Remodeling of glycoconjugates on CD44 enhances cell adhesion to hyaluronate, tumor growth and metastasis in B16 melanoma cells expressing beta1,4-N-acetylglucosaminyltransferase III.

Abstract

Beta1-4 N-acetylglucosaminyltransferase III (GnT-III) synthesizes bisecting N-acetylglucosamine structures on asparagine-linked oligosaccharides. Using B16-hm mouse melanoma cells stably expressing GnT-III activity as positive transfectants, the effect of bisecting N-acetylglucosamine on the function of CD44 was analyzed in association with adhesion to hyaluronate and tumor spread in mice. Transfection of GnT-III caused increased affinity of immunoprecipitated CD44 to erythro-agglutinating phytohemagglutinin, that preferentially recognizes bisecting N-acetylglucosamine, without affecting the surface CD44 amount, indicating an increase in bisecting N-acetylglucosamine residues on CD44 in positive transfectants. CD44-mediated adhesion to immobilized hyaluronate and the binding of fluorescence-labeled hyaluronate to the cell surface were increased in positive transfectants. The enhanced adhesion in positive transfectants was suppressed by the treatment with beta-N-acetylhexosaminidase, indicating that N-acetylglucosamine residues were responsible for the enhanced adhesion. Positive transfectants showed promoted CD44-mediated tumor growth and metastatic development in the spleen after subcutaneous inoculation into mice. These results indicate that glycosylation of CD44 due to GnT-III causes enhanced adhesion to hyaluronate, local tumor growth and metastatic growth in spleen, suggesting that the CD44-mediated adhesion and tumor spread can be modified through introduction of a glycosyltransferase gene.