Crosslinking of Cys-Mutated Human Galectin-1 to the Model Glycoprotein Ligands Asialofetuin and Laminin by Using a Photoactivatable Bifunctional Reagent

Abstract

Galectins are a group of animal lectins characterized by their specificity for β-galactosides. In our previous study, we showed that a human galectin-1 (hGal-1) mutant, in which a cysteine residue was introduced at Lys 28 , forms a covalently cross-linked complex with the model glycoprotein ligands asialofetuin and laminin by using the photoactivatable sulfhydryl reagent benzophenone-4-maleimide (BPM). In the present study, we used several hGal-1 mutants in which single cysteine residues were introduced at different positions and examined their ability to form a covalent complex with asialofetuin or laminin by using BPM. We found that the efficiency of formation of the cross-linked products differed depending on the positions of the cysteine introduced and also on the ligand used for crosslinking. Therefore, by using different cysteine hGal-1 mutants, the chances of isolating different ligands for hGal-1 should increase depending on the systems and cells used.Key words galectin; ligand; crosslink; maleimide; benzophenone Galectins comprise a group of animal lectins that specifically bind to β-galactosides and are characterized by an evolutionarily conserved sequence motif at their carbohydratebinding site. [1][2][3] Galectins are involved in a wide variety of biological phenomena, including development, cell differentiation, tumor metastasis, apoptosis, RNA splicing, and regulation of immune function. 4,5) Interactions between lectins and their carbohydrate ligands play important roles in various biological systems. Although the basic sugar structure recognized by mammalian galectins, such as human galectin-1 (hGal-1), is the N-acetyllactosamine disaccharide unit (Galβ1 4GlcNAc), the sugar-binding specificity of each galectin could be enhanced by branched, repeated, or substituted glycans, 6,7) suggesting that galectins could interact with a variety of endogenous ligands possessing different carbohydrate structures. However, because the binding ability of galectins is insufficient for some sugar structures, it may not be possible to isolate some endogenous ligand glycoconjugates by conventional methods such as affinity chromatography.We previously prepared several mutants of LEC-1 (a galectin from the nematode Caenorhabditis elegans), which has two lectin domains tandemly repeated in a single polypeptide chain, 8,9) in which a cysteine residue was introduced near the sugar-binding site.10,11) Cross-linked products were formed using a photoactivatable bifunctional reagent, benzophenone-4-maleimide (BPM), [12][13][14] when the mutant Q38C was incubated with a model glycoprotein ligand, asialofetuin. 9) BPM 12) reacts with the sulfhydryl groups of cysteine residues via its maleimide moiety. Upon ultraviolet irradiation, a reactive species that can form covalent bonds with neighboring groups is generated. We used the same method to form a cross-linked product between hGal-1 and N-acetyllactosmine-containing glycoproteins via BPM. 15) Lys 28 in hGal-1, at a location that corresponds to Gln 38 of LEC-1, was selected f...

Regulation of M1-type and M2-type macrophage polarization in RAW264.7 cells by Galectin-9

“…Galectin is a family of lectin that specifically binds to the glycoprotein-β-galactose residue and is present in a wide variety of organisms and is widely distributed in the nucleus, cytoplasm and extracellular matrix (10). Galectin has a variety of biological functions, such as cell adhesion, cell growth regulation, apoptosis, inflammatory response, immune regulation (10,11). Recent report had showed that macrophages with upregulated Galectin-3 participate in liver cirrhosis through production of both M1-and M2-related factors (12).…”

Proximity Tagging Identifies the Glycan-Mediated Glycoprotein Interactors of Galectin-1 in Muscle Stem Cells

“…22 To confirm that the fusion proteins retained their capacities for binding β-galactoside glycans, we performed an enzymelinked immunosorbent assay (ELISA) with immobilized asialofetuin as a model glycoprotein (Figure 1C and Table S1). 26,27 Both NPG1 and CPG1 bound asialofetuin in a dosedependent manner and binding was substantially reduced upon co-incubation with an excess of the soluble competitor lactose (100 mM), indicating that the interactions are glycanmediated. We observed a slight preference for NPG1 (EC 50 ∼ 32 nM) to bind asialofetuin over CPG1 (EC 50 ∼ 56 nM).…”

Purification of galectin-1 mutants using an immobilized Galactoseβ1–4Fucose affinity adsorbent

“…1). Each recombinant mGal-1 protein was subjected to affinity chromatography with asialofetuin, a glycoprotein with multiple Galb1-4GlcNAc units used as a model galectin ligand [25,29], or with synthetic Galb1-4Fuc (Fig. 2).…”

“…Recombinant mGal-1 mutants were expressed in Escherichia coli BL21 (DE3) and affinity purified using asialofetuin-Sepharose or Galb1-4Fuc-Sepharose [20,24] as described previously [25]. Briefly, E. coli cells transformed with the expression plasmids were grown at 37°C in 2Â YT medium containing 125 lg/mL ampicillin.…”