Galectin-loaded cells as a platform for the profiling of lectin specificity by fluorescent neoglycoconjugates: A case study on galectins-1 and -3 and the impact of assay setting

Abstract

The involvement of galectins as pleiotropic regulators of cell adhesion and growth in disease progression explains the interest to define their ligand-binding properties. Toward this end, it is desirable to approach in vivo conditions to attain medical relevance. In order to simulate physiological conditions with cell surface glycans as recognition sites and galectins as mediators of intercellular contacts we developed an assay using galectin-loaded Raji cells. The extent of surface binding of fluorescent neoglycoconjugates depended on the lectin presence and the type of lectin, the nature of the probes' carbohydrate headgroup and the density of unsubstituted beta-galactosides on the cell surface. Using the most frequently studied galectins-1 and -3, application of this assay led to rather equal binding levels for linear and branched oligomers of N-acetyllactosamine. A clear preference of galectin-3 for alpha1-3-linked galactosylated lactosamine was noted. In parallel, a panel of 24 neoglycoconjugates was tested as inhibitors of galectin binding from solution to N-glycans of surface-immobilized asialofetuin. These two assays differ in presentation of the galectin and ligand, facilitating identification of assay-dependent properties. Under the condition of the cell assay, selectivity among oligosaccharides for the lectins was higher, and extraordinary affinity of galectin-1 to 3'-O-sulfated probes in a solid-phase assay was lost in the cell assay. Having introduced and validated a cell assay, the comprehensive profiling of ligand binding to cell-surface-presented galectins is made possible.

Galectins: Structures, Binding Properties and Function in Cell Adhesion

“…Binding assays can be subdivided regarding the presentation of the different binding partners: 1) the glycan structure is immobilised, 2) the galectin is immobilised and 3) both binding partners are soluble. The chosen assay format influences the data generated as each assay set-up has its own advantages and disadvantages (Rapoport et al, 2008): Assays in which one of the binding partners is immobilised raise the problem that the amount of this ligand is not completely known. Moreover it is possible that side interactions with the surface occur or that the conformation and flexibility of the bound partner differ slightly from its soluble parameters.…”

“…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.…”

“…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).…”

“…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…”