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Principles of Glycan Recognition Lecture 18, Chapter 4 April 27, 2004 Jeff Esko Slide 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) Slide 3 Overview Thermodynamic issues Affinity and avidity Examples of glycan-protein interactions Techniques for identifying glycan ligands Slide 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) Slide 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 Slide 6 Solid Phase Binding G P [G] Bound Wash Apparent K d 50% 100% Slide 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 Slide 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 Slide 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 M to mM K d = k -1 /k 1 = 10 -3 -10 -6 M Slide 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 Slide 11 Thermodynamic Values G = -RTlnK a = H-T S Notice that entropic term is positive. Why? Slide 12 Binding Involves Desolvation Slide 13 Cholera Toxin Binds to GM 1 Binding occurs to the terminal sugars H-bonding Hydrophobic Interactions No electrostatic interaction Slide 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 Slide 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 Slide 16 Multivalency Most glycan binding proteins have multiple binding sites or oligomerize to achieve multivalency Multivalency can facilitate cell-cell interactions directly or indirectly Slide 17 P-Selectin/PSGL-1 Interaction Selectins involved in recruitment of leukocytes in lymph nodes and inflamed tissues Slide 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 M, 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 Slide 19 Solid phase synthesis using sulfated tyrosine and GalNAc-Thr Enzymatic synthesis of glycan using recombinant enzyme Lappnen et al. (2000) JBC 275:39569 - 10-20 M - 0.65 M - - sulfate, ~30 M - glycan, >100 M Slide 20 Somers et al. (2000) Cell 103:467 Slide 21 Slide 22 Lappnen et al. (2000) JBC 275:39569 Slide 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 Slide 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 Slide 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 Slide 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 Slide 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 Slide 28 Determining the Specificity of Binding Housman & Mrksich Chem. Biol. 9: 443 (2002) Carbochips Slide 29 Slide 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 (M 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