No abstract

Binding of a series of synthetic multivalent carbohydrate analogs to the Man/Glc-specific lectins concanavalin A and Dioclea grandiflora lectin was investigated by isothermal titration microcalorimetry. Dimeric analogs possessing terminal ␣-D-mannopyranoside residues, and di-, tri-, and tetrameric analogs possessing terminal 3,6-di-O-(␣-D-mannopyranosyl)-␣-D-mannopyranoside residues, which is the core trimannoside of asparaginelinked carbohydrates, were selected in order to compare the effects of low and high affinity analogs, respectively. Experimental conditions were found that prevented precipitation of the carbohydrate-lectin cross-linked complexes during the isothermal titration microcalorimetry experiments. The results show that the value of n, the number of binding sites on each monomer of the lectins, is inversely proportional to the number of binding epitopes (valency) of each carbohydrate. Hence, n values close to 1.0, 0.50, and 0.25 were observed for the binding of mono-, di-, and tetravalent sugars, respectively, to the two lectins. Importantly, differences in the functional valency of a triantennary analog for concanavalin A and D. grandiflora lectin are observed. The enthalpy of binding, ⌬H, is observed to be directly proportional to the number of binding epitopes in the higher affinity analogs. For example, ⌬H of a tetravalent trimannoside analog is nearly four times greater than that of the corresponding monovalent analog. Increases in K a values of the multivalent carbohydrates relative to monovalent analogs, known as the "multivalency effect," are shown to be due to more positive entropy (T⌬S) contributions to binding of the former sugars. A general thermodynamic model for distinguishing binding of multivalent ligands to a single receptor with multiple, equal subsites versus binding to separate receptor molecules is given.Carbohydrate-protein interactions are involved in a wide variety of biological functions including cellular growth, recognition, adhesion, cancer metastasis, bacterial and viral infections, and inflammation (1, 2). The specificity of these interactions has been an active area of research due, in part, to efforts at designing therapeutic analogs of carbohydrates (3, 4). However, attempts to design high affinity analogs for specific carbohydrate-binding proteins (lectins) have been difficult due to the intrinsic low affinity of carbohydrates in many cases (5, 6). For example, the affinity constants (K a ) for the binding of simple mono-and oligosaccharides to most lectins are between 10 3 and 10 6 M Ϫ1 (7,8). This range of K a values is too low for effective drug design. However, many naturally occurring carbohydrates and glycoconjugates including glycoproteins and glycolipids are multivalent (2) which results in their increased avidity for lectins (9). As a consequence, there has been considerable interest in designing multivalent or "clustered" carbohydrate analogs for high affinity binding to target lectin receptors (10, 11). Thus, it is important to understand the thermodyna...

Our previous isothermal titration microcalorimetry (ITC) studies of the binding of synthetic multivalent carbohydrates to the Man/Glc-specific lectins concanavalin A (ConA) and Dioclea grandiflora lectin (DGL) showed negative binding cooperativity that was due to the carbohydrate ligands and not the proteins [Dam, T. K., et al. (2002) Biochemistry 41, 1351-1358]. The negative cooperativity was associated with the decreasing functional valence of the carbohydrates upon progressive binding of their epitopes. The present study also shows negative cooperativity in the ITC binding data of asialofetuin (ASF), a glycoprotein that possesses nine LacNAc epitopes, to galectin-1, -2, -3, -4, -5, and -7, and truncated, monomer versions of galectin-3 and -5, which are members of a family of animal lectins. Although the observed K(a) values for binding of ASF to the galectins and two truncated forms are only 50-80-fold greater than that of LacNAc, analysis of the data in terms of the relationship between the observed macroscopic free energy of binding and the decreasing microscopic free energies of binding of the epitopes shows that the first LacNAc epitope of ASF binds with approximately 6000-fold higher affinity than the last epitope. Thus, the microscopic binding constants of the galectins for the first epitope(s) of ASF are in the nanomolar range, with a gradient of decreasing binding constants of the remaining epitopes. The results indicate that the above galectins bind with fractional, high affinities to multivalent glycoproteins such as ASF, independent of the quaternary structures of the galectins. These findings have important implications for the binding of galectins to multivalent carbohydrate receptors.

The innate immune response of multicellular organisms is initiated by the binding of soluble and membrane-bound host molecules including lectins to the surface of pathogenic organisms. Until recently, it was believed that the epitopes recognized by host molecules were uniquely associated with the pathogenic organisms. Hence, the term pattern recognition receptors (PRRs) was used to describe their binding specificities. However, with an expanding number of lectin classes including C-type lectins, siglecs, and galectins recognized as PRRs, it is apparent that many of the glycan epitopes recognized on foreign pathogens are present in the host and involved in cellular functions. Hence, the molecular basis for pattern recognition by lectins of carbohydrate epitopes on pathogens is in question. A number of studies indicate that the density and number of glycan epitopes in multivalent carbohydrates and glycoprotein receptors determine the affinity of lectins and their effector functions. This paper reviews lectins that are involved in innate immunity, mechanisms of enhanced affinity and cross-linking of lectins with density-dependent glycan epitopes, density-dependent recognition of glycan receptors by lectins in host systems and lectin-glycan interactions in foreign pathogens. Evidence indicates that lectin pattern recognition in innate immunity is part of a general mechanism of density-dependent glycan recognition. This leads to a new definition of lectin receptor in biological systems, which considers the density and number of glycan epitopes on the surface of cells and not just the affinity of single epitopes.