Involvement of murine [beta]-1,4-galactosyltransferase V in lactosylceramide biosynthesis

Human [beta]-1,4-galactosyltransferase ([beta]-1,4-GalT) V was shown to be involved in the biosynthesis of N-glycans, O-glycans and lactosylceramide (Lac-Cer) by in vitro studies. To determine its substrate specificity, enzymatic activity and its products were analyzed using mouse embryonic fibroblast (MEF) cells from [beta]-1,4-GalT V (B4galt5)-mutant mice. Analysis of expression levels of the [beta]-1,4-GalT I-VI genes revealed that the expression of the [beta]-1,4-GalT V gene in B4galt5^sup ^sup +/-^^- and B4galt5^sup ^sup -/-^^-derived MEF cells are a half and null when compared to that of B4galt5^sup ^sup +/+^^-derived MEF cells without altering the expression levels of other [beta]-1,4-GalT genes. These MEF cells showed no apparent difference in their growth. When [beta]-1,4-GalT activities were determined towards GlcNAc[beta]-S-pNP, no significant difference in its specific activity was obtained among B4galt5^sup ^sup +/+^^-, B4galt5^sup ^sup +/-^^- and B4galt5^sup ^sup -/-^^-derived MEF cells. No significant differences were obtained in structures and amounts of N-glycans and lectin bindings to membrane glycoproteins among B4galt5^sup ^sup +/+^^-, B4galt5^sup ^sup +/-^^- and B4galt5^sup ^sup -/-^^-derived MEF cells. However, when cell homogenates were incubated with glucosylceramide in the presence of UDP-[^sup 3^H]Gal, Lac-Cer synthase activity in B4galt5^sup ^sup +/-^^- and B4galt5^sup ^sup -/-^^-derived MEF cells decreased to 41% and 11% of that of B4galt5^sup ^sup +/+^^-derived MEF cells. Consistent with this, amounts of Lac-Cer and its derivative GM3 in B4galt5^sup ^sup -/-^^-derived MEF cells decreased remarkably when compared with those of B4galt5^sup ^sup +/+^^-derived MEF cells. These results indicate that murine [beta]-1,4-GalT V is involved in Lac-Cer biosynthesis. [PUBLICATION ABSTRACT]