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

Toru Hiruma,Akira Togayachi,Kayo Okamura,Takashi Sato,Norihiro Kikuchi,Yeon-Dae Kwon,Aya Nakamura,Katsuya Fujimura,Masanori Gotoh,Kouichi Tachibana,Yasuko Ishizuka,Toshiaki Noce,Hiroshi Nakanishi,Hisashi Narimatsu

doi:10.1074/jbc.m310614200

Abstract

We found, using a BLAST search, a novel human gene (GenBank trade mark accession number BC029564) that possesses beta3-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 beta1,3-glycosyltransferase motifs, which are widely conserved in the beta1,3-galactosyltransferase and beta1,3-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) beta1-O-benzyl was used as an acceptor substrate. The enzyme product was determined to have a beta1,3-linkage by NMR spectroscopic analysis, and was therefore named beta1,3-N-acetylgalactosaminyltransferase-II (beta3GalNAc-T2). The acceptor substrate specificity of beta3GalNAc-T2 was examined using various oligosaccharide substrates. Galbeta1-3(GlcNAcbeta1-6)GalNAcalpha1-O-para-nitrophenyl (core 2-pNP) was the best acceptor substrate for beta3GalNAc-T2, followed by GlcNAcbeta1-4GlcNAcbeta1-O-benzyl, and GlcNAcbeta1-6GalNAcalpha1-O-para-nitrophenyl (core 6-pNP), among the tested oligosaccharide substrates. Quantitative real time PCR analysis revealed that the beta3Gal-NAc-T2 transcripts was restricted in its distribution mainly to the testis, adipose tissue, skeletal muscle, and ovary. Its putative orthologous gene, mbeta3GalNAc-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. beta3GalNAc-T2 efficiently transferred GalNAc to N-glycans of fetal calf fetuin, which was treated with neuraminidase and beta-galactosidase. However, it showed no activity toward any glycolipid examined. Although the GalNAcbeta1-3GlcNAcbeta1-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.

Highlights

We found, using a BLAST search, a novel human gene (GenBankTM accession number BC029564) that possesses ␤3-glycosyltransferase motifs
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
Gal to the GalNAc-␤-R residue of GM2 resulting in the synthesis of GM1 (8). ␤3Gal-T5 exhibits the strongest activity for Gal transfer to galactose (Gal) to the Nacetylglucosamine (GlcNAc) among ␤3Gal-Ts, and is restrictively expressed in colon, intestine, stomach, and pancreas (1). ␤3Gal-T5 is responsible for the expression of the sialyl Lewis a antigen epitopes, a famous tumor marker, in cancer cells derived from such tissues (9). ␤3Gal-T6 catalyzes a Gal␤1–3Gal linkage and is responsible for the synthesis of Gal␤1–3Gal␤1– 4 xylose (Xly)␣-O-(Ser/Thr), the core structure of proteoglycans (4)

Summary

EXPERIMENTAL PROCEDURES

Materials—UDP-[14C]GalNAc was purchased from Amersham Biosciences (Amersham Place, UK) and UDP-[3H]GalNAc from ICN Biomedicals (Irvine, CA). N-Acetylgalactosaminyltransferase Assay—The basic reaction mixture for assaying GalNAc-T activity contained 14 mM HEPES buffer, pH 7.4, an appropriate concentration of UDP-GalNAc, 10 mM MnCl2, 0.4% Triton CF-54, a suitable amount of acceptor substrate, and the purified enzyme. Determination of Products of ␤3GalNAc-T2 with Mass Spectrometry (MS)—GlcNAc-Bz (10 nmol) was incubated with ␤3GalNAc-T2 in 20 ␮l of a basic reaction mixture containing 2.5 mM UDP-GalNAc to produce the reaction product. Determination of Products of ␤3GalNAc-T2 using 1H NMR Spectra— GlcNAc-Bz (640 nmol) was incubated with ␤3GalNAc-T2 in 1 ml of a basic reaction mixture containing 2.5 mM UDP-GalNAc to produce the reaction product. [14C]UDP-GalNAc (50 nCi) and the oligosaccharides as the acceptor substrates were added to 10 ␮l of the basic reaction mixture. Gal␤1–4GalNAc-␣-pNP Gal␤1–3GlcNAc-␤-pNP GlcNAc␤1–4GlcNAc-␤-Bz Gal␤1–3GalNAc␣(core1)-pNP Gal␤1–3(GlcNAc␤1–6)GalNAc␣(core2)-pNP GlcNAc␤1–3GalNAc␣(core3)-pNP GlcNAc␤1–6GalNAc␣(core6)-pNP

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RESULTS

DISCUSSION