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Glycobiology Advance Access originally published online on January 20, 2006
Glycobiology 2006 16(5):431-439; doi:10.1093/glycob/cwj079
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© The Author 2006. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

A specific detection of GlcNAcß1-6Man{alpha}1 branches in N-linked glycoproteins based on the specificity of N-acetylglucosaminyltransferase VI

Tae Watanabe2, Hideyuki Ihara2, Eiji Miyoshi2, Koichi Honke3, Naoyuki Taniguchi2 and Tomohiko Taguchi1,2

2 Department of Biochemistry, Osaka University Graduate School of Medicine, B1, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan and 3 Department of Molecular Genetics, Kochi University Medical School, Kochi 783-8505, Japan

1 To whom correspondence should be addressed; e-mail: tom_taguchi{at}biochem.med.osaka-u.ac.jp

Received on December 1, 2005; revised on January 12, 2006; accepted on January 15, 2006

Malignant transformation is often accompanied by an aberrant glycosylation profile of the cell surface—in particular, the production of GlcNAcß1-6Man{alpha}1 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 GlcNAcß1-6Man{alpha}1 structure for activity, when a pyridylaminated free N-glycan is used as the acceptor substrate. Human asialo-agalacto {alpha}1-acid glycoprotein (AGP), which is known to contain GlcNAcß1-6Man{alpha}1 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 GlcNAcß1-6Man{alpha}1 structure were not, thus corroborating the specificity of the assay. Several proteins from human serum after pretreatment with sialidase and ß-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.

Key words: {alpha}1-acid glycoprotein / cancer cells / GlcNAcß1-6Man{alpha}1 branch / GnT V / GnT VI


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