Thermodynamic binding studies of bivalent oligosaccharides to galectin-1, galectin-3, and the carbohydrate recognition domain of galectin-3.

Galectins are a growing family of animal lectins with common consensus sequences that bind beta-Gal and LacNAc residues. There are at present 14 members of the galectin family; however, certain galectins possess different structures as well as biological properties. Galectin-1 is a dimer of two homologous carbohydrate recognition domains (CRDs) and possesses apoptotic and proinvasive activities. Galectin-3 consists of a C-terminal CRD and an N-terminal nonlectin domain implicated in the oligomerization of the protein and is often associated with antiapoptotic activity. Because many cellular oligosaccharide receptors are multivalent, it is important to characterize the interactions of multivalent carbohydrates with galectins-1 and -3. In the present study, binding of bovine heart galectin-1 and recombinant murine galectin-3 to a series of synthetic analogs containing two LacNAc residues separated by a varying number of methylene groups, as well as biantennary analogs possessing two LacNAc residues, were examined using isothermal titration microcalorimetry (ITC) and hemagglutination inhibition measurements. The thermodynamics of binding of the multivalent carbohydrates to the C-terminal CRD domain of galectin-3 was also investigated. ITC results showed that each bivalent analog bound by both LacNAc residues to the two galectins. However, galectin-1 shows a lack of enhanced affinity for the bivalent straight chain and branched chain analogs, whereas galectin-3 shows enhanced affinity for only lacto-N-hexaose, a naturally occurring branched chain carbohydrate. The CRD domain of galectin-3 was shown to possess similar thermodynamic binding properties as the intact molecule. The results of this study have important implications for the design of carbohydrate inhibitors of the two galectins.

Galectin-3 precipitates as a pentamer with synthetic multivalent carbohydrates and forms heterogeneous cross-linked complexes.

Galectin-3 is unique among the galectin family of animal lectins in its biological activities and structure. Most members of the galectin family including galectin-1 possess apoptotic activities, whereas galectin-3 possesses anti-apoptotic activity. Galectin-3 is also the only chimera type galectin and consists of a nonlectin N-terminal domain and a C-terminal carbohydrate-binding domain. Recent sedimentation equilibrium and velocity studies show that murine galectin-3 is a monomer in the absence and presence of LacNAc, a monovalent sugar. However, quantitative precipitation studies in the present report indicate that galectin-3 precipitates as a pentamer with a series of divalent pentasaccharides with terminal LacNAc residues. Furthermore, the kinetics of precipitation are fast, on the order of seconds. This indicates that although the majority of galectin-3 in solution is a monomer, a rapid equilibrium exists between the monomer and a small percentage of pentamer. The latter, in turn, precipitates with the divalent oligosaccharides, resulting in rapid conversion of monomer to pentamer by mass action equilibria. Mixed quantitative precipitation experiments and electron microscopy suggest that galectin-3 forms heterogenous, disorganized cross-linking complexes with the multivalent carbohydrates. This contrasts with galectin-1 and many plant lectins that form homogeneous, organized cross-linked complexes. The results are discussed in terms of the biological properties of galectin-3.