Galectins are a group of animal lectins characterized by their specificity for b-galactosides and an evolutionarily conserved sequence motif in the carbohydrate-binding site. Galectins are involved in a wide variety of biological phenomena, including development, cell differentiation, tumor metastasis, apoptosis, RNA splicing, and regulation of immune functioning. [1][2][3][4][5][6] Galectins can be classified into three types on the basis of their molecular architecture: proto, chimera, and tandem-repeat types. LEC-1, the 32-kDa galectin of the nematode, Caenorhabditis elegans (C. elegans), was the first example of a tandem repeat-type galectin composed of two homologous regions. 7,8) A variety of pyridylaminated oligosaccharides were used as analytes in frontal affinity chromatography, 9) and the result showed that the two lectin domains of LEC-1 have different sugar-binding profiles.10) This finding suggests that the endogenous ligand glycoconjugates for the two domains of LEC-1 differ. However, it is difficult to confirm this finding, especially when the binding ability of each domain is not adequately strong. We developed a new approach to overcome this problem. We prepared LEC-1 mutants in which a cysteine residue was introduced near the binding site of the Nterminal half domain; after incubation with a model glycoprotein ligand, asialofetuin, cross-linked products were formed using a photoactivatable bifunctional reagent, benzophenone-4-maleimide (BPM). Among the five LEC-1 mutants produced, one of them (LEC-1 Q38C) produced the cross-linked product.11) This method was also applied to human galectin-1 (Gal-1). At first, all cysteine residues were substituted with Ser residues, and Lys 28 of the resultant cysteine-less Gal-1 was substituted with a cysteine residue. The position of Lys 28 corresponds to Gln 38 of LEC-1. Crosslinked products between hGal-1 K28C and asialofetuin via BPM were observed. 12) In the present study, we produced several LEC-1 mutants, each with a cysteine substitution in the C-terminal lectin domain (Ch) to detect interactions with asialofetuin. Two mutants, K177C and S268C, produced cross-linked products. This procedure is very promising for capturing and identifying endogenous ligands for each lectin domain of the tandem repeat-type galectin, LEC-1.
MATERIALS AND METHODS
ChemicalsRabbit anti-bovine fetuin polyclonal antibody was purchased from Chemicon International (Temecula, CA, U.S.A.). BPM and bovine fetuin were purchased from Sigma (St. Louis, MO, U.S.A.). Sialidase and b-galactosidase were purchased from Seikagaku Co. (Tokyo, Japan).Construction of Mutant Lectin Genes A cysteine residue was introduced using site-directed mutagenesis of LEC-1 cDNA (LEC-1 contains no cysteine residues).
8)Residues subjected to substitution were selected on the basis of the X-ray crystal structure of LEC-1 in complex with galactose.13) To facilitate cross-link formation, hydrophilic amino acid residues with side chains extending from the surface of the protein toward the bound galactose but not...