Glycobiology Advance Access published online on January 22, 2003
Glycobiology, doi:10.1093/glycob/cwg044
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© 2003 Oxford University Press
ORIGINAL ARTICLES
1 Department of Biochemistry, Osaka University Medical School/Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan A rat intestinal
Revised on December 25, 2002
Accepted on January 5, 2003
Purification and cDNA cloning of UDP-GlcNAc:GlcNAc
1-3Gal1-4Glc(NAc)-R [GlcNAc to Gal]1,6N-acetylglucosaminyltransferase from rat small intestine: A major carrier of dIGnT activity in rat small intestine
2 Department of Biochemistry, Osaka University Medical School/Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan; Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520, USA
3 Biomolecular Characterization Division, RIKEN, Wako, Saitama 351-0198, Japan
4 Institute of Biotechnology, University of Helsinki, FIN-00014 Helsinki, Finland
1,6N-acetylglucosaminyltransferase (1-6GnT) responsible for the formation of the 1,6-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 (GlcNAc1-3Gal1-4Glc-PA) was used as an acceptor substrate, and the reaction product was GlcNAc1-3(GlcNAc1-6)Gal1-4Glc-PA. The purified enzyme catalyzed the conversion of the polylactosamine acceptor GlcNAc1-3'LacNAc (LacNAc : Gal1-4GlcNAc) into GlcNAc1-3'(GlcNAc1-6')LacNAc (dIGnT activity), whereas it could not transfer GlcNAc to LacNAc1-3'LacNAc (cIGnT activity). This enzyme could also convert mucin core 1- and core 3-analogs, Gal1-3GalNAc
1-O-pNP (pNP : paranitrophenyl) and GlcNAc1-3GalNAc1-O-pNP into Gal1-3(GlcNAc1-6)GalNAc1-O-pNP (C2GnT activity) and GlcNAc1-3(GlcNAc1-6)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 1-6GnTs (C2GnT-Ms), indicating that this enzyme is the rat orthologue 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 orthologue 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.
Key words: 1,6N-acetylglucosaminyltransferase, I antigen, mucin core 2 structure, mucin core 4 structure, poly-N-acetyllactosamine
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