Involvement of galectin-3 with VCAM-1 in growth regulation of Mouse BALB/3T3 cells

Abstract β-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 β-1,4-galactosylation of N-glycans increases predominantly in a 100 K protein band on lectin blot analysis in combination with digestions by diplococcal β-galactosidase and N-glycanase. When cells at 100% density were treated with jack bean β-galactosidase, the incorporation of 5-bromo-deoxyuridine (BrdU) 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 BrdU in a dose-dependent manner, but the inhibition was prevented with haptenic sugar. 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.