Glycobiology and Extracellular Matrices
A Novel Human β1,3-N-Acetylgalactosaminyltransferase That Synthesizes a Unique Carbohydrate Structure, GalNAcβ1-3GlcNAc*

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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 β1,3-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. Galβ1-3(GlcNAcβ1-6)GalNAcα1-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 β3Gal-NAc-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 GalNAcβ1-3GlcNAcβ1-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.

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This work was performed as part of the R&D Project of the Industrial Science and Technology Frontier Program (R&D for Establishment and Utilization of a Technical Infrastructure for Japanese Industry) supported by the New Energy and Industrial Technology Development Organization (NEDO). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.