title: Engineered oligosaccharyltransferases with greatly relaxed acceptor-site specificity.
abstract: The Campylobacter jejuni protein glycosylation locus (pgl) encodes machinery for asparagine-linked (N-linked) glycosylation and serves as the archetype for bacterial N-linked glycosylation. This machinery has been functionally transferred into Escherichia coli, enabling convenient mechanistic dissection of the N-linked glycosylation process in this genetically tractable host. Here we sought to identify sequence determinants in the oligosaccharyltransferase PglB that restrict its specificity to only those glycan acceptor sites containing a negatively charged residue at the -2 position relative to asparagine. This involved creation of a genetic assay, glycosylation of secreted N-linked acceptor proteins (glycoSNAP), that facilitates high-throughput screening of glycophenotypes in E. coli. Using this assay, we isolated several C. jejuni PglB variants that could glycosylate an array of noncanonical acceptor sequences, including one in a eukaryotic N-glycoprotein. These results underscore the utility of glycoSNAP for shedding light on poorly understood aspects of N-linked glycosylation and for engineering designer N-linked glycosylation biocatalysts.
authors.affiliation: School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York, USA.
authors.forename: Anne A
authors.initials: AA
authors.lastname: Ollis
authors.affiliation: Proteomics and Mass Spectrometry Core Facility, Cornell University, Ithaca, New York, USA.
authors.forename: Sheng
authors.initials: S
authors.lastname: Zhang
authors.affiliation: Glycobia Inc., Ithaca, New York, USA.
authors.forename: Adam C
authors.initials: AC
authors.lastname: Fisher
authors.affiliation: School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York, USA.
authors.forename: Matthew P
authors.initials: MP
authors.lastname: DeLisa
mesh_terms: D000595:Amino Acid Sequence; D001216:Asparagine / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016123:Campylobacter jejuni / Q000737:chemistry* / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006031:Glycosylation; D006602:Hexosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; D001216:Asparagine; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase