RRC ID 36188
Author Srichaisupakit A, Ohashi T, Misaki R, Fujiyama K.
Title Production of initial-stage eukaryotic N-glycan and its protein glycosylation in Escherichia coli.
Journal J Biosci Bioeng
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.
Volume 119(4)
Pages 399-405
Published 2015-4-1
DOI 10.1016/j.jbiosc.2014.09.016
PII S1389-1723(14)00352-1
PMID 25449758
MeSH Campylobacter jejuni / enzymology Campylobacter jejuni / genetics Disaccharides / biosynthesis Disaccharides / chemistry Escherichia coli / genetics Escherichia coli / metabolism* Gene Expression Glycopeptides / analysis Glycopeptides / chemistry Glycopeptides / metabolism Glycosylation Glycosyltransferases / genetics Glycosyltransferases / metabolism Hexosyltransferases / genetics Hexosyltransferases / metabolism Mass Spectrometry Membrane Proteins / genetics Membrane Proteins / metabolism Periplasm / metabolism Plasmids / genetics Polysaccharides / analysis Polysaccharides / biosynthesis* Protein Processing, Post-Translational Saccharomyces cerevisiae / enzymology Saccharomyces cerevisiae / genetics
IF 2.366
Times Cited 7
Yeast BY4741?