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
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).
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