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 Lectin definition usually restricted to proteins that share primary sequence homologies Broaden definition to include any carbohydrate binding protein since the interactions are similar

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

Hyaluronan (HA) Synthesized at plasma membrane, extruded from cell 4 3 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

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

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

Hyaladherens Aggrecan Versican Neurocan Link Protein Brevican TSG-6 CD44 LYVE-1 = Link Module Link Module shared in common with with hyaladherens Aggrecan membersw involved in tissue integrity Aggrecan in cartilage Versican in conncetive tissues (Fibroblasts) Neurocan and Brevican in brain matrix TSG-6 Tumor necrosis factor induced gene 6(inlammatory protein of unknown function CD44 Cell attachment, turnover of HA, migration of leukocytes, multiple isoforms based on splicing and glycosylation LYVE-1Lymph vessel endothelium, HA receptor for degradatipon

Aggrecan fomrs link-protein stablizied complexes with HA, load bearing function

Members deduced by squence homologies Postion of four conserv ed cys residues, plus other sequences SS reers to structural motifs, a-helices and b-sheets

Link Module consists of two a-helices and two triple-stranded anti-parallel b-sheets #-D structure of TSG-1 determined by NMR Cosensus fold Only identified in vertebrates. Notice also that HA is relatively recent invention since Drosophila does not make it. Similar orientation toi C-type lectins, but no Ca2+ Notive alswo that Day and Prestwich (2001) JBC 277:4585

Residues determined by mutagenesis Binding site is actually gernated by folding of different segments of the chain, bringing key residues into proximity. Notice postively charged residues and aromatics www.glycoforum.gr.jp/science/hyaluronan/HA16/HA16E.html

Other HA Binding Proteins Inter-a-inhibitor Chondroitin Sulfate HC-1 HC-2 Bikunin RHAMM No consensus motif exists, although it was hypthesized that BX7B is common is found in several of these proteins. Also found in thousands of other proteins. Layilin, a recently characterized transmembrane protein with homology to C-type lectins, is a membrane-binding site for talin in peripheral ruffles of spreading cells. Similar to CD44 in that regard SPACR, SPACRCAN, CDC37, CD38, Layilin and others

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

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 Advantages Disadvantages Maccarana & Lindahl (1993) Glycobiology 3:271

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

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- Consider a protein -helix, 3.4 residues per turn would place basic residues on the same side of the helix Segments could fold as b-strands, which places the side chains on alternating faces of the polypeptide The consensus is predictive, not diagnostic. As many proteins don’t have this motif as do

FMDV Depression that defines binding site for heparin is made up of segments from all three major capsid proteins Foot and Mouth disease virus Fry et al. (1999) Embo J 18:543

Antithrombin-Heparin Show crustal structure Binding occurs by a two step mechanism, intial weak interaction followed by a conformational change, then tight binding. KD ~ 2.5 x 10-10 M DG ~ 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 Heparin Only about 30% of the chains will bind to antithrombin Gross composition of high affinity and low affinity chains are nearly identical Only a 5-mer for factor Xa, which has no binding site for heparin 6S 6S a4 b4 a4 a4 NS NS3S 2S NS

Large family of heparin-binding growth factors (24+ members) Proteoglycan Heparan sulfate FGF FGF Signaling Event Mitogenesis Large family of heparin-binding growth factors (24+ members) Multiple tyrosine kinase receptors exist (4 genes, multiple spliced forms) Heparin acts as a coreceptor, effectively lowering the concentration of ligand needed for a response Binding to matrix heparan sulfate retains and stabilizes 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 Therefore GAG chain acts as a template or scaffold on which ligand and receptor anneal Catalytic

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

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

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)