A specific detection of GlcNAcβ1-6Manα1 branches in N-linked glycoproteins based on the specificity of N-acetylglucosaminyltransferase VI

Tae Watanabe, Hideyuki Ihara, Eiji Miyoshi, Koichi Honke, Naoyuki Taniguchi, Tomohiko Taguchi

Research output: Contribution to journalArticlepeer-review

4 Citations (Scopus)

Abstract

Malignant transformation is often accompanied by an aberrant glycosylation profile of the cell surface - in particular, the production of GlcNAcβ1-6Manα1 branches in N-linked glycoproteins. To identify the target glycoproteins, we show a method using recombinant chicken N-acetylglucosaminyltransferase VI (GnT VI) and radiolabeled uridine (5′-)diphosphate-GlcNAc. The assay exploits the fact that GnT VI has a strict requirement for the GlcNAcβ1-6Manα1 structure for activity, when a pyridylaminated free N-glycan is used as the acceptor substrate. Human asialo-agalacto α1-acid glycoprotein (AGP), which is known to contain GlcNAcβ1-6Manα1 branches in its N-linked glycan chains, was radiolabeled when reacted with GnT VI, whereas human asialo-agalacto transferrin and bovine fetuin, neither of which contains a GlcNAcβ1-6Manα1 structure were not, thus corroborating the specificity of the assay. Several proteins from human serum after pretreatment with sialidase and β-galactosidase could be detected using the assay. One was identified as AGP from its mobility on SDS-PAGE, demonstrating the potential of this assay even with crude materials. Furthermore, this method could detect a protein that was also positively stained with leukoagglutinating phytohemagglutinin (L4-PHA) using glycoproteins prepared from WiDr human colon cancer cells. This method should provide a useful complement to the current method, which relies on the specificity of L4-PHA.

Original languageEnglish
Pages (from-to)431-439
Number of pages9
JournalGlycobiology
Volume16
Issue number5
DOIs
Publication statusPublished - 2006 May

Keywords

  • Cancer cells
  • GlcNAcα1-6Manα1 branch
  • GnT V
  • GnT VI
  • α1-acid glycoprotein

ASJC Scopus subject areas

  • Biochemistry

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