Recognition Mechanism of Galectin-4 for Cholesterol 3-Sulfate

Hiroko Ideo,Akira Seko,Katsuko Yamashita

doi:10.1074/jbc.m703770200

Abstract

Galectin-4 binds to glycosphingolipids carrying 3-O-sulfated Gal residues, and it co-localizes on the cell surface of human colonic adenocarcinoma cells with glycosphingolipids carrying SO(-)(3)-->3Galbeta1-->3(GalNAc) residues (Ideo, H., Seko, A., and Yamashita, K. (2005) J. Biol. Chem. 280, 4730-4737). In the present study, it was found that galectin-4 also binds to cholesterol 3-sulfate, which has no beta-galactoside moiety. This characteristic of galectin-4 is unique within the galectin family. The site-directed mutated galectin-4-R45A had diminished binding ability toward cholesterol 3-sulfate, suggesting that Arg(45) of galectin-4 is indispensable for cholesterol 3-sulfate recognition. Gel filtration and chemical cross-linking experiments revealed that some galectin-4 exists as dimers, and this multivalency seemed to enhance its avidity for cholesterol 3-sulfate binding. Cholesterol 3-sulfate and sulfatide co-existed with galectin-4 in detergent-insoluble fractions of porcine esophagus and intestine, respectively. These results suggested that not only sulfated glycosphingolipids but also cholesterol 3-sulfate are endogenous ligands for galectin-4 in vivo.

Highlights

Priority Area(s) 14082208 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan
It was found that galectin-4 binds to cholesterol 3-sulfate, which could not be separated from SM4 and SM3 by the TLC solvent system in our previous paper [2], cholesterol 3-sulfate does not have a sugar moiety
Galectin-4 Binding to Cholesterol 3-Sulfate—Galectin-4 binds to glycosphingolipids carrying 3-O-sulfated Gal residues derived from colon epithelial cells [2]

Summary

Introduction

Priority Area(s) 14082208 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. To determine the binding mechanism of cholesterol 3-sulfate, we prepared full-length galectin-4 and site-directed mutants of the N- and C-domains, based on comparisons of the amino acids in the S3 ␤-sheets of various galectins, and we examined their binding to various ligands. Fifty ␮l of galectin-4 (1 ␮g/ml) with various concentrations of inhibitors in 1% BSA in PBS were applied to cholesterol 3-sulfate-coated plates (1 ␮g/well), and the relative binding abilities of galectin-4 were measured using anti-His6 antibody.

Results

Conclusion