Galectin-3 alters the lateral mobility and clustering of beta 1-integrin receptors

Glycoprotein receptors are influenced by myriad intermolecular interactions at the cell surface. Specific glycan structures may interact with endogenous lectins that enforce or disrupt receptor-receptor interactions. Glycoproteins bound by multivalent lectins may form extended oligomers or lattices, altering the lateral mobility of the receptor and influencing its function through endocytosis or changes in activation. In this study, we have examined the interaction of Galectin-3 (Gal-3), a human lectin, with adhesion receptors. We measured the effect of recombinant Gal-3 added exogenously on the lateral mobility of the alpha 5 beta 1 integrin on HeLa cells. Using single-particle tracking (SPT) we detected increased lateral mobility of the integrin in the presence of Gal-3, while its truncated C-terminal domain (Gal-3C) showed only minor reductions in lateral mobility. Treatment of cells with Gal-3 increased beta 1-integrin mediated migration with no apparent changes in viability. In contrast, Gal-3C decreased both cell migration and viability. Fluorescence microscopy allowed us to confirm that exogenous Gal-3 resulted in reorganization of the integrin into larger clusters. We used a proteomics analysis to confirm that cells expressed endogenous Gal-3, and found that addition of competitive oligosaccharide ligands for the lectin altered the lateral mobility of the integrin. Together, our results are consistent with a Gal-3-integrin lattice model of binding and confirm that the lateral mobility of integrins is natively regulated, in part, by galectins

Mutational Analysis at Asn-41 in Peanut Agglutinin - A Residue Critical For The Binding Of The Tumor-Associated Thomsen-Friedenreich Antigen

Peanut agglutinin is a clinically important lectin due to its application in the screening of mature and immature thymocytes as well as in the detection of cancerous malignancies. The basis for these applications is the remarkably strong affinity of the lectin for the tumor associated Thomsen-Friedenreich antigen (T-antigen) and more so due to its ability to distinguish T-antigen from its cryptic forms. The crystal structure of the complex of peanut agglutinin with T-antigen reveals the basis of this specificity. Among the contacts involved in providing this specificity toward T-antigen is the watermediated interaction between the side chain of Asn-41 and the carbonyl oxygen of the acetamido group of the second hexopyranose ring of the sugar molecule. Sitedirected mutational changes were introduced at this residue with the objective of probing the role of this residue in T-antigen binding and possibly engineering an altered species with increased specificity for T-antigen,of the three mutants tested, i.e. N41A, N41D, and N41Q, the last one shows improved potency for recognition of T-antigen. The affinities of the mutants can be readily explained on the basis of the crystal structure of the complex and simple modeling. In particular, the change of asparagine to glutamine could lead to a direct interaction of the side chain with the sugar while at the same time retaining the water bridge. This study strengthens the theory that in lectins the nonprimary contacts generally made through water bridges are involved in imparting exquisite specificity