Involvement of Galectin-3 with Vascular Cell Adhesion Molecule-1 in Growth Regulation of Mouse BALB/3T3 Cells

Tomomi Tadokoro,Masahiko Ikekita,Tosifusa Toda,Hiroko Ito,Takeshi Sato,Ryunosuke Nakatani,Yu Hamaguchi,Kiyoshi Furukawa

doi:10.1074/jbc.m109.063339

Abstract

beta-Galactose residues on N-glycans have been implicated to be involved in growth regulation of cells. In the present study we compared the galactosylation of cell surface N-glycans of mouse Balb/3T3 cells between 30 and 100% densities and found the beta-1,4-galactosylation of N-glycans increases predominantly in a 100-kDa protein band on lectin blot analysis in combination with digestions by diplococcal beta-galactosidase and N-glycanase. When cells at 100% density were treated with jack bean beta-galactosidase, the incorporation of 5-bromodeoxyuridine into the cells was stimulated in a dose-dependent manner, suggesting the involvement of the galactose residues in growth regulation of cells. A galactose-binding protein was isolated from the plasma membranes of cells at 100% density by affinity chromatography using an asialo-transferrin-Sepharose column and found to be galectin-3 as revealed by mass spectrometric analysis. The addition of recombinant galectin-3 into cells at 50% density inhibited the incorporation of 5-bromodeoxyuridine in a dose-dependent manner, but the inhibition was prevented with haptenic sugar. An immunocytochemical study showed that galectin-3 is present at the surface of cells at 100% density but not at 30% density where it locates inside the cells. Several glycoproteins bind to a galectin-3-immobilized column, a major of which was identified as vascular cell adhesion molecule (VCAM)-1. Immunocytochemical studies showed that some galectin-3 and VCAM-1 co-localize at the surface of cells at 100% density, indicating that the binding of galectin-3 secreted from cells to VCAM-1 is one of the pathways involved in the growth regulation of Balb/3T3 cells.

Highlights

Cell surface carbohydrates bound to proteins and lipids play important roles in many biological events including cell-to-cell and cell-to-substratum interactions [1,2,3]
The results indicate that the galectin-3 added inhibits the cell growth by binding to the cell surface galactose residues
The present study shows that the galactosylation and high level branching of N-glycans at the cell surface increase markedly in a 100-kDa protein when Balb/3T3 cells are grown to confluency as revealed by lectin blot analysis

Summary

Introduction

Cell surface carbohydrates bound to proteins and lipids play important roles in many biological events including cell-to-cell and cell-to-substratum interactions [1,2,3]. A dose-dependent incorporation of BrdUrd into cells was observed by the ␤-galactosidase treatment (Fig. 2B), and RCA-I-binding disappeared, from a 100-kDa protein band of the cell surface glycoproteins, and heat-inactivated enzyme did not affect cell growth (data not shown).

Results

Conclusion