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

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Presentation on theme: "Glycosaminoglycan-Binding Proteins Lecture 25, Chapter 29 May 11, 2004 Jeff Esko."— Presentation transcript:

1 Glycosaminoglycan-Binding Proteins Lecture 25, Chapter 29 May 11, 2004 Jeff 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 GlcNAcGlcA   Hyaluronan GalNAcGlcA   4S 2S IdoA Chondroitin/Dermatan Sulfate     6S NSNS3S2SNS  GlcNAcGlcA IdoA 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 Hyaluronan (HA) GlcNAcGlcA 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 

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

7 Aggrecan and CD44: Hyaluronan Binding Proteins

8 Aggrecan Versican Neurocan Link Protein Brevican TSG-6 CD44LYVE-1 = Link Module Hyaluronan-Binding Proteins (HABPs) Aggrecan FamilyTissue architecture, stability Link proteinStabilizes aggrecan-HA aggregates CD44Cell adhesion TSG-6Inflammation LYVE-1Clearance

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

10 The two  -helices and two triple-stranded anti- parallel  -sheets make up the Link Module 55 44 11 33 66 22 11 22 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

12   4S 2S  NS 2S  6S NS 2S   6S NSNS3S2SNS   Heparin Binding Proteins

13 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 NS2S CO 2 -  6S NS 6S NS 6S NS2S  6S NS 6S NS2S  = GlcNAc = GlcA

14 Sugar Conformation  Most sugars prefer the 4 C 1 conformation  IdoA which is formed by epimerization of GlcA has the 1 C 4 or 1 S 0 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  -helix (3.4 residues/turn) or a  -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 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 10 4 Only need a heparin pentasaccharide to activate O O O OH NHSO 3 - OSO 3 - O COO - OH OSO 3 - O O O OSO 3 - NHSO 3 - ±OSO 3 - O COO - OH OH O OH NAc OSO 3 - O O

17 Antithrombin-Heparin K D ~ 2.5 x M  G ~ 13.3 kcal/mol

18 Heparin-Antithrombin A D 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)

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

20 Contribution of Individual Groups to Affinity Atha et al. (1985) Biochemistry 24:6723 Blue numbers refer to kcal binding deduced by altering the glycan groups Red numbers refer to kcal binding deduced by mutating amino acids

21  NS 2S  6S NS 2S   6S NSNS3S2SNS  

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

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

24 FGF-Heparin Hexasaccharide Crystal structure shows surface binding 119 KRTGQYKLGSKTGPGQK 135

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

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

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

28 FGF-2 Activation Sequence FGF-2 Binding Domain FGF-2 Binding Domain Receptor Binding Domain If symmetric dimer structure is correct:  NS 2S  6S NS 2S  NS 2S  = GlcA= IdoA= GlcNAc

29 Expression of “Active” Heparan Sulfates FGF alone FGF-2 plus AP- tagged receptor - FGF + FR1Heparinase 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/FGFR1FGF1/FGFR2 FGF Mulloy & Linhardt (2001) COSB 11:623 Its never simple!

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   4S 2S  NS 2S  6S NS 2S   6S NSNS3S2SNS  

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