title: Purification and cDNA cloning of UDP-GlcNAc:GlcNAcbeta1-3Galbeta1-4Glc(NAc)-R [GlcNAc to Gal]beta1,6N-acetylglucosaminyltransferase from rat small intestine: a major carrier of dIGnT activity in rat small intestine.
abstract: A rat intestinal beta1,6N-acetylglucosaminyltransferase (beta1-6GnT) responsible for the formation of the beta1,6-branched poly-N-acetyllactosamine structure has been purified to apparent homogeneity by successive column chromatographic procedures using an assay wherein pyridylaminated lacto- N-triose II (GlcNAcbeta1-3Galbeta1-4Glc-PA) was used as an acceptor substrate and the reaction product was GlcNAcbeta1-3(GlcNAcbeta1-6)Galbeta1-4Glc-PA. The purified enzyme catalyzed the conversion of the polylactosamine acceptor GlcNAcbeta1-3'LacNAc into GlcNAcbeta1-3'(GlcNAcbeta1-6') LacNAc (dIGnT activity), but it could not transfer GlcNAc to LacNAcbeta1-3'LacNAc (cIGnT activity). This enzyme could also convert mucin core 1 and core 3 analogs, Galbeta1-3GalNAcalpha1-O-paranitrophenyl (pNP) and GlcNAcbeta1-3GalNAcalpha1-O-pNP, into Galbeta1-3(GlcNAcbeta1-6) GalNAcalpha1-O-pNP (C2GnT activity) and GlcNAcbeta1-3(GlcNAcbeta1-6)GalNAcalpha1-O-pNP (C4GnT activity), respectively. Based on the partial amino acid sequences of the purified protein, the cDNA encoding this enzyme was cloned. The COS-1 cells transiently transfected with this cDNA had high dI/C2/C4GnT activities in a ratio of 0.34:1.00:0.90, compared with non- or mock-transfected cells. The primary structure shows a significant homology with human and viral mucin-type core 2 beta1-6GnTs (C2GnT-Ms), indicating that this enzyme is the rat ortholog of human and viral C2GnT-Ms. This is the first identification and purification of this enzyme as a major carrier of dIGnT activity in the small intestine. This rat ortholog should mostly be responsible for making distal I-branch structures on poly-N-acetyllactosamine sequences in this tissue, as well as making mucin core 2 and core 4 structures, given that it also has high C2/C4GnT activities.
authors.affiliation: Department of Biochemistry, Osaka University Medical School/graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Korekane
authors.forename: Tomohiko
authors.initials: T
authors.lastname: Taguchi
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Sakamoto
authors.forename: Koichi
authors.initials: K
authors.lastname: Honke
authors.forename: Naoshi
authors.initials: N
authors.lastname: Dohmae
authors.forename: Heidi
authors.initials: H
authors.lastname: Salminen
authors.forename: Suvi
authors.initials: S
authors.lastname: Toivonen
authors.forename: Jari
authors.initials: J
authors.lastname: Helin
authors.forename: Koji
authors.initials: K
authors.lastname: Takio
authors.forename: Ossi
authors.initials: O
authors.lastname: Renkonen
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Taniguchi
mesh_terms: D000595:Amino Acid Sequence; D000606:Amino Sugars / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis* / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D007421:Intestine, Small / Q000201:enzymology / Q000378:metabolism*; D008969:Molecular Sequence Data; D009077:Mucins / Q000378:metabolism; D017351:N-Acetylglucosaminyltransferases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D051381:Rats; D016415:Sequence Alignment; D013379:Substrate Specificity; D014162:Transfection
chemical_list: D000606:Amino Sugars; D018076:DNA, Complementary; D009077:Mucins; D011134:Polysaccharides; C066929:polylactosamine; D017351:N-Acetylglucosaminyltransferases; C053024:beta-1,3-galactosyl-O-glycosyl-glycoprotein beta-1,6-acetylglucosaminyl transferase; C052254:alpha-1,6-mannosylglycoprotein beta 1,6-N-acetylglucosaminyltransferase