Multivalent Protein–Carbohydrate Interactions: Isothermal Titration Microcalorimetry Studies

Abstract

A wide variety of cellular and pathological processes are mediated by carbohydrate–protein interactions. These interactions generally require high-affinity binding. However, carbohydrate-binding proteins (lectins) typically show low affinities for simple mono- and oligosaccharides. Higher affinity interactions occur when lectins that are oligomeric proteins, bind to the carbohydrate chains of cell surface glycolipids and glycoproteins, which possess multiple binding epitopes. As a consequence, considerable attention is given toward understanding the underlying mechanisms responsible for the enhanced affinity of multivalent carbohydrates for lectins. Insight into the thermodynamic basis for the enhanced affinities of multivalent (clustered) glycosides binding to the lectins concanavalin A (ConA) and Dioclea grandiflora (DGL) is obtained by isothermal titration microcalorimetry (ITC). ITC measurements provide direct determinations of binding enthalpy, ΔH, the association constant, Ka, and the number of binding sites of the protein, n. From measurements of Ka, the free energy of binding, ΔG, can be calculated. The entropy of binding, ΔS, is obtained from ΔH and ΔG. Thus, ITC measurements can determine the complete thermodynamics of binding of a carbohydrate to a lectin. ConA and DGL are mannose/glucose-specific lectins with similar binding specificities. They possess relatively high affinities for the monovalent trisaccharide 3, 6-di-O-(α-D-mannopyranosyl)-α-D-mannopyranoside as compared with mannose. Synthetic multivalent clustered glycosides bearing multiple terminal mannose or mannotriose residues show increased affinities for ConA and DGL up to nearly 100-fold as assessed by enzyme-linked lectin assay and hemagglutination inhibition. To gain insight into the thermodynamic basis for the enhanced affinities of these multivalent saccharides, binding of synthetic dimeric analogs of α-D-mannopyranoside and di-, tri-, and tetrameric analogs of 3,6-di-O-(α-D-mannopyranosyl)-α-D-mannopyranoside to ConA and DGL was studied by ITC. The results show that ITC yields important thermodynamic insight into the mechanism(s) of enhanced affinities of these multivalent carbohydrates for these two lectins. The results also show that the negative cooperativity that occurs during binding is associated with the multivalent carbohydrates and not the proteins, and that entropy effects play a dominant role in the enhanced affinities of these ligands.