Cloning of a novel member of the UDP-galactose:β-N-acetylglucosamine β1,4-galactosyltransferase family, β4Gal-T4, involved in glycosphingolipid biosynthesis

Abstract

A novel putative member of the human UDP-galactose:β-N- acetylglucosamine β1,4-galactosyltransferase family, designated β4Gal-T4, was identified by BLAST analysis of expressed sequence tags. The sequence of β4Gal-T4 encoded a type II membrane protein with significant sequence similarity to other β1,4-galactosyltransferases. Expression of the full coding sequence and a secreted form of β4Gal-T4 in insect cells showed that the gene product had β1,4-galactosyltransferase activity. Analysis of the substrate specificity of the secreted form revealed that the enzyme catalyzed glycosylation of glycolipids with terminal β-GlcNAc; however, in contrast to β4Gal-T1, -T2, and -T3, this enzyme did not transfer galactose to asialo- agalaeto-fetuin, asialo-agalacto-transferrin, or ovalbumin. The catalytic activity of β4Gal-T4 with monosaccharide acceptor substrates, N- acetylglucosamine as well as glucose, was markedly activated in the presence of α-lactalbumin. The genomic organization of the coding region of β4Gal- T4 was contained in six exons. All intron/exon boundaries were similarly positioned in β4Gal-T1, -T2, and -T3. β4Gal-T4 represents a new member of the β4-galactosyltransferase family. Its kinetic parameters suggest unique functions in the synthesis of neolactoseries glycosphingolipids.