Mutational Tuning of Galectin-3 Specificity and Biological Function

Abstract

Galectins are defined by a conserved β-galactoside binding site that has been linked to many of their important functions in e.g. cell adhesion, signaling, and intracellular trafficking. Weak adjacent sites may enhance or decrease affinity for natural β-galactoside-containing glycoconjugates, but little is known about the biological role of this modulation of affinity (fine specificity). We have now produced 10 mutants of human galectin-3, with changes in these adjacent sites that have altered carbohydrate-binding fine specificity but that retain the basic β-galactoside binding activity as shown by glycan-array binding and a solution-based fluorescence anisotropy assay. Each mutant was also tested in two biological assays to provide a correlation between fine specificity and function. Galectin-3 R186S, which has selectively lost affinity for LacNAc, a disaccharide moiety commonly found on glycoprotein glycans, has lost the ability to activate neutrophil leukocytes and intracellular targeting into vesicles. K176L has increased affinity for β-galactosides substituted with GlcNAcβ1–3, as found in poly-N-acetyllactosaminoglycans, and increased potency to activate neutrophil leukocytes even though it has lost other aspects of galectin-3 fine specificity. G182A has altered carbohydrate-binding fine specificity and altered intracellular targeting into vesicles, a possible link to the intracellular galectin-3-mediated anti-apoptotic effect known to be lost by this mutant. Finally, the mutants have helped to define the differences in fine specificity shown by Xenopus, mouse, and human galectin-3 and, as such, the evidence for adaptive change during evolution.

Galectins: Structures, Binding Properties and Function in Cell Adhesion

“…Due to this mutation galectin-8 N-CRD favours lactose structures over LacNAc type II structures in the binding site. Thereby different biological functions of galectin-8 in contrast to other galectins such as galectin-3 are regulated (Salomonsson et al, 2010). Specific binding preferences resulting from the differences in amino acid sequence will be discussed in chapter 4 in more detail.…”

“…The labelling of galectins can also alter the binding specificities. It is in most cases done by random chemical modification of specific functional groups such as amino or thiol functionalities (Carlsson et al, 2007;Patnaik et al, 2006;Rapoport et al, 2008;Salomonsson et al, 2010;Song et al, 2009b;Stowell et al, 2008a;Stowell et al, 2008b). Although this labelling is assumed not to influence binding specificity or inactive galectins are removed after the labelling reaction, binding and oligomerisation still might be slightly affected.…”

“…Moreover lot-specific aberrations between different labelling reactions occur. Labelled galectins are for example used for glycan arrays and ELISA-type set-ups (Blixt et al, 2004;Carlsson et al, 2007;Rapoport et al, 2008;Salomonsson et al, 2010;Song et al, 2009b;Stowell et al, 2008a) while fluorescence labelled glycans are used in frontal affinity chromatography or fluorescence polarisation (Carlsson et al, 2007;Hirabayashi et al, 2002;Salomonsson et al, 2010;Sörme et al, 2004). Assays using both binding partners in its soluble form overcome most of the mentioned problems.…”

“…Different galectins thereby show high affinity to specific disaccharides. Galectin-3, galectin-1 and the C-terminal CRD of galectin-8 bind preferentially LacNAc units of type I and type II while the N-terminal CRD of galectin-8 shows highest affinity for lactose (Carlsson et al, 2007;Ideo et al, 2011;Salomonsson et al, 2010). Extensions of the bound galactose moiety effect glycan binding in dependence on the galectin.…”

“…Extensions of the bound galactose moiety effect glycan binding in dependence on the galectin. 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).…”