Glycobiology Advance Access originally published online on August 10, 2007
Glycobiology 2007 17(11):1175-1182; doi:10.1093/glycob/cwm087
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
New oligosaccharyltransferase assay method
2 Division of Structural Biology, Medical Institute of Bioregulation, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan
3 Life Science Laboratory, Analytical and Measuring Instruments Division, Shimadzu Corporation, 1 Nishinokyo-Kuwabaracho, Nakagyo-ku, Kyoto 604-8511, Japan
1 To whom correspondence should be addressed: Tel: +81-92-642-6968; Fax: +81-92-642-6764; e-mail: kohda{at}bioreg.kyushu-u.ac.jp
Received on June 14, 2007; revised on July 28, 2007; accepted on August 7, 2007
We developed a new in vitro assay for oligosaccharyltransferase (OST), which catalyzes the transfer of preassembled oligosaccharides on lipid carriers onto asparagine residues in polypeptide chains. The asparagine residues reside in the sequon, Asn-X-Thr/Ser, where X can be any amino acid residue except Pro. We demonstrate the potency of our assay using the OST from yeast. In our method, polyacrylamide gel electrophoresis is used to separate the glycopeptide products from the peptide substrates. The substrate peptide is fluorescently labeled and the formation of glycopeptides is analyzed by fluorescence gel imaging. Two in vitro OST assay methods are now widely used, but both the methods depend on previous knowledge of the oligosaccharide moiety: One method uses lectin binding as the separation mechanism and the other method uses biosynthetically or chemoenzymatically synthesized lipid-linked oligosaccharides as donors. N-linked protein glycosylation is found in all three domains of life, but little is known about the N-glycosylation in Archaea. Thus, our new assay, which does not require a priori knowledge of the oligosaccharides, will be useful in such cases. Indeed, we have detected the OST activity in the membrane fraction from a hyperthermophilic archaeon, Pyrococcus furiosus.
Key words: enzyme assay method / fluorescence detection / N-linked protein glycosylation / oligosaccharyltransferase
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M. Abu-Qarn, A. Giordano, F. Battaglia, A. Trauner, P. G. Hitchen, H. R. Morris, A. Dell, and J. Eichler Identification of AglE, a Second Glycosyltransferase Involved in N Glycosylation of the Haloferax volcanii S-Layer Glycoprotein J. Bacteriol., May 1, 2008; 190(9): 3140 - 3146. [Abstract] [Full Text] [PDF]
|
|