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

Abstract

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.

Pre-B cell receptor binding to galectin-1 modifies galectin-1/carbohydrate affinity to modulate specific galectin-1/glycan lattice interactions

“…1c) and the a3 sialyl-N-Acetyllactosamine (a3 SiaLacNAc) belonging to group (ii) (Fig. 1c), for which a relative decrease and increase in GAL1 binding, respectively, are observed in the presence of the l5-UR domain (binding of these two glycans to GAL1 has been demonstrated previously 31,32 ). In our experimental conditions, the dissociation constant (K D ) of GAL1/LNnT interaction obtained is 51±6 mM, whereas when GAL1 is bound to the l5-UR domain, the K D increases to 130±10 mM, that is, a 2.5-fold decrease in binding affinity of LNnT for GAL1 in the presence of l5-UR (Supplementary Table 1).…”

Dimeric Galectin-1 Binds with High Affinity to α2,3-Sialylated and Non-sialylated Terminal N-Acetyllactosamine Units on Surface-bound Extended Glycans

“…that human and bovine dGal-1 bind with relatively poor affinity to extended PLs and that binding affinity is independent of the length of the glycan (44,45). These results were based on isothermal titration microcalorimetry and frontal affinity chromatography, in which glycans are free in solution.…”

“…dGal-1 also binds to glycopeptides containing PL sequences (40,41), glycolipids (42), and neoglycoproteins (43). In contrast to these studies, recent data have suggested that dGal-1 may not recognize PL structures with higher affinity compared with its recognition of short non-extended structures (44,45). Interestingly, dGal-1 has been suggested to bind primarily to nonreducing terminal LN residues rather than to mid-chain LN residues within a PL chain (42,43,46).…”

“…For example, dGal-1 from different sources has been shown to bind ␣2,3-sialylated (but not ␣2,6-sialylated) glycans (30,40,42,44,45,47). Most of these studies were based on indirect sugar inhibition assays with simple non-extended ligands such as sialylated LN and lactose.…”

“…More direct binding measurements have been carried out recently with human and bovine dGal-1. Isothermal titration microcalorimetry was used to show that bovine dGal-1 has the same affinity for LN and ␣2,3-sialyl-LN (44). Bovine dGal-1 has also been shown to bind to immobilized neoglycolipids containing ␣2,3-sialylated and non-sialylated lacto-N-tetraose (Gal␤1-3GlcNAc␤1-3Gal␤1-4Glc) by TLC overlay and microwell binding assays (42).…”