A Novel Human β1,3-N-Acetylgalactosaminyltransferase That Synthesizes A Unique Carbohydrate Structure, GalNAcβ1-3GlcNAc

Abstract

We found, using a BLAST search, a novel human gene (GenBank™ accession number BC029564) that possesses β3-glycosyltransferase motifs. The full-length open reading frame consists of 500 amino acids and encodes a typical type II membrane protein. This enzyme has a domain containing 131,3-glycosyltransferase motifs, which are widely conserved in the β1,3-galactosyltransferase and β1,3-N-acetylglucosaminyltransferase families. The putative catalytic domain was expressed in human embryonic kidney 293T cells as a soluble protein. Its N-acetylgalactosaminyltransferase activity was observed when N-acetylglucosamine (GlcNAc) β1-O-benzyl was used as an acceptor substrate. The enzyme product was determined to have a β1,3-linkage by NMR spectroscopic analysis, and was therefore named β1,3-N-acetylgalactosaminyltransferase-II (β3GalNAc-T2). The acceptor substrate specificity of β3GalNAc-T2 was examined using various oligosaccharide substrates. Gal-β1-3(GlcNAcβ1-6)GalNAcα 1-O-para-nitrophenyl (core 2-pNP) was the best acceptor substrate for β3GalNAc-T2, followed by GlcNAcβ1-4GlcNAcβ1-O-benzyl, and GlcNAcβ1-6GalNAcα1-O-para-nitrophenyl (core 6-pNP), among the tested oligosaccharide substrates. Quantitative real time PCR analysis revealed that the β3GalNAc-T2 transcripts was restricted in its distribution mainly to the testis, adipose tissue, skeletal muscle, and ovary. Its putative orthologous gene, mβ3GalNAc-T2, was also found in a data base of mouse expressed sequence tags. In situ hybridization analysis with mouse testis showed that the transcripts are expressed in germ line cells. β3GalNAc-T2 efficiently transferred GalNAc to N-glycans of fetal calf fetuin, which was treated with neuraminidase and β-galactosidase. However, it showed no activity toward any glycolipid examined. Although the GalNAcβ 1-3GlcNAcβ1-R structure has not been reported in humans or other mammals, we have discovered a novel human glycosyltransferase producing this structure on N- and O-glycans.