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)
Top Related