title: Production of initial-stage eukaryotic N-glycan and its protein glycosylation in Escherichia coli.
abstract: N-Glycosylation is a ubiquitous protein post-translational modification mechanism in eukaryotes. In this work, a synthetic pathway containing glycosyltransferases from Saccharomyces cerevisiae was introduced to Escherichia coli to synthesize lipid-linked mannosyl-chitobiose (Man-GlcNAc2) and trimannosyl-chitobiose (Man3-GlcNAc2). Transfer of Man3-GlcNAc2 onto a model periplasmic protein occurred in the engineered E. coli cell using oligosaccharyltransferase PglB from Campylobacter jejuni. Mass spectrometric analysis of the fluorescently labeled N-glycan indicated a glycan signal composed of 2 HexNAc and 3 Hex residues. The reversed-phase HPLC analysis suggested that the Hex residues were α1,3-, α1,6- and β1,4-linked mannoses. These results indicated that the constructed system synthesizes a Man3-GlcNAc2, identical to that observed in an early eukaryotic dolichol pathway. Finally, glycopeptide mass spectrometry confirmed the transfer of the assembled glycan moiety onto an engineered glycosylation motif of recombinant maltose binding protein. Surprisingly, the Man3-GlcNAc2 structure but not Man-GlcNAc2 was transferred onto maltose binding protein. This work showed that PglB protein might be able to accommodate the transfer of the further engineered glycan with greater complexity.
authors.affiliation: International Center for Biotechnology, Osaka University, Suita, Osaka 565-0871, Japan.
authors.forename: Akkaraphol
authors.initials: A
authors.lastname: Srichaisupakit
authors.affiliation: International Center for Biotechnology, Osaka University, Suita, Osaka 565-0871, Japan.
authors.forename: Takao
authors.initials: T
authors.lastname: Ohashi
authors.affiliation: International Center for Biotechnology, Osaka University, Suita, Osaka 565-0871, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Misaki
authors.affiliation: International Center for Biotechnology, Osaka University, Suita, Osaka 565-0871, Japan. Electronic address: fujiyama@icb.osaka-u.ac.jp.
authors.forename: Kazuhito
authors.initials: K
authors.lastname: Fujiyama
mesh_terms: D016123:Campylobacter jejuni / Q000201:enzymology / Q000235:genetics; D004187:Disaccharides / Q000096:biosynthesis / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006020:Glycopeptides / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism; D006602:Hexosyltransferases / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D019897:Periplasm / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011134:Polysaccharides / Q000032:analysis / Q000096:biosynthesis*; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics
chemical_list: D004187:Disaccharides; D006020:Glycopeptides; D008565:Membrane Proteins; D011134:Polysaccharides; C032438:chitobiose; D016695:Glycosyltransferases; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase
keywords: Campylobacter jejuni; Escherichia coli; Eukaryotic N-glycosylation; Glycosyltransferase; N-Glycan analysis; PglB; Saccharomyces cerevisiae