Interaction profile of galectin-5 with free saccharides and mammalian glycoproteins: Probing its fine specificity and the effect of naturally clustered ligand presentation

Cell-surface glycans are functional docking sites for tissue lectins such as the members of the galectin family. This interaction triggers a wide variety of responses; hence, there is a keen interest in defining its structural features. Toward this aim, we have used enzyme-linked lectinosorbent (ELLSA) and inhibition assays with the prototype rat galectin-5 and panels of free saccharides and glycoconjugates. Among 45 natural glycans tested for lectin binding, galectin-5 reacted best with glycoproteins (gps) presenting a high density of Galβ1-3/4GlcNAc (I/II) and multiantennary N -glycans with II termini. Their reactivities, on a nanogram basis, were up to 4.3 × 10², 3.2 × 10², 2.5 × 10² and 1.7 × 10⁴ times higher than monomeric Galβ1-3/4GlcNAc (I/II), triantennary-II (Tri-II), and Gal, respectively. Galectin-5 also bound well to several blood group type B (Galα1-3Gal)- and A (GalNAcα1-3Gal)- containing gps. It reacted weakly or not at all with tumor-associated Tn (GalNAcα1-Ser/Thr) and sialylated gps. Among the mono-, di-, and oligosaccharides and mammalian glycoconjugates tested, blood group B-active II (Galα1-3Gal β1-4GlcNAc), B-active II β1-3 L (Galα1-3Galβ1-4GlcNAc β1-3Galβ1-4Glc), and Tri- II were the best. It is concluded that (1) Galβ1-3/4GlcNAc and other Galβ1-related oligosaccharides with α1-3 extensions are essential for binding, their polyvalent form in cellular glycoconjugates being a key recognition force for galectin-5; (2) the combining site of galectin-5 appears to be of a shallow-groove type sufficiently large to accommodate a substituted β-galactoside, especially with α-anomeric extension at the non-reducing end (e.g., human blood group B -active II and B -active II β1-3 L); (3) the preference within β-anomeric positioning is Galβ1-4 ≥ Galβ1-3 > Galβ1-6; and (4) hydrophobic interactions in the vicinity of the core galactose unit can enhance binding. These results are important for the systematic comparison of ligand selection in this family of adhesion/ growth-regulatory effectors with potential for medical applications.