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

2. 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

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

5. Hyaluronan (HA) Synthesized at plasma membrane, extruded from cell4 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

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

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

8. 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

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

10. 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

11. 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

13. 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

14. 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

15. Affinity Chromatography
- High affinity interactions have Kd values of and require about 1 M NaCl to elute
- Low affinity interactions have Kd values of 10-4 to 10-6 and elute at M NaCl
Lo Affinity
High Affinity
NaCl

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

17. 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

18. 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

20. 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

21. 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

22. 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

23. 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

24. 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 M DG ~ 13.3 kcal/mol

25. 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

26. 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

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

28. FGF-2 Binding Sequence
Frequency: sites / chain

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

30. 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

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

32. 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 (?)

33. 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)