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 TM accession number BC029564) that possesses ␤3-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 ␤1,3-glycosyltransferase motifs, which are widely conserved in the ␤1,3-galactosyltransferase and ␤1,3-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) ␤1-O-benzyl was used as an acceptor substrate. The enzyme product was determined to have a ␤1,3-linkage by NMR spectroscopic analysis, and was therefore named ␤1,3-Nacetylgalactosaminyltransferase-II (␤3GalNAc-T2). The acceptor substrate specificity of ␤3GalNAc-T2 was examined using various oligosaccharide substrates. Gal-␤1-3(GlcNAc␤1-6)GalNAc␣1-O-para-nitrophenyl (core 2-pNP) was the best acceptor substrate for ␤3GalNAc-T2, followed by GlcNAc␤1-4GlcNAc␤1-O-benzyl, and GlcNAc␤1-6GalNAc␣1-O-para-nitrophenyl (core 6-pNP), among the tested oligosaccharide substrates. Quantitative real time PCR analysis revealed that the ␤3Gal-NAc-T2 transcripts was restricted in its distribution mainly to the testis, adipose tissue, skeletal muscle, and ovary. Its putative orthologous gene, m␤3GalNAc-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. ␤3GalNAc-T2 efficiently transferred GalNAc to N-glycans of fetal calf fetuin, which was treated with neuraminidase and ␤-galactosidase. However, it showed no activity toward any glycolipid examined. Although the GalNAc␤1-3GlcNAc␤1-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.

A novel β1,3‐N‐acetylglucosaminyltransferase (β3Gn‐T8), which synthesizes poly‐N‐acetyllactosamine, is dramatically upregulated in colon cancer

“…Three b3-glycosyltransferase (b3-GT) motifs, XIRX(S/T)W(G/L/M), (F/Y)-XXXXDXD and (E/D)DVXXGX, which we found in a previous study, are commonly encoded in b3-GTs that combine two sugars with a b1,3-linkage [1][2][3][4]. These specific motifs are shared by b3Gal-Ts, b3GalNAc-Ts and b3Gn-Ts.…”

“…Quantitative analysis of the b3Gn-T8 transcript in human tissues and cell lines by real-time PCR For quantification of the b3Gn-T8 transcript, we employed the real time PCR method, as described in detail previously [4]. Total RNA from various human tissues was purchased from Clontech.…”

“…To determine a donor and acceptor substrate for b3Gn-T8, all combinations of nucleotide sugars and monosaccharides were screened by a method described previously [4].…”

“…In the past, we succeeded in the molecular cloning of a series of b3-GT genes. Thirteen human b3-GTs, i.e., five b3Gal-Ts, two b3GalNAc-Ts and six b3Gn-Ts, have been identified and reported [1][2][3][4][5][6][7][8].…”

Cloning and Characterization of β1,3-Glycosyltransferase Family with a β3GT Motifs

“…Our group cloned the glycosyltransferase capable of synthesizing the GalNAcβ1-3GlcNAc structure and designated it b3GalNAc-T2 (26). This structure has not been found in humans and other mammals, although it has been reported in insect glycolipids.…”