Molecular cloning and characterization of a novel UDP-Gal:GalNAc(alpha) peptide beta 1,3-galactosyltransferase (C1Gal-T2), an enzyme synthesizing a core 1 structure of O-glycan.
Recently, a UDP-Gal:GalNAc(alpha) peptide beta1,3-galactosyltransferase (core 1 synthase 1; C1Gal-T1) has been purified from rat liver and its complementary DNA cloned from several species. We isolated a second candidate for core 1 synthase from a Colo205 cDNA library and named it C1Gal-T2. The deduced amino acid sequence of C1Gal-T2, having 26% homology to C1Gal-T1, showed a topology typical of a type II membrane protein. Real time PCR analysis revealed that the expression of C1Gal-T2 transcripts was widespread in many tissues and of relatively high level in salivary gland, stomach, small intestine, kidney, testis, thymus, and spleen. LSC cells, having no core 1 synthase activity, were transfected stably with the C1Gal-T2 gene. Their microsome fraction showed beta1,3-galactosyltransferase activity toward GalNAc-alpha-para-nitrophenyl and GalNAc(alpha)1 peptides resulting in the synthesis of the core 1 structure. The core 1 synthesizing activity of C1Gal-T2 was also determined by flow cytometry and lectin blotting using the LSC cells stably expressing C1Gal-T2. Finally, LSC cells, and Jurkat cells that also lack the core 1 synthase activity, were found to have null alleles of C1Gal-T2. These results indicated that C1Gal-T2 is the second candidate for core 1 synthase that plays an important role in synthesizing O-glycans in digestive organs.
D000595:Amino Acid Sequence; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D019992:Databases as Topic; D005091:Exons; D020224:Expressed Sequence Tags; D005434:Flow Cytometry; D005700:Galactosyltransferases / Q000737:chemistry* / Q000235:genetics*; D015723:Gene Library; D006801:Humans; D019169:Jurkat Cells; D037102:Lectins; D008969:Molecular Sequence Data; D009154:Mutation; D016366:Open Reading Frames; D010455:Peptides / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013347:Subcellular Fractions; D014018:Tissue Distribution; D014162:Transfection; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D037102:Lectins; D010455:Peptides; D011134:Polysaccharides; D005700:Galactosyltransferases; C470633:UDP-galactose N-acetylgalactosamine peptide beta1,3-galactosyltransferase