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Glycobiology Advance Access published online on January 20, 2006
Glycobiology, 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
Received December 1, 2005
Revised January 12, 2006
Accepted January 15, 2006

Article

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

Tae Watanabe 1, Hideyuki Ihara 1, Eiji Miyoshi 1, Koichi Honke 2, Naoyuki Taniguchi 1, and Tomohiko Taguchi 1 *

1 Department of Biochemistry, Osaka University Medical School, Osaka, 565-0871, Japan
2 Department of Molecular Genetics, Kochi University Medical School, Kochi, 783-8505, Japan

* To whom correspondence should be addressed.
Tomohiko Taguchi, E-mail: tom_taguchi{at}biochem.med.osaka-u.ac.jp


  Abstract

Malignant transformation is often accompanied by an aberrant glycosylation profile of the cell surface, in particular, the production of GlcNAc{beta}1-6Man{alpha}1- branches in N-linked glycoproteins. In order to identify the target glycoproteins, here we show a method using recombinant chicken N-acetylglucosaminyltransferase VI (GnT VI) and radiolabeled UDP-GlcNAc. The assay exploits the fact that GnT VI has a strict requirement for the GlcNAc{beta}1-6Man{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 GlcNAc{beta}1-6Man{alpha}1- branches in its N-linked glycan chains was radiolabeled when reacted with GnT VI, whereas human asialo-agalacto trasnferrin and bovine fetuin, neither of which contains a GlcNAc{beta}1-6Man{alpha}1- structure were not, thus corroborating the specificity of the assay. Several proteins from human serum after pretreatment with sialidase and {beta}-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 which 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.

Keywords: {alpha}1-acid glycoprotein/GlcNAc{beta}1-6Man{alpha}1- branch/GnT V/GnT VI/cancer cells.
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