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Glycobiology Advance Access published online on June 3, 2009
Glycobiology, doi:10.1093/glycob/cwp075
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© The Author 2009. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Establishment of a real-time analytical method for free oligosaccharide transport from the ER to the cytosol

Yoshimi Haga1,2, Kiichiro Totani3, Yukishige Ito4 and Tadashi Suzuki1,2,*

1 Glycometabolome Team, RIKEN Advanced Science Institute, Wako, Saitama 351-0198, Japan
2 Core Research for Evolutionary Science and Technology (CREST), Japan Science and Technology Agency (JST), Kawaguchi, Saitama 332-0012, Japan
3 Department of Materials and Life Science, Seikei University, Tokyo 180-8633, Japan
4 Synthetic Cellular Chemistry Laboratory, RIKEN Advanced Science Institute, Wako, Saitama 351-0198, Japan

Corresponding author: Tadashi Suzuki, Glycometabolome Team, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan Phone: +81-48-467-9626; Fax: +81-48-467-9626 E-mail: tsuzuki_gm{at}riken.jp

Received on February 17, 2009; accepted on May 24, 2009

During N-glycosylation of proteins, significant amounts of free unconjugated glycans are also generated in the lumen of the endoplasmic reticulum (ER). These ER-derived free glycans are translocated into the cytosol by a putative transporter on the ER membrane for further processing. However, the molecular nature of the transporter remains to be determined. Here, we report the establishment of a novel assay method for free oligosaccharide transport from the ER lumen using chemically synthesized fluorescence-labeled N-glycan derivatives. In this method, fluorescence-labeled glycan substrates were encapsulated inside mouse liver microsomes, followed by incubation with the cytosol and a fluorescence-quenching agent (anti-fluorophore antibody). The rate of substrate efflux was then monitored in real time by the decrease in the fluorescence intensity. The present data clearly demonstrated that the oligosaccharide transport activity under the current assay conditions was both ATP- and cytosol-dependent. The transporter activity was also found to be glycan structure-specific, because free glucosylated glycans were unable to be transported out of the microsomes. This new assay method will be a useful tool for identifying the transporter protein on the ER membrane.

Key words: free oligosaccharide / endoplasmic reticulum / N-glycan catabolism / transporter


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