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

Abstract

Galectin-1 is a member of the galectin family of glycan-binding proteins and occurs as an ϳ29.5-kDa noncovalent homodimer (dGal-1) that is widely expressed in many tissues. Here, we report that human recombinant dGal-1 bound preferentially and with high affinity (apparent K d ϳ 2-4 M) to immobilized extended glycans containing terminal N-acetyllactosamine (LN; Gal␤1-4GlcNAc) sequences on poly-N-acetyllactosamine (PL; (-3Gal␤1-4GlcNAc␤1-) n ) sequences, complex-type biantennary N-glycans, or novel chitin-derived glycans modified to contain terminal LN. Although terminal Gal residues are important for dGal-1 recognition, dGal-1 bound similarly to ␣3-sialylated and ␣2-fucosylated terminal LN, but not to ␣6-sialylated and ␣3-fucosylated terminal LN. The binding specificity of human recombinant dGal-1 was similar to that observed with purified bovine heart-derived dGal-1. Unexpectedly, dGal-1 bound free ligands in solution with relatively low affinity and displayed no preference for extended glycans, indicating that dGal-1 preferentially recognizes extended glycans only when they are surface-bound, such as found on cell surfaces. Human dGal-1 also bound to both native and desialylated human promyelocytic HL-60 cells with similar affinity as observed for immobilized long chain PL. Binding to these cells was reduced upon treatment with endo-␤-galactosidase, which cleaves PL sequences, indicating that cell-surface PLs are ligands. To test the role of dimerization in dGal-1 binding, we examined the binding of a mutated form of dGal-1 that weakly dimerizes (monomeric Gal-1 (mGal-1)) and a covalently dimerized (chemically cross-linked) form of mGal-1 (cd-mGal-1). dGal-1 and cd-mGal-1 had similar affinities that were both ϳ3.5-fold higher for immobilized PL than observed for mGal-1, suggesting that dGal-1 acts as a dimer to cross-link terminal LN units on immobilized PL. These results indicate that dGal-1 functions as a dimer to recognize LN units on extended PLs on cell surfaces.

Glycomics of bone marrow-derived mesenchymal stem cells can be used to evaluate their cellular differentiation stage

“…In general, poly-LacNAcs are thought to act as scaffolds presenting glycan epitopes to lectins in a multivalent fashion. For example, the galectins, a family of β-galactoside binding lectins involved in a wide range of adhesion and signaling phenomena [41], have higher affinity for poly-LacNAcs than for single LacNAc units [42][43][44]. Galectins are hypothesized to modulate their biological functions according to fine specificities for target glycans [44].…”

Galectins: Structures, Binding Properties and Function in Cell Adhesion

“…In contrast galectin-1 recognises single LacNAc units presented at the non-reducing terminus of glycans not showing preference for extended poly-LacNAc glycans (Leppänen et al, 2005). Most authors agree that galectin-1 is not able to bind internal galactose moieties in poly-LacNAc-glycans (GlcNAc-3-Gal-4-GlcNAc) (Leppänen et al, 2005;Stowell et al, 2004;Stowell et al, 2008a) but depending on the assay set-up some publications report affinity to this sugar unit (Di Virgilio et al, 1999;Zhou & Cummings, 1993). These different results prove the importance of evaluation of the test set-up and critical examination of the measured binding data.…”

“…Galectin-3 tolerates due to its enlarged binding pocket extensions at the galactose 3`-OH-group for example repetitive LacNAc (type II) -structures (poly-LacNAc), showing even higher affinities for repetitive LacNAc structures compared to single LacNAc units (Hirabayashi et al, 2002;Rapoport et al, 2008;Salomonsson et al, 2010). In contrast galectin-1 recognises single LacNAc units presented at the non-reducing terminus of glycans not showing preference for extended poly-LacNAc glycans (Leppänen et al, 2005). Most authors agree that galectin-1 is not able to bind internal galactose moieties in poly-LacNAc-glycans (GlcNAc-3-Gal-4-GlcNAc) (Leppänen et al, 2005;Stowell et al, 2004;Stowell et al, 2008a) but depending on the assay set-up some publications report affinity to this sugar unit (Di Virgilio et al, 1999;Zhou & Cummings, 1993).…”

“…Galectin-3 and galectin-8 C-CRD show high affinity for blood-group antigens (Hirabayashi et al, 2002;Yamamoto et al, 2008) Rapoport et al 2002. Symbols according to the consortium of functional glycomics (Brewer, 2004;Carlsson et al, 2007;Dell, 2002;Hirabayashi et al, 2002;Ideo et al, 2003;Ideo et al, 2011;Leppänen et al, 2005;Patnaik et al, 2006;Rabinovich & Toscano, 2009;Rapoport et al, 2008;Salomonsson et al, 2010;Stowell et al, 2004;Stowell et al, 2008a;Yamamoto et al, 2008) www.intechopen.com…”

Recent Progress and New Perspectives in Lymphoma Glycobiology

“…The number of adherent cells to galectin -1 increased dramatically with Vibrio cholerae neuraminidase treatment but not with Newcastle disease virus neuraminidase treatment. These data suggest that α2,6 -linked sialic acid on the cell surface prevents HBL -2 cells from adhesion to galectin -1. α2,6 -linked sialic acid inhibits galectin -1 binding to T cells 27) , and galectin -1 binds α2,3 -sialylated and non -sialylated terminal N -acetyllactos amine units, but not α2,6 -sialylated ones 59) . These data suggest that the type of sialylation linkage affects the binding ability of galectin -1 to terminal residues of oligosaccharides.…”