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Glycosaminoglycan-Binding Proteins Lecture 25, Chapter 29 May 11, 2004 Jeff Esko 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 1923) • 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 Glycosaminoglycans (GAG) b4 b3 b4 b3 b4 b3 b4 b3 b4 b3 GlcNAc GlcA Hyaluronan IdoA 4S b4 b3 4S b4 b3 4S b4 b3 4S b4 b3 4S b4 4S b3 2S GalNAc GlcA Chondroitin/Dermatan Sulfate a4 b4 GlcNAc GlcA a4 b4 a4 b4 6S a4 NS Heparin/Heparan Sulfate b4 6S IdoA a4 NS3S a4 2S NS 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 Hyaluronan (HA) b4 b3 b4 b3 b4 b3 b4 b3 b4 b3 n≥1000 GlcNAc GlcA • 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 Hyaluronan (HA) Bent, helical, relatively stiff structures Day & Sheehan (2001) COSB 1:1617 Fragments are potent signaling molecules Aggrecan and CD44: Hyaluronan Binding Proteins Hyaluronan-Binding Proteins (HABPs) Aggrecan Versican Link Protein Neurocan TSG-6 Brevican CD44 Aggrecan Family Link protein CD44 TSG-6 LYVE-1 LYVE-1 = Link Module Tissue architecture, stability Stabilizes aggrecan-HA aggregates Cell adhesion Inflammation Clearance 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 The two a-helices and two triple-stranded antiparallel b-sheets make up the Link Module b5 b4 b3 a2 Day and Prestwich (2001) JBC 277:4585 b2 b6 b1 a1 • 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 Heparin Binding Proteins GAG partner Oligosaccharide Hyaluronan Heparin/ heparan sulfate a4 6S Dermatan sulfate a4 b4 NS 6S a4 b3 a4 a4 NS3S b4 Heparin/ heparan sulfate Heparin/ heparan sulfate b3 a4 b4 NS 6S a4 NS 4S 2S 4S Antithrombin NS a4 a4 NS a3 2S 6S Aggrecan a4 NS a4 b4 2S Protein 2S a4 2S b4 6S a4 NS a3 2S FGF-2 4S 2S b4 2S Lipoprotein lipase Heparin cofactor II 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 6S NS a4 a4 2S 6S NS a4 a4 2S 6S a4 a4 NS 2S 6S NS NS CO2 6S CO2 - B 2S 2S - NS a4 = GlcA = GlcNAc A a4 2S 6S NS a4 2S 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- 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 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 OSO3O O OH O O NAc OSO3- ±OSO3- COO- O O OH OH OSO3 - O NHSO3- COO OH O O O OSO3- OH O NHSO3- Antithrombin-Heparin KD ~ 2.5 x 10-10 M DG ~ 13.3 kcal/mol Heparin-Antithrombin D A 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) Jin et al. (1997) PNAS 94:14683 D 3.6 kcal 2.1 kcal D E E 1.8 kcal F G 6.9 kcal P H 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 2.8 1.7 0.4 0 5.1 3.7 3.6 2.1 Atha et al. (1985) Biochemistry 24:6723 6.9 Protein GAG partner Aggrecan Hyaluronan Antithrombin Heparin/ heparan sulfate FGF-2 Lipoprotein lipase Oligosaccharide a4 6S a4 b4 NS 6S a4 b3 a4 a4 NS3S b4 Heparin/ heparan sulfate Heparin/ heparan sulfate b3 a4 b4 NS 6S NS a4 NS a4 NS a4 2S 2S a4 6S NS a4 2S a4 2S 6S NS a4 2S Heparin versus Heparan Sulfate The difference between heparin and heparan sulfate is quantitative not qualitative Characteristic Sulfate/hexosamine Heparan sulfate 0.8 - 1.8 Heparin 1.8-2.4 GlcN N-sulfates IdoA content Solubility in 2 M KAc at ph 5.7, 4ÞC Site of synthesis Size Binding to Antithrombin 40-60% 30-50% Yes •5% 8 70% • No Virtually all cells 10-70 kDa 0-0.3% Mast cells 10-12 kDa ~30% Heparan Sulfate Proteoglycans: Co-receptors and Signaling Molecules •Wnts •TGF-b/BMPs •HGF •HB-EGF •Hedgehog •FGF •VEGF •Angiopoietin Heparan sulfate FGF FGF Signaling Event Mitogenesis FGF-Heparin Hexasaccharide Crystal structure shows surface binding 119KRTGQYKLGSKTGPGQK135 FGF/FGF Receptor Co-crystals Plotnikov et al. Cell 98:641 (1999) FGF2/FGFR1 Heparin FGF FGF Mulloy & Linhardt (2001) COSB 11:623 • Symmetric structure • Heparin interacts with both ligands and receptors • Two heparin oligosaccharides present in crystal Potential Docking Site for Heparin Top View Top View with basic residues shaded blue Side View FGF-2 Activation Sequence If symmetric dimer structure is correct: b4 a4 NS b4 6S 6S a4 a4 a4 a4 a4 a4 a4 a4 a4 NS 2S NS 2S NS 2S NS 2S NS Receptor Binding Domain FGF-2 Binding Domain = GlcNAc 6S = GlcA = IdoA b4 a4 NS 2S FGF-2 Binding Domain Expression of “Active” Heparan Sulfates - FGF + FR1 Heparinase FGF-2 plus APtagged 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) FGF2/FGFR1 FGF FGF1/FGFR2 FGF FGF FGF Its never simple! Mulloy & Linhardt (2001) COSB 11:623 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 GAG partner Oligosaccharide Hyaluronan Heparin/ heparan sulfate a4 6S Dermatan sulfate a4 b4 NS 6S a4 b3 a4 a4 NS3S b4 Heparin/ heparan sulfate Heparin/ heparan sulfate b3 a4 b4 NS 6S a4 NS 4S 2S 4S Antithrombin NS a4 a4 NS a3 2S 6S Aggrecan a4 NS a4 b4 2S Protein 2S a4 2S b4 6S a4 NS a3 2S FGF-2 4S 2S b4 2S Lipoprotein lipase Heparin cofactor II
