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

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

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

5. P + G PG K eq = [PG] eq / [P] eq [G] eq 1/ K a = K d [PG] eq = [G] total in left compartment - [G] right [P] eq = [P] init - [PG] eq [PG] eq [G] eq [P] eq Written this way K eq = K association

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

7. Scatchard Analysis If ~90% saturation is achieved, then data can be analyzed by Scatchard plot B/F = K a n - K a B http://biowww.chem.utoledo.edu/eqDial/Intro B = Bound ligand/lectin F = Free ligand n = stoichiometry of binding KaKa B, Bound Ligand 12 B/F, Bound/Free n

8. 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 K d values P + G PG k1k1 k -1 K d = k -1 /k 1 P G

9. Binding Constants  G = -RTlnK a (RT at room temperature = 0.6 kcal/mol  G values range from ~ -4 to -8 kcal  Most monovalent carbohydrate bind with K a values of µM to mM K d = k -1 /k 1 = 10 -3 -10 -6 M

10. 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 (  H) Ligand Concentration

11. Thermodynamic Values  G = -RTlnK a =  H-T  S Notice that entropic term is positive. Why?

12. Binding Involves Desolvation

13. Cholera Toxin Binds to GM 1 Binding occurs to the terminal sugars  H-bonding  Hydrophobic Interactions  No electrostatic interaction

14. Affinity and Avidity Binding of GM 1 is relatively low affinity Cholera toxin exists as a AB 5 complex, each B subunit binds to a single molecule of GM 1 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, Ca 2+, one EGF repeat followed by complement regulatory repeats Lectin domain binds weakly to sialyl Lewis X (sLe X, K d ~ 8 mM,  G ~ -3 kcal/mol) Preferred ligand is a glycoprotein, PSGL-1 expressed on leukocytes (K d ~ 0.8 µM,  G ~ -8.4 kcal/mol) PSGL-1 has many O-linked carbohydrate chains, but... ….binding site consists of one O-linked chain with sLe x 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 - 0.65 µM - - sulfate, ~30 µM - glycan, >100 µM

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

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

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

24. 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 Identification and Purification of Glycan Ligands TLC Plate Overlay

25. 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 K d and non- binding fraction San Antonio & Lander (2001)Meth Mol Biol. 171:401 Identification and Purification of Glycan Ligands Affinity Co-Electrophoresis

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

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

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

30. 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