Purification and cDNA cloning of UDP-GlcNAc:GlcNAc[beta]1-3Gal[beta]1-4Glc(NAc)-R [GlcNAc to Gal][beta]1,6N-acetylglucosaminyltransferase from rat small intestine: a major carrier of dIGnT activity in rat small intestine

A rat intestinal [beta]1,6N-acetylglucosaminyltransferase ([beta]1-6GnT) responsible for the formation of the [beta]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 (GlcNAc[beta]1-3Gal[beta]1-4Glc-PA) was used as an acceptor substrate and the reaction product was GlcNAc[beta]1-3(GlcNAc[beta]1-6)Gal[beta]1-4Glc-PA. The purified enzyme catalyzed the conversion of the polylactosamine acceptor GlcNAc[beta]1-3'LacNAc into GlcNAc[beta]1-3'(GlcNAc[beta]1-6') LacNAc (dIGnT activity), but it could not transfer GlcNAc to LacNAc[beta]1-3'LacNAc (cIGnT activity). This enzyme could also convert mucin core 1 and core 3 analogs, Gal[beta]1-3GalNAc[alpha]1-O-paranitrophenyl (pNP) and GlcNAc[beta]1-3GalNAc[alpha]1-O-pNP, into Gal[beta]1-3(GlcNAc[beta]1-6) GalNAc[alpha]1-O-pNP (C2GnT activity) and GlcNAc[beta]1-3(GlcNAc[beta]1-6)GalNAc[alpha]1-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 [beta]1-6GnTs (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.