title: Relaxed acceptor site specificity of bacterial oligosaccharyltransferase in vivo.
abstract: A number of proteobacteria carry the genetic information to perform N-linked glycosylation, but only the protein glycosylation (pgl) pathway of Campylobacter jejuni has been studied to date. Here, we report that the pgl gene cluster of Campylobacter lari encodes for a functional glycosylation machinery that can be reconstituted in Escherichia coli. We determined that the N-glycan produced in this system consisted of a linear hexasaccharide. We found that the oligosaccharyltransferase (OST) of C. lari conserved a predominant specificity for the primary sequence D/E-X(-1)-N-X(+1)-S/T (where X(-1) and X(+1) can be any amino acid but proline). At the same time, we observed that this enzyme exhibited a relaxed specificity toward the acceptor site and modified asparagine residues of a protein at sequences DANSG and NNNST. Moreover, C. lari pgl glycosylated a native E. coli protein. Bacterial N-glycosylation appears as a useful tool to establish a molecular description of how single-subunit OSTs perform selection of glycosyl acceptor sites.
authors.affiliation: Department of Biology, Institute of Microbiology, ETH Zürich, Wolfgang-Pauli-Strasse 10, Zürich, Switzerland.
authors.forename: Flavio
authors.initials: F
authors.lastname: Schwarz
authors.forename: Christian
authors.initials: C
authors.lastname: Lizak
authors.forename: Yao-Yun
authors.initials: YY
authors.lastname: Fan
authors.forename: Susanna
authors.initials: S
authors.lastname: Fleurkens
authors.forename: Michael
authors.initials: M
authors.lastname: Kowarik
authors.forename: Markus
authors.initials: M
authors.lastname: Aebi
mesh_terms: D016123:Campylobacter jejuni / Q000201:enzymology*; D044863:Campylobacter lari / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology; D006031:Glycosylation; D006602:Hexosyltransferases / Q000737:chemistry* / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; D011134:Polysaccharides; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase