Glycobiology Advance Access originally published online on January 22, 2003
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Glycobiology, 2003, Vol. 13, No. 5 387-400
© 2003 Oxford University Press
Purification and cDNA cloning of UDP-GlcNAc:GlcNAcß1-3Galß1-4Glc(NAc)-R [GlcNAc to Gal]ß1,6N-acetylglucosaminyltransferase from rat small intestine: a major carrier of dIGnT activity in rat small intestine
3 Department of Biochemistry, Osaka University Medical School/graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
4 Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06520, USA
5 Biomolecular Characterization Division, Riken, Wako, Saitama 351-0198, Japan
6 The Institute of Biotechnology, University of Helsinki, FIN-00014 Helsinki, Finland
Received on November 28, 2002; revised on December 25, 2002; accepted on January 5, 2003
A rat intestinal ß1,6N-acetylglucosaminyltransferase (ß1-6GnT) responsible for the formation of the ß1,6-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 (GlcNAcß1-3Galß1-4Glc-PA) was used as an acceptor substrate and the reaction product was GlcNAcß1-3(GlcNAcß1-6)Galß1-4Glc-PA. The purified enzyme catalyzed the conversion of the polylactosamine acceptor GlcNAcß1-3'LacNAc into GlcNAcß1-3'(GlcNAcß1-6') LacNAc (dIGnT activity), but it could not transfer GlcNAc to LacNAcß1-3'LacNAc (cIGnT activity). This enzyme could also convert mucin core 1 and core 3 analogs, Galß1-3GalNAc
1-O-paranitrophenyl (pNP) and GlcNAcß1-3GalNAc1-O-pNP, into Galß1-3(GlcNAcß1-6) GalNAc1-O-pNP (C2GnT activity) and GlcNAcß1-3(GlcNAcß1-6)GalNAc1-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 ß1-6GnTs (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.
1 Present address: High Throughput Factory, RIKEN Harima Institute 1-1-1, Kouto, Mikazuki-cho, Sayo-gun, Hyogo 679-5148, Japan
2 To whom correspondence should be addressed; e-mail: proftani{at}biochem.med.osaka-u.ac.jp
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