Purification and cDNA cloning of UPDP-GlcNAc:GclNAcβ1-3Galβ1-4Glc(NAc)-R [GlcNAc to Gall]β1,6N-acetyglucosaminyltransferase from rat small intestine: A major carrier of dIGnt activity in rat small intestine

Hiroaki Korekane, Tomohiko Taguchi, Yoshihiro Sakamoto, Koichi Honke, Naoshi Dohmae, Heidi Salminen, Suvi Toivonen, Jari Helin, Koji Takio, Ossi Renkonen, Naoyuki Taniguchi

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7 Citations (Scopus)

Abstract

A rat intestinal β1,6N-acetylglucosaminyltransferase (β1-6GnT) responsible for the formation of the β1,6-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 (GlcNAcβ1-3Galβ1-4Glc-PA) was used as an acceptor substrate and the reaction product was GlcNAcβ1-3(GlcNAcβ1-6 Galβ1-4Glc-PA. The purified enzyme catalyzed the conversion of the polylactosamine acceptor GlcNAcβ1-3′LacNAc into GlcNAcβ1-3′(GlcNAcβ1-6′) LacNAc (dIGnT activity), but it could not transfer GlcNAc to LacNAcβ1-3′LacNAc (cIGnT activity). This enzyme could also convert mucin core 1 and core 3 analogs, Galβ1-3GalNAcα1-O-paranitrophenyl (pNP) and GlcNAcβ1-3GalNAcα1-O-pNP, into Galβ1-3(GlcNAcβ1-6) GalNAcα1-O-pNP (C2GnT activity) and GlcNAcβ1-3(GlcNAcβ1-6)GalNAcα1-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 β1-6GnTs (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.

Original languageEnglish
Pages (from-to)387-400
Number of pages14
JournalGlycobiology
Volume13
Issue number5
DOIs
Publication statusPublished - 2003 May 1

Keywords

  • I antigen
  • Mucin core 2 structure
  • Mucin core 4 structure
  • Poly-N-acetyllactosamine
  • β1,6N-acetyglucosaminyltransferase

ASJC Scopus subject areas

  • Biochemistry

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