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Essentials of Glycobiology April 14, 2002 Jeff Esko

Essentials of Glycobiology April 14, 2002 Jeff Esko. Glycosaminoglycan-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

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Essentials of Glycobiology April 14, 2002 Jeff Esko

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  1. Essentials of GlycobiologyApril 14, 2002Jeff Esko Glycosaminoglycan-Binding Proteins

  2. 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 Types of Carbohydrate-Binding Proteins

  3. HA binding proteins Methods for characterizing GAG-binding proteins Conformation and sequence considerations Examples of heparin-binding proteins Overview

  4. b 4 b 3 b 4 b 3 b 4 b 3 b 4 b 3 b 4 b 3 Hyaluronan (HA) n≥1000 GlcNAc GlcA GlcNAc GlcA GlcNAc GlcA GlcNAc GlcA GlcNAc GlcA Synthesized at plasma membrane, extruded from cell Present as capsule in some bacteria

  5. Physical Properties Interglycosidic H-bonding restricts rotations across glycosidic bonds Promotes rapid recovery after mechanical perturbations

  6. Hyaluronan (HA) Day & Sheehan (2001) COSB 1:1617

  7. Aggrecan Versican Link Protein Neurocan TSG-6 Brevican CD44 LYVE-1 = Link Module Hyaladherens

  8. Link Module consists of two a-helices and two triple-stranded anti-parallel b-sheets b5 b4 b3 a1 b2 b6 a2 b1 Day and Prestwich (2001) JBC 277:4585

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

  10. Other HA Binding Proteins Inter-a-inhibitor Chondroitin Sulfate HC-1 HC-2 Bikunin RHAMM SPACR, SPACRCAN, CDC37, CD38, Layilin and others

  11. Lo Affinity High Affinity Affinity Chromatography - 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 NaCl

  12. Affinity Chromatography on Immobilized GAG -Prepare column by covalently linking GAG to activated resin -Bind sample, elute with ligand, pH, chelators

  13. 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 Non-binding fraction San Antonio & Lander (2001)Meth Mol Biol. 171:401

  14. Protein Oligosaccharide Filter Binding Assay • 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

  15. NS 2S CO2- 6S CO2- NS 2S Conformational Considerations NS and 2S groups are on the same side COO- locations depend on whether its GlcA or IdoA GAG chains assume helical configurations, which causes charged residues to alternate across the helix

  16. 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

  17. 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- Do Consensus Sequences Exist?

  18. 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

  19. Antithrombin-Heparin KD ~ 2.5 x 10-10 M DG ~ 13.3 kcal/mol

  20. Antithrombin-Heparin Interactions Thrombin • Binding sequence for antithrombin is a pentasaccharide, a minor subfraction • Need an 18-mer to inactivate thrombin • Heparin acts as a template Antithrombin Heparin 6S 6S a4 b4 a4 a4 NS NS3S 2S NS

  21. Signaling Event Mitogenesis Proteoglycan Heparan sulfate FGF FGF

  22. FGF-Heparin Hexasaccharide • Crystal structure shows surface binding 119KRTGQYKLGSKTGPGQK135

  23. FGF-2 Binding Sequence Frequency: 4-8 sites / chain

  24. FGF/FGF Receptor Co-crystals Plotnikov et al. Cell 98:641 (1999)

  25. Potential Docking Site for Heparin Top View Top View with basic residues shaded blue Side View

  26. FGF2/FGFR1 FGF1/FGFR2 FGF FGF FGF FGF Mulloy & Linhardt (2001) COSB 11:623

  27. FGF-2 Activation Sequence If symmetric dimer structure is correct: Receptor Binding Domain FGF-2 Binding Domain FGF-2 Binding Domain Frequency: < 1 site / chain (?)

  28. Expression of Cell Type Specific Heparan Sulfates - FGF + FR1 Heparinase FGF-2 plus AP-tagged receptor FGF alone 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)

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