Molecular cloning and characterization of a novel UDP-GlcNAc:GalNAc-peptide beta1,3-N-acetylglucosaminyltransferase (beta 3Gn-T6), an enzyme synthesizing the core 3 structure of O-glycans.
The core 3 structure of the O-glycan, GlcNAcbeta1-3GalNAcalpha1-serine/threonine, an important precursor in the biosynthesis of mucin-type glycoproteins, is synthesized by UDP-N-acetylglucosamine:GalNAc-peptide beta1,3-N- acetylglucosaminyltransferase (beta3Gn-T; core 3 synthase). The core 3 structure is restricted in its occurrence to mucins from specific tissues such as the stomach, small intestine, and colon. A partial sequence encoding a novel member of the human beta3Gn-T family was found in one of the data bases. We cloned a complementary DNA of this gene and named it beta3Gn-T6. The putative amino acid sequence of beta3Gn-T6 retains the beta3Gn-T motifs and is predicted to comprise a typical type II membrane protein. The soluble form of beta3Gn-T6 expressed in insect cells showed beta3Gn-T activity toward GalNAcalpha-p-nitrophenyl and GalNAcalpha1-serine/threonine. The beta1,3-linkage between GlcNAc and GalNAc of the enzyme reaction product was confirmed by high performance liquid chromatography and NMR analyses. beta3Gn-T6 effectively transferred a GlcNAc to the GalNAc residue on MUC1 mucin, resulting in the synthesis of a core 3 structure. Real time PCR analysis revealed that the beta3Gn-T6 transcript was restricted in its distribution, mainly to the stomach, colon, and small intestine. We concluded that beta3Gn-T6 is the most logical candidate for the core 3 synthase, which plays an important role in the synthesis of mucin-type O-glycans in digestive organs.
Iwai
Toshie
T
Laboratory of Gene Function Analysis, Institute of Molecular and Cell Biology, National Institute of Advanced Industrial Science and Technology (AIST), Open Space Laboratory C-2, 1-1-1 Umezono, Japan.
Inaba
Niro
N
Naundorf
Andreas
A
Zhang
Yan
Y
Gotoh
Masanori
M
Iwasaki
Hiroko
H
Kudo
Takashi
T
Togayachi
Akira
A
Ishizuka
Yasuko
Y
Nakanishi
Hiroshi
H
Narimatsu
Hisashi
H
D000595:Amino Acid Sequence; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D018076:DNA, Complementary; D006801:Humans; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D011134:Polysaccharides / Q000096:biosynthesis* / Q000737:chemistry; D012333:RNA, Messenger / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D018076:DNA, Complementary; D011134:Polysaccharides; D012333:RNA, Messenger; D017351:N-Acetylglucosaminyltransferases; C454460:UDP-GlcNAc GalNAc-peptide beta1,3-N-acetylglucosaminyltransferase