New oligosaccharyltransferase assay method.
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.
Kohda
Daisuke
D
Division of Structural Biology, Medical Institute of Bioregulation, Kyushu University, Maidashi 3-1-1, Fukuoka 812-8582, Japan. kohda@bioreg.kyushu-u.ac.jp
Yamada
Masaki
M
Igura
Mayumi
M
Kamishikiryo
Jun
J
Maenaka
Katsumi
K
D001681:Biological Assay / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D006020:Glycopeptides / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D006602:Hexosyltransferases / Q000032:analysis*; D007700:Kinetics; D008565:Membrane Proteins / Q000032:analysis* / Q000302:isolation & purification; D008954:Models, Biological; D019711:Pyrococcus / Q000166:cytology / Q000201:enzymology
D006020:Glycopeptides; D008565:Membrane Proteins; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase