Eur. J. Biochem. 188.73-82 (1990) 0 FEBS 1990 Chemical characterization of the regularly arranged surface layer glycoprotein of Clostridium thermosaccharolyticum D120-70 Eleonora ALTMAN ', Jean-Robert BRISSON', Paul MESSNER2 and Uwe 9. SLEYTR' ' Division of Biological Sciences, National Research Council, Ottawa, Canada Zentrum fur Ultrastrukturforschung und Ludwig Boltzmann Institut fur Ultrastrukturforschung, Universitat fur Bodenkultur, Wien, Austria (Received August 8/0ctober 2, 1989) - EJB 89 0995 Clostridium therrnosaccharolyticum D120-70 possesses as its outermost cell envelope' layer a square-arranged array of glycoprotein molecules. SDS/polyacrylamide gel electrophoresis of the purified surface layer showed a broadened band in the molecular mass range of about 115 kDa which, upon periodic acid/Schiff staining, gave a positive reaction. After proteolytic degradation of this material, two glycopeptide fractions were obtained. One- and two-dimensional nuclear magnetic resonance studies, together with methylation analysis and periodate oxidation, were used to determine the structures of the polysaccharide portions of these glycopeptides. The combined chemical and spectroscopic evidence suggests the following structures : --+3)-P-~-Manp-(l+4)-a-~-Rhap-(l-+ 3)-a-~-Glcp-(l-+4)-a-~-Rhap-(l+ 6 2 1 t 1 t P-D-G~c~ a-D-Galp 4 1 (a-D-Galp),., . and --+ 4)-fl-~-GlcpNAc-(l+ 3)-P-~-ManpNAc-(l-+ t Regularly arranged surface layers have been found in nearly every taxonomic group of walled eubacteria and rep- resent an almost universal feature of archaebacterial cell enve- lopes [l, 21. The chemical composition of the surface layer glycoprotein of the archaebacterium Halobacterium halobium has been described by Wieland and coworkers in great detail In the course of the structural characterization of the sur- face layers of the taxonomically closely related thermophilic eubacteria Clostridium thermosaccharolyticum D120-70 and Clostridium thermohydrosulfuricum Ll l l-69 [4], chemical analysis provided first evidence for the presence of glyco- protein subunits in the surface layer arrays of these strains [5]. Since then the structures of the carbohydrate portions of surface-layer glycoproteins of C. thermohydrosulfuricum Llll-69 [6] and Bacillus stearothermophilus NRS 2004/3a [7, 81 have been elucidated and the nature of the linkage region of one of these glycopeptides has been described [9]. We now report on the glycans of the surface-layer glycoprotein of C. thermosaccharolyticum D120-70. ~31. Correspondence to E. Altman, Division of Biological Sciences, National Research Council of Canada, Ottawa, Canada K1A OR6 Abbreviations. COSY, correlated spectroscopy; relay COSY, relayed coherence transfer spectroscopy; 2D, two-dimensional ; CHORTLE, carbon-hydrogen correlations from one-dimensional polarization transfer spectra by least-square analysis; NOESY, nu- clear Overhauser enhancement spectroscopy. MATERIALS AND METHODS Growth of bacteria and isolation o f the glycopeptide C. thermosaccharolyticum D120-70 was grown as described and the surface-layer glycoprotein was isolated from clean cell walls by extraction with 5 M guanidine hydrochloride [5]. Upon thorough dialysis of this extract three times against 2 1 distilled water, self-assembly products consisting of pure surface-layer glycoprotein subunits were formed [5]. The glycopeptides were obtained after pronase digestion (1 5% pronase) as described previously [8]. Low-molecular-mass degradation products were removed by gel filtration on a Bio- Gel P-4 column (1.5 x 95 cm) equilibrated in 0.1 mM sodium chloride. Fractions (2.5 ml) giving positive tests for hexose [lo] were combined, concentrated and applied to a Bio-Gel P- 30 column (1 x 113 cm). The fractions (1 ml) were assayed for hexose and those giving positive reaction were combined and lyophilized. Analytical methods Hexoses and rhamnose were determined colorimetrically as described [ll]. Monosaccharides and amino sugars were analyzed by GLC of their alditol acetates [12] after hydrolysis of the sample with 4 M trifluoroacetic acid at 125°C for 1 h. The protein content was estimated by the method of Lowry [I31 with ovalbumin as a standard. Amino acids were quantified after hydrolysis of the samples in 4 M hydrochloric
Transcript
Eur. J . Biochem. 188.73-82 (1990) 0 FEBS 1990
Chemical characterization of the regularly arranged surface layer glycoprotein of Clostridium thermosaccharolyticum D120-70 Eleonora ALTMAN ', Jean-Robert BRISSON', Paul MESSNER2 and Uwe 9 . SLEYTR' ' Division of Biological Sciences, National Research Council, Ottawa, Canada
Zentrum fur Ultrastrukturforschung und Ludwig Boltzmann Institut fur Ultrastrukturforschung, Universitat fur Bodenkultur, Wien, Austria
(Received August 8/0ctober 2, 1989) - EJB 89 0995
Clostridium therrnosaccharolyticum D120-70 possesses as its outermost cell envelope' layer a square-arranged array of glycoprotein molecules. SDS/polyacrylamide gel electrophoresis of the purified surface layer showed a broadened band in the molecular mass range of about 115 kDa which, upon periodic acid/Schiff staining, gave a positive reaction. After proteolytic degradation of this material, two glycopeptide fractions were obtained. One- and two-dimensional nuclear magnetic resonance studies, together with methylation analysis and periodate oxidation, were used to determine the structures of the polysaccharide portions of these glycopeptides. The combined chemical and spectroscopic evidence suggests the following structures :
Regularly arranged surface layers have been found in nearly every taxonomic group of walled eubacteria and rep- resent an almost universal feature of archaebacterial cell enve- lopes [l , 21. The chemical composition of the surface layer glycoprotein of the archaebacterium Halobacterium halobium has been described by Wieland and coworkers in great detail
In the course of the structural characterization of the sur- face layers of the taxonomically closely related thermophilic eubacteria Clostridium thermosaccharolyticum D120-70 and Clostridium thermohydrosulfuricum Ll l l-69 [4], chemical analysis provided first evidence for the presence of glyco- protein subunits in the surface layer arrays of these strains [5]. Since then the structures of the carbohydrate portions of surface-layer glycoproteins of C. thermohydrosulfuricum Llll-69 [6] and Bacillus stearothermophilus NRS 2004/3a [7, 81 have been elucidated and the nature of the linkage region of one of these glycopeptides has been described [9]. We now report on the glycans of the surface-layer glycoprotein of C. thermosaccharolyticum D120-70.
~31.
Correspondence to E. Altman, Division of Biological Sciences, National Research Council of Canada, Ottawa, Canada K1A OR6
Abbreviations. COSY, correlated spectroscopy; relay COSY, relayed coherence transfer spectroscopy; 2D, two-dimensional ; CHORTLE, carbon-hydrogen correlations from one-dimensional polarization transfer spectra by least-square analysis; NOESY, nu- clear Overhauser enhancement spectroscopy.
MATERIALS AND METHODS
Growth of bacteria and isolation of the glycopeptide
C. thermosaccharolyticum D120-70 was grown as described and the surface-layer glycoprotein was isolated from clean cell walls by extraction with 5 M guanidine hydrochloride [5]. Upon thorough dialysis of this extract three times against 2 1 distilled water, self-assembly products consisting of pure surface-layer glycoprotein subunits were formed [5]. The glycopeptides were obtained after pronase digestion (1 5% pronase) as described previously [8]. Low-molecular-mass degradation products were removed by gel filtration on a Bio- Gel P-4 column (1.5 x 95 cm) equilibrated in 0.1 mM sodium chloride. Fractions (2.5 ml) giving positive tests for hexose [lo] were combined, concentrated and applied to a Bio-Gel P- 30 column (1 x 113 cm). The fractions (1 ml) were assayed for hexose and those giving positive reaction were combined and lyophilized.
Analytical methods Hexoses and rhamnose were determined colorimetrically
as described [ll]. Monosaccharides and amino sugars were analyzed by GLC of their alditol acetates [12] after hydrolysis of the sample with 4 M trifluoroacetic acid at 125°C for 1 h. The protein content was estimated by the method of Lowry [I31 with ovalbumin as a standard. Amino acids were quantified after hydrolysis of the samples in 4 M hydrochloric
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acid at 100 C for 16 h on a Biotronic 5000 amino acid analyzer. Amino sugars were separated and quantified by high-performance anion-exchange chromatography with pulsed amperometric detection on a system consisting of a Dionex Bio-LC gradient pump, CarboPac PA-I column (4.6 x 250 mm) and a model PAD2 detector, using 15 mM sodium hydroxide with 150 mM sodium hydroxide wash be- tween the injections. The absolute configuration of glycoses was established by capillary GLC of their trimethylsilylated (-)-2-butyl glycosides according to the method of Gerwig ~141.
M e th y la t ion analysis
Samples were methylated according to the Hakomori pro- cedure as previously described [15]. Methylated polysac- charides were treated with anhydrous hydrogen fluoride at 22°C for 2 h and then hydrolyzed with 2 M trifluoroacetic acid at 100°C for 2 h as described by Jansson et al. [16]. Methylated oligosaccharides were hydrolyzed with 4 M trifluoroacetic acid at 125 "C for 1 h. Methylation analyses were made according to previously reported conditions [17].
Periodate oxidation
A solution of the polysaccharide (18.6 mg) in distilled water (1.5 ml) was treated with 0.1 M sodium metaperiodate (1.5 ml) in the dark for 6 days at 4°C. Excess periodate was reduced by the addition of ethylene glycol (100 pl) and the oxidized polymer was reduced with sodium borohydride (40 mg). After 16 h at 22"C, the cooled solution was neutral- ized with dilute acetic acid, dialyzed until salt-free, and lyophilized.
Smith-type hydrolysis of the periodate-oxidized and re- duced polymer was effected with 0.5 M trifluoroacetic acid at 22 "C for 48 h and the degradation products were fractionated on a column (1.6 x 87 cm) of Bio-Gel P-2 (200 - 400 mesh).
Nuclear magnetic resonance spectroscopy
Proton-decoupled I3C-NMR 125-MHz spectra were re- corded at 300K and 330K for a 25-kHz spectral width using a n/2 pulse and a 32 K data set on a Bruker AM500 spectrometer. Chemical shifts are expressed relative to external 1,4-dioxane ( l%, 67.4 ppm). The 'H-NMR spectra at 500 MHz were recorded at 300 K and 330 K using a spectral width of 2.5-kHz, a 7c/2 pulse and a 16K data set for a digital resolution of 0.3 Hz/point. Proton chemical shifts are expressed relative to internal acetone (0.1%, 6 2.225 ppm). Coupling constants are reported in hertz.
Proton homonuclear correlated two-dimensional (2D) NMR experiments (COSY) [18] and two- and three-step relay COSY [19] were performed at 300 K and 330 K using the standard software provided by Bruker (DISNMR) ; all exper- iments were done in the magnitude mode. Heteronuclear 13C- 'H shift correlation experiments were done on a Bruker AM500 spectrometer using CHORTLE (carbon-hydrogen correlations from one-dimensional polarization-transfer spectra by least-squares analysis) technique [20]. Nuclear Overhauser enhancements (NOE) were performed in the dif- ference mode with sequential irradiation of each line in a multiplet [21, 221. Phase-sensitive two-dimensional NOESY experiment was performed according to Bodenhausen et al. [23]. Data was processed using FTNMR program (Hare Re- search, Inc.). Spin simulations were performed using the LAOCNS program in conjunction with FTNMR.
Other methods
SDSjPAGE was carried out according to Laemmli [24] on 10% slab gels [25]. Freeze-etching and thin-sectioning electron microscopy was performed as previously described [4].
RESULTS
Surface layer structure
Freeze-etched preparations of intact cells of C. thermo- saccharolyticum showed the presence of a square surface-layer lattice (Fig. 1 a), as already demonstrated by Sleytr and Thorne [5]. Thin sections confirmed the location of the surface layer as the outermost cell envelope component (Fig. 1 b). Upon isolation of the surface-layer glycoprotein from clean cell wall preparations by extraction with guanidine hydrochloride and thorough dialysis, the surface-layer subunits recrystallized into self-assembly products which frequently had the shape of open-ended single or double-layer cylinders of flat sheets (Fig. Ic). SDSjPAGE and Coomassie blue staining of the SDS- soluble whole cell extract (Fig. Id , lane a) and the surface- layer self-assembly products (Fig. Id , lane b) showed a broadened band in the molecular mass range of 115 kDa which gave positive periodic acid/Schiff-staining reaction (Fig. 1 d, lane c). The lattice constant of 11 nm of the self-assembly products was identical to that found previously on intact cells [5]. This material was used for the subsequent glycopeptide preparations.
Chemical characterization of the glycopeptide fraction
Upon pronase digestion and removal of the degradation products by gel-filtration on a Bio-Gel P-4 column, the ma- terial eluting at the column void volume was eventually sepa- rated on a Bio-Gel P-30 column and showed a single broad peak with an apparent molecular mass of about 40 kDa. Amino acid analysis of this material revealed a small peptide portion (Asx, Thr, Ser, Glu, Gly, Ala, in total 10 pg/mg) and the amino sugars of the carbohydrate moiety (GlcNAc, GalNAc, and ManNAc).
Carbohydrate analysis of the hydrolysis products by high- performance anion-exchange chromatography, followed by GLC analysis of the derived alditol acetates and the trimethylsilylated derivatives of their (-)-2-butyl glycosides, revealed the presence of L-rhamnose, D-mannOSe, D-glucose, D-galaCtOSe, 2-amino-2-deoxy-D-glucose, 2-amino-2-deoxy-~- galactose and 2-amino-2-deoxy-~-mannose in the ratios 1.5 : 1 .O: 2.2: 1 .O : 1.5 : 1 .O: 0.7, with ribose and arabinose found in negligible amount (z lo%, relative to internal inositol).
The 'H-NMR (500 MHz, 330 K) spectrum of the glycan revealed a complex structure with at least ten proton signals of different intensities in the anomeric region, four signals corresponding to the N-acetyl groups at 2.019, 2.022, 2.050 and 2.069 ppm, and signals for two rhamnose terminal methyl groups at 6 1.326 and 1.374ppm (Fig. 2a). The methylated and hydrolyzed glycan afforded a product, that, after reduction (NaBD,) and acetylation, gave GLC-MS (program B) results (Table 1) consistent with NMR evidence, indicating a complex structure with at least three branch points. To obtain further structural data, a periodate oxidation followed by reduction (NaBH4), mild acid hydrolysis of the acetal linkages in the resulting polyalcohol, and subsequent gel filtration chromatography on Bio-Gel P-2, was employed. This allowed isolation of two structurally significant fragments : a modified polysaccharide & and an oligosaccharide B.
75
Fig. 1. Clzuracterizution of the surface layer of Clostridium thermosaccharolyticum 0120-70. Electron micrographs of (a) a freeze-etched preparation of the intact cell, (b) a thin sectioned cell showing the typical cell envelope profile, and (c) negatively stained surface-layer self- assembly products. Bars = 100 nm. SDSjPAGE (d) of the SDS-soluble cell extract (lane a) and the surface layer self-assembly products (lanes b, c); Coomassie blue staining (lanes a, b) and periodic acid/Schiff staining (lane c). S, surface-layer; pg, peptidoglycan; cm, cytoplasmic membrane
N A c m n m n
bc m n 6b 6c a d e f Y Z
, I I I
5.2 4.8 2.0 1 .6 P P m P P m
Fig. 2. polysaccharide A
H-NMR spectrum of the anomeric and NAc regions, recorded at 330 K . (a) Native glycan (a mixture of I, E, and 12); (b) modified
The modified polysaccharide A (Kav 0.02) on hydrolysis (125 MHz, 300 K) G/ppm = 175.2 (NH@CH3), 175.1 afforded 2-amino-2-deoxy-~-g~ucose and 2-amino-2-deoxy-~- ( N H g C H 3 ) , 100.4 (anomeric C), 98.7 (anomeric C), 23.2 mannose (1 : 1). Partial 'H-NMR (500 MHz, 330 K) G/ppm = (NHCOS3),22.9 (NHCOB3) . The methylation analysis 4.828 (d, lH, J1,2 = 1.0 Hz), 4.594 (d, lH, J1,z = 8.2 Hz), by GLC/MS methods (program B) gave 2-deoxy-2-(N- 2.050 (s, 3H), 2.022 (s, 3H) (Fig. 2b). Partial 13C-NMR methylacetamido)-3,6-di-O-methyl-~-glucose and 2-deoxy-2-
76
Table 1. GLCIMS of the products of methylation analysis of the native glycan and its Smith degradation products tGM is the retention time relative to 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-~-glucitol
I ,3,4,5-Tetra-O-acetyl-2-deoxy-6-O-methyl- 2-(N-methyIacetamido)-~-[l -'H]mannitol
0.90 1 .oo 1.07 1.17 1.37 1.44 2.21
3.42
3.83
3.87
4.59
0.74 1 .oo 0.87 0.67 1.03
0.82
0.09
0.04
0.08
0.10
-
3 9 3 7 597- I I 1
P I I 1 I , I I
1 8 'I+ 2 1 9;
I !
I ! I t # I , I I , , I , I
I (
! I ' '-321 ' L l l J
Fig. 3. GLC/MS of the methylated trisaccharide glycoside with some primary and secondary fragments
(N-methylacetamido)-4,6-di-O-methyl-~-mannose (0.9 : 1 .O), demonstrating that the polymer obtained after periodate oxi- dation has the following structure:
-4)-p-~-GlcpNAc-(l --f 3)-P-~-ManpNAc-(l+
The anomeric configuration of the component monosaccha- rides was confirmed by NMR analysis (see later).
The second product of Smith degradation, oligosaccharide - B (Kav 0.47), on hydrolysis afforded I-deoxyerythritol, L- rhamnose, D-mannose and D-glucose. The 'H-NMR (500 MHz, 300 K) spectrum of this trisaccharide glycoside contained three anomeric proton resonances at G/ppm =
and 4.819 (d, IH, J1,2 = 1.0 Hz); one signal for CH3 of 6- deoxyglycose at 6 = 1.356 ppm (d, 3H, J5,6 = 6.4 Hz) and one signal for CH3 I-deoxyerythritol at 1.198 ppm (d, 3H, J5,6 = 6.5 Hz). The 13C-NMR (125 MHz, 300 K) showed signals at 100.5 (anomeric C), 100.4 (anomeric C), 96.9 (anomeric C), 18.1 (CH3 of rhamnose) and 17.6 ppm (CH3 of 1-deoxyerythritol). GLC-MS analysis of permethylated trisaccharide glycoside by electron impact (EI) confirmed the presence of the terminal nonreducing hexosyl residue and a C4 fragment (1-deoxytetritol) (Fig. 3). Primary ions of the A series [26], characteristic of the terminal nonreducing hexosyl
residue, were m/z 219 (aAJ and 187 (aA2). Those of the 1- deoxytetritol moiety were m/z 117 (ald), 177 (c-ald J1), 321 (c-ald), 495 (b-c-ald). GLC/MS analysis in the chemical ion- ization mode gave fragments which were consistent with the trisaccharide glycoside structure: base peak at m/z 135 (ald . H20), a peak at m/z 219 (a-A'), 393 (a-b-Al), 597 (a-b-c-A') and parent peak (M + 1) at m/z 731.
GLC/MS (program B) analysis of the reduced (NaBD4) and acetylated hydrolysis products obtained from methylated trisaccharide glycoside identified 1,4,5-tri-O-acetyl-2,3-di- O-methyl[l-2H]rhamnitol, 1,5-di-O-acety1-2,3,4,6-tetra-O- methyl[1-2H]glucitol, and 1,3,5-tri-O-acety1-2,4,6-tri-O- methyl[l-2H]mannitol (Table 1). GLC/MS analysis in the EI mode confirmed that the hexosyl residue was linked to the 1- deoxytetritol (m/z 321, c-ald). The following structure was assigned since the glucopyranosyl residue formed the nonreducing end of this trisaccharide glycoside.
CHzOH I
I
I CH3
t~-~-Gl~p-(1+4)-cc-~-Rhap-(l-+ 3)-p-~-Manp-(l --f 3) - OCH
HOCH
77
3f 5f 4f 2f 6f 6'f
A J MM f ) 2e 6e 6'e 3-4e 5e
m e ) rz 8'd
4c c)
3,5,2b 4b A b)
5a
4.2 4.0 3.8 3.6 3.4 A I a )
I I
PPm
Fig. 4. Observed ' H - N M R spectrum of the native glycan at 330 K (g) and the contributions to the simulated ' H - N M R spectrum for the polysaccharide (a- f). (a) a-D-Galp-(l+ residue; (b) +4)-a-~-Rhap-(l-+ residue; (c) +4)-u-~-Rhap-(l ---t residue; (d) +3)-a-~-GIcp-(l-+
2 t
residue; (e) +3)-P-~-Manp-(l+ residue; ( f ) for the j -~-Glcp- ( l+ residue 6 t
a ) 3m 6m 6'm
I I 1 I
4.2 4.0 3.8 3.6 3.4 PPm
Fig. 5 . Sum of the simulated ' H - N M R spectra for the polysaccharides I , and EI of the native glycan (f) and contributions to the simulated spectrum (a, b, d, e). (a) +3)-P-~-ManpNAc-(l + residue; (b) -+4)-P-~-GlcpNAc-(l --t residue; (d) a-D-Galp-(l-+ residue; (e) +~)-P-D- ManpNAc-(I+ residue. Observed 'H-NMR spectra at 330 K are shown in (c) for the modified polysaccharide - A and in (g) for the native
4 t
glycan
The anomeric configuration of each monosaccharide and their sequences were confirmed by NMR analysis.
Methylation data of the native glycan indicated the pres- ence of branching rhamnopyranosyl and mannopyranosyl residues (Table 1). GLCIMS analysis of methylated (Table 1) confirmed that branching occurred through position 0-2 of the rhamnopyranosyl residue and position 0-6 of the mannopyranosyl residue. Detection of the 2-amino-2-deoxy- D-galactose in both the hydrolysis and the methylation analy- sis products of the native glycan (Table 1) was consistent with existence of the N-glycosidic linkage region in the glycoprotein
In summary, this combined evidence suggested that the native glycan was a complex mixture containing three different polysaccharides (I, 11, IIJ : a polysaccharide 5 susceptible to Smith degradation to give trisaccharide glycoside B, a modi-
[91.
fied polysaccharide A (IJJ and a modified polysaccharide & substituted by either glucopyranosyl or galactopyranosyl resi- dues at position 0-4 of the 2-acetamido-2-deoxy-P-~- mannopyranosyl residue (III).
These structural conclusions were confirmed by sub- sequent one- and two-dimensional NMR analysis of the native glycan and the two products of Smith degradation, A and B (Figs 4 and 5).
NMR studies
Assignments of the proton resonances in the native glycan (a mixture of I, and IIJ were made from COSY [18] and two- and three-step relay COSY [19] experiments. A system of residue notation based on the sequence of anomericprotons in the 1D 'H-NMR spectrum was adopted for each of the three
78
Table 2. Proton chemical shifts and coupling constants for polysaccharide I Shifts were measured in ppm (k 0.005 ppm) at 330 K in D 2 0 with acetone as internal reference (2.225 ppm). Jl,*, J 2 , 3 , J3.4, J4,5, J 5 , 6 , J6 ,6
and J 5 . 6 . , values in Hz (T 0.5 Hz) are enclosed in parentheses
Shift ( J ) for Unit
H-I H-2 H-3 H-4 H-5 H-6 H-6’
~
3.852 (10.0)
(3.5) 4.087
5.131 (3.8)
(1.6) 5.127
4.031 (1 .O)
(10.0) 3.627
4.225 (4.2)
(6.4) 4.108
3.747 (12.3) 1.376
3.747 (8.6)
a x-D-Galp-(l+
b +4)-cc-~-Rhap-(l+ 2 T
c +4)-~-~-Rhap-( l+ 5.087 (1.6) 5.072 (4.1) 4.891 (1 .O)
4.050 (3.5)
(9.8)
(3.2)
3.674
4.295
3.945 (9.5) 3.762 (9.3) 3.737 (9.8)
3.697 (9.5) 3.490 (9.3) 3.737 (10.2)
1.326
3.834 (12.3) 4.212 (11.5)
3.767 (5.0) 3.929 (6.7)
3.329 (9.2)
3.511 (9.4)
3.402 (9.4)
3.456 (2.3)
3.920 (12.4)
3.736 (6.1)
Table 3. Proton chemical shifts and coupling constants for polysaccharide E I Shifts were measured in ppm (t- 0.005 ppm) at 330 K in DzO with acetone as internal reference (2.225 ppm). J1.2r J 2 , 3 , J3,4, J4,5r J5,6.r J6,6. and J5,6. , values in Hz (t- 0.5 Hz) are enclosed in parentheses
Table 4. Proton chemical shifts and coupling constantsfor the modijkdpolysaccharide A andpolysaccharide Shifts were measured in ppm (k 0.005 ppm) at 330 K in D20 with acetone as internal reference (2.225 ppm). J l , z , J2,3r J3,4r J4,5, J 5 , 6 . J6 6’
and J5,6. values in Hz (& 0.5 Hz) are enclosed in parentheses
Unit Shift ( J ) for
H-I H-2 H-3 H-4 H-5 H-6 H-6’ N Ac
m +3)-P-~-ManpNAc-(l+ 4.828 4.651 4.029 3.607 3.456 3.908 3.802 2.050
components. Thus, in order of descending chemical shifts pyranose residues in polysaccharide I were labelled 5-f, in polysaccharide II-n, and in polysaccharide 111 5-5 (Fig. 2a). Connectivities were traced via cross-peaks to each anomeric and 6-deoxy resonance, which provided convenient
spectral windows for analysis. The majority of proton reso- nances were assigned in this manner and then refined by simulating the ‘H-NMR spectrum (Tables 2-4). The se- quence and the position of the glycosidic linkages in each component was established by phase-sensitive NOESY exper-
79
M
-
d)
c )
2d 4b i d
- zc 3d
n - b ) 3e 2e 2b
_ i l l c . - 2b 2a
a) _ - - I I , I I
5.2 4 . 8 4 . 4 4.0 3 . 6 PPm
Fig. 6. Cross-section through NOESY spectrum of the native glycan (a mixture of I , sections for: (a) H-la, (b) H-lb, (c) H-lc, (d) H-Id, (e) H-le, (9 H-If. Observed 'H-NMR spectrum of the native glycan is shown in (g)
and EI) for the polysaccharide 1 resonances. Cross-
I l Y 3-42 1 2 Y 3Y b )
a ) V
I I I I
5.2 4.8 4 . 4 4.0 3 . 6 P P m
Fig. I. Cross-section through NOESY spectrum of the native glycan (a mixture of I , sections for: (a) H-lx, (b) H-ly, (c) H-lz. Observed 'H-NMR spectrum of the native glycan is shown in (d)
and KI) for the polysaccharide EI resonances. Cross-
iment [23] on the native glycan, which also provided infor- mation on the anomeric configuration of each glycose unit. The two-dimensional NOE cross-sections for the poly- saccharide I resonances (Fig. 6) correlated the anomeric pro- ton H-la with H-2a and H-2b which required that unit a was linked through an a1,2 bond to residue b. Similarly, H-lb correlated with H-2b, H-2e and H-3e, indicating that residue b was a-linked to residue e. However, the position of the linkage could not be established unambiguously and was de- termined by 2D NMR analysis performed on the trisaccharide glycoside (see later). Correlation of anomeric proton H-lc with H-2c and H-3d indicated that unit c was linked through an a1,3 bond to residue d. Similar arguments were used to establish that unit d was LY 1,4-linked to unit b, unit e was p 1,4- linked to unit c, and unit f was p1,6-linked to unit e. Thus, for the polysaccharide I, NOE data was consistent with the sequence
-b-e-c-d- . I 1 a f
Similarly, from the two-dimensional NOESY experiment on the native glycan mixture, cross-sections through the
polysaccharide 111 resonances (Fig. 7) correlated the anomeric proton H-lx with H-2x and H-4y indicating that the unit x was a 1 ,Clinked to unit y. In addition, anomeric proton H-lz could be correlated with both H-2y and H-3y, the largest NOE being to H-2y, leaving the linkage position ambiguous, and in the case of H-1 y, almost coincident H-3z/H-4z signals prevent- ed assignment of the linkage position. In both cases the pos- ition of the linkage was deduced from methylation analysis of the native glycan. The anomeric configuration and the sequence of the glycosidic linkages in the modified poly- saccharide A obtained after Smith degradation of the native glycan was confirmed by NOE difference experiment, which required complete assignment of all proton resonances via a COSY experiment. Strengly coupled H-2n, H-3n and H-4n proton resonances prevented their unambiguous assign- ment and their chemical shifts could be determined only by simulating the 'H-NMR spectrum of until a good agree- ment between the observed and calculated spectra could be achieved. Although the NOES produced upon saturation of each anomeric proton residue provided the anomeric con- figuration and sequence information, they failed to determine the position of the linkage due to the presence of more than
80
Table 5 . Proton chemical shifts and coupling constants for the trisaccharide glycoside Shifts were measured in ppm (k 0.005 ppm) at 330 K in D 2 0 with acetone as internal reference (2.225 ppm). Jl,z, J2,3r J3,4 , J4 ,5 , J5 ,6 , J6,6 and J5 ,6s , values in Hz are enclosed in parentheses (& 0.5 Hz). For unit c* J1 ,z , Jz ,3 , J3 ,4 , J4,4. and J3,4. values are given
Fig. 8. NOE difference spectra for the trisaccharideglycoside &. (a) On saturation of the H-ld*; (b) on saturation of the H-lb*; (c) saturation of the H-le*; (d) off-resonance control spectrum, measured at 300 K
one interresidue NOE upon saturation of each anomeric res- onance. The linkage position was determined from the methylation data (Table 1). Equal proton chemical shifts for the modified polysaccharide A and the polysaccharide 11 dem- onstrated their structural identity. Similarly, the NOE patterns for the modified polysaccharide A and the backbone of the polysaccharide were identical, indicating their structural relatedness. The combined chemical and NMR evidence per- mits the structure of polysaccharides and 111 to be unam- biguously established:
(n) (m) -4)-B-~-GlcpNAc-(l+3)-~-~-ManpNAc-( I -+ (IJ)
(z) (Y) -4)-P-~-GlcpNAc-(l+3)-P-~-ManpNAc-(l+ @I)
4 t 1
(4 a-D-Galp .
The remaining problem was to confirm the sequence of polysaccharide I. This was achieved by two-dimensional NMR analysis of trisaccharide glycoside @ obtained after Smith degradation of a polysaccharide I. The residues in were, for convenience, labelled according to their order in polysaccharide I and differentiated by an asterisk (*). The proton resonances of were readily assigned from the COSY experiment and then refined (Table 5) by simulating the 'H- NMR spectrum until a good agreement between the observed and calculated spectrum was achieved. The proton chemical shifts were confirmed by I3C-'H shift correlation experiment CHORTLE [20], which also served to establish the 13C-NMR assignments (Table 6). Examination of I3C chemical shifts and comparison of these with literature values [27] indicated which pyranose rings are involved in glycosidic linkages. Thus those I3C atoms that experienced significant deshielding were the putative linkage sites: C-4b*, C-3e* and C-3c*. The mono- saccharide sequence was determined by one-dimensional NOE difference experiment [21, 221 (Fig. 8) which established the linkage sequence d*-b*-e*-c*. Based on the chemical evidence and one- and two-dimensional NMR analysis the structure of the polysaccharide I was established as a polymer of branched hexasaccharide repeating units each with the structure:
(d) [b)
eubacterial surface layers [6 - 81 reveals a similar heterogeneity to that already observed in the protein portions of these mol- ecules [25,28]. The functional significance of the carbohydrate portions of the proteins has yet to be determined; however, formation of the glycan chains consisting of regular repeating units might resemble that of the 0-antigens of lipopoly- saccharides of Gram-negative bacteria and may follow a simi- lar biosynthetic pathway. Labelling experiments of the glycan chain of Clostridium thermohydrosuljiuricum L111-69 with polycationic ferritin have shown that the glycan chains pro- trude out of the surface layer [30]. Such spatial arrangement modulates the hydrophobicity of the bacterial cell surface and this feature would therefore be responsible for the interaction of the bacterium with its environment.
In contrast to the very limited data on functional aspects of prokaryotic glycoproteins, a considerable amount of knowledge has accumulated on eukaryotic glycoproteins (for review see [31- 341). For example, the glycan moieties can play important roles in (a) protection of the polypeptide against proteolytic degradation, (b) maintenance of protein confor- mation and stability, and (c) surface or intercellular recog- nition and cell adhesion phenomena. All these functions could also be relevant for prokaryotic organisms.
(e) (C) _ I
-3)-a-~-Gkp-( 1 -+4)-a-~-Rhap-(l+3)-fl-~-Manp-(l+4)-a-~-Rhap-( + 2 t 1
a-D-Galp
(a)
Based on the integration of the anomeric resonances in the 'H-NMR spectrum of the native glycan the relative ratio of 1:II:III was 2: 1 : 1, respectively.
DISCUSSION
The results of this investigation confirmed the early as- sumption of Sleytr and Thorne [5] that covalently linked carbohydrate moieties are present in surface layers of thermophilic Clostridia. Combined chemical and NMR evi- dence revealed two complex glycan chain structures for the glycopeptides derived from pronase digestion of the purified surface-layer preparation of C . thermosaccharolyticum D120- 70.
Structure elucidation of the carbohydrate portion of a glycopeptide was based upon a complete assignment of 'H- NMR chemical shifts for each coupled spin system of the individual monomer residues, using COSY and relay COSY techniques, followed by NOESY and NOE difference exper- iments to determine linkage position and sequence of sugar residues in the native glycan and its Smith degradation frag- ments. The results of this study clearly show limitations of the NMR approach for determination of linkage positions in cases where a system of strongly coupled or overlapping pro- ton resonances is present. Conventional methylation analysis was applied in these instances, illustrating the effective combi- nation of classical methods with high-resolution NMR tech- niques as the most practical approach to structural analysis.
Structurally diverse glycans were found on several Bacillus and Desuljotomaculum strains [28, 291, thereby demostrating
6
1 fl-~-Glcp
t
(0 The obvious diversity of both the constituent protein sub-
units and the glycosylation of surface layers, even among closely related strains, coincides with the generally observed, nonconservative, character of bacterial surface molecules (e.g. exopolysaccharides, lipopolysaccharides) [35, 361.
We thank Drs F. M. Unger and D. R. Bundle for valuable dis- cussions and Dr H. Konig for the amino acid analysis. We also thank Dr Dennis Hare for providing us with a copy of FTNMR and Mr F. P. Cooper for the GLC/MS analyses. This work was supported in part by grants from Chembiomed Ltd (Edmonton, Alberta, Canada) and the Osterreichisches Bundesministerium fur Wissenschajt und Forschung.
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