Glycosaminoglycan-Binding Proteins Lecture 25, Chapter 29 May 11, 2004 Jeff Esko

1. Glycosaminoglycan-Binding Proteins Lecture 25, Chapter 29May 11, 2004Jeff Esko

2. Types of Glycan-Binding Proteins • Glycosyltransferases and modifying enzymes • Antibodies induced by carbohydrate antigens • Animal Lectins: P,C,S,R,L and I-type (Lectures 19-23) • Plant Lectins: Con A, PHA, WGA, Ricin, and many others (Lecture 24) • Glycosaminoglycan-binding proteins (Lecture 25) • Bacterial adhesins and Viral hemagglutinins (Lecture 26) Lectin - term usually restricted to proteins that share primary sequence homologies

3. GlcNAc GlcA Hyaluronan IdoA 4S 4S 4S 4S 4S 4S b4 b3 b4 b3 b4 b3 b4 b3 b4 b3 2S GalNAc GlcA Chondroitin/Dermatan Sulfate b4 b3 b4 b3 b4 b3 b4 b3 b4 b3 IdoA 6S 6S b4 a4 b4 a4 a4 a4 b4 a4 b4 a4 NS NS3S 2S NS GlcNAc GlcA Heparin/Heparan Sulfate Glycosaminoglycans (GAG)

4. GAG Binding Proteins • Hyaluronan-binding proteins have a binding motif called the Link module • Chondroitin sulfate binds to many proteins, but with low affinity, no apparent fold • Hundreds of heparin binding proteins exist and do not generally sort into families of genes related through a common fold • Dermatan sulfate binds to many of the same proteins as heparin

5. b4 b3 b 4 b 3 b 4 b 3 b 4 b 3 b 4 b 3 Hyaluronan (HA) n≥1000 • Synthesized at plasma membrane, extruded from cell • Abundant in skeletal tissues, synovial fluid, skin, elevated in expanding tissues (morphogenesis, invasion) • Interesting biophysical properties (hydration, viscous solutions, resiliency) • Present as capsule in some bacteria GlcNAc GlcA

6. Hyaluronan (HA) Bent, helical, relatively stiff structures Fragments are potent signaling molecules Day & Sheehan (2001) COSB 1:1617

7. Aggrecan and CD44:Hyaluronan Binding Proteins

8. Aggrecan Versican Link Protein Neurocan TSG-6 Brevican = Link Module CD44 LYVE-1 Hyaluronan-Binding Proteins (HABPs) Aggrecan Family Tissue architecture, stability Link protein Stabilizes aggrecan-HA aggregates CD44 Cell adhesion TSG-6 Inflammation LYVE-1 Clearance

9. SS: b = b-sheet a = a-helix • Members deduced by sequence homologies • Note position of four conserved Cys residues, plus other amino acids in consensus sequence

10. The two a-helices and two triple-stranded anti-parallel b-sheets make up the Link Module b5 b4 b3 a1 b2 b6 a2 b1 Day and Prestwich (2001) JBC 277:4585

11. Binding site is actually generated by folding of different segments of the chain, bringing key residues into proximity • Notice positively charged residues and aromatics www.glycoforum.gr.jp/science/hyaluronan/HA16/HA16E.html

12. a4 b3 a4 b3 a4 6S 6S a4 b4 a4 a4 NS NS3S 2S NS b4 a4 b4 a4 NS NS 2S 4S 4S 4S b4 a3 b4 a3 b4 2S 2S 2S 6S 6S 6S a4 a4 a4 a4 a4 NS 2S NS 2S NS 2S Heparin Binding Proteins

13. 6S 6S 6S 6S 6S a4 a4 a4 a4 a4 a4 a4 a4 a4 NS 2S NS 2S NS 2S NS 2S NS 2S Conformational Considerations = GlcA = GlcNAc GAG chains assume helical configurations, which causes charged residues to alternate across the helix NS 2S NS and 2S groups are on the same side COO- locations depend on whether its GlcA or IdoA CO2- 6S CO2- NS 2S

14. 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

15. 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- • Spacing would place basic residues on the same face of an a-helix (3.4 residues/turn) or a b-strand (alternating faces) • It turns out that most binding proteins do not fit this pattern and binding site is composed of positive residues contributed by different segments of the protein

16. O Antithrombin • Antithrombin, a serpin (serine protease inhibitor) • Inactivates proteases involved in coagulation (Factors IIa and Xa) • Blocks coagulation • Antithrombin deficiency results in thrombosis (clot formation) • Heparin binds to antithrombin, alters its conformation, and enhances rate of inactivation of Xa and IIa by a factor of 104 • Only need a heparin pentasaccharide to activate - - ±OSO3- - O S O O S O C O O 3 3 O O O O O - C O O - O H O S O O H O O O H O H 3 O O O - - - NAc O H N H S O O S O N H S O 3 3 3

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

18. Heparin-Antithrombin • Binding site for heparin is in a cleft formed by two helices • Binding is oriented, with pentasaccharide in cleft and flanking chain to the non-reducing side extending up and over the protein • An 18-mer is actually needed to inactivate thrombin, so it acts like a template to approximate antithrombin-thrombin • Interaction with thrombin does not require specific oligosaccharide sequence (low affinity) D A

19. Jin et al. (1997) PNAS 94:14683 D 3.6 kcal D E 2.1 kcal E 1.8 kcal F G 6.9 kcal H P

20. 2.8 1.7 0.4 5.1 0 3.7 Contribution of Individual Groups to Affinity • Blue numbers refer to kcal binding deduced by altering the glycan groups • Red numbers refer to kcal binding deduced by mutating amino acids 1.8 3.6 2.1 6.9 Atha et al. (1985) Biochemistry 24:6723

21. a4 b3 a4 b3 a4 6S 6S a4 b4 a4 a4 NS NS3S 2S NS b4 a4 b4 a4 NS NS 2S 6S 6S 6S a4 a4 a4 a4 a4 NS 2S NS 2S NS 2S

22. Heparin versus Heparan Sulfate The difference between heparin and heparan sulfate is quantitative not qualitative

23. Wnts • TGF-/BMPs • HGF • HB-EGF • Hedgehog • FGF • VEGF • Angiopoietin Signaling Event Mitogenesis Heparan Sulfate Proteoglycans: Co-receptors and Signaling Molecules Heparan sulfate FGF FGF

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

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

26. FGF2/FGFR1 Heparin • Symmetric structure • Heparin interacts with both ligands and receptors • Two heparin oligosaccharides present in crystal FGF FGF Mulloy & Linhardt (2001) COSB 11:623

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

28. a4 a4 b4 a4 NS NS 2S b4 a4 b4 a4 NS NS 2S = GlcNAc = GlcA = IdoA 6S 6S 6S a4 a4 a4 a4 a4 NS 2S NS 2S NS 2S FGF-2 Activation Sequence If symmetric dimer structure is correct: a4 Receptor Binding Domain FGF-2 Binding Domain FGF-2 Binding Domain

29. Expression of “Active” 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)

30. FGF2/FGFR1 FGF1/FGFR2 FGF FGF FGF FGF Its never simple! Mulloy & Linhardt (2001) COSB 11:623

31. 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

32. a4 b3 a4 b3 a4 6S 6S a4 b4 a4 a4 NS NS3S 2S NS b4 a4 b4 a4 NS NS 2S 4S 4S 4S b4 a3 b4 a3 b4 2S 2S 2S 6S 6S 6S a4 a4 a4 a4 a4 NS 2S NS 2S NS 2S