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Comparison of Oligosaccharyltransferase Assay Methods Using a Fluorescent Peptide  

Kim, Seong-Hun (Integrative Omics Research Center, Korea Research Institute of Bioscience and Biotechnology)
Publication Information
Korean Journal of Microbiology / v.46, no.1, 2010 , pp. 96-103 More about this Journal
Abstract
Oligosaccharyltransferase (OTase) catalyzes the transfer of a lipid-linked oligosaccharide (LLO) to the nascent polypeptide. Most eukaryotes have an OTase composed of a multisubunit protein complex. However, the kinetoplastid Leishmania major and the bacterium Campylobacter jejuni have only a single subunit for OTase activity, Stt3p and PglB, respectively. In this study, a new in vitro assay for OTase was developed by using a fluorescent peptide containing N-glycosylation sequon, Asn-Xaa-Thr/Ser, where Xaa can be any amino acid residue except Pro. L. major Stt3p and C. jejuni PglB as a model OTase enzyme demonstrated the formation of glycopeptides from a fluorescent peptide through OTase activities. For separation and measurement of the glycopeptides produced by the OTases, Tricine-SDS-PAGE, a lectin column and fluorospectrophotometer, and HPLC were applied. Comparison of these assay methods for analyzing a fluorescent glycopeptide showed HPLC analysis is the best method for separation of glycopeptides and nonglycosylated peptides as well as for quantify the peptides than other methods.
Keywords
fluorescent peptide; N-glycosylation; lipid-linked oligosaccharide; oligosaccharyltransferase; PglB; Stt3p;
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