Essentials of Glycobiology

April 14, 2002

Jeff Esko

Glycosaminoglycan-Binding Proteins

Types of Carbohydrate-Binding Proteins

• Glycosyltransferases and modifying enzymes

• Plant Lectins: Con A, PHA, WGA, Ricin, and many others

• Animal Lectins: P,C.S, and I-type, chaperone proteins

• Glycosaminoglycan-binding proteins

• Antibodies induced by carbohydrate antigens

• Bacterial adhesins

• Viral hemagglutinins

Overview

HA binding proteins

Methods for characterizing GAG-binding proteins

Conformation and sequence considerations

Examples of heparin-binding proteins

Hyaluronan (HA)

GlcNAc GlcA GlcNAc GlcA GlcNAc GlcA GlcNAc GlcA GlcNAc GlcA

n≥1000

Synthesized at plasma membrane, extruded from cell

Present as capsule in some bacteria

Physical Properties

Interglycosidic H-bonding restricts rotations across glycosidic bondsPromotes rapid recovery after mechanical perturbations

Hyaluronan (HA)

Day & Sheehan (2001) COSB 1:1617

Aggrecan

Versican

NeurocanLink Protein

BrevicanTSG-6

CD44 LYVE-1 = Link Module

Hyaladherens

Link Module consists of two -helices and two triple-stranded anti-parallel -sheets

54

1

3

6

2 1

2

Day and Prestwich (2001) JBC 277:4585

www.glycoforum.gr.jp/science/hyaluronan/HA16/HA16E.html

Other HA Binding Proteins

SPACR, SPACRCAN, CDC37, CD38, Layilin and others

Inter--inhibitor

HC-1

ChondroitinSulfate

BikuninHC-2

RHAMM

Affinity Chromatography

Lo Affinity

High Affinity

NaC

l

- High affinity interactions have Kd values of 10-7-10-9

and require about 1 M NaCl to elute

- Low affinity interactions have Kd values of 10-4 to 10-6

and elute at 0.15 - 0.5 M NaCl

Affinity Chromatography on Immobilized GAG

-Prepare column by covalently linking GAG to activated resin

-Bind sample, elute with ligand, pH, chelators

Affinity Co-Electrophoresis

• Pour acrylamide gel with GAG binding protein in gel

• Electrophorese radioactive GAG

• Binding of chains to protein retards their mobility

• Measure Kd and non-binding fraction

San Antonio & Lander (2001)Meth Mol Biol. 171:401

Non-binding fraction

Filter Binding Assay

Protein

Oligosaccharide

Mix oligosaccharides with protein in solution

Collect by filtration on nitrocellulose membranes

Only oligosaccharides bound to protein stick to filter

Solution equilibrium conditions are achieved

Vary salt, divalent cations, pH, competitors

Maccarana & Lindahl (1993) Glycobiology 3:271

Conformational Considerations

GAG chains assume helical configurations, which causes charged residues to alternate across the helix

NS and 2S groups are on the same side

COO- locations depend on whether its GlcA or IdoA

NS

6S

2S

NS 2S

CO2-

CO2-

Sugar Conformation

Most sugars prefer the 4C1 conformation

IdoA which is formed by epimerization of GlcA has the 1C4 or 1S0 conformation

The greater conformational flexibility means that the sulfate and carboxylates can shift position more readily

Greater binding possibilities and induced fit

Do Consensus Sequences Exist?

Generally, GAG binding proteins contain clustered Lysine and Arginine residues

In 1989, Cardin and Weintraub proposed a consensus sequence for heparin binding proteins, B = basic residue

-XBBXBX- -XBBBXXBX-

FMDV

Depression that defines binding site for heparin is made up of segments from all three major capsid proteins

Fry et al. (1999) Embo J 18:543

Antithrombin-Heparin

KD ~ 2.5 x 10-10 M G ~ 13.3 kcal/mol

Antithrombin-Heparin Interactions

Binding sequence for antithrombin is a pentasaccharide, a minor subfraction

Need an 18-mer to inactivate thrombin

Heparin acts as a template

ThrombinAntithrombin

Heparin

6S

NS NS3S NS2S

6S

Signaling EventMitogenesis

FGF

Heparan sulfate

Proteoglycan

FGF

FGF-Heparin Hexasaccharide

Crystal structure shows surface binding

119KRTGQYKLGSKTGPGQK135

FGF-2 Binding Sequence

Frequency: 4-8 sites / chain

FGF/FGF Receptor Co-crystals

Plotnikov et al. Cell 98:641 (1999)

Potential Docking Site for Heparin

Top View

Top View with basic residues shaded blue

Side View

FGF2/FGFR1 FGF1/FGFR2

FGFFGF

FGF FGF

Mulloy & Linhardt (2001) COSB 11:623

FGF-2 Activation Sequence

Frequency: < 1 site / chain (?)

FGF-2 BindingDomain

FGF-2 BindingDomain

Receptor BindingDomain

If symmetric dimer structure is correct:

Expression of Cell Type Specific Heparan Sulfates

FGF alone

FGF-2 plus AP-tagged receptor

- FGF + FR1 Heparinase

Locate all HS by antibody staining

K= keratinocytes, BM = basement membrane, V = blood vessel, FR1-AP = alkaline phosphatase fusion to FGF receptor-1, 3G10 = monoclonal antibody to heparinase treated HS

Chang et al. FASEB J. 14:137 (2000)