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Principles of Glycan Recognition

Principles of Glycan Recognition. Lecture 18, Chapter 4 April 27, 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 19-23)

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Principles of Glycan Recognition

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  1. Principles of Glycan Recognition Lecture 18, Chapter 4April 27, 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)

  3. Overview • Thermodynamic issues • Affinity and avidity • Examples of glycan-protein interactions • Techniques for identifying glycan ligands

  4. P + G PG = Protein (P) = Glycan (G) Equilibrium-Dialysis G P Set up dialysis and allow equilibrium to occur (days). Measure G in both chambers

  5. P + G P•G [P]eq Keq = [P•G]eq / [P]eq[G]eq [G]eq [P•G]eq [P]eq= [P]init - [P•G]eq [P•G]eq = [G]total in left compartment - [G]right Written this way Keq = Kassociation 1/ Ka = Kd

  6. Wash Apparent Kd Solid Phase Binding G 100% P Bound 50% [G]

  7. Scatchard Analysis If ~90% saturation is achieved, then data can be analyzed by Scatchard plot Ka B/F, Bound/Free B/F = Kan - KaB n 1 2 B, Bound Ligand B = Bound ligand/lectin F = Free ligand n = stoichiometry of binding http://biowww.chem.utoledo.edu/eqDial/Intro

  8. k1 P + G P•G k-1 Surface Plasmon Resonance • Immobilize glycan on sensor chip • Flow analyte (protein) across chip • Measure change in refractive index • Measure on and off rate constants, determine Kd values G P Kd = k-1/k1

  9. Binding Constants • Most monovalent carbohydrate bind with Ka values of µM to mM Kd = k-1/k1 = 10-3-10-6 M DG = -RTlnKa (RT at room temperature = 0.6 kcal/mol \ DG values range from ~ -4 to -8 kcal

  10. Ligand Concentration Isothermal Titration Calorimetry (ITC) • When carbohydrate binds, heat is released and temperature rises • Calculate how much heat has to be added to reference cell to achieve same temperature change, get top panel • Replot data to get binding isotherm (bottom panel) • Calculate area under curve to get change in heat/mol = enthalpy (DH)

  11. Thermodynamic Values DG = -RTlnKa = DH-TDS Notice that entropic term is positive. Why?

  12. Binding Involves Desolvation

  13. Cholera Toxin Binds to GM1 Binding occurs to the terminal sugars • H-bonding • Hydrophobic Interactions • No electrostatic interaction

  14. Affinity and Avidity • Binding of GM1 is relatively low affinity • Cholera toxin exists as a AB5 complex, each B subunit binds to a single molecule of GM1 • Low affinity interactions give rise to high avidity if clustering occurs

  15. Another Example: Mannose Binding Protein • Mannose binding protein is trimeric (C-type lectin) and each subunit binds to mannose • Arrangement allows the receptor complex to bind to microbial cell surfaces and cell wall fragments

  16. Multivalency • Most glycan binding proteins have multiple binding sites or oligomerize to achieve multivalency • Multivalency can facilitate cell-cell interactions directly or indirectly

  17. P-Selectin/PSGL-1 Interaction Selectins involved in recruitment of leukocytes in lymph nodes and inflamed tissues

  18. P-selectin/PSGL-1 • P-selectin expressed on platelets and endothelial cells • N-terminus contains lectin domain, Ca2+, one EGF repeat followed by complement regulatory repeats • Lectin domain binds weakly to sialyl Lewis X (sLeX, Kd ~ 8 mM, DG ~ -3 kcal/mol) • Preferred ligand is a glycoprotein, PSGL-1 expressed on leukocytes (Kd ~ 0.8 µM, DG ~ -8.4 kcal/mol) • PSGL-1 has many O-linked carbohydrate chains, but... • ….binding site consists of one O-linked chain with sLex and sulfated tyrosines

  19. Solid phase synthesis using sulfated tyrosine and GalNAc-Thr Enzymatic synthesis of glycan using recombinant enzyme Lappänen et al. (2000) JBC 275:39569 - - 10-20 µM - - glycan, >100 µM - sulfate, ~30 µM 0.65 µM

  20. Somers et al. (2000) Cell 103:467

  21. Lappänen et al. (2000) JBC 275:39569

  22. Protein Oligosaccharide Identification and Purification of Glycan Ligands • 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 Filter Binding Assay Maccarana & Lindahl (1993) Glycobiology3:271

  23. Identification and Purification of Glycan Ligands TLC Plate Overlay Separate oligosaccharides by TLC or paper chromatography Block plate and overlay with protein ligand 1 = Glycolipid preparation 2 = Standards containing HNK-1 3,4 = overlay with mAb to HNK-1 http://www.glycotech.com/protocols/Proto5.html

  24. Identification and Purification of Glycan Ligands Affinity Co-Electrophoresis • Pour acrylamide gel with GAG binding protein in gel • Electrophorese different amounts of radioactive GAG chains • Binding of chains to protein retards their mobility • Measure Kd and non-binding fraction San Antonio & Lander (2001)Meth Mol Biol. 171:401

  25. Identification and Purification of Glycan Ligands Affinity Chromatography • Prepare column by covalently linking protein to resin • Bind sample • Elute with ligand, NaCl, pH, chelators

  26. Identification and Purification of Glycan Ligands Frontal Affinity Chromatography A column is prepared with immobilized lectin A mixture of oligosaccharides is passed continuously through the column Palcic et al. (2003) Methods Enzymol. 362:369

  27. Determining the Specificity of Binding Carbochips Housman & Mrksich Chem. Biol. 9: 443 (2002)

  28. Summary • Protein-Glycan interactions are guided by the same principles as other macromolecular interactions • Hydrogen bonding • Hydrophobic interactions • Electrostatic interactions • Most monovalent interactions between proteins and glycans are low affinity (µM to mM) • High specificity can be achieved, but not all interactions are totally selective • Most interactions are multivalent, usually involving oligomerization of the lectin or multiple types of interactions (protein-protein as well as protein-carbohydrate) • Many binding techniques for identifying glycan ligands are available • Carbohydrate arrays are new and should become more useful as facile methods for producing compound libraries improve

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