plants

play a key role in the defence against insect invasion.

_________________________________________________________________ 32


1.9 Applications of plant lectins

Plant lectins have gained increasing interest in the last couple of decades, as they are

useful tools in the structural and functional studies of complex carbohydrates such as

glycoproteins. Other applications of plant lectins involve the expression of their genes

in transgenic plants for the production of large quantities of lectin and to develop insect

resistant crops (Van Damme et. al., 1997).

Plant lectins also can be used for the detection, isolation and characterisation of

glycoconjugates from a number of different sources. Specific carbohydrate structures

can be targeted and their isolation can be achieved using these lectins. Lectins can also

be used to induce specific processes in animal or human cells. The activation of

lymphocytes with mitogenic lectins and the expression of cytokines and interleukins are

a few more functions of lectins. At this stage, the concanavilin A and peanut

hemagglutinin lectins have been found to cause these cellular reactions.

Lectins are used as diagnostic tools and some of these uses include the typing of the red

blood cells (ABO system), the tracing of aberrant glycosylation of glycoproteins, and

the histochemical staining of carbohydrates. Potential therapeutic uses of lectins

include immunomodulation and cancer therapy with immunotoxins (Peumans & Van

Damme, 1998).

_________________________________________________________________ 33


1.10 Serine Protease Inhibitors

One of the two major proteins that were isolated from the seeds of G.robusta was shown

to be a protease inhibitor. There are four different families of protease inhibitors that

exist in plants and they include the serine protease inhibitor family, the cysteine

protease inhibitors, the metallo-protease inhibitors and the aspartic protease inhibitors.

Protease inhibitors are characterised into these families based on the amino acids

involved within the reactive site of the inhibitor. Table 1.5 lists the families of protease

inhibitors and the protease that they inhibit.

Table 1.5: Families of plant protein protease inhibitors. Family Protease inhibited

Serine protease inhibitors Trypsin and chymotrypsin Soybean trypsin inhibitor (Kunitz) family Bowman-Birk family Barley trypsin inhibitor family Potato inhibitor I family Potato inhibitor II family Squash inhibitor family Ragi I-2/maize trypsin inhibitor family Serpin family Cysteine protease inhibitors Papain, cathepsin B, H, L (phytocystatins) Metallo-protease inhibitors Carboxypeptidase A, B Aspartic protease inhibitors Cathepsin D

Taken from Koiwa, 1997.

Serine protease inhibitors are the largest group of protease inhibitors isolated from

plants. There are eight subgroups within this family that are characterised based on their

sequence homology, the location of their reactive site and their structural characteristics

such as the positioning of disulfide bonds. The majority of these inhibitors have been

_________________________________________________________________ 34


extracted from seeds. Two of these protease inhibitors, the Kunitz and Bowman-Birk,

have been extensively characterised (Kunitz 1947; Bowman 1946, 1993; Birk 1961,

1974, 1976, 1985). These inhibitors were first discovered in soybeans and were found

to differ from each other in size, primary sequence, three-dimensional structure and

enzyme inactivation properties (Birk, 1976).

The Kunitz-type (Soybean trypsin inhibitor, SBTI) inhibitor is made up of 181 amino

acids, contains 2 disulfide bridges and inhibits trypsin. The Bowman-Birk protease

inhibitor (BBI) is comprised of 71 amino acids, contains 7 disulfides bridges (located

between C8-C62, C9-C24, C12- C58, C14-C22, C32-C39, C36-C51 & C41-C49) and

inactivates both trypsin and chymotrypsin at two independent sites (Birk, 1976). For

the inhibition of trypsin, the presence of a lysine or arginine in the reactive site is

required. For chymotrypsin, the presence of tyrosine, phenylalanine, leucine or

methionine will inactive this protease (Laskowski & Kato, 1980).

Figure 1.15 shows a number of residues that are conserved between all of the members

of the Bowman Birk family. All of the cysteine residues were conserved throughout the

family and these residues were involved in forming disulfide bridges (Figure 1.15). It

was therefore thought that these residues play an important role in stabilising the

structure of the proteins by forming disulfide bridges.

_________________________________________________________________ 35


1 10 20 30 P4 P3

1BBI -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- D D E S S K P C C D Q C A C 1PI2 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- D E Y S K P C C D L C M C IBB3_DOLAX -- -- -- -- -- -- -- -- D H H H S T D E P S E S S K P C C D E C A C IBB4_DOLAX -- -- -- -- -- -- -- -- -- H E H S S D E S S E S S K P C C D L C T C IBB_PHAAU -- -- -- -- -- -- -- -- -- -- -- -- S H D E P S E S S E P C C D S C D C IBB2_PHAAN -- -- -- -- -- -- S V H H Q D S S D E P S E S S H P C C D L C L C IBB3_SOYBN M C I L S F L K S D Q S S S Y D D D E Y S K P C C D L C M C IBB2_SOYBN -- -- -- M E L N L F K S D H S S S D D E S S D P C C D L C M C

1 2 3 4 31 40 50 60 P2 P1 P1

' P2'

P3'

P4'

P4 P3 P2 P1 P1'

1BBI T K S N P P Q C R C S D M R L N S C H S A C K S C I C A L S PI-II T R S M P P Q C S C E D R I -- N S C H S D C K S C M C T R S IBB3_DOLAX T K S I P P Q C R C T D V R L N S C H S A C S S C V C T F S IBB4_DOLAX T K S I P P Q C G C N D M R L N S C H S A C K S C I C A L S IBB_PHAAU T K S I P P E C H C A N I R L N S C H S A C K S C I C T R S IBB2_PHAAN T K S I P P Q C Q C A D I R L D S C H S A C K S C M C T R S IBB3_SOYBN T R S M P P Q C S C E D I R L N S C H S D C K S C M C T R S IBB2_SOYBN T A S M P P Q C H C A D I R L N S C H S A C D R C A C T R S

4 2 5 6 5 7 61 70 80 90 P2' P3

' P4'

1BBI Y P A Q C F C V D I T D F C Y E P C K P S E D D K E N -- -- -- PI-II Q P G Q C R C L D T N D F C Y K P C K S R D D -- -- -- -- -- -- -- IBB3_DOLAX I P A Q C V C V D M K D F C Y A P C K S S H D D -- -- -- -- -- -- IBB4_DOLAX E P A Q C F C V D T T D F C Y K S C H N N A E K D -- -- -- -- -- IBB_PHAAU M P G K C R C L D T D D F C Y K P C E S M D K D -- -- -- -- -- -- IBB2_PHAAN M P G Q C R C L D T H D F C H K P C K S R D K D -- -- -- -- -- -- IBB3_SOYBN Q P G Q C R C L D T N D F C Y K P C K S R D D -- -- -- -- -- -- -- IBB2_SOYBN M P G Q C R C L D T T D F C Y K P C K S S D E D D D -- -- -- --

7 6 3 1

Figure 1.15: Sequential alignment of the members of the Bowman-Birk inhibitor

family.

Numbers underneath cysteine residues represent the conserved disulfide-bonding pattern for the

family. The scissle bond or P1P1’ site is highlighted in cyan. The serine residues involved in

the inhibition of proteases are highlighted in yellow (for trypsin) and red (for chymotrypsin).

_________________________________________________________________ 36


The reactive site of the inhibitor is made up of the scissle bond or P1-P1' bond

(nomenclature derived by Schechter & Berger, 1967) which is highlighted in cyan in

Figure 1.15. In the P1 site, a basic amino acid residue is found and this residue forms a

salt bridge with an acidic amino acid within the S1 pocket of trypsin (Koepke et al,

2000). Trypsin will bind to these residues under normal conditions and hydrolyse the

polypeptide chain at the carboxyl end of this residue. The inhibitor functions by having

the basic amino acid residues on the surface of the molecule, which allows the trypsin to

bind to the residue and blocks the protease’s binding site. This prevents the protease

from binding to other proteins and their function is therefore inhibited and is referred to

as competitive inhibition.

Residues at position P3' and P4' of the trypsin binding region were found to be

conserved within the Bowman-Birk family. These residues are involved in the

formation of a turn between two β-sheet structures. A threonine at the P2 site is highly

conserved throughout all Bowman-Birk inhibitors. A number of substitutional

experiments at this P2 site have shown that threonine is important for the functioning of

the inhibitor (McBride et. al, 1998). A standard inhibitory kinetic assay was established

to determine the effects of residue substitution at the P2 position on their ability to

inhibit the protease (McBride et. al., 1998). It was found that threonine had the lowest

KI value, followed by serine with a 20 fold difference in inhibition when compared to

threonine. It was also found that this site had a preference for small residues as large

aliphatic and aromatic side chain residues were found to have large KI values. The

presence of negatively charged residues such as aspartic acid and glutamic acid also

resulted in poor inhibition. This could be due to the unfavourable electrostatic

interactions within the catalytic triad of the protease (McBride et. al, 1998). The

_________________________________________________________________ 37


threonine residue is conserved throughout the Bowman-Birk inhibitor family and has

been shown to be important in the functioning of the inhibitor (McBride et. al, 1998).

The reactive site for chymotrypsin contains a serine in the P1' position and a conserved

leucine residue at P1. Chymotrypsin will mostly bind to hydrophobic residues such as

phenylalanine or leucine in the P1 position. The preference for these residues within

chymotrypsin is due to an uncharged serine residue in the S1 pocket (Koepke et. al,

2000). At position P3', the residue proline is conserved throughout the family. Like the

trypsin-binding region, this position is important in forming the turn between two β-

sheet structures.

Structurally, the Bowman-Birk inhibitor has been described as a “bow-tie” motif as it

contains two independent binding sites. The structure of the inhibitor is shown in

Figure 1.16 (Werner & Wemmer, 1992). Each inhibitory domain is defined to contain a

canonical structural motif which is made up of a β-hairpin or antiparallel β-sheet with a

cis-proline containing type VI turn (Werner & Wemmer, 1992). According to the

structural characterisation of proteins (SCOP) database, the Bowman-Birk inhibitor

superfamily belong to the knottin (or small inhibitors, toxins, lectins) fold family which

is defined as “ disulphide-bound fold; contains beta-hairpin with two adjacent

disulphides” (Murzin et. al, 1995).

_________________________________________________________________ 38


___________________________________________

Figure 1.16: The structure of the Bowman-Birk in

The two inhibitory sites are found in the loop regio

structures (in red). Taken from the RSCB PDB prote

Like plant lectins, plant protease inhibitors are also th

plants from insect invasion. Normally, the leaves of

protease inhibitors. It was found that there was an

inhibitors localised within the leaves of plants after

1.10.1 Functional Role of Serine Protease Inhibitors

echanically damaged (Green & Ryan, 1972). In

production of protease inhibitors within the rest of th

nd

993). Feeding trials using protease inhibitors have

Chymotrypsin binding site

m

possibly due to signals being sent from the wou

1

of insects which was thought to be due to the inte

proteases found the gut of the insect (Koiwa, et.

inhibitors play an important role in the defence of the

Trypsin binding site

______________________ 39

hibitor.

n between the antiparallel β sheet

in database (1BBI).

ought to function in the defence of

plants contain very low levels of

increase in the levels of protease

they were attacked by insects

or

addition to this, an increase in the

e plant was also seen and this was

site to the phloem (Pearce et. al.,

shown to slow or stop the growth

raction of the inhibitors with the

al., 1997). Therefore, protease

plant against insect invasion.


1.10.2 Applications of Serine Protease inhibitors

Certain protease inhibitors have the ability to prevent or suppress cancer-induced cells

(Kennedy, 1998a). The Bowman-Birk protease inhibitor (BBI) derived from soybeans

was found to be a very effective anticarcinogenic agent. The BBI has been extensively

studied both as a purified BBI and as a concentrated form of BBI (BBIC). The BBIC

was produced by treating the soybean flour with acetone and purified using the Birk

purification procedure described in detail elsewhere (Birk 1976). The chymotrypsin-

press carcinogenesis in three

ifferent species (mice, rats and hamsters)(Kennedy 1998b). The BBIC was also found

to suppress several cancers in different organ and tissue types including the colon, liver,

lung, esophagus, cheek pouch and cell of hematopoietic origin and human trials have

begun using the concentrated form of Bowman-Birk protease inhibitor (Kennedy,

1998b). Specific animal models have been created that shows the suppressive effect of

BBI and or BBIC on carcinogenesis (Kennedy, 1998b).

binding site within the inhibitor was found to be responsible for the anticarcinogenic

effect seen in these experiments (Kennedy 1998b). The dose rates of BBIC showed the

protein to have a high chymotrypsin inhibitory activity (100mg/g) while the trypsin

inhibitory activity was more than half (40mg/g) than that seen for chymotrypsin thus,

suggesting the chymotrypsin inhibitory site is important for function (Kennedy, 1998a).

Both the BBI and BBIC proteins were found to sup

d

_________________________________________________________________ 40


1.11 Initial Research

Researchers at the Australian Red Cross Blood Service of Queensland (ARCBS-QLD)

are investigating the isolation of lectins, from a number of different species, that result

in the agglutination of granulocytes. Approximately 700 different species of native

Australian and exotic plants were screened for reactivity towards granulocytes. Of

these, only 36 species were reactive with granulocytes and not with red blood cells

(Minchinton, 1997b). Eight of these species were chosen for further study based on the

availability of the seeds.

A number of preliminary bioassays and characteristics were determined on these 8

different species and these results are shown in Table 1.6. Of these 8 species, the lectins

isolated from Caragana and Arbus are commercially available. The lectin from

Hernandia moerenhoutiana has been isolated by a colleague at the ARCBS- QLD

(Clarke, 1997).

Grevillea robusta was one of the 8 short listed species to be investigated in the future.

The plant was chosen for this study because of: (1) the size of the protein, (2) the

availability of the crude material (ie seeds), (3) the ability to specifically target white

blood cells and not red blood cells and (4) the presence of two major proteins that were

found to fall within the molecular weight range of interest.

_________________________________________________________________ 41


Table 1.6: Preliminary biological characteristics of the 8 species of plants. Plant species MW in kDa of SDS Sugar specificity Potential antigen target Potential antigen target PAGE bands (crude - defined by monoclonal defined by human reduced) antibodies antibodies

Grevillea robusta 27, 21, <10 man> malt CD15,CD18, CD65 HNA-1a, HNA-2a NB2* Hernandia moerenhoutianna 28, 26, 24, 23, 16, man=fuc>malt CD15,CD16, CD18, HNA-2a,HNA-4a NB2* 14.5, 14 CD65 Lablab purpureus sweet rongai 78/74,35/31/28.5, gal=galNAc CD11b,CD15, CD65 HNA-1a, NB2*, SL* 19,<10 Erythrina speciosa 84, 76, 28 galNAc>gal >fuc CD15, CD65 HNA-1a Vicia sativa 94, 54, 39, 16 glc=man CD11b,CD15, CD16, HNA-2a CD18, CD24, CD65 Caragana arborescens 100, 25, 13 gal>galNAc incomplete incomplete Lathyrus lirsutus 85, 54, 39 glc>malt incomplete incomplete Arbrus precatorius 58, 54, 39 gal>galNAc CD11b,CD15, CD16, HNA-1a, HNA-1b CD43

* Indicates the neutrophil antigen has not been characterised.

The major proteins identified in the crude extract of G.robusta were found to have a

molecular weight of approximately 7000 Da and therefore would provide an excellent

candidate for structural studies using nuclear magnetic resonance (NMR) spectroscopy.

The preliminary studies revealed the crude extract from G.robusta contained lectin like

properties that were specific towards white blood cells rather than red blood cells. This

species of plant was chosen for further investigation because of at least 1 bioactive

protein with a molecular weight of 7000 Da, which was amenable to NMR studies.

_________________________________________________________________ 42


1.12 Aims and Expected Outcomes

There were two main aims to this project; (1) the purification and characterisation of a

proteins from the seeds of a native Australian species that fell within the 10 – 30 kDa

molecular weight range and (2) the determination of the structure of the protein from

that species. The initial focus was to target the lectin identified in the seeds of the

native Australian plant, Grevillea robusta. This focus was altered as the project

progressed due to limited amounts of lectin found within the seeds (less than 5% of the

total protein content) and the discovery of a serine protease inhibitor (major

representative within the crude extract). Therefore, the first aim was to isolate and

characterise the lectin and serine protease inhibitor from the seeds of G.robusta. The

purified lectin would provide an alternative tool in the characterisation of neutrophil

antigens while the serine protease inhibitor may possess anticarcinogenic properties as it

has a high level of similarity with the Bowman-Birk inhibitor.

As the serine protease inhibitor was very similar to the Bowman-Birk inhibitor (both

sequentially and in function), it was decided that the 3D structure of the inhibitor would

be determined. In addition, the 3D structure of the Bowman Birk inhibitor had already

been determined by NMR spectroscopy (Werner & Wemmer, 1992), thus providing an

excellent starting platform for the study of the serine protease inhibitor from G.robusta.

The second aim was to determine the structure of the serine protease inhibitor from the

seeds of G.robusta.

_________________________________________________________________ 43

Chapter 2 Extraction of Proteins from Grevillea robusta _________________________________________________________________________________

Chapter 2 Extraction of Proteins from Grevillea

robusta

_________________________________________________________________ 44

Chapter 2 Extraction of Proteins from Grevillea robusta _________________________________________________________________________________

Chapter 2 Extraction of proteins from Grevillea robusta

2.1 Introduction

Grevillea robusta (or Silky Oak tree) is a member of the Proteaceae family and is

native to Australia where it is localised in the rainforests of Queensland and northern

New South Wales but has also been found in other neighbouring countries. It is

characterised as a tall, straight tree (seen in Figure 2.1) that contains golden

toothbrush-like flowers, seen only during spring. The leaves are pinnate with lobed

segments that resemble a fern and the undersides of the leaves contain silky hairs.

Preliminary experiments have shown the G.robusta seeds to contain a lectin with an

approximate molecular weight of 7000Da with a sugar specificity for

mannose>maltose (Minchinton, 1997b). As stated in the previous chapter, the lectin

from G.robusta has the potential to be used as a tool in characterising neutrophil

antigens and therefore, the purification and characterisation of this protein would be

advantageous.

_________________________________________________________________ 45

Chapter 2 Extraction of Proteins from Grevillea robusta _____________________________________________________________________________________

Figure 2.1: Grevillea robusta or Silky Oak tree.

_________________________________________________________________ 46


2.2 Ammonium sulfate precipitation of crude proteins.

G.robusta seeds were ground and soaked overnight in phosphate buffered saline

(PBS) pH 7.3 (0.15M NaCl, 11mM sodium phosphate). Ammonium sulfate was

added to the filtered supernatant in two stages resulting in the proteins being

precipitated. SDS (18% separating gel with 3% β-mercaptoethanol) and native

(13% separating gel) PAGE were used to visualise these extracted proteins (Figure

2.2). These precipitated proteins will be referred to as the “crude extract” for the

remainder of the thesis.

Figure 2.2: SDS (A) and Native (B) PAGE of the crude

The gels were stained with Coomassie Brilliant Blue R-250. (

(5% stacking gel) pH 8.8 in the presence of 3% β-mercaptoeth

Lane 1: crude extract from G.robusta. (B) 13% native separati

8.9. Lane 2: crude extract from G.robusta.

_______________________________________________

2

extract from G.robusta.

A) 18% SDS separating gel

anol. M: LMW standards.

ng gel (3% stacking gel) pH

__________________ 47


SDS PAGE (under reducing conditions, 3% β-mercaptoethanol) was used to

determine the approximate molecular weight of proteins of the crude extract. The

ratio, between the migration of the protein band and the migration of the dye front,

was determined for each of the standards and samples. These values were plotted

against the log of the standard molecular weight and the molecular weight of the

crude extract was calculated using this graph. SDS PAGE of the crude extract

revealed two major bands below 14.4 kDa and a minor band at 21 kDa. The

approximate molecular weight for the major bands was calculated to be 8700 Da &

7000 Da (upper and lower bands respectively).

Native PAGE was used to show the migration of the crude extract under non-

reducing conditions. This form of electrophoresis allows the proteins to migrate

through the gel in their native or “true” state, which is dependent on the overall

charge of the protein, and to a lesser extent, its size. Native PAGE of the crude

extract, seen in Figure 2.2B, showed 6 bands (labelled SO1-SO5 & I). The major

bands were labelled SO1 and SO2. The remaining 3 bands were of equal intensity

and were labelled SO3, SO4, SO5 & I. Due to the increase in the number of

proteins seen using native PAGE compared to SDS PAGE, it was decided to use

native PAGE to identify the proteins during each purification step.

_________________________________________________________________ 48


2.3 N-terminal sequencing of the crude extract

SDS PAGE of the crude extract revealed 2 major species of proteins with a

molecular weight of approximately 7000 Da. Native PAGE of the same sample

showed 5 bands suggesting the crude extract might contain a number of isoforms.

Isoforms are proteins that contain sequences that are homologous with each other

but differ in their primary sequence by a few amino acid residues.

N-terminal sequencing is useful in determining whether a sample contains isoforms

and was used to determine whether isoforms were present in the crude extract of

G.robusta. The five bands seen on native PAGE were transferred onto Sequi-Blot

PVDF membrane for 1 hour at 100V using BioRads Mini Trans-blot equipment.

Coomassie Blue stained bands were cut from the PVDF membrane, washed and sent

to the University of Queensland Biochemistry Department and sequenced for a fee.

Table 2.1 shows the sequencing results of the proteins seen in the crude extract.

Table 2.1: N-terminal sequences of the crude extract from G.robusta.#

SO1 E/ L T N A R W S -- -- -- -- -- -- -- -- -- -- -- -- -- S

SO2 G G E E A D W (C) E D D V V T T S (C) S I P P

SO3 (C) L P N I (C) I S S D L D -- -- -- -- -- -- -- -- --

SO4 L G P/ N/ I/ (C) D/ S S/ D/ L/ -- V/ -- -- -- -- -- -- -- -- S E W I E G ? ?

SO5 S L D (C) I (C) I (C) S D L -- -- -- -- -- -- -- -- -- --

#The one letter code is used to represent the amino acids. Cysteine residues are destroyed

during the sequencing process and are seen as a blank cycle. Therefore the cysteine residues

are placed in brackets.

_________________________________________________________________ 49


The N-terminal sequencing results revealed 5 different sequences (as seen in Table

2.1) which suggested that the additional bands seen on native PAGE were different

proteins rather than isoforms. The preliminary sequencing of the crude extract from

G.robusta was inconclusive due to the lack of resolution between the bands seen on

native PAGE and further purification of this extract will be required to provide good

sequencing results.

2.4 Bioassays

Functional bioassays were developed using granulocytes to identify lectins and they

were the granulocyte agglutination test (GAT), granulocyte immunofluorescence

test (GIFT) and the sugar-blocking GIFT. These bioassays will be discussed in

detail throughout this section. White blood cells were separated from red blood cells

and platelets using EDTA collection tubes and centrifugation. As granulocytes are

heavier than monocytes and lymphocytes (a density greater than 1.077g/ml)

(Olofsson et. al., 1980), granulocytes were further separated from the white blood

cells by using a density gradient and centrifugation. Neutrophils have a very short

life span (they only survive 1-4 days after being released from the bone marrow), the

bioassays were set up immediately after the cells were harvested (Glasser, 1988).

Even though granulocytes are made up of three different cell types, the results seen

in the bioassays are due to the neutrophils response to the addition of the testing

sample and not from the other cells (basophils or eosinophils) (Glasser, 1988).

_________________________________________________________________ 50


2.4.1 Agglutination

Agglutination distinguishes lectins from other sugar binding macromolecules such

as glycosidases and glycosyltransferases (Lis & Sharon, 1986). The binding of the

lectin to the cells sugar surface receptors results in the activation and migration of

neighbouring cells to the site of infection and it is this migration of neighbouring

cells that causes agglutination. For agglutination to occur, the lectin must form

multiple cross bridges with the cells suggesting a number of sugar binding sites are

required within the lectin (Lis & Sharon, 1986).

Agglutination can be prevented by the size and the number of carbohydrate binding

sites within the lectin, the number and accessibility of receptor sites on the cell

surface, the fluidity of the membrane and the metabolic state of the cells. External

conditions such as temperature, concentration of the cells and the mixing of cells

may also affect agglutination (Lis & Sharon, 1986). All of these factors need to be

taken into consideration when setting up the bioassays.

2.4.2 Granulocyte Agglutination test (GAT)

The granulocyte agglutination test (GAT) is a functional bioassay where the amount

of agglutination is determined by visualisation using a reverse phase microscope.

Granulocytes were diluted to a concentration of 5 x 106 cells/ml in granulocyte

resuspension solution (GRS) to provide a confluent monolayer of cells in the bottom

of a TERASAKI well. The protein sample to be tested was placed (in duplicate)

into each well of the TERASAKI plate under paraffin oil and granulocytes were

_________________________________________________________________ 51


added to the sample (a ratio of 1:3 (v/v) cells to protein). The plate was incubated at

30°C for 4 hours to allow the agglutination to occur.

The level of agglutination was scored by eye using a reverse phase microscope. No

agglutination (a score of 0) had a confluent layer of cells while for a +1 score, one or

two cells were seen to be interacting with each other, however, the majority of the

cells were still confluent. Lines of cells or small clusters of cells with a large

number of “single” cells gave a score of +2 and as these clusters become larger and

less “single” cells were seen the score was increase to +3. Samples containing large

clusters of cells that look like bunches of grapes with very few “single” cells

resulted in a score of +4. A positive for the GAT bioassay for this work was a score

of 2+ or better.

2.4.3 Granulocyte immunofluorescence Test (GIFT)

The binding of a lectin to the surface glycoproteins on granulocytes can be identified

using flow cytometry. Flow cytometry detects the level of fluorescence of labelled

cells as they flow past a laser beam and can distinguish between red blood cells,

platelets and granulocytes as each cell type has a different physiological make up ie

size and density.

In order to detect the lectin-cell binding, a fluorescent label is attached to the lectin.

To do this, a cofactor (biotin) is required that will bind to the conjugated

fluorochrome (FITC) and also binds to the protein/lectin. Biotin is a common

reagent used for the immunolabeling of antigens in histochemical, blotting and

_________________________________________________________________ 52


multiwell assays. It irreversibly binds to the lectin/protein through an amide group

(such as lysine) as seen in Figure 2.3.

Figure 2.3: The biotinylation of proteins.

The biotin binds irreversibly to the primary amine groups of lysine in a protein.

_________________________________________________________________ 53


Biotin has a high affinity for avidin (and its derivatives). Unfortunately this affinity

can be too strong and non-specific binding to the membrane can occur. To get

around this problem a derivative of avidin, such as streptavidin, is used to reduce the

amount of non-specific binding. Avidin and streptavidin are commercially available

unconjugated and conjugated to enzymes, fluorochromes and colloidal gold for the

use in a number of immunoassays.

Avidin is a homotetramic protein that contains four identical subunits and each of

these subunits has the capacity to bind one molecule of biotin and therefore, one

avidin molecule will bind 4 biotin molecules. Streptavidin is derived from

Streptomyces avidinii and can also bind up to 4 biotin molecules. It is this form of

avidin that is used in this bioassay.

The flow cytometer is set up to detect granulocytes based on its size and density. A

schematic diagram outlining the GIFT bioassay is shown in Figure 2.4. The level of

fluorescence detected by the flow cytometer is the mean channel fluorescence

(MCF) and is calculated from the peaks seen in the histogram (Figure 2.4D). The

larger the MCF value, the stronger the lectin binds to the granulocyte.

_________________________________________________________________ 54


Figure 2.4: The schematic representation of the GIFT bioassay.

(A) The biotin labelled lectin protein is added to the granulocyte. (B) The lectin binds to the

specific sugar receptor on the surface of the granulocyte. (C) The streptavidin-FITC label is

added which binds to the biotin on the lectin protein. (D) The flow cytometer detects the

level of fluorescence and places it in a histogram.

Each batch of granulocytes harvested will have a different MCF value for the blank

control value on the flow cytometer. The Relative MCF (Rel. MCF) allows the

results to be quantitated between experiments. By taking the MCF value for the

sample and dividing it against the MCF value for the blank, the relative MCF of the

sample is determined. Any Rel. MCF value that is above 4 is regarded as a positive

for binding in this bioassay.

Lectin

Biotin

Streptavidin-FITC

Granulocyte Sugars

A B C

Log Fluorescence

Number of cells counted

D

_________________________________________________________________ 55


The sample to be tested in the GIFT bioassay is usually diluted to various

concentrations to identify the point of saturation with the granulocytes. This

information provides the optimal working conditions for the sample in which will be

used in other bioassays. The concentration on the sample is also important for

getting good and useful results in the GIFT bioassay. If the protein sample is too

concentrated, the cells will aggregate or agglutinate rendering the experiment

useless, resulting in variation throughout the experiment. It must be noted that the

principle behind the GIFT assay is to study the binding of the lectin to the surface of

the cells. If the cells have agglutinated, there is no way of determining this binding

and future inhibitory experiments used to determine specificity will also be invalid.

2.4.4 Sugar blocking granulocyte immunofluorescence test

Sugar-blocking studies were undertaken to determine the sugar specificity of the

crude extract. The sugar blocking GIFT bioassay provides information on which

sugar the lectin is targeting on the surface of the granulocyte and provides

information for the purification of the lectin proteins from the crude extract.

Lectins are sugar binding proteins that contain at least two binding sites (in the GAT

bioassay, two or more binding sites are required for agglutination to occur). An

inhibitory effect can been detected by the flow cytometer if the sugar binding sites

within the lectin are blocked with their specific sugar before the sample is added to

the cell suspension. Under these conditions, the lectin would not be able to bind to

the surface of the granulocyte and therefore a decrease in the MCF value would be

seen.

_________________________________________________________________ 56


As the aim of this bioassay is to block all sites within the lectin, thus preventing the

lectin from binding to the surface of the granulocytes, it is important to ensure that

the lectin concentration is not too high. If too many binding sites are available even

the highest concentration of sugar will not be sufficient to block all of the sites.

Therefore, the GIFT bioassay should be completed prior to the sugar blocking

bioassay where the sample is diluted to determine the point of saturation of the lectin

on the granulocytes. Once this point is known, the dilution determined is used for

the sugar blocking GIFT bioassay.

The biotinylated proteins were added to eight different sugars at three different

concentrations (0.1M, 0.25M, 0.5M). The ratio of protein to sugar was 1:4 (relative

v/v) and the samples were incubated for 30 minutes, which ensured all of the

binding sites within the protein were filled with the specific sugar. The sugars used

were a combination of monosaccharides (fucose, galactose, N-acetylgalactosamine,

glucose, N-acetylglucosamine & mannose) and disaccharides (lactose & maltose).

Figure 2.5 shows the schematic flow chart of this bioassay.

_________________________________________________________________ 57


A

Figure 2.5: Schematic representation of the sugar blocking GIFT bioassay.

(A) The lectin binds to the granulocyte surface without the addition of sugar. (B) Sugar is

added which blocks the binding site of the lectin and prevents it from binding to the surface

of the granulocyte. (C) The migration of the peak detected by the flow cytometer is an

indication of sugar specificity for the lectin.

The sugar specific for the lectin will occupy its binding site and prevent it from

binding to the surface of the granulocyte. The flow cytometer is set up to detect the

granulocyte and the level of fluorescence. The decrease in MCF indicates the lectin-

binding site being occupied by its specific sugar as seen in Figure 2.5B. The peak in

the histogram will move towards the left indicating the successful blocking of the

lectin-binding site (Figure 2.5C).

Biotin - Lectin

BStreptavidin-FITC

No binding

Sugars

Granulocyte

B

C

Number of cells counted

A

Log Fluorescence

_________________________________________________________________ 58


2.4.5 Bioassay results for the crude extract of G.robusta

The crude extract from the ground seeds of G.robusta were found to be reactive with

granulocytes in both the GAT and GIFT bioassays as seen in Table 2.2. The extract

was reactive in the GAT to a dilution of 1/8 and the GIFT bioassay revealed strong

binding to the granulocyte surface receptors, as seen by the increase in the MCF

value.

Table 2.2: The GAT and GIFT results of the crude extract from G.robusta.#

GAT GIFT Dilution Score Rel. MCF Neat 4+ -- 1/2 3/4+ 23.61 1/4 3/4+ 23.16 1/8 3+ --

# The GAT bioassay is scored from 0-4+ and the Rel. MCF value indicated strong binding in the GIFT bioassay.

The sugar specificity of the crude extract was determined using the sugar blocking

GIFT assay. The crude extract was diluted ¼ with Bornes BSA buffer and 8

different sugars were added (using different concentrations) to the crude extract

prior to mixing with the neutrophils. The results are shown in Table 2.3.

_________________________________________________________________ 59


Table 2.3: Sugar-blocking GIFT results of the crude extract from G.robusta.#

Sugar Rel. MCF value with Rel. MCF value with 0.25M sugar 0.5M sugar

fucose 25.34 23.62 galactose 20.96 19.14

N-acetylgalactosamine 26.55 24.83 glucose 27.16 28.71

N-acetylglucosamine 25.17 25.26 lactose 34.22 22.84 maltose 26.03 25.43 mannose 16.98 10.09

#Only two different sugar concentrations are shown. All sugars are D-sugars unless

otherwise indicated. The sugar specific for the lectin in G.robusta is shown in bold.

The Relative MCF values are determined by dividing the MCF value obtained by

the sample with the MCF value obtained for the blank. In this case, the blank

sample would be the diluted crude extract incubated with no sugar. The crude

extract incubated with mannose showed the largest decrease in Rel. MCF value (ie.

a shift to the left was seen in the histogram) when compared to the other sugars

(Refer to Table 2.3). These results were different from the preliminary findings

listed in Table 1.6, where the crude extract was shown to be specific for man>malt.

However, in this work, it is evident that the crude extract was not specific for

maltose, as the Rel. MCF values were high (refer to Table 2.3). Therefore in this

work, the crude extract from Grevillea robusta was found to be specific for only

mannose.

_________________________________________________________________ 60


2.5 Conclusion

The crude extract from G.robusta contained at least 5 different proteins. The crude

extract was reactive in both GAT and GIFT bioassays and the sugar-binding GIFT

assay revealed the proteins to be specific for mannose only. This result was

different from the preliminary findings (Refer to section 1.11; Minchinton, 1997b)

where it was suggested that the crude extract from G.robusta were specific for

mannose> maltose. Native PAGE was established as the method of choice for the

visualisation for future purification stages.

_________________________________________________________________ 61

Chapter 3 Purification & Characterisation of a lectin isolated from the seeds of G.robusta

_____________________________________________________________________________________

Chapter 3 Purification & characterisation of a lectin

isolated from the seeds of G.robusta

_________________________________________________________________ 62


_____________________________________________________________________________________

Chapter 3 Purification and characterisation of a lectin isolated from

the seeds of Grevillea robusta.

3.1 Introduction

Lectins are ubiquitous in nature and can be relatively easy to isolate from the natural

source. The functional role and biochemical characteristics of these lectins are

discussed in Chapter 1 (sections 1.5 to 1.9) and will not be discussed here. The most

successful method used for the purification of lectins from crude plant extracts is

affinity chromatography (Padma et al, 1999, Moreira et al, 1997, Machuka et al, 1999)

Therefore, it was the method of choice to isolate the protein from G.robusta. Affinity

chromatography allows proteins to be separated on the basis of their specificity for a

subunit and many lectins have been isolated using affinity chromatography. A large

number of affinity chromatography resins with different sugar specificities are

commercially available.

_________________________________________________________________ 63


_____________________________________________________________________________________

3.2 Purification of the lectin from G.robusta

The lectin in G.robusta has been shown to be specific for mannose>maltose

(Minchinton, 1997). A mannose-agarose column (10ml) was used and equilibrated in

PBS pH 7.3. An aliquot (1ml) of crude extract was injected onto the column and the

column was washed several times to ensure all of the non-binding material had been

removed from the column. The elution buffer (0.5M mannose in PBS pH 7.3) was

added. It was found that the lectin in the crude extract did not bind to the column as no

peak was seen after the addition of the elution buffer.

The lack of binding to the column could result from the lectin not containing a

functional or intact binding site, due the metals ions being lost during the extraction

stage. Another possibility involves the sugar specificity of the lectin. Some lectins

require more complex sugars, such as oligosaccharides or polysaccharides that are

attached to the resin before they will bind.

Other lectins (such as those in the legume family) require metal ions to form their

binding site (Sharon & Lis, 1990; Rini, 1995). This could be the case with the lectin

from G.robusta. During the extraction process, metal ions could have been removed by

the addition of an acid or Na-EDTA. If these metal ions have been removed, the lectin-

binding site will no longer be formed thus rendering the protein inactive and unable to

bind to sugars. Sharon and Lis (1990) showed that these metal ions could be re-

introduced to the lectin in order to re-fold the binding site. However, the metal ions

must be added in a specific order to ensure the correct refolding of the binding site.

_________________________________________________________________ 64


_____________________________________________________________________________________

To test this hypothesis, metal ions were added to the crude extract (50µl of 0.5M metal

ion solutions/ 100µl of protein solution), in the order of Mn2+ and then Ca2+, and

allowed to incubate for 30 minutes (Sharon & Lis; 1990). This mixture was injected

onto the mannose-agarose column to determine whether the binding site of the lectin

was inactive or the specificity of the lectin required more complex structures for its

binding. The column was run using the same conditions as before. The addition of

metal ions to the crude extract did not improve the binding of the lectin to the resin

(results not shown).

Therefore it was proposed that the lectin from G.robusta did not bind to the mannose-

agarose resin due to its specificity for more complex sugar molecules rather than

monosaccharides. An oligosaccharide-agarose affinity chromatography resin (mannan-

agarose resin) was purchased and used in the attempt to isolate this lectin. This resin,

which is made up of many mannose units bound together, was equilibrated with PBS

pH 7.3. The crude extract from G.robusta was injected onto this column and washed

with PBS pH 7.3 until the UV trace returned to the baseline. This peak represented the

unbound material (P1). The bound protein (P2) was eluted using 0.5M mannose in PBS

pH 7.3 as seen in Figure 3.1. Previous experiments using the crude extract from

G.robusta have shown the extract to contain a lectin that was specific for mannose and

therefore it was used to elute the lectin from the column.

_________________________________________________________________ 65


_____________________________________________________________________________________

Figure 3.1: Affinity chromatography of the crude extract from G.robusta.

A mannan-agarose column was equilibrated in PBS pH 7.3 and 0.5M mannose in PBS pH 7.3

was used to elute the bound proteins. Two peaks were seen and labelled P1 & P2.

When comparing the sizes of the two peaks seen in Figure 3.1, it could be safely stated

that the lectin protein isolated from G.robusta could constitute approximately less than

5% of the total protein content. These results are consistent with a number of lectins

that have been isolated from seed material (Rüdiger, 1998; Moreira, R, et. al., 1998).

Native PAGE was used to identify the proteins within the eluted fractions in the attempt

to identify the fraction containing the lectin (Figure 3.2). P1 contained all of the bands

seen in the crude extract (SO1-SO5) while P2 contained the SO1 band with a few faint

bands (SO3 & SO5). Therefore, the lectin was identified as the SO1 band seen on

native PAGE.

_________________________________________________________________ 66


_____________________________________________________________________________________

Figure 3.2: Native PAGE of the eluted fractions.

Crude extract was prepared in PBS pH 7.3. Lane 1: P1. Lane 2: bound protein, P2.

3.3 Bioassays

d there was evidence of some binding in the GIFT assay. The absence of

that all of the lectin was removed from the crude extract.

Review g the bioas o ation was found that the amount

of mate i uir r the lutinatio granulocytes was much less for

the eluted lectin when comp ude extract. As the amount of lectin found

in the c ude extract is less than 5% of e total p functioning ability of the

A 13% separating gel (with a 3% stacking gel) was made and stained with Coomassie Blue. C:

The two peaks from the mannan-agarose column were diluted to 1:20 and tested in the

GAT and GIFT bioassays to determine which fraction contained the lectin. The GAT

bioassay showed the lectin to be present in P2 and this was confirmed with the GIFT

bioassay. The results are shown in Table 3.1.

The protein concentration was determined using the Bradford protein estimation

protocol (refer to section 10.3). P2 was found to agglutinate neutrophils in the GAT

bioassay an

agglutination and low Rel. MCF values were seen for P1 which indicated the lectin was

not present in this sample and

in say and protein c ncentr results it

rial or prote n req ed fo agg n of

ared with the cr

r th rotein, the

_________________________________________________________________ 67


_____________________________________________________________________________________ lectin would be im sev nteracting proteins within this

extract. Therefore, it was not surprising to find the purified lectin to result in a stronger

reaction with the gra yte en co ared to the crude extract.

able 3.1: GAT and GIFT bioassay results of the proteins eluted from the

mannan-agarose column. #

paired as there would be eral i

nuloc s wh mp

T

Dilution [Protein] GAT GIFT (mg/ml) Score Rel. MCF Crude extract Neat 2.40 1+ 5.23 1:2 1.20 3+ 4.35 1:5 0.48 1+ 1.95 P1 Neat 1.48 0 2.65 1:2 0.74 0 1.83 1:5 0.30 0 1.25 1:10 0.15 0 1.02 1:20 0.07 0 1.02 P2 Neat 0.65 2+ 12.33 1:2 0.33 2+ 9.45 1:5 0.13 1+ 6.01 1:10 0.07 0 1.47 1:20 0.03 0 1.16

#The GAT score is graded from 0 – 4+ and the Rel.MCF values above 4 indicate a positive result

in the GIFT bioassay.

The sugar specificity of the lectin was confirmed by using the sugar blocking GIFT

bioassay. The fraction containing the lectin (P2) (final concentration of 0.33 mg/ml)

was mixed with 8 different sugars (in a 1:4 v/v ratio of protein to sugar) using two

different sugar concentrations (0.25 M & 0.5 M). Both monosaccharides and

disaccharides were chosen for the study to determine whether the protein had a

preference for simple or complex sugars. The results of the sugar blocking GIFT assay

are shown in Table 3.2.

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_____________________________________________________________________________________ Table 3.2: Confirmation of the sugar specificity of the lectin.†

Rel. MCF Rel. MCF 0.25M sugar 0.5M sugar lectin & buffer 28.74 -- fucose 25.83 24.47 galactose 29.42 * N-acetylgalactosamine 28.83 21.65 glucose 29.61 24.47 N-acetylglucosamine 29.03 20.78 lactose 28.83 22.62 maltose 28.74 25.73 mannose 21.75 16.70

† The bold value indicated the sugar that the lectin was specific for. A decrease in the Rel.MCF

value indicates blocking of the carbohydrate-binding site within the lectin. * The sample was

inconclusive.

To determine the sugar specificity of the lectin, a decrease in the Rel. MCF value would

be detected using a flow cytometer. This was seen in the sample incubated with 0.25 M

and 0.5 M mannose. These results confirmed the previous findings when the crude

extract was tested. Also there were no other sugars that blocked the binding site of the

lectin and it was therefore concluded that the lectin isolated from G.robusta was specific

for mannose only and not mannose/glucose or mannose/maltose as previously suggested

(Minchinton, 1997b).

_________________________________________________________________ 69


_____________________________________________________________________________________

3.4 N-terminal sequencing of the lectin.

The purified lectin was N-terminally sequenced to determine whether it was similar to

the previously determined GR1 protein. The sample was sequenced three times and

these results were compared with the GR1 protein sequence as seen in Figure 3.3.

Lectin Seq1 -- ?L/ N/ E/ A/ D/ ? P/ E D D V V T ? ? P I P -- ?V Q D R E ? W Lectin Seq2 G? G? N E A/ D/ W C? E D D V V T R -- -- -- -- R E Lectin Seq3 ?G/ G? D/ E A D W C? E D D V V T R/ -- -- -- -- ?A E T Lectin Seq4 ? ? P E A D ? S E D D V ? T/ ? A -- -- -- G GR1 protein G G E E A D W C E D C V C T R S I P P

Figure 3.3: N-terminal sequencing results of the lectin.

The results were compared with the cDNA-determined sequence of GR1 protein. The lectin

was sequenced 3 times (Lectin Seq1-3) and lectin Seq4 represents the minor band (SO3) seen

on native PAGE. The yellow highlights indicated similarities between the GR1 protein and the

lectin.

The regions of similarity between the N-terminal sequence and the GR1 protein are

highlighted in Figure 3.3. Three separate sequencing experiments were carried out

which corresponds to the three sequences in the figure labelled as lectin Seq 1-3. The

sequence labelled Lectin Seq4 corresponds to the contaminating band seen on native

PAGE (SO3) as seen in Figure 3.2.

There were similarities between the N-terminally derived sequences for the lectin and

the GR1 protein. The conserved residues (indicated in yellow within the figure)

demonstrated a 50% homology throughout all of the N-terminal sequences. While there

is insufficient evidence to state the lectin and the GR1 protein are the same protein;

_________________________________________________________________ 70


_____________________________________________________________________________________ there is sufficient data to suggest there are significant regions of similarity between the

two proteins. The level of similarity between the two proteins will not be determined

until the full amino acid sequence of the lectin has been defined. Unfortunately, the

same strategy used to determine the sequence of the GR1 protein cannot be replicated as

the N-terminal sequences for the GR1 protein and the lectin are too similar. Another

method will have to be applied to determine this sequence, such as sequencing using

digestive proteases and liquid chromatography-mass spectroscopy (LC-MS).

The lectin Seq4 was found to contain 50% sequence identity with the lectin Seq 1-3

proteins and 44% sequence identity with the known GR1 protein. Therefore the band

corresponding to SO3 could be a product of carboxy-terminal cleaving of the GR1

protein as the N-terminal sequencing results showed substantial homology with the

lectin Seq1-3 N-terminal sequences.

_________________________________________________________________ 71


_____________________________________________________________________________________

3.5 Conclusion

The lectin was purified from the seeds of G.robusta by using affinity chromatography.

Previous attempts to use this method were unsuccessful due to the lectins’ lack of

specificity for monosaccharides. A multi-branched mannose matrix was used to bind

the lectin from the crude extract and the lectin was eluted using mannose. Native PAGE

revealed the lectin to correspond to the band SO1. The amount of lectin protein isolated

from the crude extract of G.robusta was estimated to be less than 5% and this was

determined by the results from the purification and by native PAGE. These results were

consistent with other lectins isolated from seeds. Bioassays identified the fraction

containing the lectin and also aided in the quantitation of this lectin. The specificity of

the eluted lectin was confirmed with the sugar-blocking GIFT bioassay. This bioassay

also confirmed the lectin from G.robusta to belong to the monocot mannose binding

family. N-terminal sequencing of the lectin revealed a 50% sequence identity with the

previously determined GR1 protein. The full amino acid sequence of the lectin will

have to be determined using alternative methods due to the similarity of the N-terminal

sequences and the GR1 protein. The 3D structure of the lectin could not be determined

because the total lectin concentration within the seeds was very low. In order to

increase the protein concentration required for NMR spectroscopy, a number of

different experiments, such as recombinant protein expression, would need to be

completed. The lectin isolated from the seeds of Grevillea robusta was named the GR2

protein.

_________________________________________________________________ 72

Chapter 4 Purification of the GR1.HPLC protein ____________________________________________________________________________________

Chapter 4 Purification of the GR1.HPLC protein

_________________________________________________________________ 73


Chapter 4 Purification of the GR1.HPLC protein

4.1 Introduction

Affinity, ion exchange (IEX), gel filtration (GF), hydrophobic interaction

chromatography (HIC), hydroxyapatite (CHT II), and reverse phase high-pressure liquid

chromatography (RP-HPLC) were used in the purification of the proteins from the crude

extract of G.robusta. All of the chromatography methods were completed using the

Biologic HR chromatography system from Bio-Rad Laboratories with the exception of

the RP-HPLC chromatography, where the Waters HPLC system was used. The

Biologic HR system is a medium pressure liquid chromatography system that contains

an in-line UV detector (wavelengths 254 & 280nm) and in-line conductivity cell. Both

of these detectors were useful in monitoring and identifying eluting proteins in each

chromatography method.

_________________________________________________________________ 74


4.1 Initial Purification of the Proteins from the Crude Extract of G.robusta

4.1.1 Gel Filtration (GF) Chromatography

Gel filtration chromatography separates proteins on the basis of their size. The resin is

made up of tiny beads that contain pores that allow proteins/compounds to enter and

exit. A number of different pore sizes are available to accommodate any size protein

molecule or macromolecule.

SDS PAGE of the crude extract from G.robusta revealed 2 major bands of

approximately 8700 Da & 7000 Da and several other bands of approximately 14 kDa,

28kDa (Figure 3.2A). A sephacryl S-200 HR resin (Pharmacia) (with a pore size of

5000 – 250000 Da) was used to separate the higher molecular weight proteins from the

lower molecular weight proteins. A S-200 column (45ml) was made and equilibrated in

PBS pH 7.3 at a flow rate of 1.5ml/min. An aliquot (1ml) of crude extract was injected

onto the column. Three peaks were detected (by absorbance at 280nm) as seen in

Figure 4.1. SDS PAGE and native PAGE was used to identify these eluted proteins

(Figure 4.2).

_________________________________________________________________ 75


Figure 4.1: Eluted proteins from G.robusta using gel filtration chromatography.

The column was run in PBS pH 7.3 at a flow rate of 1.5ml/min. UV (at a wavelength of

280nm) was used to monitor the elution profile. Three peaks were identified and labelled Pk1-

Pk3.

Two major bands were seen on SDS PAGE for Pk2. This pattern of bands was seen for

the crude extract. This fraction was run on native PAGE (under non-denaturing

conditions) that resulted in the 5 bands seen previously and these bands are labelled

SO1-5 (Figure 4.2B). The GAT bioassay identified the lectin protein to be in Pk2

(Figure 3.1: GAT results are not shown).

Gel filtration chromatography separated the higher molecular weight proteins from the

lower molecular weight proteins (8700Da & 7000Da) and provided information on the

location of the lectin protein. However, the separation of these proteins (two major

bands on SDS PAGE and five bands on native PAGE) required other chromatography

methods.

_________________________________________________________________ 76


Figure 4.2: SDS PAGE (A) and Native PAGE (B) of eluted GF.S200.Pk2 from gel

An 18% SDS separating (5% stacking) gel at pH 8.8 in the presence of 3% β-mercaptoethanol.

M: Low molecular weight standar

filtration chromatography.

ds. Lane 1: crude extract from G.robusta. Lane 2: Pk2 from

e S-200 column. (B) A 13% native separating (3% stacking) gel at pH 8.9 under non-reducing

conditions. Bands are labelled SO1- SO5. Lane 3: crude extract. Lane 4: Pk2 from the S-200

ine groups (-N+(CH3)3) on the resin allows the binding of

th

column.

4.1.2 Ion Exchange (IEX) Chromatography

Ion exchange chromatography separates proteins on the basis of their net charge and is

available in both positively (anion) and negatively (cation) charged functional groups.

These functional groups are covalently linked to a solid support (matrix) to provide the

anion and cation exchanger.

A strong anion exchange resin (Q-sepharose FF, Amersham Pharmacia Biotech) was

used where the quaternary am

negatively charged proteins. These bound proteins can be removed from the resin by

the addition of a competing ion such as Cl- from NaCl. A Q-sepharose FF column

(10ml) was made and equilibrated in 20 mM Tris at pH 8.0. One ml of crude extract

_________________________________________________________________ 77


from G.robusta was added and a salt gradient (0-15%, 15-40%, 40-100% 2 M NaCl in

20 mM Tris pH 8.0) was used to elute bound proteins. Each step in the gradient had an

isocratic flow step between each stage, which was equivalent to one column volume.

This allowed the weaker binding proteins to elute during each step without being

contaminated with proteins that eluted later. The elution profile is shown in Figure 4.3.

Eluting peaks were detected by A280 nm. Three peaks were detected and the non-binding

proteins or positively charged proteins were located in Pk1 and Pk2 & Pk3 eluted with

approximately 280 mM and 600 mM NaCl respectively. The irregular shape of Pk2

suggested that this fraction contained multiple proteins and therefore, this method must

be further modified to improve resolution of these bound proteins. This cross

contamination of Pk2 & Pk3 was due to insufficient resolution during the 15-40%

gradient step and this was improved by reducing the concentration of the elution buffer

and eluting the proteins over a shorter gradient.

Figure 4.3: Anion exchange chromatography of eluted proteins from G.robusta.

A step gradient (0-15%, 15-40%, 40-100%) 2 M NaCl in 20 mM Tris pH 8.0 at a flow rate of

1ml/min was used to elute bound proteins. Three peaks were identified and labelled (Pk1-3).

_________________________________________________________________ 78


The salt concentration of the elution buffer was decreased to 1M NaCl and more steps

were added (0-14%, 14-35%, 35-55%, 55-100% 1 M NaCl in Tris pH 8.0) to allow

better separation of the proteins in Pk2. The pH, sample volume injected, flow rate

(1ml/min) and buffer composition remained the same. The proteins eluted under these

conditions are shown in Figure 4.4 revealing five peaks (Pk1- Pk5).

Figure 4.4: The elution profile of the crude extract after modifications to the

Proteins were eluted using the gradient 0-14%, 14-35%, 35-55%, 55-100% 1 M NaCl in 20 mM

Tris pH 8.0. T

elution buffer and gradient.

he flow rate was 1 ml/ min. Five peaks were detected and labelled Pk1-Pk5.

SDS PAGE was used to visualise the eluted proteins. These chromatography steps were

completed before the native PAGE results were established as the tool for identifying

the eluted proteins. Pk2 eluted with 140 mM NaCl and contained the two major bands

characteristic of the crude extract from G.robusta ie the proteins of interest. Pk3 also

contained these bands but they were very faint (results not shown) and this fraction

eluted with 300 mM NaCl. Therefore, the proteins of interest were located in Pk2 that

eluted within the range of 140-300 mM NaCl. This method needs to be improved in

order to separate these proteins from each other and this could be done by reducing the

_________________________________________________________________ 79


concentration of the elution buffer (from 1 M to 0.5 M) or by increasing the length of

the gradient step (14-35% B).

Using the same column, the eluting salt concentration was further decreased (to 0.5 M

NaCl), the step gradient was altered (0-12%, 12-23%, 23-35%, 35-100%) and the flow

rate was increased to 1.5 ml/min. The increase in the flow rate was due to the increase

in the time taken to run the experiment due to the additional step in the gradient. One ml

of crude extract was injected onto the column (as before) and the results are shown in

Figure 4.5.

Figure 4.5: Modifications to the salt concentration, flow rate and gradient

increase in the flow rate (to 1.5 ml/min) and the addition of a step in the gradient (0-12%, 12-

conditions.

A decrease in the salt concentration of the eluting buffer (0.5 M NaCl in 20 mM Tris pH 8.0), an

23%, 23-35%, 35-100%). Five peaks were detected and labelled Pk1-5.

_________________________________________________________________ 80


The chromatograph revealed 5 peaks labelled Pk1-5 (Figure 4.5) and native PAGE was

eins being positively charged (ie unable to

ove through the gel).

used to visualise these eluted peaks (Figure 4.6). Pk2, 3 & 4 eluted with 115, 115 &

160 mM NaCl and native PAGE revealed an additional band (labelled I as seen in figure

3.5) within Pk2 along with the band SO3. Pk3 contained SO1/2 SO4/5 and Pk4

contained GR1. The non-binding protein (Pk1) did not reveal any bands on native

PAGE, which could have been due to the prot

m

Figure 4.6: Native PAGE of the eluted peaks from IEX chromatography.

A 13% discontinuous separating gel (with a 3 % stacking gel) was made under non-reducing

conditions. The gel was stained with Coomassie Blue. C: crude extract. Lanes 1-5: Pks1-5.

Two small-scale experiments were completed to determine whether all of the proteins in

G.robusta could be further separated using ion exchange chromatography. The gradient

was further modified and the composition of the buffer was changed. Native PAGE

was used to identify the fractions eluted during each experiment. The conditions and

results of each experiment are listed below.

odified from 0-12% to 0-17%, which would improve the separation

A Q-sepharose FF column (10ml) was made and equilibrated in 20 mM Tris pH 8.0.

The gradient was m

_________________________________________________________________ 81


of the proteins between Pk2 and Pk3 as seen in Figure 4.5. The buffer composition and

elution buffer (0.5 M NaCl in 20 mM Tris pH 8.0), the flow rate (1.5 ml/ min) and the

volume of sample injected (1ml) remained the same. Five peaks were detected and the

results are shown in Figure 4.7.

Figure 4.7: Adjustment of the gradient conditions using a 10ml Q -sepharose FF

he flow rate was 1.5 ml/min and gradients used were 0-17%, 17-23%, 23-35%, 35-100% 0.5

M NaCl in 20 mM Tris pH 8.0. Five peaks were detected and labelled Pk1-5.

he increase in the gradient resulted in the separation of Pk3 from Pk4 (eluted with 100

n of the proteins from G.robusta when compared to the previous

onditions.

column.

T

T

mM & 115 mM respectively); however, the gradient was not sufficient to separate Pk2

from Pk3 (eluted with 85 mM & 100 mM respectively). Pk2 contained a split peak and

two fractions were taken and identified (labelled Pk2A & Pk2B). Native PAGE showed

Pk2A to contain the proteins SO3 and I where Pk2B contained both of these proteins

plus SO1. Pk3 contained SO1/2 and SO4 and Pk4 contained GR1. This method did not

improve the separatio

c

_________________________________________________________________ 82


Reviewing all chromatography methods and conditions, it was concluded that the

proteins from G.robusta were of similar charge. Purification of these proteins in one

step was not possible and each fraction would have to be further processed. It was

noted that specific proteins detected by native PAGE would elute together. The

isolation of these groups of proteins was the focus of the initial purification step of the

crude extract from G.robusta.

4.2 Large scale preparation of the crude extract from G.robusta

The best conditions for the isolation of three groups of proteins were identified and

applied to a larger scale purification protocol. Tris (20 mM, pH 8.0) was chosen as the

buffer for it was found that phosphate buffers could chelate metal ions from proteins

aintain

biological activity.

The large scale Q-sepharose FF method used the gradient 0-12%, 12-23%, 23-35%, 35-

100% with 0.5 M NaCl in 20 mM Tris pH 8.0 as the eluting buffer as it provided the

best separation of the proteins. A Q-sepharose column (40 ml) was used and the flow

rate was increased to 4 ml/min (due to the increase in column volume) and 6 mls of

sample was injected onto the column. Six peaks were seen and are shown in Figure 4.8.

Qseph.8.Pk2 required very little NaCl to elute from the column (60mM) while

Qseph.8.Pk3 & Pk4 required 115 mM and 150 mM NaCl to elute. However,

Qseph.8.Pk5 & Pk6 required 175 mM and 450 mM NaCl to elute from the column.

Native PAGE of these two fractions revealed they contained the bands that

(Rüdiger, 1998). As some lectins require metal ions to be present for their functioning

(Sharon & Lis, 1988; Loris et. al., 1998), the buffering system was changed to m

_________________________________________________________________ 83


corresponded to the SO2 and SO5 proteins and no lectin activity was detected within

these fractions (refer to Table 4.1).

Figure 4.8: Large scale Q-sepharose column at pH 8.0.

The gradient used was 0-12%, 12-23%, 23-35% 35-100% 0.5 M NaCl/20 mM Tris-HCl pH 8.0

Native PAGE results showed the Qseph.8.Pk2 fraction to contain the proteins SO3 and

I. Interestingly, the Qseph.8.Pk1 fraction contained lectin activity but did

at a flow rate of 4 ml/min. Seven peaks were detected and labelled Qseph.8.Pk1-Pk7.

not show any

ands on the gel. This could be due to the levels of protein for the Qseph.8.Pk1 fraction

Lectins only constitute up

to 10% o the total pro within eeds and therefore the amount of protein that

correspo c be very lo ithin th tion a ay not be detected

on nativ s lectin isolated from e seeds of G.robusta

was dete e the total seed content. This could explain the

absence of a band on native PAGE.

The second possibility involves the overall charge of the protein. If the protein was

positively charged it would not migrate through the gel and will not be detected. It was

later found that this was not the reason for the absence of band on native PAGE. The

b

were well below the detection limits for the staining system.

f tein content s

nds to the le tin would w w e frac nd m

e PAGE. A seen in chapter 5, the th

rmined to b less than 5% of

_________________________________________________________________ 84


lectin was isolated (refer to Chapter 3) from the seeds of G.robusta and was identified

The GAT bioassay was used to identify the fractions containing the lectin within each

eluted fraction. Qseph.8.Pk1 & Qseph.8.Pk2 were found to agglutinate granulocytes in

assay are shown in Table 4.1. From these

preliminary results shown in Table 4.1, Qseph.8.Pk2 was used to determine the sugar

specificity of the lectin proteins. This fraction was diluted to 1:50 and tested in the

GAT and GIFT bioassays to determine the saturation point of the lectin binding to the

granulocyte receptors. This information provided the working solution for the sugar

blocking GIFT bioassay. The results of the titre are shown in Table 4.2.

obusta seeds. #

Native PAGE GAT score

as the band corresponding to SO1 on native PAGE.

the GAT bioassay and therefore contained the lectin proteins. A summary of these

results for both native PAGE and the GAT bio

Table 4.1: Native PAGE and GAT bioassay results of eluted fraction from the

large-scale purification of proteins from G.r

pH 8.0 Neat 1:2 1:4 Qseph.8.Pk1 no bands 3+ 2/3+ 2/3+ Qseph.8.Pk2 I, SO3 3+ 2/3+ 1/2+ Qseph.8.Pk3 SO1, GR1, SO4, SO5 0 0 0 Qseph.8.Pk4 SO1, GR1, SO4 0 0 0 Qseph.8.Pk5 GR1,SO4, SO5 0 0 0 Qseph.8.Pk6 GR1, SO5 0 0 0

ge of 0-4+.

#A 13% separating (with a 3% stacking gel) native gel was made and stained with Coomassie

Brilliant Blue. The GAT bioassay was graded from a ran

_________________________________________________________________ 85


Table 4.2: Saturation point on granulocytes using the Qseph.8.5.Pk2 fraction. #

Dilution GAT score CF Rel.M1:2 2/3+ .97 331:5 1+ .55 17

1:10 0 8.73 1:15 0 6.57 1:20 0 5.34 1:50 0 2.83

#The GAT was scored from e 4+ indica rong agglutina Rel. MCF value

above 4 indicated positive binding in the GIFT.

saturation for the lectin-granulocyte

inding was between 1:2 and 1:5. If the 1:15 dilution were used, it would be difficult to

disaccharides) at three different concentrations (0.1 M, 0.25 M, &

.5 M) to determine the specificity of each sample. Each sugar was incubated with the

0-4+ wher ted st tion. A

From Table 4.2, the dilution chosen for the sugar blocking GIFT bioassay for the G.

robusta lectin was 1:10 because the point of

b

distinguish the difference between a positive and negative result as this dilution gave a

Rel. MCF value of 6.57. On the other extreme, if the 1:5 dilution was used, there would

be too many binding sites to fill with sugar and effective blocking of those sites might

not occur, thus providing inaccurate results.

The sugar blocking GIFT bioassay used eight different sugars (a combination of

monosaccharides &

0

diluted lectin solution (1 part lectin & 4 parts sugar) prior to adding the mixture to the

granulocytes. The results are shown in Table 4.3.

_________________________________________________________________ 86


Table 4.3: Sugar-blocking GIFT results of Qseph.8.Pk2. †

Pk2 (1:10) Pk2 (1:10) 0.5M Sugar MCF Rel. MCF

L-fucose 0.946 6.66 Galactose 0.91 5.83 N-acetylgalactosamine 1.09 6.99 glucose 1.13 7.24 N-acetylglucosamine 1.14 7.31 lactose 0.94 6.03 maltose 0.856 5.49 mannose 0.633 4.06 No block 1.4 8.97

† All sugars are D-sugars unless otherwise indicated. The decrease in Relative MCF (and MCF)

hese results were consistent with the preliminary results obtained for the crude extract.

The method for the large-scale purification of G.robusta proteins was further modified

by increasing the pH from 8.0 – 8.5 (to increase the binding of the proteins to the

column). Figure 4.9 shows the elution pattern for the proteins when the pH is changed

(9 peaks were detected and labelled Qseph.8.5.Pk1 – Qseph.8.5.Pk9).

was detected in the mannose sample (shown in bold).

Mannose was found to block the sugar-binding site of the proteins in Qseph.8.Pk2.

T

A large number of lectins are specific for mannose/glucose, as the only difference

between the two sugars is the orientation of the OH group around C2 in the ring. The

lectin in G.robusta was found to be specific for mannose only, suggesting the

orientation of the OH group at the C2 of the sugar is important in the binding of the

sugar to the lectin.

_________________________________________________________________ 87


Figure 4.9: Large scale Q-sepharose column at pH 8.5.

The pH of the buffers were increased to 8.5 and the column was run with a flow rate of 4

ml/min using the gradient 0-12%, 12-23%, 23-35%, 35-100% 0.5 M NaCl/ 20 mM Tris-HCl pH

8.5. Nine peaks were detected and labelled Qseph.8.5.Pk1-Qseph.8.5.Pk9.

Native PAGE and GAT bioassay were used to identify and characterise the proteins

eluted using this method at pH 8.5. The samples were diluted to 1:8 for the GAT

bioassay and to 1:4 in the GIFT. Fractions Qseph.8.5.Pk1 through to Pk6 returned

positive results to the GAT and GIFT bioassay for the presence of lectins. Table 4.4

summarises these results from native PAGE and the GAT and GIFT bioassays for the

fractions eluted from the column.

_________________________________________________________________ 88


Table 4.4: Summary of native PAGE and bioassay results of the proteins eluted #

Native PAGE GAT GIFT

from the large-scale Q-sepharose column at pH 8.5. 13% separating gel Score Rel. MCF Rel. MCF 3% stacking gel 1:2 1:4 1:2 1:4 Crude extract All bands 3+/4+ 3+/4+ 23.61 23.16 Qseph.8.5.Pk1 Blank 2+/3+ 2+ 34.18 21.71 Qseph.8.5.Pk2 Blank 3+ 3+ 64.56 46.20 Qseph.8.5.Pk3 Blank 2+ 1+/2+ 16.08 8.92 Qseph.8.5.Pk4 I, SO1 3+ 2+/3+ 52.97 -- Qseph.8.5.Pk5 SO1,SO3, SO5 2+ 1+/2+ 42.66 24.49 Qseph.8.5.Pk6 SO1,SO4 1+ 0 27.47 21.39 Qseph.8.5.Pk7 SO1,GR1,SO4,SO5 0 0 9.87 5.51 Qseph.8.5.Pk8 GR1,SO4, SO5 0 0 14.11 9.68 Qseph.8.5.Pk9 SO4 1+/2+ 0 21.08 13.48 #Bands were stained with Coomassie brilliant blue on native PAGE. The GAT bioassay was

scored from 0-4+. A positive result is a score of 2+ or better. A Rel. MCF value greater than 4,

dicates some interaction or binding of the protein to the surface structures of the granulocyte.

H 8 (Qseph.8.Pk4). A

umber of runs were completed using the large-scale method at pH 8.5 to collect

enough material for further purification.

in

Agglutination was detected in the fractions, Qseph.8.5.Pk1-Qseph.8.5.Pk6, and all of

the eluted peaks were found to bind to the surface of the granulocytes as seen with the

GIFT results. Qseph.8.5.Pk4 contained the bands, I & SO1, which were the same as

those seen in Qseph.8.Pk2. From these results, it was concluded that the fractions

containing the lectin were Qseph.8.Pk2 and Qseph.8.5.Pk4. No agglutination was

detected in Qseph.8.5.Pk7 however, this fraction did bind to the granulocytes surface

structures detected by the GIFT bioassay. Native PAGE indicated that this fraction is

made up of mainly the GR1 protein with some minor bands (SO1, SO4 and SO5) which

was similar to the fraction 4 from the large-scale method at p

n

_________________________________________________________________ 89


4.3 Further Purification of G.robusta proteins

Each fraction eluted from the large-scale Q sepharose method (at pH 8.5) was further

purified using a number of different chromatography techniques. The fractions that

were further investigated were Qseph.8.5.Pk4, Qseph.8.5.Pk5/6 and Qseph.8.5.Pk7

from the large-scale method at pH 8.5. All of these fractions were positive in the GIFT

and GAT bioassays except for Qseph.8.5.Pk7, which did not agglutinate cells in the

GAT bioassay. Fraction Qseph.8.5.Pk4 and the fraction containing the

seph.8.5.Pk5/6) were the first two groups to be studied.

raphy supports and

lectrophoretic techniques. It is available in two forms, crystalline and spherical. There

are two different types of spherical CHT-type I and type II. CHT-type I has a high

finity for acidic proteins but provides better

(Q

Hydroxyapatite chromatography was used in the attempt to separate the proteins in

Qseph.8.5.Pk4. Hydroxyapatite is a form of calcium phosphate (Ca5(PO4)3OH)2) which

has been used in the separation of a number of proteins, enzymes, nucleic acids and

macromolecules (Bio-Rad Laboratories, 2001). Hydroxyapatite allows the separation of

molecules that are usually homogenous to other chromatog

e

protein binding capacity and has a high affinity for acidic proteins while the CHT-type

II resin has a lower binding capacity and af

resolution of proteins that eluted at lower NaP concentrations.

_________________________________________________________________ 90


4.3.1 Further purification of the GR1.Qseph and SO4/5.Qseph proteins.

A CHT II column was used to separate the SO1/2 and SO4/5 proteins. The column was

equilibrated in 10mM sodium phosphate buffer (NaP) pH 6.9 and 0.6ml of SO1/2 &

SO4/5 mixture was injected onto the column. A gradient of 0-100% 400 mM NaP pH

6.9 was used to elute bound proteins at a flow rate of 0.8 ml/min. Three peaks were

detected using UV (at a wavelength of 280 nm) and native PAGE was used to identify

these eluted peaks (Figure 4.10).

Native PAGE revealed CHTII.Pk1 to contain the SO4/5 proteins while CHTII.Pk2 and

CHTII.Pk3 both contained the SO1/2 proteins. Therefore, the SO4/5 proteins could be

separated from the SO1/2 proteins using the CHT II column. Due to the low protein

concentration, these samples were not tested for biological activity. Other workers took

on the project to isolate the lectin proteins from Qseph.8.5.Pk4 and Qseph.8.5.Pk5/6

however; their work did not result in the purification of the lectin.

_________________________________________________________________ 91


Figure 4.10: Hydroxyapatite chromatography of the proteins SO1/2 and SO4/5

derived from the Q sepharose method.

continuous gradient was used (0-100% 400 mM Na Phosphate buffer pH 6.9) at a flow rate of

0.8 ml/min. Three peaks were detected and labelled (CHTII.Pk1-Pk3).

as further

urified using reverse phase high-pressure liquid chromatography (RP-HPLC) which

A

4.3.2 Further purification of the GR1.HPLC protein.

The final fraction, Qseph.8.5.Pk7, which contained the proteins GR1/4, w

p

separates proteins on the basis of their hydrophobic nature. The resin is made up of

silica that can contain a number of bonded phases (C-4, C-8, & C-18) that vary

depending on the hydrophobicity of the sample (C-18 is used for hydrophilic samples).

The sample is injected onto the column in water and a gradient applied using a

hydrophobic solvent such as acetonitrile to elute the proteins from the column.

_________________________________________________________________ 92


A Rainin C-18 analytical column was used to separate the GR1/4 proteins. The column

was equilibrated in 0.1% TFA/ Milli-Q water and acetonitrile/0.1% TFA was the eluting

olvent. A continuous gradient (0-100% acetonitrile) was used initially to determine

where the proteins would elute and this resulted in two peaks eluting at approximately

Using this information, the method was fine-tuned to establish the best conditions for

eins. A small gradient of 25-32% acetonitrile/ 0.1% TFA

ver a period of 10 minutes was found to provide effective separation of these proteins

s

25% acetonitrile/0.1% TFA (results not shown).

the separation of the two prot

o

as seen in Figure 4.11. Two peaks were seen and native PAGE showed HPLC.Pk2 to

correspond to the GR1 protein (results not shown).

Figure 4.11: RP-HPLC of GR1/4 proteins using the gradient 25-32%

The column was equilibrated at 25% acetonitrile/TFA and the gradient was increased over a

period of 10 minutes t

acetonitrile/0.1% TFA.

o 32%. Two main peaks were detected using UV at a wavelength of

and labelled Pk1 and Pk2. 280nm

_________________________________________________________________ 93


The method was altered to determine whether the proteins could be eluted using an

isocratic flow rate. This was important as there was a large volume of protein sample to

proce protein could be purified in a shorter tim An isoc ic flow rate

at 29% a / 0.1% FA w ed to arate fract seen he gradient

ethod (Pk1 & Pk2). The results of this method are shown in Figure 4.12.

in corresponding to the GR1 protein. This

action was concentrated using an ultra filtration stirred cell (membrane cut off of 3000

Da). The GR1 protein isolated using this method is referred to as the GR1.HPLC

protein.

ss, and the more e. rat

cetonitrile T as us sep the ions in t

m

Native PAGE was used to analyse all of the peaks that eluted from the column and the

two main peaks were labelled HPLC.Pk1 & HPLC.Pk2. The fraction labelled

HPLC.Pk2 contained a number of peaks which were the result of the protein interacting

with the resin as it was eluting under the isocratic flow rate. Native PAGE revealed the

fraction HPLC.Pk2 to contain the prote

fr

Figure 4.12: RP-HPLC using an isocratic flow rate at 29% acetonitrile/TFA 0.1%.

Two peaks were detected and labelled HPLC.I.Pk1 & HPLC.I.Pk2.

_________________________________________________________________ 94


4.3.3 Bioassays

Bioassays were completed on the purified GR1.HPLC protein. In both the GAT and

GIFT bioassays, no agglutination or binding was seen for the purified GR1.HPLC

protein. However, the starting material prior to RP-HPLC (Qseph.8.5.Pk7) indicated

some binding to granulocytes in the GIFT bioassay and no agglutination in the GAT

bioassay. It was proposed that the solvents used in the isolation of the GR1.HPLC

protein altered the binding site or structure of the protein, which inhibits their ability to

n experiment was set up to determine whether this was the case. The GAT bioassay

Table 4.5: GAT bioassay and the biological effects of the solvents on the crude

extract. #

Neat 1:2 1:4 1:10 1:50 1:100

bind to their specific sugar.

A

was used to determine the effects of the addition of solvents (the same solvents used for

the isolation of GR1.HPLC protein) on the crude extract. The crude extract was used

for this bioassay as it provided a strong positive result to agglutination and any decrease

in agglutination could be easily identified. The solvents (acetonitrile/0.1% TFA and

0.1% TFA/water) were diluted to 1:100 with water and added to the crude extract (a

control was also used (diluted to 1:5) to ensure the assay was working). The results are

shown in Table 4.5.

Crude extract (1:5) 4+ -- -- -- -- -- Acetonitrile/TFA 0 0 0 1+ 3+ 3+/4+

TFA/water 0 0/1+ 3+ 3+ 3+ 3+ #The bioassay is scored from 0-4+ where 4+ represents strong agglutination.

_________________________________________________________________ 95


No agglutination was detected for the extract in acetonitrile/TFA and TFA/water, until

the dilution reached 1:10 for acetonitrile/TFA and 1:2 for TFA/water (as seen in Table

3.5). For agglutin % or less of tr e/TFA 0% or less for

TFA/water would be required. fo runn he 4.Q m e on a

colu r the current conditio (29% ceto /TF 71% FA/w r) would

result in decreased biological activity.

.3.4 Purification of the GR1.HighQ protein

Another method was required that maintained the biological activity of the

purification process. A different anion exchange resin

ng this method will be referred to as the

ation to occur, 10 acetoni il or 5

There re, ing t GR1/ seph ixtur

mn unde ns a nitrile A, T ate

4

GR1/4.Qseph proteins during the

was used to further separate the GR1/4.Qseph proteins. A High Q column (10ml;

strong anion resin from Bio-Rad Laboratories) was made and equilibrated in 20 mM

Tris pH 8.5. Proteins were separated using a gradient of 0-30% 0.5 M NaCl (in 20 mM

Tris pH 8.5) over 10mls. Five peaks were seen and are shown in Figure 3.13. Native

PAGE was used to identify the eluted proteins. HighQ.Pk4 eluted at 110 mM NaCl and

contained the GR1 band while HighQ.Pk5 contained the SO4 band and eluted with 175

mM NaCl. The GR1 protein isolated usi

GR1.HighQ protein.

_________________________________________________________________ 96


Figure 4.13: The separation of GR1/4.Qseph proteins using a High Q column.

The proteins were eluted from the column using a gradient of 0-30% 0.5 M NaCl in 20 mM Tris

pH 8.5 over 20 minutes. The peaks are labelled HighQ.Pk1- Pk5.

GAT and GIFT bioassays were completed on the eluted fractions from the High Q

column (HighQ.Pk1-Pk5). As determined earlier, the GR1/4.Qseph mixture provided a

negative result in the GAT bioassay. Eluted fractions were tested undiluted while the

starting material and the crude extracts were diluted to 1:10. The bioassay results are

shown in Table 4.6. Agglutination was not seen in any of the eluted fractions in the

GAT bioassay. This was to be expected, as the starting material did not agglutinate

cells. The GIFT bioassay did reveal some binding in HighQ.Pk4 (GR1.HighQ).

_________________________________________________________________ 97


Table 4.6: The GAT and GIFT bioassay results of the eluted fractions using High-

in. GIFT

Q res #

GAT Score Rel.MCF

Sample Neat 1:2 1:5 1:10 Neat 1:2 1:5 1:10

Crude extract 3+ 3+ 2+/3+ 2+ 31.17 26.02 20.78 17.57 Qseph.8.5.Pk7 0 0 0 0 10.39 9.09 5.01 2.63 HighQ.Pk1 0 -- -- -- 10.00 -- -- -- HighQ.Pk2 0 -- -- -- 4.49 -- -- -- HighQ.Pk3 0 -- -- -- 7.96 -- -- -- HighQ.Pk4 0 -- -- -- 6.29 -- -- -- HighQ.Pk5 0 -- -- -- 2.59 -- -- --

# + +

value indicates the level of binding of the lectin to the surface of the neutrophil.

4.4 N-terminal Sequencing

more conclusive N-terminal sequencing

results. All of the bands seen on native PAGE were sent for sequencing; however, some

of the samples did not return a sequence as their N-terminal regions were blocked with

sugars or there was very little sample present. The proteins that did return sequences

are summarised in Figure 4.14.

SO1.High Q

The GAT is scored between 0-4 where 4 represents strong agglutination. The Rel.MCF

Preliminary N-terminal sequencing results using the crude extract provided a number of

inconclusive results. The separation of proteins during the initial ion exchange

chromatography step (Q-sepharose) provided

G G D E R E F ? E D D V V T T S/ I/ P P R -- -- -- -- --

R R

GR1 "Crude" G G E E A D W (C) E D D V V T T S (C) S I P P -- -- -- --

GR1.Qseph G G E E A D (C) (C) E E D V V Q C T I V -- -- -- -- -- -- --

GR1.High Q G G E -- A D F (C) E D D V V T -- R I I/ P P ? K R ? T

P

SO4.Qseph S I P P E A D R E T D S V V V V K -- -- -- -- -- -- -- --

Figure 4.14: N-terminal sequences of proteins seen on native PAGE.

between N-terminal sequences.

Overlapping residues are shown in red.

The yellow highlighted residues indicate similar residues

_________________________________________________________________ 98


The samples used for the sequencing of the proteins were taken at a number of different

stages of purification. The similarities in residues are highlighted in yellow. The

SO1.HighQ sequence was obtained when the SO1 protein was purified using High-Q

resin. The GR1 “Crude” sequence is the GR1 band taken from the crude extract. The

GR1.Qseph and SO4.Qseph are the GR1 and SO4 bands seen in the fraction

Qseph.8.5.Pk7. The GR1.HighQ is the purified GR1 protein after running it down the

High-Q column. N-terminal sequencing results of the SO4 protein revealed a region

that overlapped with the sequencing results from GR1 protein (shown in red). It was

roposed that the SO4 protein was a cleaved product of GR1 protein. p

_________________________________________________________________ 99


4.5 Determination of the full amino acid sequence and sequence alignment

studies of the GR1.HPLC protein.

or a

d o

used to produce degenerate primers providing the full amino acid e u f the GR1

and SO4 proteins. The results and methods used to determ e th u mino a

ence are t o e

ote t

ore

h r

compared, it was found that there were differences as shown in Figure 4.15. It was

concluded from these results that the GR1 protein has under e s m o of post-

lational m e

blue.

S -- - -- -- -- -- E D

R1cDNA C V C T R S I P P R C R C T D S S V C T K C V C

R F D A F C P -- -- -- -- -- --

A fellow w ker used cDNA techniques to determine the full mino acid sequence of

the GR1 an SO4 pr teins (Clague et. al., 1999). The N-terminal sequencing data was

s q ence o

in e f ll a cid

sequ ou lined in Appendix A. Fr m these sequencing r sults, it was found that

the SO4 pr in was 100% homologous (by sequence alignmen ) with the GR1 protein

and theref it was concluded that the SO4 protein was a degraded product of the GR1

protein. W en the full amino acid sequence and the N-te minal sequences were

gon o e f rm

trans odification within the se d.

GR1cDNA M A V A K V A L M I T L M V L L F V A T L P A PGR1 N-term -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --

GR1cDNAGR1 N-term

T --

A --

T -

S --

N P --

F --

G P F --

R --

P S --

GG G

G EE

EE

AA D

D WW

CC

E D

GGR1 N-term D V V T T S I P P R D R E T D S V V V V K -- -- --

GR1cDNA Y L T V P A A M R P Y C E S M A SGR1 N-term -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --

Figure 4.15: Comparison between the full amino acid sequence and the N-terminal

sequencing results for the GR1 protein. The N-terminal sequencing results obtained for the GR1.HPLC protein is shown in red while the

sequencing results obtained for the SO4 protein are shown in

GR1cDNA I G S L Q S Y NGR1 N-term -- -- -- -- -- -- -- --

_________________________________________________________________ 100


The sequencing alignment program called BLAST (Basic local alignment search tool)

hibitors from the Bowman-Birk inhibitor family. The

equences were aligned and shown in Figure 4.16.

as a

protein when comp re the her members of the Bowman-Birk f m y ithin the

in

position and the cysteine residues located in front of the reactive site of the

o d position. I w s th re ore

ought that these residues play an important role in stabilising the structure of the

was used to determine whether there were any similarities between the amino acid

sequence of the GR1 protein and other known proteins (Altschul et. al., 1990). BLAST

(from NCBI) searches the Brookhaven Protein Database and Swissprot databases and

provides information on the level of homology of the unknown protein with a number of

known proteins. The results of this search found the GR1 protein to be similar to a

number of serine protease in

s

It w found th t the cysteine residues were highly conserved throughout the GR1

a d to ot a il . W

tryps -binding region, all but one of the cysteine residues were located in the same

chym trypsin binding region were foun to be in the same t a e f

th

proteins by forming disulfide bridges.

_________________________________________________________________ 101


1 10 20 30

GR1 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- G G E E A D W C E D C V C


’ P1GR1 T R

31 P1 P1 40 50 P1 60’

S I P P R C R C T D S -- -- -- -- -- -- S V C T K C V C Y L T


61 70 80 90

GR1 V P A A M R P Y C E S M A S R F D A F C P I G S L Q S Y N --

1BBI Y P A Q C F C V D I T D -- -- -- -- -- -- F C Y E P C K P S E D D PI-II Q P G Q C R C L D T N D -- -- -- -- -- -- F C Y K P C K S R D D -- IBB3_DOLAX I P A Q C V C V D M K D -- -- -- -- -- -- F C Y A P C K S S H D D IBB4_DOLAX E P A Q C F C V D T T D -- -- -- -- -- -- F C Y K S C H N N A E K IBB_PHAAU M P G K C R C L D T D D -- -- -- -- -- -- F C Y K P C E S M D K D IBB2_PHAAN M P G Q C R C L D T H D -- -- -- -- -- -- F C H K P C K S R D K D IBB3_SOYBN Q P G Q C R C L D T N D -- -- -- -- -- -- F C Y K P C K S R D D -- IBB2_SOYBN M P G Q C R C L D T T D -- -- -- -- -- -- F C Y K P C K S S D E D

Figure 4.16: Sequence alignment of the GR1 protein with a number of protease

inhibitors from the Bowman-Birk protease inhibitor family.

Cysteine residues are boxed and the reactive site is shown by the P1-P1’ scissle bond (in blue).

The trypsin-binding site is highlighted in yellow and the chymotrypsin-binding site is shown in

red. 1, GR1 (Grevillea robusta inhibitor). 2, 1BBI (Bowman-Birk Inhibitor, Werner &

Wemmer, 1992). 3, PI-II Glycine max, Chen et. al., 1992). 4, 1BB3_DOLAX & 5,

1BB4_DOLAX (Dolichos axillaris inhibitor, Jourbert et. al., 1979). 6, 1BB_PHAAU

(Phaseolus aureus (mung bean) inhibitor, Zhang et. al., 1982). 7, 1BB2_PHAAN (Phaseolus

angularis (adzuki bean) inhibitor, Kiyohara et. al., 1981). 8, 1BB3_SOYBN & 9,

1BB2_SOYBN (Glycine max, Joudrier et. al., 1987).

_________________________________________________________________ 102


The Bowman Birk inhibitor contains 14 cysteine residues that form 7 disulfide bridges

while the GR1 protein has only 10 cysteine residues that could form 5 disulfide bridges.

The disulfide-bonding pattern for the Bowman-Birk inhibitor is shown in Figure 4.17.

However on closer inspection of the sequence alignment of the two proteins, it was

thought that the GR1 protein might form 3-5 disulfide bonds due to the positioning of

these residues (Figure 4.17).

1 10 P4 P3 P2 P1 P1’ P2’ P3’ P4’ 30GR1 -- G G E

1BBI D D E S

E A D W C E D C V C T R S I P P R C R C T D -- -- -- --

S K P C C D Q C A C T K S N P P Q C R C S D M R L N

40 31 P4 P3 P2 P1 P1’ P2’ P3’ P4’ 50 60

1BBI S C H S A C K S C I C A L S Y P A Q C F C V D I T D -- -- -- --

1BBI -- -- F C Y E P C K P S E D D K E N

The scissle bond for both trypsin and chymotrypsin (P1 & P1') are highlighted in yellow. The disulfide

ridges found in the Bowman-Birk inhibitor are shown in different colours. The conserved cysteine

within this protein.

3' and P4' of the trypsin binding region were

und to be conserved within the Bowman-Birk family and the GR1 protein. A

threonine at the P2 site is highly conserved throughout all Bowman-Birk inhibitors and

onserved throughout

the Bowman-Birk inhibitor family and has been shown to be important in the

GR1 S -- -- S V C T K C V C Y L T V P A A M R P Y C E S M A S R F

61 70

GR1 D A F C P I G S L Q S Y N -- -- -- --

Figure 4.17: Position of the disulfide bridges of the Bowman-Birk inhibitor and

sequence alignment with the GR1 protein.

b

residues in the GR1 protein are also coloured providing an insight into the possible disulfide bond pattern

Within the trypsin binding region, the P1' residue (highlighted in yellow in Figure 4.18)

is conserved throughout the Bowman-Birk family and this was also found to be true for

the GR1 protein. Residues at position P

fo

this was also true for the GR1 protein. The threonine residue is c

_________________________________________________________________ 103


functioning of the inhibitor. Therefore, the presence of this residue in the same position

ight be important for the function of this protein within the GR1 protein suggests it m

(McBride et. al, 1998).

The reactive site for chymotrypsin contains a serine in the P1' position and a conserved

serine. It is possible that the P1-P1' region of the GR1 protein functions in a different

conserved throughout the family and this was also seen in the GR1 protein. Like the

leucine residue at P1. The GR1 protein does not have the conserved serine residue in

the P1’ position, instead, it contains a threonine residue, which could substitute for the

manner to other Bowman-Birk inhibitors. At position P3', the residue proline is

trypsin-binding region, this position may be important in forming the turn between two

β-sheet structures within the GR1 protein.

_________________________________________________________________ 104


4.6 Mass Spectroscopy (MS) of the GR1.HPLC protein

n was injected (Figure 3.18). Utilising the

ass spectrometry software, these 4 ion peaks were related to each other (charged

pecies are shown in red in the Figure 4.18) and the molecular weight of the GR1.HPLC

protein was calculated to be 6669 Da.

Mass spectrometry was used to determine the molecular weight of the GR1.HPLC

protein. Mass spectrometry is a useful tool in providing structural information about the

molecule. The principle behind this form of spectroscopic technique is that a small

amount of sample is bombarded with a high-energy electron beam resulting in a

molecular ion (M+) being produced. The instrument is set up to detect the mass to

charge ratio of the molecular ions, which therefore determines the molecular weight of

the molecule.

A single quadruple electrospray mass spectrometer (Fisons instruments) was used to

determine the molecular weight of the GR1 protein has a detection limit of 1 charge per

2000 Da. SDS PAGE and gel filtration chromatography suggested the GR1 protein to

have an approximate molecular weight of 7000 Da. This spectrometer could still be

used to predict the molecular weight of the protein by detecting the multiply charged

species of the protein.

Positive electrospray was used and a number of ion peaks were detected on the

spectrometer after the GR1.HPLC protei

m

s

_________________________________________________________________ 105

Chapter 4 Purification of the GR1.HPLC protein ____________________________________________________________________________________ Chapter 4 Purification of the GR1.HPLC protein ____________________________________________________________________________________

___________________________

Figure 4.18: Positive electrospra

There are 4 ion peaks that correspo

calculated to be 6669Da using the M

The program Peptide Mass in ExP

of the cDNA encoded GR1 prot

values obtained were in the redu

7285.31 Da. This value is very

GR1.HPLC protein (6669 Da).

some proteolysis. A number of

entered into the Peptide Mass p

actual value for the GR1.HPLC

(LQSYN) at the C-terminus was

mass of 6679.67 Da which w

GR1.HPLC protein. It should b

GR1.HPLC protein (without the

GR1.HPLC protein contains 10 cy

___________________________

[M]/6+

___________

the GR1

y of the GR1

nd to

assLynx softw

ASy was use

ein (Wilkins

ced form and

different from

It was therefo

residues were

rogram until

protein. It w

removed fro

as 10 Da of

e noted that

5 residues) w

steine residu

___________

[M]/5+

________________

.HP

.HPLC protein.

LC protein. The

are provided with the

d to calculate the th

et. al, 1997; Wilkin

the average mass

the experimental v

re concluded that t

subtracted from th

a value was obtain

as found that whe

m the sequence it

f the actual mass

the theoretical ma

as determined in a

es and therefore has

________________

[M]/4+

[M]/7+

___________ 106

molecular weight was

spectrometer.

eoretical mass and pI

s et. al, 1998). The

was calculated to be

alue obtained for the

he protein undergoes

e C-terminus and re-

ed that matched the

n the last 5 residues

provided an average

determined for the

ss calculated for the

reduced state. The

the potential to form

___________ 106


5 disulfide bonds. If all of the cysteine residues were involved in forming disulfide

the

ctual mass of the protein and thus giving a mass of 6679 Da. Therefore, all cysteine

nvolved in forming disulfide bridges and the

protein undergoes post-translational modifications at the C-terminus within the seed.

bonds and the protein was fully reduced, an additional 10 Da would be added to

a

residues within the GR1.HPLC protein are i

_________________________________________________________________ 107


4.7 Conclusion

A number of different proteins were identified in the crude extract from G.robusta. A

large-scale chromatography method was established which partially separated these

proteins into three different groups. Two of these groups of proteins were reactive in

both the GIFT and GAT bioassays while the other group was only reactive in the GIFT

bioassay. Initial work on the isolation of the lectin protein was started and was taken

over by co-workers. The group of proteins that was reactive only in the GIFT bioassay

contained the GR1/4.Qseph proteins. These proteins were successfully separated using

two different methods, RP-HPLC and ion exchange chromatography. The GR1.HPLC

d had the 5 C-terminal residues removed by post-

anslational modification. The full amino acid sequence was determined and sequence

protein was N-terminally sequenced and this information was used to generate

degenerative primers to determine the full amino acid sequence of the protein. Mass

spectrometry determined the molecular weight of the GR1.HPLC protein to be 6669 Da.

The cDNA encoded GR1 protein was found to contain 5 disulfide bridges (identified by

comparing the molecular weights of the theoretical and experimental masses using the

Peptide Mass program in ExPASy) an

tr

alignment studies have shown the GR1 protein to belong to the Bowman-Birk

subfamily of serine protease inhibitors.

_________________________________________________________________ 108

Chapter 5 Purification and Characterisation of the GR1.GF protein ______________________________________________________________________

Chapter 5 Purification and characterisation of the GR1.GF

protein.

_________________________________________________________________ 109

Chapter 5 Purification and Characterisation of the GR1.GF protein _____________________________________________________________________________________

Chapter 5 Purification & characterisation of the GR1.GF protein

he full amino acid sequence of the GR1.HPLC protein suggested the protein to be

5.1 Introduction

A serine protease inhibitor was isolated from G.robusta using ion exchange

chromatography and RP-HPLC. Serine protease inhibitors are one of four different

protease inhibitors identified in nature. The characterisation of these inhibitors is based

on the amino acids involved within the reactive site (refer to section 1.9 for more

information).

T

cleaved during the extraction process (leaving a minor contaminant seen on native

PAGE) (section 4.5). This chapter will outline the extraction of the serine protease

inhibitor from the seeds of G.robusta without proteolysis, the isolation and

characterisation of the GR1 protein.

_________________________________________________________________ 110


5.2 Extraction of the proteins from the seeds of G.robusta.

s of proteolysis during the extraction stage, protease inhibitors

ferent protease inhibitors to ensure all proteases found naturally in seeds

would be inactive. A commercially available mixture of protease inhibitors was used

which contained the following protease inhibitors: 4-(2-aminoethyl)benzenesulfonyl

fluoride (AEBSF), trans-epoxysuccinyl- L-leucylamido(4-guanidino)butane (E-64),

bestatin, leupeptin, aprotinin and sodium EDTA. PMSF was also added to the buffer.

Ground seeds were soaked in the extraction buffer containing protease inhibitors (TBS

+ PI’s) overnight at 4ºC. Ammonium sulfate was used as before (refer to section 3.2) in

two stages to precipitate the proteins from solution. Once precipitated, the proteins

s

the

and corresponding to SO4 was the result of protein degradation.

To minimise the chance

(PI’s) were added to the TBS buffer. The extraction buffer contained TBS and a wide

range of dif

were resuspended in milliQ water and initially dialysed in milliQ water (2 change

every 30 mins) and then against TBS + PI buffer pH 7.8 (2 changes every 30 mins) to

remove excess ammonium sulfate.

Native PAGE was used to visualise the extracted proteins. The results are shown in

Figure 5.1. Native PAGE revealed an absence of the band in the crude extract prepared

with protease inhibitors, which corresponded, to SO4. These findings supported the

initial findings from the N-terminal sequencing and cDNA sequencing data that

b

_________________________________________________________________ 111


_________________________________________________________________ 112

Figure 5.1: Native PAGE of the crude extracts processed without and with

protease inhibitors.

The gel was stained with Coomassie Brilliant Blue. Lane 1: crude extract processed with PBS

pH 7.3; Lane 2: crude extract processed with TBS + PI’s pH 7.8. The arrow shows the absence

of the SO4 band in Lane 2.

5.3 Purification of the GR1.GF protein from the crude extract

The crude extract (containing protease inhibitors) was further purified using gel

filtration chromatography. A superdex 75 prep grade gel matrix was used to separate

the proteins from the crude extract. Superdex 75 is made up of highly cross-linked

porous agarose beads and covalently bound dextrin (Pharmacia LKB Biotechnology,

1991). The combination of dextran and cross-linked agarose stabilises the matrix,

physically and chemically. The pore size for the superdex 75 resin is in the range of

3000 – 70 000Da, which will provide adequate separation of the proteins found within

G.robusta.

A 20cm superdex 75 column (ID of 2.5cm) was made and equilibrated with TBS + PI’s

pH 7.8. Calibration markers were used to determine whether the column was packed

evenly and provided information on the approximate molecular weight of the eluted

proteins. The proteins used to calibrate the column were Blue dextran (Mr = 2000000


Da), bovine serum albumin (BSA) (66000 Da), carbonic anhydrase (29000 Da),

cytochrome (12400 Da) and aprotini Da).

The approximate molecular weights of eluted pro s were de paring

the ratio of /Vo of the unknown proteins against the Ve/Vo of the standard proteins.

The void volume of the column (Vo) was identified by blue dextrin (Mr of 2000000 Da)

while the r inder of the standard teins, eluted at specific volumes (Ve). A

eights of the unknown proteins were determined by plotting their Ve/Vo values onto

c n (6500

tein termined by com

Ve

ema pro

calibration curve was created where the logarithms of the molecular weight of the

standard proteins were plotted against their respective Ve/Vo values. The molecular

w

this graph.

One ml of crude extract (prepared with protease inhibitors) was injected onto the

column at a flow rate of 1.5ml/min and the column was run in 100% extraction buffer

(+ protease inhibitors) pH 7.8. Five peaks were detected (Figure 5.2) and native PAGE

(Figure 5.3) identified the proteins within each eluted fraction.

_________________________________________________________________ 113


Figure 5.2: Gel filtration chromatography of the crude extract (containing

rotease inhibitors).

Five peaks were detected and labelled F1-F5. The flo ate wa . m and one ml of

crude extract was injected t h .

p

w r s 1 5 l/min

on o t e column

Figure 5.3: Native PAGE of eluted fractions from gel filtration chromatography.

A 13% separating gel (with a 3% stacking gel) was made and stained with Coomassie Blue.

pH 7.8. Lanes 3-7: eluted fractions from gel filtration chromatography labelled F1-F5.

Lane 1: crude extract prepared with PBS pH 7.3. Lane 2: crude extract prepared with TBS + PI

_________________________________________________________________ 114


Native PAGE showed the F2 fraction to contain all of the corresponding bands (SO1-

SO5). PAGE also revealed Fraction (F3) to contain a single band and this band was

thought to correspond to the GR1 protein. Due to a single band being present on the

Native PAGE, no additional purification experiments were applied to this fraction.

5.4 Bioassays

he crude extract was prepared with and without protease inhibitors and the fractions

ry

little binding to granulocytes and thus a negative result.

T

eluted from the gel filtration column were tested for lectin activity. The results from the

GAT and GIFT bioassays are shown in Table 5.1. The GAT bioassay of the crude

extract containing protease inhibitors indicated the activity of the lectin was maintained

when the proteins were extracted using protease inhibitors in the buffer. The GIFT

bioassay was not as conclusive as the GAT bioassay as the relative MCF value for the

crude sample prepared with protease inhibitors was below 4, suggesting none or ve

_________________________________________________________________ 115


Table 5.1: GAT and GIFT bioassay results.

GAT GIFT Dilution Score Rel. MCF Crude extract in Neat 2+ 16.60 PBS 1:2 1+ 14.47 Crude extract in TBS + PI's Neat 4+ 4.68 1:2 3+ 3.23 F1 N 3+ 17.48 eat 1:2 2+ 15.53 F2 Neat 0 6.64 1:2 0 6.67 F3 Neat 0 3.67 1:2 0 2.71 F4 Neat 0 3.26 1:2 0 3.25

The bioassay tested both crude extracts (i.e. with and without protease inhibitors) and the

fractions that eluted from the gel filtration chromatography. The GAT bioassay was scored

from 0-4+ and a Rel. MCF value above 4 indicates binding in the GIFT bioassay.

The lectin was identified in fraction 1 (F1) as seen in Table 5.1, however, no significant

not mean the lectin is

protein content within the seed and therefore, only a small amount of lectin would be

purified. The GIFT bioassay confirmed the presence of the lectin within this fraction by

porting a high relative MCF value. Agglutination was not seen in the remainder of the

bands were detected on native PAGE. The absence of bands does

not present but rather the total concentration of the lectin within this sample was too low

to be detected by commercial staining. Lectins only constitute up to 10% of the total

re

fractions and the GIFT bioassay revealed some granulocyte binding in fraction 2 (F2).

The peak containing the GR1 protein (F3) did not agglutinate the granulocytes in the

GAT nor was there any evidence of the proteins binding to granulocytes in the GIFT

assay. Therefore the GR1 protein purified using gel filtration chromatography did not

contain any lectin-like properties and will be referred to from this point on as the

GR1.GF protein.

_________________________________________________________________ 116


5.5 N-terminal sequencing of the GR1.GF protein

The GR1.GF protein was N-terminally sequenced to determine whether it was the same

protein as the one purified using reverse phase chromatography (section 3.4.2). The

sequencing results are shown in Figure 5.4. The N-terminal sequencing results for the

GR1.GF protein revealed similarities with the previously identified GR1.HPLC and

GR1.HighQ proteins. Differences were seen between the three N-terminal sequences.

The GR1.GF protein was found to have a cysteine residue instead of a glycine at

position 2 and an asparagine was found in the place of an aspartic acid at positions 6 &

11 (shown in red in Figure 5.4).

GR1.HPLC G G E E A D W (C) E D D V V T T S (C) I P GR1.HighQ G G E E A D F (C) E E D V V T C T I -- --GR1.GF G (C) E E A N W (C) E E N V V T T -- -- -- --GR1 (full sequence) G G E E A D W C E D C V C T R S I P P

Figure 5.4: N-terminal sequencing homology of the eluted GR1 proteins.

Amino acids are shown using the one letter code. Conserved regions are highlighted (in yellow)

and amino acid residues that differed from the previously determined N-terminals are shown in

red. The full amino acid sequence (derived from cDNA techniques) was used to compare the

N-terminal sequences.

The cysteine residue in the GR1.GF sequence is in brackets as this residue is destroyed

during the sequencing process. To obtain an N-terminal sequence, the amino acid

residues of the protein are hydrolysed and this often results in the modification of these

residues. The addition of strong regents such as hydrochloric acid has been found to

deaminate the amino acids asparagine and glutamine into their respective acids

(Davidson, 1997). Therefore, it is difficult to determine which residue is an aspartic

acid or an asparagine.

_________________________________________________________________ 117


The N-terminal sequences did not differ from the sequence derived from full sequence

derived from DNA techniques. The regions that are homologous with the full sequence

are highlighted in yellow in Figure 5.4. Therefore, N-terminal sequencing provided a

good and reasonably accurate prediction on the amino acid sequence for the GR1

protein.

The three-boxed peak values are charged species of the same protein. These species

exist because the mass spectrometer measures the ratio between the molecular weight or

mass and the charge of the protein. The mass spectrometer used in this experiment was

the same one used to identify the mass of the GR1 protein isolated using HPLC

(GR1.HPLC; refer to section 4.7 for more details). Key ion peaks that corresponded to

the HPLC purified GR1 protein were the same as those seen in Figure 5.5.

5.6 Mass Spectroscopy of the GR1.GF protein

The GR1.GF protein had a molecular weight of 6669 Da, which was determined using

mass spectroscopy. The GR1.HPLC protein also had a molecular weight of 6669 Da.

The mass spectrum for the GR1.GF protein is shown in Figure 5.5. Despite the

GR1.GF protein N-terminal sequence differing by two amino acids, the calculated

molecular weights for both proteins were the same. It was therefore assumed that the

two proteins isolated by different chromatography methods were the same proteins.

_________________________________________________________________ 118



_________________________________________________________________ 119

btained for the GR1.GF prFigure 5.5: Mass spectrum o otein.

Positive electrospray of the sample reveals multiple ion peaks that equate to the molecular

weight of the protein. The boxed peaks show the related peaks in this trace.

5.7 Serine protease inhibition assays

Protease inhibitors that belong to the Bowman-Birk inhibitor family are able to

inactivate both the trypsin and chymotrypsin proteases (independently). The trypsin-

binding site of the GR1 protein was very similar with the protease inhibitors from the

Bowman-Birk family and there were some regions of homology in the chymotrypsin-

binding region. An inhibition assay was set up by a co-worker at the ARCBS to

determine whether the GR1 protein (using the GR1.GF sample) belonged to the

Bowman-Birk family by inactivating trypsin and chymotrypsin (Clague, 2001).

[M]/5+

[M]/6+

[M]/4+

_________________________________________________________________ 119


It was found that the GR1.GF protein inactivated both trypsin and chymotrypsin and

was therefore c ember of the Bowman-Birk family. The KI values for

the inactivation of trypsin and chymotrypsin were 1.37 x 10-9 M and 4.8 x 10-9 M

respectively (Clague, 2001; Appendix B). On comparison with the Bowman-Birk

protease inhibitor (Werner & Wemmer, 1991), where the KI values for trypsin and

chymotrypsin were 5 x 10-9 M and 5.2 x 10-9 M respectively, the GR1.GF protein was

ion of trypsin (Figure B1-

A) does not fit the data points on the graph. This could explain why the inhibitory

haracterised as a m

found to be similar in its ability to inactivate these proteases.

A fellow co-worker, as a part of her Masters research, generated the inhibition curves

for the GR1.GF protein (seen in Figure B1 {Appendix B}) (Clague et. al., 1999, 2001).

It should be noted that the curve corresponding to the inactivat

results obtained for trypsin were lower than that seen for the Bowman-Birk inhibitor.

_________________________________________________________________ 120


5.8 Conclusion

The preparation of the crude extract using protease inhibitors revealed an absence of the

protein band SO4 on native PAGE. These results supported the initial sequencing

results that the SO4 protein was a degradation product of the GR1 protein. Using gel

filtration chromatography, the GR1 protein was purified from the crude extract

containing protease inhibitors (labelled GR1.GF). Bioassays ensured the crude extract

prepared with protease inhibitors maintained lectin activity and was located in the first

fraction after gel filtration chromatography (GF.F1). The GR1.GF protein did not

agglutinate cells in the GAT bioassay nor did it bind to the granulocytes in the GIFT

bioassay. N-terminal sequencing of the GR1.GF protein showed differences in only two

amino acids when compared with the other GR1 proteins (GR1.HPLC & GR1.HighQ).

Mass spectrometry determined the molecular weight of the GR1.GF protein to be 6669

Da, which was identical to the results obtained for the GR1.HPLC and GR1.HighQ

proteins.

_________________________________________________________________ 121

Chapter 6 NMR Assignment of the GR1 protein from Grevillea robusta ____________________________________________________________________________________

Chapter 6 NMR assignment of the GR1 protein from

Grevillea robusta

________________________________________________________________________________ 122


Chapter 6 NMR assignment of the GR1 protein from Grevillea

robusta

6.1 Introduction

The GR1 protein was purified and characterised using HPLC and gel filtration

chromatography, mass spectroscopy, N-terminal sequencing and PAGE as described in

Chapters 3 & 4. Functional assays (Clague et. al., 1999) showed the GR1 protein could

hibit both trypsin and chymotrypsin independently and the Ki values were

in the inhibitory effects for both the GR1 protein and Bowman-Birk

hibitor could suggest a similar structure/function relationship. However, sequence

alignment showed a number of differences between the two proteins especially within

the proposed chymotrypsin-binding site. Key residues involved in forming the reactive

site towards chymotrypsin within the GR1 protein were C-X-C-X-L-T/S-X-P-A-X,

where L and T/S correspond to the P1-P1′ of the scissile bond.

in

comparable to those obtained for the Bowman-Birk inhibitor.

The Bowman-Birk inhibitor (BBI) is a member of the serine protease inhibitor family

and has been described as a double-headed protein that inhibits both trypsin and

chymotrypsin independently. It is a 71 amino acid protein that contains 14 cysteine

residues that form 7 disulfide bridges. It was first isolated from soybeans over 40 years

ago and to date, a large number of proteins isolated from plants have been characterised

as members of the Bowman-Birk serine protease superfamily (Birk, 1985).

The similarities

in

________________________________________________________________________________ 123


Within the Bowman-Birk inhibitor, the positioning of the cysteine residues plays an

important role in stabilising the overall structure of the protein through the formation of

disulfide bridges. These bridges aid in the formation of the antiparallel β-sheet and

turn structures seen for the trypsin and chymotrypsin binding sites. In the

chymotrypsin-binding region of the GR1 protein, the cysteine residues are not

conserved relative to the Bowman-Birk inhibitor and therefore the disulfide-bonding

pattern will be different, and could result in a different structure. Due to the similar

inhibitory effect of the GR1 protein and differences within the chymotrypsin-binding

site, the structure of the GR1 protein was determined using NMR spectroscopy. This

chapter will briefly discuss the basic principles behind protein NMR spectroscopy and

outline the steps used to assign the GR1 structure.

________________________________________________________________________________ 124


6.2 NMR Spectroscopy

The protein samples were dissolved in 18% CD3CN /H2O pH 3.5 and the spectra was

collected using a Varian INOVA 600MHz spectrometer at both 303 and 288 K. These

conditions were chosen because the structure of the Bowman-Birk inhibitor was solved

using NMR spectroscopy under these conditions (Werner & Wemmer, 1991). One

dimensional homonuclear and 2D homonuclear and heteronuclear experiments, DQF-

COSY, TOCSY, NOESY & HSQC were acquired to provide data for the assignment of

the protein. Coupling constants could be measured using DQF-COSY spectra.

6.2.1 One dimensional NMR experiments

One-dimensional experiments were used to optimise the experimental conditions so the

experiment would yield the best dispersion of peaks in the amide region. In order to

achieve this, a number of different experimental conditions (including temperature and

pH) were trialed to provide the best peak dispersion. Figure 6.1 shows an example of

the amide region of the 1D spectrum obtained for the GR1 protein.

________________________________________________________________________________ 125


________________________________________________________________________________ 126

Figure 6.1: 1D spectrum of the amide region for the GR1 protein.

One-dimensional and 2D experiments (DQF-COSY and TOCSY) were acquired on

both GR1 proteins (GR1.HPLC and GR1.GF) at 303K. The amide region of the 1D

spectrum showed narrower peaks for the GR1.HPLC when compared with the GR1.GF

sample and this is shown in Figure 6.2. The broadening of the amide 1D peaks in the

GR1.GF sample was evident and could be due to protein aggregation or due to a

number of impurities within the sample. The 2D experiments confirmed the presence

of aggregation and impurities (in the form of additional peak patterns) within the

GR1.GF sample (results not shown). However, the solution structure of the GR1

protein was determined using the GR1.GF sample as this sample was biologically

active.


Fi

(B

6.2

To

ac

4

sta

__

A

g

)

.

q

st

g

__

B

ure 6.2: The 1D NMR spectra for the amide region of the (A) GR1.HPLC and

GR1.GF proteins

2 Two dimensional NMR experiments

sequentially assign the GR1 protein, a number of 2D NMR experiments were

uired these included the DQF-COSY, TOCSY and NOESY experiments. There are

ages within a 2D experiment; the preparation, evolution, mixing and detection

es as seen in Figure 6.3 (Wüthrich, 1986). The preparation stage involves a delay

____________________________________________________________________________ 127


time that allows the nuclei to establish thermal equilibrium. At the end of this stage,

ne or morel rf pulses are applied to create the desired coherence.

Figure 6.3: The schematic representation of the NOESY experiment.

The P1, P2 & P3 are the pulses; t1 is the evolution time; τm is the mixing time; the FID is

ransform (FT) is applied to the time

omain data, which produces the 2D-frequency spectrum.

o

t2 t1

P1 P2

Delay

P3

τm

Preparation Evolution Mixing Detection

recorded during t2.

During the evolution stage (defined by the evolution time t1), coherence transfer is

achieved where the nuclei may adopt a different spin state and it is these differences

that allow the chemical shift values for the individual spins to be identified or labelled.

One or several rf pulses are applied in the mixing stage over a period of time denoted as

the mixing time (τm). The detection stage results in the free induction decay (FID)

being acquired and stored as time domain data. Repeating the same experiment a

number of times while increasing the evolution time after each experiment creates the

second dimension. A two-dimensional Fourier T

d

________________________________________________________________________________ 128


6.2.2.1 Correlated spectroscopy (COSY) and Double quantum filtered COSY (DQF-

COSY)

COSY is the simplest of the homonuclear 2D NMR experiments and is used to identify

pairs of protons involved in scalar coupling. The experiment is made up of two 90°

pulses (P1 & P2), separated by an evolution time t1 (Figure 6.4A). This experiment

was not used to assign the GR1 protein. Instead the Double Quantum Filtered COSY

(DQF-COSY) experiment was used which provided better data for the assignment of

the protein since it removes any singlets from the acquired data.

The DQF-COSY is an extension of the COSY experiment where it involves an

shor

r the (A) COSY and (B) DQ

(A) P1 & P2 are 90° pulses separated by the evolution time (t

mixing stage is shown by ∆. The FID is recorded during t2 in bo

DQF-COSY spectra are used to generally detect spin-spin

constants in individual amino acid residues in a protein, a

provide the dihedral angle restraints for structure calculati

additional 90° pulse (P3) in the mixing phase of the experiment that is separated by a

t delay (∆) from P2 (Figure 6.4B). The additional pulse converts the double

quantum coherence into single quantum coherence prior to detection.

t1

Prep Evolution Mix Detect

3

Prep E

Figure 6.4: Pulse sequence fo

_________________________________________________________

t1

F-COSY experi

1). (B) The short d

th experiments.

3JNH-Hα and 3JHα-H

nd these coupling

ons. The HN-Hα

volution Mix

_________________

t2

P1
P2
t2

P1
P2 P
∆

A
B
ments.

elay in the

β coupling

constants

region of

Detect

______ 129


DQF-COSY spectra for the GR1 protein is shown in Figure 6.5. In theory, all of the

HN-Hα connectivities of the protein should be seen within this region. For GR1, not

all of the HN-Hα peaks were observed and this was due to a number of overlapping

peaks in the TOCSY/ NOESY spectra. Alternatively, peaks could be absent from the

spectra if the peaks of interest are located around the same frequency as the water that

is being suppressed.

Figure 6.5: The HN-Hα region of the DQF-COSY experiment for the GR1

.2.2.2 Total correlation spectroscopy (TOCSY)

he TOCSY experiment allows through bond connectivities between protons to be

the second pulse with a sequence called

MLEV-17 or spin lock (Bax & Davis, 1985), during the mixing stage of the experiment

protein.

6

T

determined that are not restricted to 2 or 3 bonds. The TOCSY experiment differs

from the COSY experiment by replacing

________________________________________________________________________________ 130


(Figure 6.6). This MLEV-17 sequence causes the magnetisation to become “spin-

locked” in one axis, which results in all spins in a coupled system to have the same

Figure 6.6: The pu

6.2.2.3 Nuclear Ove

The spectra obtained

the protein to be det

This experiment ide

less than 5 Å. The

time (τm), which all

diagram of the NO

relaxation, the magn

dependent on the l

frequency. Once the spin lock is removed and the FID is collected, the cross peaks are

detected for all spins in their respective coupled system such as the side-chain protons

in an amino acid.

The four stages are s

during t2.

n

___________________

P1

lse sequence for

rhauser Enhanc

from the homo

ermined by visu

ntifies the proto

NOESY exper

ows the pairs of

ESY experime

etisation of the

ength of the m

hown. The evolu

n

________________

M

2
t1
the TOCSY exper

ement spectroscopy

nuclear NOESY ex

alising through spa

ns that are in close

iment contains thre

protons to undergo

nt is shown in Fi

protons is transferre

ixing pulse and th

tion time is represen

___________________

t

LEV-17

n
Preparatio Evolutio Mixing Detectio
iment.

(NOESY)

periment allows the sequence of

ce connectivities of the protons.

proximity to each other, usually

e pulses (P1-P3) and a mixing

cross-relaxation. A schematic

gure 6.7A. During this cross

d and the rate of this transfer is

e size of the molecule being

ted by t1 and the FID is recorded

__________________________ 131


investigated (Roberts, 1993). Figure 6.7B is an example of the HN-HN region of the

NOESY spectrum obtained for the GR1 protein.

Figure 6.7 he (A) sc

the HN-HN region of t

(A) P1, P2 & P3 are rf pu

The HN-HN region of the

B

n

: T

_______________________

P1

hematic pulse s

he NOESY spec

lses applied. The

NOESY spectrum

n

________________

P2

eq

tra for the

mixing time

for the GR1

m

uence for

___________

P3

t1
τ
GR1 prot

is shown b

protein wi

the NOE

__________

t2
Preparatio Evolutio Mixing Detection
A

ein.

etween P2 & P3 by τm. (B)

th a mixing time of 200ms.

SY experiment and (B)

____________________ 132


6.3 The 1H NMR Assignment of the GR1 protein

6.3.1 Solvent suppression

The NMR assignment of the GR1 protein was achieved by using a combination of

DQF-COSY, TOCSY and NOESY experiments. Using the full amino acid sequence of

the protein derived from cDNA techniques and by following the approach outlined by

Roberts (1993), the spin systems of the individual amino acids within the protein were

on

pressing water. It involves a

continuous irradiation at a low power rf at the same frequency of the solvent peak

during the relaxation delay (Roberts, 1993) as shown in Figure 6.8A. In this work, the

presaturation sequence was used in the DQF-COSY experiments.

assigned. The majority of the protein structures determined by NMR spectroscopy are

dissolved in H2O or H2O/D2O mixtures that allow the researcher to study the structure

of the protein in solution. However, the proton peak for water is many times more

intense than the protein’s protons (approximately 10 000 times) and therefore this

signal needs to be suppressed. To overcome this, a number of solvent suppressi

sequences have been developed to reduce the intensity of the water protons and allow

the proteins’ protons to be visualised.

Presaturation is the easiest and effective way of sup

________________________________________________________________________________ 133P1

P2 P1 P2 A B


Figure 6.8: Schematic representation of the water suppression sequences for (A)

(A) The long low power rf pulse applied during the relaxation delay is shown in P1. P2

corresponds to the 90° pulse applied after this delay. (B) P1 corresponds

presaturation and (B) WATERGATE.

to a non-selective 90°

ulse; P2 is the selective 180° pulse or the pulse trains targeting water. G1 & G2 correspond to

the gradient pulses that are used to suppress the solvent.

Water suppression using gradient tailored excitation or WATERGATE is the second

sequence applied to suppress the water peak in the protein NMR sample. The water is

suppressed by using two short gradient pulses of the same amplitude, separated by a

selective 180° rf pulse (Figure 6.8B). Prior to the first gradient pulse, a non-selective

90° rf (P1) is applied which results in all of the coherences being excited. These

coherences are dephased by the first gradient pulse (G1) but can be rephased by the

second gradient pulse (G2) only if they are flipped by 180° from the selective rf pulse

2) (Sklenář et. al., 1993). During this stage, application of the second gradient pulse

ing the initial WATERGATE sequence has been improved (Liu et

l; 1998). The selective 180° rf pulse has been replaced by the addition of a number of

different pulse trains, denoted W, that consist of 3, 4, or 5 pulses (Sklenář et al., 1993;

p

(P

(G2) results in the water signal being further dephased. By the time the receiver

acquires the data, there is little or no water signal observed (Sklenář et. al., 1993; Liu

et. al., 1998). The WATERGATE sequence was used to remove the water in the

TOCSY and NOESY 2D experiments in this work.

Water suppression us

a

________________________________________________________________________________ 134


Liu, 1998). The pulse train used in this work was the 3-9-19 or W3, where 6 pulses are

applied during P2 (Figure 6.8B). The W3 is made up of the sequence 3α-τ-9α-τ-19α-τ-

19α-τ-9α-τ-3α where 26α = 180° pulse where τ was the time between the pulses, which

govern the width of the excitation region. These modifications to the original

WATERGATE sequence improved the suppression of the resulting data by narrowing

the suppressed region, ie the water, and allowing the proteins peaks to be observed (Liu

et al., 1998). There are several other methods of water suppression but since they were

not used in this work, they will not be discussed.

6.3.2 Spin system identification

R1 protein was to

efine the chemical shifts that correspond to each proton. The random coil chemical

shift values obtained by Wüthrich (1986) were used only as a guide in the assignment

f the amino acid spin systems of the GR1 protein.

+

groups are located in section A and the Hα to side-

chain connectivities are found in section C.

Spin system assignments were made using the data collected from the DQF-COSY and

TOCSY experiments. The first stage in the assignment of the G

d

o

The 2D spectrum was divided into 4 regions where each region provided information

for the assignment of the protein as seen in Figure 6.9. The HN to side-chain cross

peaks is found within section B. The aromatic protons, the NH3 of lysine and the NH

groups of arginine and amide NH

________________________________________________________________________________ 135


__________

Figure 6.

The four s

obtained fr

Many am

have more

experimen

spin syste

Roberts (1

Glycine w

formation

peaks eac

were dete

experimen

A

_

9:

e

o

in

t

m

9

o

h

c

t

B

_____________________________

An example of a 2D spectru

ctions are boxed in blue and lab

m the GR1 protein.

o acids have unique spin syst

complex spin systems and req

, to assign them. The assignm

s for each individual amino

93).

as identified in the Hα-HN

f an AMX spin system. The t

containing 4 antiphase comp

ted within the DQF-COSY sp

s such as the TOCSY and NO

C

B

________________________________________ 136

m.

elled A-C in red. This is the TOCSY spectrum

ems that can be easily identified, while others

uire different experiments such as the NOESY

ent strategy used for the identification of the

acid for the GR1 protein was outlined by

region of the DQF-COSY spectrum by the

wo protons at the Cα formed two DQF-COSY

onents. Not all of the HN-Hα connectivities

ectrum for this work and therefore additional

ESY were used to aid in their assignment.


The methyl groups of alanine and threonine were found in the same area in the DQF-

COSY and TOCSY spectra. The methyl groups for both alanine and threonine were

found to be between 1-2 ppm and correlated to the Hα of alanine and the Hα & Hβ of

threonine at chemical shift values between 4-4.5 ppm.

The methyl resonances of valine Hγ, isoleucine Hγ2, isoleucine Hδ and leucine Hδ were

found within the same region as the methyl groups of alanine and threonine, but they

were well separated in the DQF-COSY and TOCSY spectra. The two-methyl

resonances of valine and leucine, with chemical shift values between 0.5-1.0 ppm

correlated to the Hβ for Val and Hγ for Leu with chemical shift values between 1.5-2.5

ppm. The methyl connectivities of Hγ2 and Hδ within isoleucine correlated with each

Connectivities were seen in the TOCSY spectrum between the Hα and 2 Hβ protons for

the amino acids serine, cysteine, tryptophan, phenylalanine, tyrosine, and aspartic acid.

The 2 Hβ peaks for the cysteine and aromatic amino acids were found between 2.8-3.6

ppm and for serine these peaks were located downfield between 3.5-4.0 ppm. The

confirmation of the aromatic and asparagine residues were determined by visualising

connectivities in the NOESY spectrum between the Hβ protons and the ring protons for

the aromatic residues or side chain amide protons for asparagine.

he remaining amino acids including lysine, arginine, glutamine, glutamic acid and

ied by their Hα-Hβ-Hγ-Hδ connectivities in the

other and occurred at chemical shift values between 0.5-1.0 ppm.

T

proline all contain similar Hα chemical shift values that were found to be between 4.3-

4.5ppm and each of these Hα protons correlated to a pair of Hβ protons that were found

between 1.7-2.2 ppm in the TOCSY spectrum. Proline residues do not contain a HN

chemical shift value and were identif

________________________________________________________________________________ 137


TOCSY/ NOESY spectra. Connectivities between the Hγ-Hδ of arginine and Hδ-Hε of

for the Hδ of Arg and

Hε of Lys and 1.7-2.1ppm for the Hγ of Arg and Hδ of Lys.

The Hα-HN regions of the DQF-COSY and TOCSY spectra were used to identify the

intra-residue spin systems of the amino acids of the protein. Figure 6.8 shows the

assignment of the residues found within the Hα-HN region of the TOCSY spectrum.

Not all of the peaks between 7.0 and 7.5 ppm in the TOCSY spectra were labelled in

Figure 6.10 as they corresponded to the side-chain N-H protons of arginine, lysine and

atic residues.

the GR1 protein.

lysine were found in the DQF-COSY spectrum at 3.1-3.6 ppm

arom

Figure 6.10: A summary of the identified spin systems in the TOCSY spectrum for

The spin systems were identified and labelled. The red lines were used for contrast in areas of

overlapping peaks. Three or more peaks were found under the red lines.

________________________________________________________________________________ 138


6.3.3 Sequential Assignment of the GR1 protein

Not all of the spin systems were identified using DQF-COSY and TOCSY experiments,

and additional experiments such as the NOESY, were used in the attempt to locate the

missing spin systems. The NOESY experiment was also used to sequentially assign

The XEASY program was used to analyse and document the peaks seen in the NOESY

spectrum (Bartels et. al., 1995). This program generates assignments and peak volume

tables that can be transferred into other programs such as DYANA, to create the three-

O

these residues by providing information on the intra-residues and inter-residue

connectivities within the protein.

Sequential assignment of the GR1 protein was obtained by looking for correlations

between adjacent spin systems, ie by viewing the Hαi-HNi+1, Hβi-HNi+1 and HNi-HNi+1

connectivities in the NOESY spectra as seen in Figure 6.11. The GR1 protein was

assigned sequentially until a proline residue (which has no amide proton) or 2 HN

resonances with similar chemical shift values (resulting in the overlapping of NOE

peaks within the spectra) were reached.

--- Ni-1 ---- CHi-1 ----- C ----- Ni ---- CHi ---- C ----- Ni+1 ---- CHi+1 ---- C ----

H H H

O O

CβHi-1 CβHi CβHi+1

Ri-1 Ri Ri+1

Figure 6.11: Sequential assignment of the protein.

________________________________________________________________________________ 139


dimensional structure for the protein. Table 6.1 summarises the assignment of the GR1

protein and Table 6.2 outlines the chemical shift values for the individual amino acids.

Ninety seven percent of the residues within the GR1 protein were assigned. The

remaining residues were not assigned due to regions of overlapping peaks in the DQF-

COSY, TOCSY and NOESY spectrums. This overlap made the definitive assignment

of the residues within the GR1 protein difficult and Figure 6.12 shows the extent of this

verlap in the TOCSY spectra.

Figure 6.12: The TOCSY spectra for the GR1 protein that shows the regions of

overlapping peaks.

ach boxed area corresponds to at least 3 different residues.

In attempts to overcome this problem, the experimental conditions were altered,

including reducing the temperature, adjusting the pH and varying the mixing times.

After reducing the temperature to 288K and adjusting the pH from 3.5 to 2.0, there was

very little difference within the overlapped regions. These overlapping peaks are also

o

E

________________________________________________________________________________ 140


apparent in the 13 C heteronuclear single-quantum correlation (HSQC) experiment,

especially within the region corresponding to the side-chain protons, but again very

little information could be obtained from this experiment (spectrum not shown).

The assignment of the GR1 protein was continued even though the overlapping peaks

were not assigned. The first residue was not assigned as no NOE connectivity was seen

between the Hα of residue 1 and HN of residue 2, and residues 18 & 31 could not be

assigned due to overlapping peaks.

C -Val-Cys and Cys-Arg-Cys, provided the starting point in

deciphering the NOESY spectra for the protein. The spin systems for the cysteine and

6.3.3.1 Sequential assignment of Residues 1-29

The first section of the GR1 protein to be assigned were residues 1 to 29 and the

connectivity plot is shown in Figure 6.13. Specific amino acid sequences within this

region, such as ys

valine residues were identified and the surrounding residues were sequentially assigned

using Hαi-HNi+1, Hβi-HNi+1 and HN-HN connectivities.

A break in the connectivity occurred as a result of two proline residues, found at

positions 18 and 19. The Hα protons of these proline residues were assigned from the

NOESY spectra. A strong NOE peak was seen between the Hα of Pro19 and HN of

Arg20 and NOE peaks were seen between the Hβ of Pro19 and HN of Arg20. Proline

18 was not completely assigned due to Hα chemical shift value for Pro18 being found

around the water peak (4.91ppm). Caution was taken with any peaks located in or

around the water peak, as it was unclear whether the peaks within this region were

________________________________________________________________________________ 141


artefacts. Peaks within this region may also be suppressed as a suppression sequence

was applied to the sample prior to acquiring data.

As a strong peak was detected between the Hα of Pro18 and Hδ of Pro19 in the

i i+1

Non-sequential connectivities were seen

Hα Pro19 & HN Ser16 suggesting these residues were involved in the

formation of a turn like structure.

This section of the GR1 protein contains 4 disulfide bridges that exist between Cys8-

Cys23, Cys11-Cys57, Cys13-Cys21 and Cys29-Cys34. Non-sequential NOE

connectivities between Hαi-Hβj of the cysteine residues identified the position of these

disulfide bonds. NOE connectivities were seen between Hα8-Hβ23, Hβ11- Hα57 and

Hα29-Hβ34. The identification of the Cys13-Cys21 disulfide bond was not

straightforward, as the Hβ protons of both cysteine residues were identical. However,

the presence of extremely large NOE peak volumes for the Hαi-Hβj and Hβi-Hβj

connectivities for these residues suggested the Hβ protons for both residues were

overlapped. Non-sequential connectivities were seen around the disulfide bridges at

HN14-HN20, Hα11-HN24, Hα11-HN23, Hα13-HN22, Hα21-HN14, HN12-HN22, HN14-Hβ20,

HN28-HN35 and Hα23-HN12.

NOESY spectrum and a peak was seen between the Hα Pro18 and Hα Ile17, the

assignment of the Hα of Proline 18 was confirmed. From this data, the cis/trans

conformation of the proline residues could be determined. Connectivities between the

Hαi-Hαi+1 of Ile17 and Pro18 showed this region to be in the cis conformation, while

the Hα -Hδ connectivities between Pro18 and Pro19 were consistent with the trans

conformation (Wüthrich et. al., 1984).

between the

________________________________________________________________________________ 142


Figure 6.13: The fingerprint region of NOESY spectra of the GR1 protein at 303

K in 18% CD3CN/ H2O.

The one letter code is used to represent the amino acid residues. The blue and red lines indicate

the sequential connectivities between the residues 6-17 and Hα19-26 respectively.

The HN-HN connectivities seen in the NOESY spectra were used in conjunction with

the Hα-HN NOE peaks to provide sequential assignment of the residues within this

section by identifying neighbouring amino acid residues within the primary sequence.

Figure 6.14 shows the HN-HN connectivities for the residues in this section. There

were three non-sequential connectivities found within the HN-HN region - between

residues Thr14 and Arg20, between Val12 and Arg22 and between Val28 and Tyr35.

l HN-HN connectivities were also seen in this region of

These connectivities were located around the disulfide bridges of Cys13-Cys21 and

Cys29-Cys46. Non-sequentia

the Bowman-Birk inhibitor between Gln11-Ser25 and Thr15-Gln21 (Werner &

________________________________________________________________________________ 143


Wemmer, 1991). These connectivities were also found near two disulfide bridges at

Cys14-Cys22 and Cys9-Cys24 in the Bowman-Birk inhibitor.

Figure 6.14: The HN-HN region of the NOESY spectra for the GR1 protein.

________________________________________________________________________________ 144


6.3.3.2 Sequential assignment of Residues 30-46

The second section to be assigned was for residues 30-46 and the HN-HN and the Hαi-

NHi+1 connectivities are shown in Figure 6.15. The identification of specific sequence

patterns and the remaining 2 valine spin systems within the protein allowed the

sequential assignment of this section. The HN-HN non-sequential connectivities within

the NOESY spectra aided in the assignment of a number of amino acid residues that

were located in overlapped regions (Figure 6.15A).

Non-sequential connectivities were detected between HN28-HN35, between Hα46-HN33,

between Hα20-HN45 and between Hα45-HN21. Disulfide bonds within this region were

located between residues Cys29-Cys34 and between Cys32-Cys46 and this was

confirmed by the presence of non-sequential connectivities between Hα29-Hβ34 and

between HN32-Hβ46.

The assignment of Pro39 was difficult, as no HN value could be determined for Ala40

in both the NOESY and TOCSY spectra. However, a strong NOE peak was seen

between Hα of Val38 and Hδ of Pro39, which allowed the proline residue to be

assigned. The second proline residue was assigned by identifying NOE peaks between

the Hα of Pro44 and HN of Tyr45 and between Hα of Arg43 and Hδ of Pro44. Both of

these proline residues were found to adopt the trans conformation as there were strong

sequential Hαi -Hδi+1 NOE cross peaks. This was a surprising find, as Pro39 was not

, the inhibitory studies completed on the GR1 protein

expected to be in the trans conformation due to its involvement in the inhibition of the

protease, chymotrypsin. However

did not show any reduction in the inhibitory effect of the protein towards chymotrypsin

________________________________________________________________________________ 145


due to the P3′ proline being in the trans-conformation. The KI values for the GR1

protein for chymotrypsin of 4.8 x 10-9M (Clague, 2001) are comparable with those

obtained for the Bowman-Birk inhibitor of 5.2 x 10-9M (Werner & Wemmer, 1991).

The assignment of the Hαi-HNi+1 NOE connectivities for this section was split into

three, due to the presence of two proline residues at positions 39 and 44. The

sequential assignments of these three regions are shown in Figure 6.15B. There were a

number of non-sequential medium ranged connectivities seen around Pro39 to Met42.

hese connectivities may be involved in forming a turn, similar to that seen for the first

T

region or trypsin binding site.

________________________________________________________________________________ 146


_

F

4

T

4

A

B

_______________________________________________________________________________ 147

igure 6.15: The (A) HNi –HN and (B) H tivities for residues 32-

thin the GR1 protein.

roline dues e the a ment of this region. Residues 32-38 are shown in green;

is in r 4-46 cyan. The one letter code was used to represent the amino acids.

i+1 α -HN connecI i+1

6 wi

wo p resi brok ssign

0-43 ed; 4 is in


6.3.3.3 Seque l assignment of Residues 47-61

ird a inal s on tha quentially assigned corresponded to the residues

. Th αi-N and H i+1 con f the remaining residues are

in F ure 6.1 This was r tforward to assign, as the

ity of rema g unas igned residu ere within this region. Specific amino

cids such s methi ine and soleucine we asily identified and assigned, as they

ad charac ristic peak patterns in the TOCSY spectra and only one was to be assigned.

position 58 was assigned by NOE connectivities between the Hα

nd Hδ of Pro58. The Hαi-HδI+1

on were assigned from the HN-HN and Hαi-HNI+1 regions of the

NOESY spectra. There were the occasional non-sequential connectivity between the

Hα53-HN57, Hβ53-HN57 and a disulfide bond was identified to exist between Cys11 and

Cys57.

ntia

The th nd f ecti t was se

48-61 e H Hi+1 Ni-NH nectivities o

shown ig 6. section elatively straigh

major the inin s es w

a a on i re e

h te

A proline residue at

of Pro58 and HN of Ile59 and between Hα of Cys57 a

connectivity resulted in Pro58 being in the trans conformation. A number of residues

within this regi

________________________________________________________________________________ 148


B

A

________________________________________________________________________________ 149

Figure 6.16: The (A) HN-HN and (B) HαI-HNi+1 connectivities for the residues 48-

61 seen in the NOESY spectra.

Residues 48-57 are shown in red and residues 59-61 are shown in blue. The one letter code for

the amino acids is used.


The summary of the sequential assignment for the amino acid residues within the GR1

protein are shown in Table 6.1 and the chemical shift values for these residues are

own in Table 6.2. Table 6.1 reveals a large number of sequential NOE connectivities

and very little medium range NOEs were seen for the GR1 protein. Interpreting the

ta seen within this table, it could be stated that there could be one region (residues 8-

27) that might contain β-sheet like structures due to the presence of a large number of

αN(i,i+1) and dβN(i,i+1) connectivities, while the second half of the GR1 protein looks

relatively disordered.

Table 6.1: The summary of the assignment of the GR1 protein.

sh

da

d

________________________________________________________________________________ 150


Table 6.2: Chemical shift assignment of the GR1 protein in 18% CD3CN/ H2O pH 3.5 at 303 K.

Chemical Shifts in ppm Residue HN Hα Hβ Others

1. Gly ND ND 2. Gly 8.70 4.19 3. Glu 8.86 4.41 2.12 γCH2 2.46,2.24 4. Glu 8.50 4.17 2.09,2.02 γCH2 2.37

7. Trp 8.16 4.61 3.34,2.85#

9. Glu 7.73 4.12 2.29,2.17 γCH 2.47

11. Cys 7.75 5.54 3.42#,2.96

13. Cys 8.87 6.25 3.21 13 4.66 4.85 γCH3 1.63 55 4.75 2.27 γCH2 1.91; δCH2 3.42

16. Ser 7.57 4.61 4.15,4.01# 17. Ile 8.17 4.37 1.96 γ CH2 1.65,1.27;γCH3 1.07; δCH3 1.06 18. Pro -- 4.91 ND ND 19. Pro -- 4.40 2.61,2.06 γCH2 2.29,2.18; δCH2 3.70 20. Arg 7.63 5.58 2.04 γCH2 1.90; δCH2 3.44; εNH 7.46 21. Cys 8.58 5.99 3.21,2.96# 22. Arg 8.88 4.85 2.01 γCH2 1.81; δCH2 3.41; δNH 7.44 23. Cys 9.27 5.72 3.37,3.04 24. Thr 8.37 4.12 4.43 γCH3 1.50 25. Asp 8.16 5.08 3.23,2.74 26. Ser 8.83 4.55 4.20, 4.38 27. Ser 8.02 4.74 4.26 28. Val 7.55 4.44 2.66 γCH3 1.40, 1.28 29. Cys 8.22 4.55 3.35. 3.23 30. Thr 7.89 4.24 3.90 γ1.27 31. Lys ND ND ND ND 32. Cys 8.82 6.38 3.30,3.20 33. Val 9.26 4.58 2.24 γCH3 1.19 34. Cys 7.95 4.87 3.44,3.11# 35. Tyr 8.83 4.08 3.69, 3.56# δH 7.14, εH 6.87 36. Leu 8.23 4.33 2.02, 1.74# γCH2 2.39; δCH3 1.15 37. Thr 7.95 4.31 4.01 γCH3 1.34 38. Val 8.10 4.73 2.57 γCH3 1.40,1.01†

39. Pro -- 4.59 2.64 γCH2 2.24,2.01; δCH2 3.81, 4.05 40. Ala ND 3.91 1.62 41. Ala 8.79 4.34 1.64 42. Met 8.26 5.01 2.41,2.20# γCH2 2.89, 2.71 43. Arg 7.66 4.28 2.06 γCH2 1.88,1.57; δCH2 3.21 44. Pro -- 4.67 2.52 γCH2 2.13; δCH2 3.93, 3.55 45. Tyr 7.53 5.71 3.56,3.21 46. Cys 8.69 6.04 3.20,2.96 47. Glu ND ND ND 48. Ser 7.67 4.62 4.28#,4.10 49. Met 8.07 4.93 2.50#,2.40 δCH2 3.00,2.79 50. Ala 7.66 4.16 1.72

5. Ala 7.94 4.09 1.03 6. Asp 8.08 4.32 2.26, 2.08

δΗ 7.14;εNH 10.43; εH 7.59; ζH 7.71, 7.27; ηH 7.47 8. Cys 8.30 4.95 3.51,3.02#

2

10. Asp 8.98 5.10 3.37,2.88

12. Val 9.21 4.58 2.23 γCH 1.20,1.16

14. Thr 9.15. Arg 8.

3

________________________________________________________________________________ 151


Residue HN Hα Hβ Others 51. Ser 8.43 4.61 4.14 52. Arg 8.25 4.31 1.72 γCH2 1.59; δCH2 3.23; εNH 7.46 53. Phe 8.67 4.90 3.82,3.24# δH 7.42; εH 7.31; ζH 7.22 54. Asp 7.71 4.39 2.88,2.79# 55. Ala 8.63 4.20 1.12 56. Phe 8.03 4.56 3.60,3.28# δH 7.53; εH 7.46 57. Cys 7.54 5.75 3.57. 3.22 58. Pro -- 4.68 2.46, 2.15 γCH2 2.28; δCH2 4.12 59. Ile 8.46 4.44 2.10 γCH2 1.75,1.45; γCH3 1.17; δCH3 1.11 60. Gly 8.56 4.21 61. Ser 7.97 4.49 4.06, 4.21

ND = not determined # Indicates Hβ protons that were stereospecifically assigned † Indicates Hγ protons of Val that were stereospecifically assigned.

Referenced to an external sample of DSS at 0ppm that was prepared in 18% CH3CN-D3/H2O

pH 3.5 at 303K.

________________________________________________________________________________ 152


6.4 Conclusion

NMR spectroscopy was used to determine the structure of the GR1 protein. One-

dimensional and homonuclear two-dimensional NMR experiments were used to

identify the spin systems of the individual amino acids and sequentially assign these

residues within the GR1 protein. The spin systems of the amino acids were identified

by the DQF-COSY and TOCSY experiments, and the sequential assignment was

achieved by looking for Hαi-HNi+1 and HNi-HNi+1 connectivities in the NOESY

spectra. Ninety seven percent of the amino acid residues within the GR1 protein were

assigned. The remaining residues were not assigned due to a number of overlapping

peaks in the homonuclear 2D spectra. Connectivities between the Hαx-αHPro and Hαx-

HδPro in the NOESY spectra (where x is the preceding residue) determined the cis/trans

conformation of the proline residues. Five proline residues were found in the GR1

protein and all proline residues adopted the trans conformation with the exception of

Proline 18 where it was found in the cis conformation. There were very few medium

and long range NOE connectivities seen for the GR1 protein. However, the

connectivities that were seen did suggest one region may contain β-sheet like structures

based on the Hαi-HNi+1 and Hβi-HNi+1 connectivities.

________________________________________________________________________________ 153

Chapter 7 Structural studies of the GR1 protein ____________________________________________________________________________________

Chapter 7 Structural studies of the GR1 protein

________________________________________________________________________________ 154


Chapter 7 Structural Studies of the GR1 protein

7.1 Introduction

This chapter outlines the strategy to predict the secondary structure of the GR1 protein

and the disulfide bond pattern for this protein. It also outlines the steps involved in

determining the final 3D structure of the GR1 protein and discusses the structural

differences between the final structure of the GR1 protein and the Bowman-Birk

inhibitor.

7.2 Secondary Structure of the GR1 protein

Prediction of secondary structure

The secondary structure of any protein can be determined by comparing the chemical

shift values for each amino acid with the random coil or theoretical chemical shift

he secondary structure for the protein can be predicted by reviewing the values

btained (random coil values – observed chemical shift values) and looking for stretches

7.2.1

values. A number of different methods were used to determine the random coil

chemical shifts, however, the shift values used in this work were derived from Wüthrich

(1986). The difference between the Hα values of the random coil chemical shift values

and the observed chemical shift values for the GR1 protein were calculated (Table 6.2

in Chapter 6) and graphed as seen in Figure 7.1.

T

o

________________________________________________________________________________ 155


of positive or negative values within this graph. Positive and negative values represent

regions of either α-helical or β-sheet like structures respectively. A continuous stretch

of 4 or more values of either negative or positive results strongly suggests the protein to

be in that conformation.

-2.00

1.00

0.50

-1.50

-1.00

-0.50

0.001 11 21 31 41 51 61

Residue number

d(rc

-obs

)

GR1 protein

Figure 7.1: Secondary structure prediction of the GR1 protein.

Wüthrich val

The values were determined by subtracting the observed Hα values from the protein with the

ues (Wüthrich, 1986). A negative result corresponds to the β-sheet conformation

and a positive value corresponds to the α-helix.

Regions of β-sheet like structures were predicted for residues 10-18, 20-23, 25-28, 44-

49 and 57-60 as they were found to contain stretches of 4 or more negative values as

seen in Figure 7.1. A number of regions contained a stretch of 3 consecutive negative

values and these regions may contain β-sheet like structures however, this can only be

final structure is determined. A disruption to the stretch of negative confirmed when the

values as seen for residues 18-20, may indicate the presence of a turn. Residues 3-6 and

________________________________________________________________________________ 156


54-56 contain a stretch of 3-4 consecutive positive values that could correspond to a α-

elical like structure. This structure may exist but can only be confirmed when the final

allel β-sheet structure, held together by a

etwork of disulfide bridges and hydrogen bonds (Werner & Wemmer, 1991; Werner &

man-Birk inhibitor.

h

structure of the GR1 protein is solved.

Secondary structure prediction was also applied to the Bowman-Birk inhibitor as this

structure was also determined by NMR spectroscopy (Werner & Wemmer 1991). As

there was significant sequence homology between the Bowman-Birk inhibitor and the

GR1 protein and the structure for the Bowman-Birk inhibitor was known, the two

sequences were aligned and the secondary structure prediction values were compared.

Figure 7.2 and Figure 7.3 show the comparison between the two proteins within the

trypsin- and chymotrypsin- binding regions of the Bowman-Birk inhibitor. The

numbering of the residues in the figures were based on the GR1 proteins’ sequence.

The trypsin-binding region showed both proteins to have a consecutive stretch of

negative values between residues 10-23 (Figure 7.2). Within the Bowman-Birk

inhibitor, this region was made up of an antipar

n

Wemmer, 1992). A disruption to the consecutive stretch of negative values as seen at

residue 19 was found to be characteristic of a turn, and this was confirmed by viewing

the structure for the Bowman-Birk inhibitor (Werner & Wemmer, 1992). Therefore,

reviewing the sequence alignment and the secondary structure prediction results, it

could be assumed that the trypsin-binding region of the GR1 protein would have very

similar structural characteristics with the Bow

________________________________________________________________________________ 157


________________________________________________________________________________ 158

: S o d comparison between the GR1 protein

(this work) and Bowman-Birk inhibitor (Werner & Wemmer, 1991) for the

trypsin-binding region

ues indicate a and

GR1 protein values are shown in

try i

e r s

not surprising to see a similar

-2

-1.5

-1

-0.5

1 16 10

0.5

1

1.5

1 6 1 2

Residue number

d(rc

-obs

)

BBIGR1

Figure 7.2 ec ndary structure pre iction

.

Negative val β-sheet conform tion α-helices are indicated by positive values.

The blue and the BBI values are shown in red.

The chymo psin-binding region of the Bowman-Birk inhib tor and the corresponding

region in th GR1 protein were compa ed and the difference are shown in Figure 7.3.

Residues 32-40 within the GR1 protein showed a number of sequence similarities with

the Bowman-Birk inhibitor, and therefore it was

functional behaviour between the two proteins. After residue 41 there was very little

sequence similarity, however, the secondary structure prediction between the two

proteins was similar. Stretches of negative values were identified for the GR1 protein

for residues 31-35 and 44-46, which suggested this region contained β-sheet-like

structures.


inhibitor (Werner & Wemmer, 1991) a

Figure 7.3: Secondary structure prediction comparison between the Bowman-Birk

nd the GR1 protein (this work) for the

hymotrypsin-binding region.

structure and the predicted α-helix like structures are represented by a positive value.

c

The GR1 protein is shown in blue and the BBI is in red. The negative values indicate a β-sheet

7.2.2 Secondary structure assignment

The sequential assignment revealed all proline residues were in the trans-conformation,

with the exception of Pro18, which was found in the cis-conformation, as there were

NOE connectivities between the HαIle17 and HαPro18 protons. The cis X-Pro peptide

bond was also seen in the Bowman-Birk inhibitor (Asn18-Pro19) and was involved in

forming the type VI turn within the trypsin-binding site (and also the chymotrypsin

binding site) of the inhibitor (Werner & Wemmer, 1992).

-2

-1.5

-1

0

0.5

1

-0.5

27 37 47

Residue number

d(rc

-obs

)

BBIGR1

________________________________________________________________________________ 159


All members of the Bowman-Birk family have been found to (a) contain a proline

residue at position P3′ and (b) have a cis- conformation between the P2′ and P3′ of the

anonical motif (McBride et. al, 1998). Several researchers have shown the highly

conserved proline residue in the P3′ position of the reactive site to be important for the

inhibition of the protease. They have also shown the level of inhibition was improved if

this proline residue was in the cis conformation rather than in trans (Brauer et. al.,

c

2002).

Hydrogen bonds were determined by detecting slowly exchanging amides. The sample

was dissolved in D2O at pH 3.5 at 288 K, and a series of 1D and TOCSY experiments

were completed over a period of 16 hours. Studying the amide protons within this

experiment provides information on the overall structure of the protein and reveals how

exposed these proteins are to the solvent (Wagner & Wüthrich, 1982). The slowly

exchanging amide hydrogens were Ala5, Cys11, Val12, Thr14, Ser16, Arg20, Arg22,

Thr37, Ser48 and Ser61. Hydrogen bonds may exist between Val12-Arg22 and Thr14-

Arg20 as NOESY connectivities were seen for these residues (shown by arrows in

Figure 7.4). Schematic representation of these potential hydrogen bonds is shown in

Figure 7.4.

Figure 7.4: Schematic representation of the hydrogen bonds between the residues

The arrows show NOE connectivities between residues. The dashed lines show possible

10-15 and 19-24.

hydrogen bonds.

________________________________________________________________________________ 160


7.3 Positioning of the disulfide bridges in the GR1 protein

The GR1 protein was characterised as a Bowman-Birk protease inhibitor based on the

sequence alignment studies (refer to section 3.6) and competitive inhibition work (refer

served cysteine residues as shown in the Figure

.5 represent the disulfide-bonding pattern within the Bowman-Birk inhibitors. Two

The GR1 protein contains 10 cysteine residues that form 5 disulfide bonds (determined

by mass spectroscopy; refer to section 3.7 for details). A number of these cysteine

residues align with those from the Bowman-Birk inhibitor family (Figure 7.5).

However the GR1 protein does not contain 4 of these cysteine residues. Therefore this

protein will mostly likely have a different disulfide pattern when compared to any of the

other members of the Bowman-Birk family.

to section 4.7). The Bowman-Birk inhibitor contains 14 cysteine residues

(approximately 20% of the total number of residues) that form 7 disulfide bridges, and

these cysteine residues are conserved within the Bowman-Birk family, suggesting they

play an important role in the function/ structure of these proteins (Gueven et al., 1998).

As all the cysteine residues within the Bowman-Birk inhibitor form disulfide bridges, it

can be assumed that these residues are important in maintaining the structure of the

inhibitor. Numbers underneath the con

7

disulfide bridges exist between the reactive sites for both trypsin (disulfide bond

numbers 2 and 4) and chymotrypsin (disulfide bond numbers 6 and 7) for the Bowman-

Birk inhibitors. The remaining 3 disulfide bridges are involved in maintaining the

overall structure of the inhibitor (Figure 7.5).

________________________________________________________________________________ 161


GR1 -- -- -- -- -- -- -- -- -- --

1 10 20 30

-- -- -- -- -- -- -- G G E E A D W C E D C V C


1 2 3 4

31 40 50 60

GR1 T R S I P P R C R C T D S -- -- -- -- -- -- S V C T K C V C Y L T


4 2 5 6 5 7

GR1 V P A A M R P Y

61 70 80 90

C E S M A S R F D A F C P I G S -- -- -- -- -- --

1BBI Y P A Q C F C V D I T D -- -- -- -- -- -- F C Y E P C K P S E D D PI-II Q P G Q C R C L D T N D -- -- -- -- -- -- F C Y K P C K S R D D -- IBB3_DOLAX I P A Q C V C V D M K D -- -- -- -- -- -- F C Y A P C K S S H D D IBB4_DOLAX E P A Q C F C V D T T D -- -- -- -- -- -- F C Y K S C H N N A E K IBB_PHAAU M P G K C R C L D T D D -- -- -- -- -- -- F C Y K P C E S M D K D IBB2_PHAAN M P G Q C R C L D T H D -- -- -- -- -- -- F C H K P C K S R D K D IBB3_SOYBN Q P G Q C R C L D T N D -- -- -- -- -- -- F C Y K P C K S R D D -- IBB2_SOYBN M P G Q C R C L D T T D -- -- -- -- -- -- F C Y K P C K S S D E D

7 6 3 1

Figure 7.5: The conservation of cysteine residues within a number of Bowman-

Conserved cysteine residues are boxed. The reactive sites for trypsin and chymotrypsin are

highlighted in yellow and red respectively. The number under each cysteine residue shows the

disulfide-bonding pattern for the Bowman-Birk inhibitor family. 1, GR1 (Grevillea robusta

Chen et. al., 1992). 4, 1BB3_DOLAX & 5, 1BB4_DOLAX (Dolichos axillaris inhibitor,

Jourbert et. al., 1979). 6, 1BB_PHA

Birk inhibitors and the GR1 protein (this work).

inhibitor). 2, 1BBI (Bowman-Birk Inhibitor, Werner & Wemmer, 1992). 3, PI-II Glycine max,

AU (Phaseolus aureus (mung bean) inhibitor, Zhang et. al.,

1982). 7, 1BB2_PHAAN (Phaseolus angularis (adzuki bean) inhibitor, Kiyohara et. al., 1981).

, 1BB3_SOYBN & 9, 1BB2_SOYBN (Glycine max, Joudrier et. al., 1987). 8

________________________________________________________________________________ 162


The disulfide-bonding pattern for the GR1 protein was identified by NMR spectroscopy

by looking for non-sequential NOE connectivities between Hαi-Hβj and Hβi-Hβj (Klaus

et. al., 1993). It was difficult to detect connectivities between the Hβi-Hβj protons, as

there was a significant number of other NOE peaks within this area. Connectivities

were however, seen for the following residues - Hα57-Hβ11, Hα29-Hβ34 and Hα8-Hβ23.

Due to both Hβ protons of Cys13 & Cys21 having identical chemical shifts, it was

difficult to assign this disulfide bond. However, the peak volumes for the Hαi-Hβj for

both residues were significantly larger than the other identified cysteine residues.

Therefore, the increase in peak volumes and the positioning of the cysteine residues

within the protein (Figure 7.5; disulfide number 4) confirmed the disulfide bond existed

between residues Cys13 and Cys21. As 4 of the 5 disulfide bridges were identified, the

final bridge existed between Cys32-Cys46.

The biochemical approach to the identification of the disulfide bonding patterns was

itations in finding the appropriate protease that

tion and identification of the cleaved fragments by

otential proteolytic cleavage sites, especially within the chymotrypsin region where the

considered however, there were lim

would cleave the GR1 protein. This approach requires the proteolytic cleavage of the

protein of interest, followed by isola

HPLC and N-terminal sequencing. The GR1 protein sequence was investigated for

p

positioning of the cysteine residues were not conserved. Ideally, the protein would need

to be cleaved between the Cys29 and Cys32 and between Cys34 and Cys46 to

determine the disulfide pattern within this region. However, there were very few

proteases that would cleave within this region due to the absence of key amino acids

such as aspartic acid, glutamic acid, lysine and arginine etc. This approach would not

provide enough data to determine the connectivities between the cysteine residues

________________________________________________________________________________ 163


within this region, and therefore, the biochemical approach to the identification of the

disulfide-bonding pattern for the GR1 protein was not used.

7.4 Three dimensional structure of the GR1 protein

7.4.1 S

Distance R1 protein were derived from the peaks assigned in the

NOESY with a mixing time of 200 ms. A number of different

mixing tim ent and, after insp ion of each of the

experime at the data acquired with a mixing time of 200ms

provided the best data to determine to structure of the GR1 protein despite the

possibility of artefacts being formed as the result of spin diffusion. Spin diffusion

occurs when there is indirect magnetisation of atoms from systems within the

vicinity o nte sity is related to the mixing time

and the d ixing time directly

tabulated

the XEASY software program. Peak volumes were defined by performing

n the NOESY spectrum. The peak

olumes were then used to generate distance restraints for calculating the structure of

tructural Restraints

restraints for the G

spectrum at 303 K

es were run for the NOESY experim ect

nts, it was decided th

other spin

f the NOE peak of interest. As the NOE i n

istance between the two interacting spins, the increased m

influences the intensity of the NOE peak and thus increases the chances of spin

diffusion occurring (Guntert 1998). All of the NOE peaks were recorded and

in

rectangular integration on each assigned peak i

v

the GR1 protein.

Stereospecific assignments were made by identifying peaks between HN-Hβ2, HN-Hβ3

and Hα-Hβ2 and Hα-Hβ3 on the β methylene protons and the γ-methyl groups of valine

________________________________________________________________________________ 164


in the NOESY spectrum. Seventeen stereospecific assignments were identified which

included one γ-methyl group of valine from the GR1 protein. Pseudoatoms were added

to the unassigned β-methylene and γ-methyl protons.

The structure predication program, dynamic algorithm for NMR applications or

DYANA (version 1.5; Güntert et. al., 1997) was used to generate the restraints from the

information generated in the XEASY program. A number of macros are written within

ino acid sequence, the

NOE peak volumes and assignment file th to create t e

s for the protein.

within DYANA was use to convert he NOE pea volumes into

r distance restraints. D on tiv os

s and the hydrogen bond w o A

distance restraints for the a ts

DYANA was used to generate the three dimensional structures of the GR1 protein. The

ANNEAL macro within DYANA uses the distance restraints generated by CALIBA to

create these structures. The ANNEAL macro was customised to calculate 100 structures

with a total of 10000 molecular dynamics steps followed by 1000 steps of minimisation.

The macro starts with 800 molecular dynamics steps at a temperature of 8.0 (Güntert et.

al., 1997). This was followed by a slow cooling stage over 9200 steps until the

temperature reached 0.0. After the molecular dynamics steps, 1000 steps of

DYANA that convert the information obtained from the NMR experiments into distance

and angle restraints. One such macro called CALIBA uses the am

s from e XEASY program h

DYANA restraint file

The CALIBA macro d t k

upper and lowe isulfide b ds connec ities, stere pecific

assignment value restraints ere added t the CALIB macro,

which calculated se addition l assignmen .

7.4.2 Structural Calculations

________________________________________________________________________________ 165


minimisation were applied to each structure. The temperature is a measure of the target

function units per degree of freedom.

7.4.2.1 Initial GR1 structures

The distance restraints for the structures generated for the GR1 proteins are shown in

Figure 7.6. Very few medium (R<5) and long-range (R>5) restraints were found

however these restraints were located throughout the whole protein suggesting the

protein had regions of well defined structure. The two regions corresponding to the

reactive sites of the protein were relatively exposed to the solvent and this was shown

by the rapid exchange of the Hα-HN protons (within the f 2hrs of the experiment).

Slowly exchanging amide protons were located surrounding the disulfide bridges

throughout the GR1 protein.

A number of preliminary DYANA structures were completed where only 10 structures

were generated to identify any major violations with the data. As there were a number

the peak volume value or by completely removing the assignment from the XEASY

database. Once all of the violating restraints were removed, the 100 initial structures

were generated with DYANA.

A total of 363 distance restraints were defined where 154 were intra-residue, 156 were

sequential, 18 were medium range and 36 were long range restraints (Figure 7.6). Due

irst

of overlapping peaks within the Hα-HN region of the NOESY spectrum, it was not

surprising to find distance restraint violations within the generated DYANA structures

as it was difficult to get an accurate peak volume value for some of the residues. The

violating distances were addressed by either reducing the rectangular integration box or

________________________________________________________________________________ 166


________________________________________________________________________________ 167

to a large number of overlapping peaks, the total number of distance restraints was

much less than expected for a protein of this size. The majority of the long- range

NOEs were located surrounding the disulfide bridges and the medium range NOEs were

found around regions that may correspond to turns.

Intra-residue connectivities are shown in white; sequential connectives are in light grey;

ROCHECK

was also used to determine the stereochemical quality of the generated structures and

these results, along with the RMSD values, are shown in the Table 7.1. For the 20

structures generated in DYANA with the lowest target function, 73.27% of their

residues were located in the most-favoured and additionally allowed regions, 21.64%

Figure 7.6: The number of NOE upper distance limits per residue in the amino

acid sequence of the GR1 protein.

medium range are in dark grey; and long-range restraints are in black.

The average global root mean square deviation (RMSD) values were determined for the

20 structures generated in DYANA with the lowest target function value, and these

values were determined for the three sections within the GR1 protein. P


were found in the generously allowed regions and 5.08% were located in the disallowed

gions. These results suggest the GR1 protein will need to be further refined before the

nal solution structure can be determined.

able 7.1: Summary of NMR restraints and structural statistics from DYANA for

ll 20 structures.

DYANA NOE distance restraints

re

fi

T

a

Total 363 Intra-residue 154 Sequential 156 Medium-range (R<5) 18 Long-range (R>5) 36 Max. NOE violation, Å 0.487 ± 0.08 Max. vdw violation, Å 0.232 ± 0.07 Residual Target Function 2.82 ± 0.48 Mean Global RMSD ‡ (Å)

4.20 ± 0.92 3.74 ± 0.90

3.51 ± 0.97 5.06 ± 1.11

Stereochemical (Ramachandran plot) quality #

Residues in disallowed regions (%) 5.08 ± 2.45

Res. 8-25 bb $ 2.85 ± 0.97 Res. 8-25 heavy Res. 30-48 bb $

Res. 30-48 heavy 5.37 ± 0.98 $Res. 48-61 bb

Res. 48-61 heavy

Residues in most/additionally allowed regions (%) 73.27 ± 5.17 Residues in generously allowed regions (%) 21.64 ± 4.27

‡ RMSD values were calculated using MOLMOL. $ bb refers to the backbone atoms N, Cα, C’.

7.5 Further refinement of the GR1 protein

The quality of the structures obtained from DYANA were assessed using the

minimisation program, SYBYL® program (Tripos Inc). Disulfide bonds and distance

restraints were added to each structure prior to minimisation to ensure these bonds were

constrained during the energy minimisation steps. A total of 8000 steps of conjugated

# Stereochemical quality was assessed using PROCHECK (Laskowski et. al, 1993).

________________________________________________________________________________ 168


gradient minimisation using the Tripos force field were applied to each of the 20

structures generated in DYANA with the lowest target function (Table 7.2). The

RMSD values were determined for the three sections within the GR1 protein and

PROCHECK was used again to determine the stereochemical quality of these refined

structures. The RMSD and PROCHECK results for the original DYANA structures and

SYBYL minimised structures are shown in Table 7.2.

Table 7.2: The summary of the 20 energy-minimised NMR structures of the GR1

DYANA SYBYL® minimisation

protein before and after SYBYL® minimisation.

R sidual Target Function 2.82 ± 0.48 -- -- -- SYBYL Total number of steps -- 2000 4000 8000 SYBYL Total energy (kcal/mol) -- 92.96 ± 17.13 76.41 ± 15.07 63.27 ± 15.60 Mean global RMSD‡ (Å) Res. 8-25 bb $ 2.85 ± 0.97 2.97 ± 0.96 2.98 ± 0.95 3.03 ± 0.93 Res. 8-25 heavy 4.20 ± 0.92 4.33 ± 0.99 4.34 ± 0.99 4.38 ± 1.00 Res. 27-48 bb $ 3.74 ± 0.90 4.11 ± 0.94 4.13 ± 0.94 4.18 ± 0.94 Res. 27-48 heavy 5.37 ± 0.98 5.58 ± 1.02 5.60 ± 1.01 5.63 + 1.00 Res. 50-61 bb $ 3.51 ± 0.97 3.05 ± 0.90 3.06 ± 0.90 3.37 ± 1.00 Res. 50-61 heavy 5.06 ± 1.11 4.58 ± 1.10 4.59 ± 1.09 5.05 ± 1.14 Stereochemical (Ramachandran Plot) quality # Residues in most/additionally allowed regions (%) 73.27 ± 5.17 82.34 ± 4.21 83.18 ± 4.33 83.46 ± 4.47 Residues in generously allowed regions (%) 21.64 ± 4.27 9.82 ± 3.31 8.64 ± 4.07 8.06 ± 3.78 Residues in disallowed regions (%) 5.08 ± 2.45 7.88 ± 3.36 8.16 ± 3.30 8.46 ± 3.08

e

‡ RMSD values were determined using MOLMOL. $ bb refers to the backbone atoms. #Stereochemical quality was assessed using PROCHECK.

The minimisation of the DYANA structures (with the lowest target function values) was

completed over three steps and these results are shown in Table 7.2. The total energy

calculated for the 20 structures decreased after each minimisation step suggesting the

structures had improved. This improvement was also seen in PROCHECK, where the

number of residues found in the most/ additionally allowed regions of the

Ramachandran plot had increased for all of the minimisation steps. There was a

________________________________________________________________________________ 169


significant reduction in the number of residues located in the generously allowed

regions (reduction of 13.5%) of the Ramachandran plot for the structures minimised

after 8000 steps. However, there was an increase in the number of residues found

within the disallowed regions from 3 to 4 residues after the final step of minimisation.

Overall, there was very little difference in the RMSD values obtained for the structures

generated before and after minimisation. After the first minimisation step, a slight

difference was seen but further minimisation steps did not result in any improvement to

these values. MOLMOL was used to visualise the structures generated before and after

minimisation and each section was individually studied.

The structures generated in DYANA and the SYBYL® minimised structures for

residues 8-25 are shown in Figure7.7. The RMSD values within this region were

consistent throughout each of the minimisation steps, however there was a slight

increase in these RMSD values when compared to the original DYANA structures, and

this increase was not apparent when the structures were visualised. The regions

surrounding the disulfide bridges overlay relatively well in both the DYANA and

SYBYL® structures. This was not unexpected as this region was heavily constrained

with distance restraints corresponding to the disulfide bridges and the non-sequential

NOE assignments. The loop region (residues 15-20) did not overlay very well

suggesting this region was more flexible within the GR1 protein.

________________________________________________________________________________ 170


___________________

Figure 7.7: Conve

are shown in red.

of constrained resid

of these residues

assignment of peak

residues surrounding

the minimised SYB

before and (B) afte

The disulfide bridges

There was very littl

GR1 protein (residu

The backbone for a

have a similar over

protein are again rel

Cys8

_________________________________________

rged structures of the residues 8-25

ues during the generation of these structur

were located within overlapping regio

volumes and therefore the generation o

the disulfide bond of Cys32-Cys46 were

YL® structures compared with the DYAN

r minimisation using SYBYL®.

formed across the region are shown in yellow

e change to the RMSD values obtained fo

e 29-48) for both the DYANA and SYB

ll 20 structures studied are shown in Fi

lapping pattern that suggests the residues

atively flexible. This flexibility could be

Cys8

A
B
Cys11

__________

of the GR

ue to the

es in DYA

ns, which

f distance

found to o

A structure

and the lo

r the seco

YL® gene

gure 7.8.

within th

d

Cys11

Cys13
Cys13 Cys21 Cys21
Cys23
Cys23
__________ 171

1 protein (A)

limited number

NA. A number

prevented the

restraints. The

verlay better in

s.

ng-range bridges

nd region of the

rated structures.

Both structures

is region of the


________________________________________________________________________________ 172

BA

steine residues that are involved in forming disulfide bonds are shown in red and yellow.

(A) 20 structures and (B) 10 structures shown.

The final region of the GR1 protein, residues 50-61, did not reveal any significant

differences between the two groups of structures generated by DYANA and SYBYL®

as seen in Figure 7.9. The RMSD values for both groups of structures revealed a slight

improvement for the minimised structures however this was not evident when the

structures were visualised.

Figure 7.8: Converged structures of the residues 29-48 of the GR1 protein (A)

before and (B) after minimisation using SYBYL®. The cy


Figure

before a

________

A
B
7.9: Converged structures for the residues 50-61 of the GR1 protein (A)

nd (B) after minimisation using SYBYL®.

________________________________________________________________________ 173


7.6 The optimised 3D structure of the GR1 protein.

The SYBYL minimisation showed the original DYANA structures were of high quality,

this conclusion was based on the stereochemical quality of the structures, the

improvement in the total energy levels after each minimisation step and the consistent

RMSD values obtained throughout the minimisation process. Figure 7.9 shows the

solution s of the GR1 protein. The structura cteristics of each of the three

regions within the GR1 protein will be discussed in detail below. The structural

similarities and differences seen within these regions will be compared with those seen

w wman-Birk inhibitor.

Figure 7.10: The solution structure of the GR1 protein.

tructure l chara

ithin the Bo

The blue regions of the ribbon represent β-sheet like structures.

________________________________________________________________________________ 174


7.6.1 The structure of section one of the GR1 protein

Residues 8-26 of the GR1 protein were found to contain a short region of antiparallel β-

sheet structures held together by two-disulfide bonds at residues Cys8-Cys23 and

al disulfide bond was located

etween Cys11 and Cys57 and this bridge is thought to play a role in maintaining the

amide experiment (refer to section 7.2.2). The other amide hydrogens

identified from this experiment did not form hydrogen bonds.

Cys13-Cys21 (shown in Figure 7.11). An addition

b

structure of the GR1 protein. The antiparallel β-sheet region within the GR1 protein

was separated by a turn that was defined by identifying Hαi-HNi+3 and Hαi-Hδi+2 NOE

connectivities between Ser16 and Pro19 and between Thr17 and Pro19 respectively.

These patterns of NOE peaks were also seen for the Bowman-Birk inhibitor and are

known to form a type VI turn (Richardson, 1981; Werner & Wemmer, 1992). This

region also contains a cis X-Pro peptide bond at residues 18 and 19 and a trans X-Pro

between residues 17 and 18. An Hα-Hα NOE connectivity existed between the Ile17-

Pro18 residues which is characteristic of a cis-peptide. Hydrogen bonds were located

between HN12-O22 and HN14-O20 in the GR1 protein as seen in the slowly

exchanging

________________________________________________________________________________ 175


Figure 7.11: Converged structures of the trypsin-binding region of the GR1

protein.

Twenty structures with the lowest target function values were overlaid. The cysteine residues

rk

hibitor (Werner & Wemmer, 1991; Werner & Wemmer, 1992). These residues are

highlighted in blue within Figure 7.12. For example, an acid residue was found after a

in s o i s a g s o ted in

between two cysteine residues (position 6) and a basic residue was found in the P1

position of the reactive site. From these results, it was predicted that this region of the

-Birk inhibitor.

involved in forming disulfide bridges are shown in yellow and red.

Residues 8-26 of the GR1 protein were found to be sequentially similar with the trypsin-

binding region of the Bowman-Birk inhibitor as seen in Figure 7.12. There were

residues within the GR1 protein that were not conserved but contained similar

characteristics (basic, acid, non-polar, polar etc) with those found in the Bowman-Bi

in

cyste e (po iti n 3 within the f gure), a m ll non char ed residue wa l ca

GR1 protein would be structurally similar to the Bowman

________________________________________________________________________________ 176


GR1 7 W C E D C V C T R S I P P R C R C T D S -- 26BBI 8 C C D Q C A C T K S N P P Q C R C S D M R 28

Figure 7.12: The sequential alignment of the trypsin-binding region of the

The residues that are homologous are shown in red and the residues that are similar and could

substitute those seen in the Bowman-Birk inhibitor are shown in blue. The one letter code was

used for the amino acid residues.

Bowman-Birk inhibitor with the GR1 protein.

The Bowman-Birk inhibitor contains two distinct binding sites called the canonical

motif that is made up of 2 antiparallel β-sheet structures separated by a type VI turn

(Werner & Wemmer, 1991; Richardson, 1981). These antiparallel β-sheet and turn

regions were seen in the Bowman-Birk inhibitor and were also seen within these regions

of the GR1 protein as seen in Figure 7.13. NOE connectivities were seen between the

Hαi-HNi+3 and Hαi-Hαi+2 for the Bowman-Birk inhibitor, which defined the type of turn

(Richardson, 1981), and the formation of this turn was aided by the presence of a cis-

peptide between Ile19-Pro20. Within the GR1 protein, these NOE connectivities were

also seen, however the cis peptide was not located at the P3' position (at residue 18) but

instead the cis peptide was located at Pro19.

________________________________________________________________________________ 177


__

Fi

BA

th

h

is

RS

h

fr

T

T

lo

th

C

re

fo

(o

w

B

al

si

Arg15

__________________________________________

gure 7.13: Structural similarities betwe

e (B) Bowman-Birk inhibitor.

e cysteine residues that form disulfide bridges

boxed and shown in magenta and green. Th

CB database (PDB ID: 1BBI; Werner and Wem

e GR1 protein had three disulfide bonds w

cated between Cys8-Cys23, Cys11-Cys57

an-Birk inhibitor had four disulf

ys9-Cys24, Cys12-Cys58 and Cys14-Cys2

sidues form bridges across the β-sheet and

r P1-P1’ site) for the GR1 protein (Arg15

ithin the figure) were found to be in a simi

om the backbone and therefore, they are

e Bowm

r both proteins within Figure 7.13. The res

irk inhibitor. In order for these proteins to f

low adequate interaction with the protease,

de chain residues involved in interacting

Lys16

Ser16

__________

en the (A)

are shown

e Bowman-

mer, 1992

ithin the

and Cys1

ide bonds

2 (Figure

the locatio

dues invo

and Ser16

lar position

able to act

i

unction, th

trypsin (W

with the p

Ser17

Cys8

Cys23

Cys13
Cys21
Cys11

Cys14

___________

GR1 after

in yellow and

Birk inhibito

).

trypsin-bind

3-Cys21 (F

located be

7.13B). Tw

n of these

lved in form

), (shown in

to those se

e residue

with nearb

es

erner & W

rotease wer

Cys22

Cys12
Cys24
__

r

in

ig

tw

o

di

i

e

s

y

e

e

Cys9

___

mi

red

wa

g r

ure

ee

o

sul

ng

ma

n fo

mu

re

mm

po

Pro20
Pro19
__________ 178

nimisation and

. The P1-P1’ site

s taken from the

egion that were

7.13A), while

n Cys8-Cys62,

f these cysteine

fides are shown

the reactive site

genta and green

r the Bowman-

st be exposed to

sidues from the

er, 1992). The

inting outwards


proteases. It was not surprising to see these residues in a similar position as the

ting trypsin by both proteins.

o loops that do not

ontain any obvious secondary structures such as α-helices or β-sheet. There was a fair

amount of flexibility around the backbone of the top 10 m d GR1 protein

ts between the residues within

f

the atoms have to move and the resulting structures are more likely to

inhibition assays revealed similar Ki values towards inhibi

7.6.2 The structure of section two of the GR1 protein

Residues 29-48 in the GR1 protein were found to contain disulfide bridges between

residues Cys32-Cys46 and Cys29-Cys34 (shown in yellow and red respectively within

Figure 7.14). These disulfide bonds resulted in the formation of tw

c

inimise

structures as shown in Figure 7.14. The presence of overlap could be due the decrease

in the number of medium and long-range distance restrain

this region. The more restraints added to the structures would limit the amount o

freedom

converge.

________________________________________________________________________________ 179


Figure 7.14: Top 10 refined structures for residues 28-48 in the GR1 protein.

bonds for residues Cys29-Cys34 and Cys32-Cys46 are shown in red and

.

cture of the chymotrypsin-binding site of the Bowman-Birk inhibitor was very

The disulfide yellow

respectively

The stru

different when compared to the GR1 protein. The sequence similarity between the two

proteins within the chymotrypsin-binding region was restricted to the reactive site

(shown in Figure 7.15 by the P1, P1’). Similarities existed between residues that

contained certain characteristics (acidic, non-polar etc) and these residues are

highlighted in Figure 7.15 along with the identical residues between both proteins.

GR1 27 -- S V C T K C V C Y L T V P A A M R P Y C E 47BBI 33 H S A C K S C I C A L S Y P A Q C F C V D I 54

Figure 7.15: The sequential similarities between the chymotrypsin-binding region

of the Bowman-Birk inhibitor and the GR1 protein.

Amino acids that were conserved between the two proteins are shown in red and residues that

could be substituted are highlighted in blue. Multicoloured lines show the disulfide-bonding

pattern within both proteins. The one letter code is used to represent the amino acids.

P1 P1’

________________________________________________________________________________ 180


The Bowman-Birk inhibitor contained three disulfide bridges between Cys32-Cys39,

ys36-Cys51 and Cys41-Cys49, which were involved in holding the β-sheet structures

gether, while the GR1 protein contains only two disulfide bridges between Cys29-

Cys34 and Cys32-Cys46. It is obvious from Figure 7.15, that these two proteins will

ave very different structure based on the positioning of the disulfide bridges. The

isulfide bonds within the Bowman-Birk inhibitor result in this region forming a “U”

haped structure that is highly restrained through these bonds. The GR1 protein on the

ther hand, will adopt an “S” like conformation that will have regions of flexibility due

the lack of the third disulfide found within the reactive site region.

The chymotrypsin-binding region of the Bowman-Birk inhibitor contained antiparallel

β-sheet structures and a type VI turn (Werner & Wemmer, 1991). The type VI turn

contained the NOE connectivities between the Hαi-HNi+3 between Ser44 and Ala47and

Hαi-Hαi+2 between Tyr45 and Ala47. A cis peptide bond was also seen between Tyr45

and Pro46 for the Bowman-Birk inhibitor and this aided the formation of the type VIb

turn. The chymotrypsin-binding region for the Bowman-Birk inhibitor is shown in

Figure 7.16A.

The GR1 protein does not contain any β-sheet like structures, instead it contains a

number of turns that are held together by two disulfide bridges to form two loops as

seen in Figure 7.16B. There are a number of small similarities between the two

proteins, however, the major difference is the presence of a disulfide bridge across this

region. The residues involved in forming the reactive site within the GR1 protein were

found to point away from the loop and therefore they are in the ideal position to act with

nearby residues from the protease as seen in Figure 7.15B.

C

to

h

d

s

o

to

________________________________________________________________________________ 181


The two proline residues found within this region of the GR1 protein were both in the

trans conformation, which differs from that seen for the Bowman-Birk inhibitor. The

proline at the P3′ position of the reactive site of the Bowman-Birk inhibitor (Pro46) was

in the cis-conformation which aids in the formation of the type VIb turn. As this proline

is conserved amongst all Bowman-Birk inhibitors including the GR1 protein, it must

play an important role in the function of the protein. The proline residue within the

GR1 protein at the P3′ position of the motif was found to be in the trans-conformation.

Therefore, as the GR1 protein was able to inhibit chymotrypsin at a similar level to that

seen for the Bowman-Birk inhibitor, it can be stated that the conformation of this

residue is not important for the functioning of the protein.

The residue numbers are shown for each protein. The disulfide bonds are labelled and shown in

Figure 7.16: The chymotrypsin-binding region of the (A) Bowman-Birk inhibitor

and (B) the minimised GR1 protein.

yellow and red. Proline residues are shown in cyan and the reactive sites within each protein are

boxed and shown in magenta and green.

A B

Cys39

Pro 46

Leu43

Ser44

Cys51Cys36

Cys41 Cys49

Leu 36Thr37

Pro39

Cys46Cys32

Cys34

Cys29

________________________________________________________________________________ 182


7.6.3 The structure of section three for the GR1 protein

The final section of the GR1 protein to be studied contained residues 50-61 which had

limited similarities with the Bowman-Birk inhibitor as seen in the Figure 7.17. There

are three residues that are identical between the two proteins, including a cysteine that

was found to form a disulfide bond with Cys11 in the GR1 protein, and the formation of

this bond was identical to that seen in the Bowman-Birk inhibitor.

GR1 48 S M A S R F D A F C P I G S 61

BBI 55 T -- -- -- -- -- D -- F C Y E P C 62

Figure 7.17: Sequence alignment of residues 50-61 from the GR1 protein with the

the Bowman-Birk inhibitor. milarities are shown in red for identical and blue for residues of similar characteristics.

or this region, the GR1 protein was defined as having a random coil like structure that

ndary structure prediction

gion

ight contain α-helical like structures however this was not the case, as seen in Figure

corresponding region inThe si

F

contained a bend between Arg52 and Phe56. The seco

completed using the chemical shift values for the GR1 protein indicated that this re

m

7.18.

________________________________________________________________________________ 183


__________________

The backbone is hig

The complete stru

in Figure 7.19. T

Figure 7.18: The

o distinct dom

structure and the r

inhibitor domain (L

tw

Ala50

_________________________________________

hlighted in blue.

ctures for the Bowman-Birk inhibitor and t

he Bowman-Birk inhibitor has a well orde

solution structure of the final section of t

ains while the GR1 protein consists of

emaining region held together by two disul

eu-Thr).

Ser61

_____________________ 184

he GR1 protein are shown

red structure that contains

he GR1 protein.

one region of secondary

fide bridges to provide the


S17
A
Figure 7.19: Structural differences

(Werner & Wemmer, 1992) and (B) thBoth structures are shown in ribbon format

The two reactive sites are highlighted in mag

K16

L43

S44

S16

________________________________________

B

______________________ 185

between the (A) Bowman-Birk inhibitor

e GR1 protein (this work). where the disulfide bridges are shown in yellow.

enta and green.

R15

L36

T37

__________________


7.7 Conclusion

The structure of the GR1 protein was determined using data obtained from NMR

techniques and computer derived simulating annealing program (DYANA {Güntert et.

al., 1997}). Distance restraints were determined by using macros within DYANA and

additional restraints such as hydrogen bonds and disulfide bonds were added. Twenty

structures with the lowest target function values were chosen from the 100 structures

generated to be the initial solution structures of the GR1 protein. Further refinement of

the structures was achieved using minimisation with a conjugated gradient within the

SYBYL® software. The RMSD values and the stereochemical quality of the structures

were determined by using the MOLMOL and the PROCHECK programs respectively.

It was found that the structures had improved after the minimisation refinement with 43

residues in the most/allowed region (38 prior to minimisation). A decrease in the

number of residues were seen in the generously allowed region from 11 to 4 residues

and an increase was seen in the number of disallowed residues (from 3 to 4 residues).

As there was not a large increase in the number of disallowed residues, it was decided

that the solution structures for the GR1 protein, would be those that had undergone

minimisation using SYBYL®.

The solution structure of the GR1 protein is shown in Figure 7.20 and the residues for

both reactive sites within the protein are shown. Both of the residues within the reactive

sites were located at the top of the loop or turn-like structure, which provided easy

access to interact with their respective proteases. The sequence homology of the GR1

protein with the Bowman-Birk inhibitor provided some insight into the possible

structure of the protein. The trypsin binding region of the GR1 protein was very similar

to that seen in the Bowman-Birk inhibitor however, this was the limit of the similarity

________________________________________________________________________________ 186


______________________ 187

the GR1 protein was similar to that seen for the Bowman-Birk inhibitor and

erefore, the cis/trans-conformation at the P3' position of the reactive site was not

residues involved in inhibiting trypsin and chymotrypsin is shown in magenta and green.

chemical and structural data from the Bowman-Birk inhibitor

(Werner & Wemmer, 1991, 1992), provides a number of reasons why there were

differences in the two structures. Firstly, the Bowman-Birk inhibitor contained 2 extra

disulfide bonds when compared to the number of disulfide bonds the GR1 protein had.

Long range disulfide bridges throughout the Bowman-Birk inhibitor provided structural

support for the inhibitor. Secondly, the assignments of the NOE peaks for the Bowman-

Birk inhibitor (Werner & Wemmer, 1991) were more straightforward as this structure

did not have as many overlapping regions. As a result of this, there was a large number

between the two proteins. The proline residue at the P3' position in both the trypsin and

chymotrypsin binding regions for the GR1 protein was found in the trans-conformation

which was different to that seen for the Bowman-Birk inhibitor family. However, the

function of

th

important for the functioning of this protein.

Figure 7.20: The solution structure of the GR1 protein.

The backbone is represented by the ribbon structure. Disulfide bonds are in yellow and the

Reviewing the bio

R15

L36

S16

T37

__________________________________________________________


of medium and long range NOE connectivities assigned for the Bowman-Birk inhibitor

and these assignmen s aid e e r o the solutio tu s providing

restraints for The ote had lar num o residues

located in overlapp regio s, w ic m ore

difficul e s

Leguminoseae m e

of the proteins within this fam ar e m n

first characterised y e lyc a t me b s of h s perfamily

have been isolated from g e n ; h n et al., 1 8 , Winter pea

al., 1981) to name a few. The GR1 protein is the first member of the

Proteaceae family to be characterised as a Bowman-Birk inhibitor.

t ed in th g ne ation f n struc re by

all of the residues. GR1 pr in a ge ber f

ing n h h ade the assignment of NOE peaks m

t. Finally, most of the m mber of the Bowman-Birk superfamily belong to the

fa ily and it could therefore be assum d that the function and structure

ily would be simil . Th Bow a -Birk inhibitor was

from so b ans (G ine m x) and o her m er t is u

mun b a (Phaseolus aureus Z a g . 9 2)

(Pisum sativum; De La Sierra et. al., 1999), Adzuki bean (Phaseolus angularis;

Kiyohara et.

________________________________________________________________________________ 188

Chapter 8 Conclusions ____________________________________________________________________________________

Chapter 8 Conclusions

________________________________________________________________________________ 189


Chapter 8 Conclusions

Two functional proteins were isolated from the seeds of Grevillea robusta: a lectin

(GR2; Chapter 5) and a serine protease inhibitor (GR1; Chapters 3 & 4). The lectin

targeted specifically white blood cell receptors and therefore could be used to

characterise the white blood cell receptors (Chapter 1). The serine protease inhibitor

was found to inactivate both trypsin and chymotrypsin independently and has potential

application in the generation of insect resistant plants or in the treatment of specific

cancers (Kennedy, 1998a; Chapter 4). These proteins were isolated and characterised

by a number of different techniques including gel electrophoresis, affinity, ion exchange

and gel filtration chromatography, mass spectroscopy, N-terminal sequencing and

specific bioassays. The extract was shown to have lectin properties and the protein was

present in low yields within the seeds of G.robusta (Chapter 2). The lectin was

Agglutination, binding and sugar specificity confirmed the isolated protein was a lectin,

which was mannose specific. The lectin isolated in G.robusta was called the GR2

protein.

A serine protease inhibitor was isolated by two different methods: (1) ion exchange &

RP-HPLC and (2) size exclusion chromatography. A number of different conditions

such as the salt concentration, the pH and the gradient applied to the column were

applied to the ion exchange column, to ensure the maximum separation of the proteins

in the crude extract. Two methods were derived due to the presence of a contaminating

protein seen on native PAGE, which was later identified by N-terminal sequencing as a

cleaved product of the GR1 protein. Therefore, to minimise the presence of

contaminating proteins, a second method was developed which involved the addition of

successfully isolated with affinity chromatography using an oligosaccharide matrix.

________________________________________________________________________________ 190

Chapter 8 Conclusions ____________________________________________________________________________________ protease inhibitors to the extraction buffers. The resulting protein was 95% pure

etermine by visualisation on native PAGE. Mass spectroscopy confirmed the proteins

isolated from the two different methods were identical and had a molecular weight of

6669Da.

he full amino acid sequence of the GR1 protein was determined using cDNA

techniques (Clague, 1999). The GR1 protein was found to have significant sequence

omology with a number of known serine protease inhibitors, especially those within

the Bowman-Birk superfamily. Within the Bowman-Birk family, the cysteine residues

nd those involved in forming the reactive site are conserved, which suggested these

ites played an important role in the structure/function within the protein. All of the

ysteine residues within the Bowman-Birk inhibitor are involved in forming disulfide

e GR1 protein, a number of

cysteine residues and other key residues were found in the same position as those seen

r the Bowman-Birk inhibitors. However, the total number of cysteine residues within

e GR1 protein was less than that seen for the Bowman-Birk inhibitors and this was

evident in the chymotrypsin-binding region, resulting in a different disulfide-bonding

pattern for the GR1 protein. Figure 8.1 outlines the similarities and differences between

the two proteins.

d

T

h

a

s

c

bridges, which provide structure to the protein. Within th

fo

th

________________________________________________________________________________ 191


1 10 P1 P1’ 20

GR1 -- G G E E A D W C E D C V C T R S I P P BBI D D E S S K P C C D Q C A C T K S N P P 21 30 40

GR1 R C R C T D -- -- -- -- S -- -- S V C T K C V BBI Q C R C S D M R L N S C H S A C K S C I 41 P1 P1’ 50 60

S M A S R F D GRI C Y L T V P A M R P Y C E BBI C A L S Y P A Q C F C V D I T D -- -- -- -- 61 70

GR1 A F C P I G S -- -- -- -- -- -- -- -- -- -- BBI -- F C Y E P C K P S E D D K E N --

Figure 8.1: Sequence alignment of the GR1 protein and the Bowman-Birk

The residues that are conserved between the two proteins are shown in red. The cysteine

residues are shown in dark blue and those that are conserved are boxed. The scissle bonds for

the two inhibitory sites within the Bowman-Birk inhibitor (BBI) are shown in blue.

inhibitor.

Competitive inhibition assays were completed and confirmed the initial findings of the

GR1 protein belonging to the Bowman-Birk superfamily of serine protease inhibitors.

The GR1 protein inhibited both trypsin and chymotrypsin with Ki values comparable to

those seen for the Bowman-Birk inhibitor.

NMR spectroscopy was used to determine the three-dimensional structure of the GR1

protein. Homonuclear experiments two-dimensional experiments were used to identify

the individual spin systems for each amino acid and was also used to sequentially assign

the residues in the GR1 protein. Ninety seven percent of the residues were assigned

within the protein and the remaining 3% were not assigned due to a number of

overlapping peaks within the 2D spectrum. The cis/trans conformations of each of the

5 proline residues were determined by looking for Hαi-Hαi+1 and Hαi-Hδi+1 NOE

________________________________________________________________________________ 192

Chapter 8 Conclusions ____________________________________________________________________________________ connectivities. Four of these residues adopted the trans conformation and Proline 18

as in the cis conformation.

The initial GR1 structures were determined by using the computer program DYANA

(version 1.5; Güntert 1997). Distance restraints, stereospecific assignments and

hydrogen bonds were calculated using the CALIBA macro within DYANA. One

hundred structures were generated using the ANNEAL macro where each structure

underwent 10000 steps of molecular dynamics followed by 1000 steps of minimisation.

Twenty of the generated GR1 structures with the lowest target function values

nderwent further refinement using the SYBYL® program. A total of 8000 steps of

conjugated gradient minimisation steps were applied over 3 stages to the DYANA

structures. The stereochemical quality of the minimised structures were determined by

ent in the total number of residues in the

on were reduced (from 11 to 4 residues)

region increase from 3 to 4 residues. As

r ructures had improved from the original

DYANA structures, the solution structures for the GR1 protein were defined as those

w

u

PROCHECK and resulted in an improvem

most/allowed region of the Ramachandran plot (from 38 to 43). The total number of

residues found in the generously allowed regi

and the number of residues in the disallowed

the ste eochemical quality of the minimised st

generated after minimisation using SYBYL®.

________________________________________________________________________________ 193


antiparallel β-sheet structures that

ere separated by a turn. The turn in the Bowman-Birk inhibitor was defined by a cis-

peptide between Asn18-Pro19. However, the cis-peptide within the GR1 protein was

located between the Pro18-Pro19 residues and therefore, the type of turn seen in the

GR1 protein was different from the Bowman-Birk inhibitor. The chymotrypsin-binding

region of the GR1 protein was very different from the Bowman-Birk inhibitor. This

region was made up of a number of turns and loops that were held together by 2

disulfide bridges. Sequence similarities existed for the key residues within the reactive

site, which explained the similar functional properties of the GR1 protein.

The GR1 protein contained one region of secondary structure that was made up of an

antiparallel β-sheet-like structure, which corresponded to the trypsin-binding region.

A

There were a number of structural similarities and differences between the GR1 protein

and the Bowman-Birk inhibitor. The trypsin-binding regions within both proteins were

sequentially and structurally similar with each other as seen in the previous chapter

(Figure 7.18). Both proteins contained regions of

w

A

se

disulfide bridges (Cys29-Cys34 & Cys32-Cys4

the Bowman-Birk inhibitor.

The cond region of the protein exposed l

the GR1 protein that were responsible for in

regions that could be easily accessible by t

conserved in both reactive sites (ie in the P3'

conformation rather than the cis-conformatio

within this family. It was thought that the cis

within the protein (McBride et. al., 1998), how

protein as the KI values obtained for this prot

_____________________________________________

B

B

_______ 194

6) to fold the protein. Both sites within

eucine 36 and threonine 37 using two

hibiting the proteases, were located in

he protease. The two proline residues

position), were found to adopt the trans-

n seen in all other protease inhibitors

-peptide improved the level of inhibition

ever, this was not the case for the GR1

ein were comparable with those seen for

____________________________

Chapter 9 Experimental _____________________________________________________________________________________

___________________________________________________________________ 195

Chapter 9 Experimental


Chapter 9 Experimental

9.1 Extraction of proteins from ground seed material.

9.1.1 Ammonium sulfate precipitation of proteins.

The crude protein sample was extracted from the seeds of G.robusta using a two-step

ammonium sulfate precipitation. Seeds were ground in a commercial grinder and

oaked in PBS at pH 7.3 (11.3 mM disodium hydrogen orthophosphate (BDH), 1.28

o hosphate (BDH), 140 mM sodium chloride (Sigma))

overnight at 4ºC slowly stirring. Ground material was removed by filtering through

cheesecloth followed by centrifugation at 48400x g for 30 minutes at 4ºC. Ammonium

sulfate (Sigma) was added to the supernatant to create a 0-40% saturation range (22.6

g/100 ml of supernatant) and incubated for a further 6 hours at 4ºC. Centrifugation

removed the precipitated material (unwanted proteins) and a 40 –80% saturation range

was created using ammonium sulfate to precipitate the proteins of interest. Precipitated

material was removed by centrifugation (48400 x g, 30 minutes at 4ºC) and the pellet

was resuspended in milli-Q water. Dialysis was used to remove excess ammonium

te ing against milli-Q water (30 minute

hanges for 3 hours) followed by dialysing against PBS pH 7.3 (30 minute changes for

4 hours). Due to the majority of proteins containing a molecular weight of 14000 Da or

below, dialysis tubing with a MWCO of 7000 Da was used (Selby Biolab).

s

mM s dium dihydrogen orthop

sulfa from the “crude” proteins by initially dialys

c

___________________________________________________________________ 196


9.1.2 Ammonium sulfate precipitation of proteins using protease inhibitors.

he proteins were extracted from the seeds of G.robusta using protease inhibitors in the

extraction buffer containing a mixture of protease inhibitors (TBS pH 7.8 (20 mM

Trizma (Sigma Aldrich), 0.15 M sodium chloride), protease inhibitor cocktail (PIC) (1.5

ml; Sigma Aldrich) and 0.25 mM PMSF (Sigma Aldrich) where they were stirred

overnight at 4ºC. The initial extraction of the crude extract followed the same method

as that outlined in section 10.1.1. Dialysis of the precipitated material was initially

against milli-Q water followed by extraction buffer containing protease inhibitors.

9.2 Polyacrylamide gel electrophoresis

The Mini Protean 3 electrophoresis equipment from Bio-Rad Laboratories was used to

prepare and run the polyacrylamide gels.

9.2.1 Sodium Dodecyl Sulfate (SDS) PAGE

An 18% separating gel with a 5% stacking gel was prepared following the method

developed by Laemmli (1970). The protein sample (20 µg) was diluted 1:2 with

reducing sample buffer (0.06M Tris-HCl, 3.2% glycerol (BDH), 2% SDS, 5% β-

mercaptoethanol (Bio-Rad laboratories) and 0.01% bromophenol blue) and heated to

100°C for 5 minutes to ensure the protein was in its reduced state. The protein was

loaded onto the gel with LMW standards (Pharmacia Biotech) and the gel was run at

constant voltage at 100 V until the dye front reached the bottom of the glass plates. The

T

extraction buffer to prevent proteolysis. Seeds were ground as before and soaked in

___________________________________________________________________ 197


LMW markers used were phosphorylase B (Mr 96 kDa), bovine serum albumin (67

kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa)

and α-lactalbumin (14.4 kDa). The gels were stained with Coomassie Brilliant Blue

(0.1% Coomassie blue R-250 in 10% acetic acid, 30% methanol in water) and destained

using 10% acetic acid and 30% methanol in water.

9.2.2 Native PAGE

Native polyacrylamide gels were prepared as outlined by Ornstein (1964) and Davis

(1964). A 13% separating gel with a 3% stacking gel was made. Samples containing

40 µg of protein were diluted 1:4 with 4 x native sample buffer (1.98 M Tris-HCl, 4%

glycerol and 0.01% bromophenol blue). The gel was run at a constant voltage of 100 V

initially and then increased to 200 V after the sample had migrated into the separating

gel. Coomassie Brilliant Blue was used to stain the gels.

9.3 Protein concentration estimation

A known protein standard was used to create a standard curve for the assay. Bovine

serum albumin (1 mg/ml)(BSA; Sigma Aldrich) was diluted to form a range of different

concentrations from 5 – 160 µg/ml. Using a 96 well microtitre plate, 100 µl of standard

(at each concentration) and 100 µl of protein sample was added to the wells (in

triplicate). To these wells, 100 µl of Bradford’s reagent was added (Sigma Aldrich)

and the plate was mixed carefully. A plate reader was used to determine the absorbance

of the samples at a wavelength of 595 nm. The results obtained for the standards were

___________________________________________________________________ 198


graphed against its respective concentration to provide a standard curve. This curve

allowed the concentration of the protein samples to be determined.

9.4 Purification of the GR1 protein - Part 1

The BioLogic HR chromatography system was used to monitor the ion exchange and

gel filtration columns. It contained an in-line conductivity and UV monitor that allowed

The Q sepharose FF column (25 x 200mm) (Amersham Pharmacia Biotech) was

equilibrated with the 20 mM Tris-HCl pH 8.5 at a flow rate of 4 ml/min. The step

gradient 0-12%, 12-23%, 23-35%, 35-100% 0.5M NaCl/ 20 mM Tris-HCl pH 8.5 was

used to separate these proteins. The proteins were detected using UV at 280 nm.

Fractions were pooled and freeze dried. Once dried, these samples were resuspended in

PBS pH 7.3 and dialyzed against this buffer to remove excess salt.

the detection of proteins and ionic strength to be recorded. The Waters HPLC system

was used to elute proteins from a reverse phase HPLC column. It contained an auto-

injection carousel and UV was used to detect eluting proteins.

A number of difference methods were applied in order to separate the proteins from

G.robusta. The methods listed below are those used to produce the final product.

9.4.1 Ion exchange chromatography (IEX)

___________________________________________________________________ 199


9.4.2 Reverse phase-HPLC

The SO2 protein eluted from ion exchange chromatography was further purified using

reverse phase HPLC. A C-18 column (Rainin; 10 x 50mm) was used and equilibrated

with 0.1% TFA in water. Acetonitrile was used to elute the proteins from the column

by applying a continuous gradient (25-32%). A second method was developed to

decrease the time between runs. An isocratic flow rate at 29% acetonitrile/ 71% TFA in

water (0.1%) was used and the eluted peaks from both methods were pooled and freeze

dried.

9.4.3 High Q chromatography

A High Q column (10ml; Bio-Rad laboratories) was prepared by equilibrating it with 20

mM Tris pH 8.5. The fraction containing the SO2/4 proteins (Qseph.8.5.Pk7) was

9.5.1 Gel filtration chromatography

Proteins were isolated using a superdex 75 column (Amersham Pharmacia Biotech) (25

x 200mm) that was equilibrated with TBS + PI pH 7.8. The crude extract prepared with

protease inhibitors was injected onto this column and the proteins were eluted using the

injected onto this column and washed with equilibrating buffer. A continuous gradient

(0-30% 0.5 M NaCl/ 20 mM Tris pH 8.5) was applied over 30 minutes at a flow rate of

2 ml/min. Fractions were pooled and freeze dried.

9.5 Purification of the GR1 protein – Part 2

___________________________________________________________________ 200


equilibration buffer at a flow rate of 2 ml/min. Eluted proteins were pooled and

concentrated by freeze-drying.

9.6 Purification of a lectin from G.robusta

The lectin from G.robusta was purified using affinity chromatography. A mannan-

agarose column (10 ml) was equilibrated with PBS pH 7.3 and the crude extract was

applied at a flow rate of 1ml/min. The column was washed three times with PBS pH

7.3 and the lectin was eluted using 0.5 M mannose in PBS pH 7.3. The fractions

ontaining the lectin were pooled and dialyzed initially against water for two 30 minutes

e pH 7.3 for a further hour. The lectin was concentrated by

freeze-drying.

9.7.1 Deglycosylation of proteins

ase II (10U/ml in 20 mM Tris-HCl pH 7.5, 25 mM NaCl) and 2 µl of O-

lycosidase (1 U/ml in 20 mM Tris-HCl pH 7.5, 25 mM NaCl) was added to the protein

and incubated at 37°C for 1 hour. Ten µl of water, 10 µl of pH adjustment buffer (0.5

M sodium phosphate dibasic) and 2 µl of PNGase F (2.5 U/ml in 20 mM Tris-HCl pH

c

chang s and then against PBS

9.7 N-terminal sequencing of proteins isolated from G.robusta

An enzymatic deglycosylation kit was purchased from Bio-Rad laboratories and used to

prepare the proteins. Twelve µl of sample (containing up to 100 µg of protein) was

diluted with 4 µl of 5x reaction buffer (250 mM sodium phosphate pH 6.0). A further 2

µl of NAN

g

___________________________________________________________________ 201


7.5, 50 mM NaCl, 1 mM EDTA) was added to the reaction vial. The sample was

incubated for a further 24 hours at 37°C. Samples were stored at 4°C until needed.

stem was packed in ice to provide better results. The

embrane was stained with Coomassie Blue in 50% methanol and destained with 50%

methanol. The bands were removed from the membrane and sent to the Protein centre

at the University of Queensland Biochemistry department to be sequenced for a fee.

9.8 Bioassays

9.7.2 Native PAGE and Electroblotting of proteins

A 13% native separating gel with a 3% stacking gel was prepared for sequencing. The

deglycosylated proteins were loaded onto the gel and run at 150 V until the dye front

was at the bottom of the glass plates. The proteins were electroblotted onto Sequi-blot

PVDF membrane (Bio-Rad laboratories) at 100 V for 90 minutes using the Bio-Rad

mini Trans blot system. The buffer used to transfer the proteins onto the membrane

(Towbin buffer; 25 mM Tris, 192 mM glycine, 20% methanol) was cooled to 4°C prior

to use and the Trans-Blot sy

m

9.8.1 Biotinylation of proteins

Samples to be tested in the GIFT were biotinylated prior to test being set-up. The

samples were dialyzed against 0.1 M sodium bicarbonate pH 8.5 for 1 ½ hours (two

changes of buffer at 45 minutes each) at room temperature using dialysis tubing with a

MWCO of 6000-8000 Da (Selby). The volume was measured for each sample and 16

µl of biotin solution (0.02 g in 1ml of DMSO) per 100 µl of sample was added to the

___________________________________________________________________ 202


protein. The sample was covered in foil and incubated at room temperature for 2 hours

rocking slowly. The excess biotin was removed by dialyzing against PBS pH 7.3 for 1-

½ hours at room temperature (two changes every 45 minutes). The sample was stored

9.8.2 Granulocyte harvest

Anti-coagulated blood was collected in 10 ml EDTA tubes and centrifuged at room

temperature for 10 minutes at 100 g. The platelet rich plasma (PRP) was removed and 2

m chloride) and 1 ml of Bornes EDTA/BSA (18 mM

orthophosphate, 9 mM sodium-EDTA, 0.15 M sodium chloride, 0.8 mM

d at room temperature for 15 minutes at 1600 x g. Granulocytes were

arvested from the middle layer of the double gradient and washed in Bornes

at -20°C.

mls of 5% Dextran (in 0.9% sodiu

disodium

sodium azide and 0.2% BSA) was added to each of the 10 ml tubes. The tubes were

mixed by inversion and incubated at a 45-degree angle for approximately 30 minutes at

37°C. The leukocyte rich plasma (LRP) was removed and transferred into graduated

plastic conical tubes (4 mls maximum volume per tube). A double density gradient was

formed using 9” glass pipettes and by placing 2.5 ml of Ficoll Paque (density of 1.077)

followed by 2.5 ml of Mono-Poly resolving medium (density of 1.114). The tubes were

centrifuge

h

EDTA/BSA three times. After the final wash, the cells were resuspended in 1ml of

Bornes EDTA/BSA and counted on a Sysmex K-100. Cells were then diluted to the

required concentration for each of the tests.

___________________________________________________________________ 203


9.8.3 Granulocyte Agglutination Test (GAT)

After counting the cells, the granulocytes were diluted to a final concentration of 5 x

6 l tion (GRS; 20 mls of Bornes EDTA/BSA,

0.1 g of EDTA & 260 µl of 30% BSA). Paraffin oil was placed into the wells of a

s read on an inverted phase

icroscope and graded depending on the strength of reaction (0-4+).

After the cells were counted, half of the cells were removed for other tests (eg the GAT)

and the remainder was centrifuged in an immufuge for 2 minutes on ‘HIGH’. The cell

button was resuspended in 1 ml of 1% paraformaldehyde and incubated for 2 minutes at

room temperature to fix the cells. The granulocytes were washed with Bornes

EDTA/BSA twice and diluted to a final concentration of 10 x 10 /ml.

Twenty-five µl of cells (at 10 x 106/ml) were pipetted into the wells of a V-bottom

microtitre plate. For control samples, 50 µl was added to the wells. The plate was

centrifuged at 3000 rpm for 2 minutes and the buffer was removed from the cell pellet

by flicking and draining onto a tissue. Each cell pellet was resuspended in 30 µl of

as

10 / m with granulocyte resuspension solu

microtest culture plate and 3 µl of test sample was placed in duplicate under this oil in

the middle of each well. One µl of cell suspension was added to the sample in each well

using a multi-dispense syringe (dispensing in 1 µl aliquot). Between samples, 3 µl of

cells were expelled and the needle wipes to prevent contamination of the samples. The

trays were incubated at 30°C for 3-4 hours. The plate wa

m

9.8.4 Granulocyte Immunofluorescence Test (GIFT)

6

biotinylated testing sample and 60 µl was added for the control samples. The plate w

___________________________________________________________________ 204


sealed and incubated at 30°C for 30 minutes. Unbound material was removed by

washing the plate twice with Bornes EDTA/BSA and centrifuging at 3000 rpm for 2

minutes. The cell pellet was resuspended in 30 µl of streptavidin-FITC (1:45 dilution in

Bornes EDTA/BSA) in each well (60 µl for control samples), sealed and incubated at

The samples to be tested were biotinylated as outlined in section 10.8.1. Different

concentrations were made up of the 8 chosen sugars (0.1 M, 0.25 M & 0.5 M). The

biotinylated sample to be tested for sugar specificity was diluted in a 1:4 (v/v) ratio of

sample to sugar and incubated at room temperature for at least 30 minutes in the dark.

Granulocytes were prepared as seen in section 10.8.2. Granulocytes were fixed with

paraformaldehyde, washed and diluted to a final concentration of 10 x 106/ml. The

remainder of the set up was the same as the GIFT bioassay (refer to section 10.8.4).

The sample used in this set up corresponded to the previously prepared sample-sugar

mixture.

room temperature for 30 minutes in the dark. The plate was washed again to remove

any excess streptavidin-FITC and 100 µl of Bornes EDTA/BSA was added to each of

the wells. The contents of the wells were mixed well and transferred into labelled flow

cytometer tubes containing 250 µl of Bornes EDTA/BSA (control tubes contained 500

µl). These tubes were vortexed prior to being read on the flow cytometer.

9.8.5 Sugar blocking GIFT

___________________________________________________________________ 205


9.9 Mass spectroscopy

single quadruple electrospray mass spectrometer (Fison instruments) was used to

etermine the molecular weight of the eluted proteins from the G.robusta. As some of

the proteins were isolated using buffers, each sample was prepared by running it

rough a small plug of C-18 resin (1ml bed) in water and eluting with 50% acetonitrile

in water. This removed any salt found in the sample that could affect the mass

pectrometer. The samples were freeze-dried and resuspended in 50%

cetonitrile/water (total volume was 200 µl). The spectrometer was set to positive

lectrospray and 3 µl of sample was directly injected into the spectrometer using 50%

acetonitrile/water. The Masslynx icromass) software program was

used to analyse the results.

9.10 NMR Spectroscopy

9.10.1 NMR measurements

All 1D and 2D spectra were recorded using a Varian INOVA 600MHz spectrometer at

either 303 K or 288 K. The GR1 protein was resuspended in 18% CD3CN/H2O or in

99.99% D2O at pH 3.5. All spectra were analysed using the program VNMR. Water

suppression was achieved using a WATERGATE sequence in the TOCSY and NOESY

experiments while a presaturation sequence was used for the suppression in the DQF-

COSY experiment. The mixing times for the NOESY spectra were 125ms and 200ms

for the D2O sample and 75 ms, 125 ms and 200 ms for the H2O sample. NOESY

spectra were recorded at 288 K and 303 K. TOCSY spectra had a mixing time of 80 ms

A

d

th

s

a

e

NT (version 3.4; M

___________________________________________________________________ 206


and were run at 288 K and 303 K for both D O and H O samples. All spectra obtained

were processed by a sine-bell window using a 60° phase shift in the F2 and F1.

lowly exchanging amides protons were determined by dissolving the protein in

leted over a period of 16 hours

mediately after the solvent was added to the protein and run again 48 hours after to

exchanged.

tereospecific assignments of the β protons were determined by identifying the HN-Hβ

and Hα-Hβ connectivities. Each of the stereospecific assignments was added to the

CALIBA macro. The disulfide bonds between Cys11-Cys23, Cys13-Cys21 and Cys32-

Cys46 were also added to the macro. Hydrogen bonds found between Thr14-Arg20 and

Val12-Arg22 from the slowly exchanging amide experiment was also added to the

macro by restraining the HN-O connectivity to 2.4 Å. The modified CALIBA macro

was run in DYANA (version 1.4) to generate the distance restraint file to be used in the

2 2

S

99.99% D2O at pH 3.5 and the experiments were run at 303 and 288 K. One-

dimensional and TOCSY experiments were comp

im

ensure all of the amides had

9.10.2 NMR distance restraints

The peaks from the TOCSY, DQF-COSY and NOESY experiments were tabulated

using the software program XEASY. Rectangular volume integration was used on the

NOESY spectra (with a mixing time of 200 ms) to select peaks and provide volumes for

each peak. The CALIBA macro within DYANA was modified and used to convert the

peak volumes into distance restraints. A series of commands were used to include the

disulfide bonds, the stereospecific assignments and the hydrogen bonds in the CALIBA

macro.

S

___________________________________________________________________ 207


generation of the structures. The CALIBA macro used for each of the experiments is

outlined in Appendix D.

set number of steps. For all structures

generated throughout this work, the following procedure was used and a full list of the

nd 1000 steps of minimisation. The calculation began with

00 molecular dynamic steps at a temperature of 8.0 (temperature was a measure of the

value. This overview file also included the restraints that were violating (upper and

lower limits and vdw) over the set limits, which was useful in determining where

potential problems could arise.

Twenty structures with the lowest target function value were visualised using the

program MOLMOL and the RMSD values were calculated within this program using

the converged structures. The stereochemical quality of each of the structures was

determined using the program PROCHECK (Laskowski et. al., 1993).

9.10.3 Structure calculations

DYANA version 1.5 was also used to generate the structures of the protein (Güntert et.

al., 1997). The macro ANNEAL was modified to include the distance restraints

generated from CALIBA and to perform a

protocols used for ANNEAL are outlined in Appendix D.

One hundred structures were generated per DYANA calculation using a total of 10000

molecular dynamics steps a

8

target function units per degree of freedom) which was followed by a slow cooling stage

for 9200 steps of molecular dynamics until it reached a temperature of 0.0. Finally the

structures were subjected to 1000 steps of minimisation. The resulting structures were

tabulated in the overview file that listed the structures with the lowest target function

___________________________________________________________________ 208


9.10.4 Further refinement

Twen of th e ith e lo st ta et function values

were further refined using the program S YL Th isu br es e a d to

each ctur d a d DY A w s als added. A total of

8000 steps of conjugated gradient min isation using the Tripos force field were

appli o each f th s r T firs wo s s co aine 000

steps of minim tion and t inal step tain 40 tep Th tal energi er

compared and the stereochemical qua determined for each structure using

PRO ECK w ere t re h r d 2.0 Å

of the generated structures

ty e structures generat d from DYANA w th we rg

YB ®. e d lfide idg wer dde

stru e and istance restr ints generate in AN a o

im

ed t o e 20 tructu es over three steps. he t t tep nt d 2

isa he f con ed 00 s s. e to es w e

lity was

CH h he solution for t ese st uctures were define as .

___________________________________________________________________ 209

Appendices ____________________________________________________________________________________

Appendices

___________________________________________________________________ 210

Appendix A ____________________________________________________________________________________

Appendix A Methodology used to determine the full amino acid

sequence of the GR1 protein from Grevillea robusta.

A-1 RNA extraction

The developing seeds of G.robusta were removed from the fruit and seed coat and

ground using a mortar and pestle under liquid nitrogen and the total RNA was extracted

using the method outlined by Cheng et. al., 1993. Absorbance readings were taken at

the wavelengths of 260 and 280nm to determine the purity and quantity of RNA

present. Electrophoresis on an RNase free 2% NuSieve agarose gel allowed the RNA to

be visualised. The RNA sample was treated with RQ1 RNase-free DNase according to

the manufacturer’s instructions to ensure the sample did not contain any DNA

contamination. The RNA was recovered using ethanol precipitation and the samples

This information has been taken from the final assessment for a coworker at the ARCBS

as part of her Masters degree (Clague et. al., 1999).

immediately placed into liquid nitrogen where they were stored. The seeds were finely

were resuspended in 50µl of RNase-free water and stored at -80°C.

___________________________________________________________________ 211

Appendix A ____________________________________________________________________________________

A-2 3’RACE

The N-terminal sequences obtained from t G.robusta seeds

were used to generate 12 degenerative, inosine containing oligonucleotide primers for

the 5’en he 3 E oligo

(dT) prim us rformed

using Superscript™ (II) RNase H- Reverse Transcriptase and the oligo (dT)20 primer

with 5µg of purified RNA in 20µl reaction.

The 3’R E R s fo d using 100 of c m tu h ontained

50mM KCl, 10mM Tris pH 8.3, 1.5mM MgCl , 0.2mM of each dNTP, 20pmol primer

B, 10pm o pliTaq

Gold™

The rea minutes and paused to add the

AmpliTa u and this

involved n 5°C for

30 secon r econds.

The next 10 cycles involved: 94°C for 30 seconds, 40°C for 30 seconds, 72°C for 30

ose gel and ethidium bromide. The DNA

d from the gel using the QIA quick gel extraction kit.

the proteins isolated from he

d of the cDNA. These primers are shown in Figure A-1. T ’RAC

er was ed for the unknown 3’end. The first strand of cDNA was pe

AC PC wa per rme µl rea tion ix re w ich c

2

ol 3’RACE primer, 1µl cDNA (equivalent to 1µg) and 2.5U f Am

DNA polymerase.

ction mixture was heated to 94°C for 10

q Gold™ DNA polymerase. The touchdown PCR protocol was sed

the first 30 cycles to consist of the following: 94°C for 30 seco ds, 5

ds with the temperature decreasing 0.5°C per cycle and 72°C fo 30 s

seconds and finally 72°C for 7 minutes. The DNA products were visualised

electrophoretically using a 2% NuSieve agar

products were purifie

___________________________________________________________________ 212

Appendix A ____________________________________________________________________________________

G2 G G A GGI GGI GAG GAG GCI GA3’ B --- --- --G --A --- --- C D

E D/E D V V T T S C I P P E GAI GAI GAT GTT GT3’

G CCI AAC ATI TGI AT

--- GAT TGI TGG

L --- --- --- --c --- ---

3'RACE universal primer 5'CGCCTAGG(T)17 3'

igure A-1: A list of the degenerative primers designed from the N-terminal

equencing results. The N-terminal sequences are in bold.

E E A D W C

--- --- --A --G --- --- --- --- --A --A --- ---

G2..cont

F --- --- --- --C ---

G3 C L P N I C S S D L D 3’

H --- --T --- --- --- G4 S I P E A D C W R C T D I TCT ATI CCI GAA GCI GA3’ J --C --- --- --- --- K CCI GAA GCI

F

s

___________________________________________________________________ 213

Appendix A ____________________________________________________________________________________

the sequencing results obtained from the 3’RACE experiment

were designed and these primers are shown in figure A-2.

GR1 5' G C C A A C A G G T T C A G A A G A T 3' GR2 5' G G C T A G G A C C A C A C C A T G 3'GR3 5' C C G G A G C A G T A G A G G A G A C 3'

igure A-2: The specific pri ers deve ped from the 3’ resu

imer l GR1 w used he o fir of cDNA

ma urer’s ctions). Two 5’RACE PCR reactions were completed

2 R3 pri indiv ly. tio ure s what was

utlined in the manufacturer’ instructions. The CR pr us 4°C for 10

inutes which was followed by 35 cycles of: 94°C for 30 seconds, 55 r 30 seconds,

2°C for 60 seconds and finally 72°C for 7 minutes. The DNA products produced

alise e 2% eve a e g se cts ified using

uick ractio

A-3 5’RACE

The 5’RACE system for rapid amplification of cDNA ends was used (Gibco). Three

primers selected from

G AG G T T

T A T

F m lo RACE lts.

The pr abeled as for t synthesis f the st strand

(following nufact instru

using the GR and G mers idual The reac n mixt used wa

o s P otocol ed was 9

m °C fo

7

were visu d on th NuSi garos el and the produ were pur

the QIA q gel ext n kit.

___________________________________________________________________ 214

Appendix A ____________________________________________________________________________________

A-4 Sequencing of the cDNA

he purified DNA products were re run on the 2% NuSieve agarose gel with

be

ined. The final PCR reaction volume of 20 µl included 200ng of template DNA,

rd primer and 8 µl of Big Dye Terminator reaction mix. The control

action contained 200ng of pGEM-3Zf(+) as template DNA ad 3.2 pmol of M13 as the

e to 25 cycles at 96°C for 10 seconds, 50°C

oducts were transferred to 1.5 ml eppendorf tubes that contained 2

hanol/5% isopropanol (v/v) and

mples were centrifuged at 21000 x g at 4°C for

The precipitated DNA pellet was washed

re centrifuged again in a microfuge at

aximum speed for 15 minutes. The supernatant was removed and the tubes were

minute to dry the sample. These samples were sequenced for a

located at the Queensland Institute of Medical Research.

e resulting sequence.

T

quantitative molecular weight markers, which allowed to amount of DNA present to

determ

3.2 pmol of forwa

re

forward primer. Both sampl s were subjected

for 5 seconds and 60°C for 4 minutes.

The resulting PCR pr

µl of 3 M sodium acetate pH 4.6 and 50 µl of 95% et

incubated on ice for 15 minutes. The sa

30 minutes and the supernatant was removed.

with 500 µl of 70% ethanol and the samples we

m

incubated at 90°C for 1

fee on the ABI Prism system

Figure A-3 shows th

___________________________________________________________________ 215

Appendix A ____________________________________________________________________________________

35 AGA GTG CAA GTG ATC AGA GAT CGA TCG AGA GAG 71

A V A K V A L M I T L 12 CT GTT GCT AAG GTG GCG TTG ATG ATA ACA CTA 107

L L F V A T L P A P 24

CTG CCT GCT CCA 143

S T A T S N P F G P F R 36 179

P S G G E E A D W C E D 48 CCA AGT GGT GGA GAG GAA GCT GAC TGG TGC GAA GAC 215

TGT GTT TGC ACA AGA TCA ATT CCT CCT CGC TGT GTT 251

C T D S S V C T K C V C 72 TGC ACT GAT TCT TCG GTG TGA ACC AAA TGT GTT TGC 287

Y L T V P A A M R P Y C 84 TAC CTA ACT GTA CCT GCT GCA ATG AGG CCT TAT TGT 323

E S M A S R F D A F C P 96 GAG TCT ATG GCT TCC AGA TTC GAT GCC TTC TGC CCC 359

I G S L Q S Y N 104 ATT GCC TCT CTT CAA TCC TAC AAC 383

TGA TCG ATG AGC TCA ACA GAA CCC TAA ATA GTC TCC 419TCT ACT GCT CCG GCT GTC AAC CAT GGT CGT GGT CCT 455AGC CAG CTT ATA TAT GCA GTT CTT TTC TAC TTT ATG 491TCT TGT ATT TCT TCT CTT AGT TTC ATC TTC TGT AAC 527CCA GTT GGC AGT TGT TAG CGA AAG TGG CTA ACA ACT 563AGT TTG TTG ATA GTT GAT AAT AAA GAG GAG ATT TTC 599ATA AAA AAA AA 610

Figure A-3: The complete amino acid sequence of the GR1 protein from the seeds

f G.robusta. The start of the N-terminal sequence is highlighted in red. The one letter code is used for the

GG GGG GGG ACC GTG TGT TTG TGT AAA CTG TAG GTG TGA

M ATG G

M V ATG GTG TTG CTC TTC GTA GCA ACA

TCG ACT GCA ACA AGC AAC CCG TTC GGG CCG TTC AGA

C V C T R S I P P R C R 60

o

amino acid residues.

___________________________________________________________________ 216

Appendix B

______________________________________________________________________________

R1 protein isolated from the seeds of G.robusta.

B-1 Trypsin and Chymotrypsin inhibition assays

he protease inhibition assays were carried out using the instructions provided by the

anufacturer (Roche). This assay used casein, resorufin-labeled, universal protease

ubstrate. The inhibition constants were calculated at pH 7.8 and at 24°C. Data was

nalysed using the GraphPad Prism software. The inhibition curves are shown in Figure

-1 for (A) trypsin and (B) chymotrypsin.

igure B-1: The inhibition curves for the GR1 p

Appendix B Enzymatic inhibitory studies of the G

T

m

s

a

B

A

F

________________________________________

B

rotein.

___________________________ 217

Appendix C

______________________________________________________________________________

Appendix C Experimental Random coil values

The experimental random coil values used are shown below and these values were taken

from Wüthrich, 1986.

Residue NH αH βH Others

Glycine Gly, G 8.39 3.97 Alanine Ala, A 8.25 4.35 1.39 Valine Val, V 8.44 4.18 2.13 Isoleucine Ile, I 8.19 4.23 1.9 γCH3 0.97, 0.94 γCH2 1.48, 1.19 γCH3 0.95 δCH3 0.89 Leucine Leu, L 8.42 4.38 1.65, 1.65 γH 1.64 δCH3 0.94, 0.90 Proline Pro, P 4.44 2.28, 2.02 γCH2 2.03, 2.03 δCH2 3.68, 3.65 Serine Ser, S 8.38 4.5 3.88, 3.88 Threonine Thr, T 8.24 4.35 4.22 γCH3 1.23 Aspartic acid Asp, D 8.41 4.76 1.84, 1.75 Glutamic acid Glu, E 8.37 4.29 2.09, 1.97 γCH2 2.31, 2.28

εCH2 3.02, 3.02 εNH3- 7.52 Arginine Arg, R 8.27 4.38 1.89, 1.79 γCH2 1.70, 1.70 δCH2 3.32, 3.32 NH 7.17, 6.62 Asparagine Asn, N 8.75 4.75 2.83, 2.75 γNH2 7.59, 6.91 Glutamine Gln, Q 8.41 4.37 2.13, 2.01 γCH2 2.38, 2.38 δNH2 6.87, 7.59 Methonine Met, M 8.42 4.52 2.15, 2.01 γCH2 2.64, 2.64 εCH3 2.13 Cysteine Cys, C 8.31 4.69 3.28, 2.96 Tryptophan Trp, W 8.09 4.7 3.32, 3.19 2H 7.24 4H 7.65 5H 7.17 6H 7.24 7H 7.5 NH 10.22 Phenylalanine Phe, F 8.23 4.66 3.22, 2.99 2,6H 7.3 3,5H 7.39 Tyrosine Tyr, Y 8.18 4.6 3.13, 2.92 2,6H 7.15 3,5H 6.86 Histidine His, H 8.41 4.63 3.26, 3.20 2H 8.12 4H 7.14

Lysine Lys, K 8.41 4.36 1.85, 1.76 γCH2 1.45, 1.45 δCH2 1.70, 1.70

___________________________________________________________________ 218

Appendix D

______________________________________________________________________________

___________________________________________________________________ 219

ppendix D Structure Calculations

-1 CALIBA and ANNEAL macros used to generate the DYANA structures

ALIBA_B.dya

ar # deletes all distance constraints ad seq SO2protein_mod.seq # read sequence ad prot /userdata/people/sarahk/xeasy_files/revnoesy.prot # read proton list

y_files/REVNOESY.peaks assigned integrated # read eak list

istance unique # keep strongest constraint for each write upl caliba_A.upl ssbond 8-23 11-57 13-21 29-34 32-46 atoms stereo HB2 7 8 11 16 21 34 35 36 42 48 49 53 54 56 toms stereo QG1 38 istance make 2.4 14 HN THR, 20 O ARG

weight=1.0 distance make 2.4 12 HN VAL, 22 O ARG weight=1.0 distance modify write upl caliba_B.upl # save upper limits write lol caliba_B.lol # save lower limits

ANNEAL_GR1D.dya

read seq SO2protein_mod.seq read upl caliba_B.upl read lol caliba_B.lol distance modify write lol GR1protein_D.lol write upl GR1protein_D.upl random_all 100 forall parallel anneal thigh=8.0 tend=0.0 steps=10000 highsteps=800 minsteps=1000 structure sort cutNo = 0 do i 1 100 if (tf (i) <= 2000) then cutNo = i end if end do structure select 1..cutNo write pdb GR1_D.pdb all

A

D

C

distance clerereread peaks /userdata/people/sarahk/xeaspcaliba d

ad

Appendix D

______________________________________________________________________________

pdb structure select ut_tf := 50 ut_upl := 0.5

overview GR1_D structures=100 ang cor hbond vdw

D-2 Stereochemical Quality of the DYANA Structures

PROCHECK was used to determine the quality of the structures generated from

DYANA. A summary of these results is shown below.

DYANA generated structures

GR_3 2.14 32.7 48.1 17.3 1.9 80.8

GR_2 1.82 17.3 59.6 19.2 3.8 76.9GR_15 3.06 28.8 46.2 19.2 5.8 75GR_16 3.08 21.2 46.2 28.8 3.8 67.4

GR_13 2.93 15.4 57.7 23.1 3.8 73.1

GR_10 2.89 21.2 40.4 30.8 7.7 61.6GR_12 2.93 25 50 21.2 3.8 75GR_18 3.37 36.5 32.7 21.2 9.6 69.2GR_5 2.52 28.8 44.2 25 1.9 73GR_6 2.79 26.9 51.9 17.3 3.8 78.8GR_19 3.43 26.9 46.2 21.2 5.8 73.1

write_all GR1_D

cccut_lol := 0.5 cut_vdw := 0.2 hb_len := 2.4 hb_ang := 0.610865

DYANA Procheck results before minimisationTF core allow gener diallow c + a

GR_14 3.01 26.9 44.2 25 3.8 71.1

GR_4 2.37 30.8 46.2 19.2 3.8 77GR1_1 1.78 34.6 44.2 15.4 5.8 78.8

Figure D-1: PROCHECK results of the

GR_20 3.44 28.8 40.4 25 5.8 69.2

Av 2.82 26.63 46.64 21.64 5.08 73.3

GR_8 2.88 23.1 51.9 23.1 1.9 75GR_7 2.86 30.8 48.1 15.4 5.8 78.9GR_11 2.90 26.9 44.2 19.2 9.6 71.1GR_17 3.36 28.8 48.1 19.2 3.8 76.9

GR_9 2.89 21.2 42.3 26.9 9.6 63.5

___________________________________________________________________ 220

Appendix D

______________________________________________________________________________

___________________________________________________________________ 221

s after (A)

000 steps and (B) 4000 steps.

D-3 Stereochemical Quality of the SYBYL® minimised structures

Procheck results after 2000 steps of minimisation

GR_14 64.35 42.3 40.4 15.4Energy Core Allow gener disall c + a

1.9 82.7GR_3 65.00 51.9 42.3 3.8 1.9 94.2GR_4 75.63 44.5 36.5 13.5 5.8 81GR1_1 79.71 42.3 40.4 7.7 9.6 82.7GR_2 80.43 38.5 46.2 7.7 7.7 84.7

GR_16 83.58 32.7 50 7.7 9.6 82.7.7 48.1 7.7 11.5 80.8.3 44.2 5.8 7.7 86.5

GR_11 91.68 34.6 50 7.7 7.7 84.6GR_17 91.73 40.4 46.2 9.6 3.8 86.6GR_13 94.73 30.8 44.2 9.6 15.4 75

GR_12 97.95 32.7 48.1 13.5 5.8 80.8

GR_5 110.39 44.5 38.5 13.5 3.8 83GR_6 111.80 44.2 40.4 7.7 7.7 84.6GR_19 119.59 48.1 26.9 15.4 9.6 75GR_20 129.70 32.7 48.1 9.6 9.6 80.8

Av 92.96 38.30 44.05 9.82 7.88 82.34

GR_15 83.58 32.7 50 7.7 9.6 82.7

GR_8 86.41 32GR_7 87.23 42

GR_9 97.49 28.8 50 13.5 7.7 78.8GR_10 97.83 34.6 46.2 7.7 11.5 80.8

GR_18 110.30 34.6 44.2 11.5 9.6 78.8

SD 17.13 6.45 5.76 3.31 3.36 4.21

Procheck results after 4000 s teps of minimisation

Energy Core allow gener disall c + aGR_3 53.332 51.9 42.3 3.8 1.9 94.2GR_14 53.8467 46.2 36.5 15.4 1.9 82.7GR_2 54.84097 38.5 48.1 3.8 9.6 86.6

GR_8 71.2816 36.5 46.2 5.8 11.5 82.7GR_15 71.8524 34.6 46.2 9.6 9.6 80.8GR_16 71.8524 34.6 46.2 9.6 9.6 80.8GR_11 72.3013 30.8 55.8 5.8 7.7 86.6GR_7 74.2422 40.4 48.1 3.8 7.7 88.5

GR_10 77.5409 30.8 50 7.7 11.5 80.8GR_9 79.2774 30.8 51.9 9.6 7.7 82.7GR_12 88.6027 32.7 44.2 17.3 5.8 76.9

GR_20 95.2538 36.5 42.3 11.5 9.6 78.8

45.005 8.645 8.165 83.18SD 15.074 6.218 5.360 4.072 3.350 4.33

GR_4 61.0314 42.3 42.3 9.6 5.8 84.6GR_1 64.5039 38.5 48.1 3.8 9.6 86.6

GR_13 74.9967 28.8 50 5.8 15.4 78.8GR_17 76.3022 42.3 44.2 7.7 5.8 86.5

GR_6 90.5669 46.2 40.4 5.8 7.7 86.6GR_5 92.2646 44.2 36.5 15.4 3.8 80.7

GR_18 97.2014 34.6 46.2 9.6 9.6 80.8GR_19 107.1231 42.3 34.6 11.5 11.5 76.9

AV 76.411 38.175

Figure D-2: PROCHECK results of the SYBYL® minimised structure

2

Appendix D

______________________________________________________________________________

PROCHECK results after 8000 steps of minimisationEnergy core allow gener disall c + a

GR_14 40.6034 44.2 38.5 13.5 3.8 82.7GR_2 41.6047 36.5 48.1 5.8 9.6 84.6

___________________________________________________________________ 222

Figure D-3: PROCHECK results after 8000 steps of minimisation using SYBYL®.

GR_4 46.0056 38.5 44.2 11.5 5.8 82.7

7.7 80.77.7 84.6

GR_10 63.0637 25 53.8 9.6 11.5 78.8GR_7 63.2059 48.1 40.4 3.8 7.7 88.5GR_15 66.228 34.6 48.1 7.7 9.6 82.7

GR_5 80.4959 44.2 38.5 11.5 5.8 82.7

GR_19 94.0553 38.5 38.5 11.5 11.5 77

63.27 37.40 46.06 8.06 8.46 83.4615.60 6.99 5.51 3.78 3.08 4.47

GR_3 44.78 51.9 42.3 3.8 1.9 94.2GR_11 45.1107 38.5 50 3.8 7.7 88.5

GR_1 49.1346 34.6 53.8 1.9 9.6 88.4GR_8 58.0681 30.8 51.9 5.8 11.5 82.7GR_9 60.6977 26.9 53.8 11.5GR_17 61.6472 42.3 42.3 7.7

GR_16 66.228 34.6 48.1 7.7 9.6 82.7GR_13 67.0577 28.8 51.9 3.8 15.4 80.7GR_20 72.8701 34.6 40.4 13.5 11.5 75GR_6 75.9208 44.2 44.2 3.8 7.7 88.4

GR_12 81.4724 32.7 50 11.5 5.8 82.7GR_18 87.2117 38.5 42.3 11.5 7.7 80.8

References ____________________________________________________________________________________

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___________________________________________________________________ 223

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