Molecular cloning and characterization of a novel human beta 1,4-N-acetylgalactosaminyltransferase, beta 4GalNAc-T3, responsible for the synthesis of N,N'-diacetyllactosediamine, galNAc beta 1-4GlcNAc.
We found a novel human glycosyltransferase gene carrying a hypothetical beta1,4-glycosyltransferase motif during a BLAST search, and we cloned its full-length open reading frame by using the 5'-rapid amplification of cDNA ends method. It encodes a type II transmembrane protein of 999 amino acids with homology to chondroitin sulfate synthase in its C-terminal region (GenBank accession number AB089940). Its putative orthologous gene was also found in mouse (accession number AB114826). The truncated form of the human enzyme was expressed in HEK293T cells as a soluble protein. The recombinant enzyme transferred GalNAc to GlcNAc beta-benzyl. The product was deduced to be GalNAc beta 1-4GlcNAc beta-benzyl based on mass spectrometry and NMR spectroscopy. We renamed the enzyme beta1,4-N-acetylgalactosaminyltransferase-III (beta 4GalNAc-T3). beta 4GalNAc-T3 effectively synthesized N,N'-diacetylgalactosediamine, GalNAc beta 1-4GlcNAc, at non-reducing termini of various acceptors derived not only from N-glycans but also from O-glycans. Quantitative real time PCR analysis showed that its transcript was highly expressed in stomach, colon, and testis. As some glycohormones contain N,N'-diacetylgalactosediamine structures in their N-glycans, we examined the ability of beta 4GalNAc-T3 to synthesize N,N'-diacetylgalactosediamine structures in N-glycans on a model protein. When fetal calf fetuin treated with neuraminidase and beta1,4-galactosidase was utilized as an acceptor protein, beta 4GalNAc-T3 transferred GalNAc to it. Furthermore, the majority of the signal from GalNAc disappeared on treatment with glycopeptidase F. These results suggest that beta 4GalNAc-T3 could transfer GalNAc residues, producing N,N'-diacetylgalactosediamine structures at least in N-glycans and probably in both N- and O-glycans.
Sato
Takashi
T
Glycogene Function Team, Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology, Open Space Laboratory Central-2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan.
Gotoh
Masanori
M
Kiyohara
Katsue
K
Kameyama
Akihiko
A
Kubota
Tomomi
T
Kikuchi
Norihiro
N
Ishizuka
Yasuko
Y
Iwasaki
Hiroko
H
Togayachi
Akira
A
Kudo
Takashi
T
Ohkura
Takashi
T
Nakanishi
Hiroshi
H
Narimatsu
Hisashi
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002240:Carbohydrate Sequence; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D030541:Databases, Genetic; D004187:Disaccharides / Q000737:chemistry* / Q000378:metabolism; D014453:Glucuronosyltransferase; D016695:Glycosyltransferases / Q000378:metabolism; D006801:Humans; D007785:Lactose / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D051379:Mice; D008957:Models, Genetic; D008969:Molecular Sequence Data; D064251:Multifunctional Enzymes; D017350:N-Acetylgalactosaminyltransferases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D016366:Open Reading Frames; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000737:chemistry; D010802:Phylogeny; D011134:Polysaccharides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013997:Time Factors; D014018:Tissue Distribution; D000097763:Polypeptide N-acetylgalactosaminyltransferase
D018076:DNA, Complementary; D004187:Disaccharides; D014453:Glucuronosyltransferase; D016695:Glycosyltransferases; D007785:Lactose; D064251:Multifunctional Enzymes; D017350:N-Acetylgalactosaminyltransferases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011134:Polysaccharides; D012333:RNA, Messenger; D011994:Recombinant Proteins; D000097763:Polypeptide N-acetylgalactosaminyltransferase; C093701:N-acetylgalactosaminyl-1-4-N-acetylglucosamine; C480161:B4GALNT3 protein, human; C412725:chondroitin synthase; C498800:CHSY1 protein, human