A novel human beta1,3-N-acetylgalactosaminyltransferase that synthesizes a unique carbohydrate structure, GalNAcbeta1-3GlcNAc.
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.
Hiruma
Toru
T
Research Center for Glycoscience (RCG), National Institute of Advanced Industrial Science and Technology (AIST), Open Space Laboratory Central-2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan.
Togayachi
Akira
A
Okamura
Kayo
K
Sato
Takashi
T
Kikuchi
Norihiro
N
Kwon
Yeon-Dae
YD
Nakamura
Aya
A
Fujimura
Katsuya
K
Gotoh
Masanori
M
Tachibana
Kouichi
K
Ishizuka
Yasuko
Y
Noce
Toshiaki
T
Nakanishi
Hiroshi
H
Narimatsu
Hisashi
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002240:Carbohydrate Sequence; D019295:Computational Biology; D006801:Humans; D017403:In Situ Hybridization; D051379:Mice; D008969:Molecular Sequence Data; D017350:N-Acetylgalactosaminyltransferases / Q000235:genetics* / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D010802:Phylogeny; D011522:Protons; D012333:RNA, Messenger / Q000032:analysis; D013379:Substrate Specificity
D009844:Oligosaccharides; D011522:Protons; D012333:RNA, Messenger; C000594712:B3GALNT2 protein, human; D017350:N-Acetylgalactosaminyltransferases