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- Title
Engineered oligosaccharyltransferases with greatly relaxed acceptor-site specificity.
- Authors
Ollis, Anne A; Zhang, Sheng; Fisher, Adam C; DeLisa, Matthew P
- 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.
- Subjects
OLIGOSACCHARYLTRANSFERASE; LEWIS acidity; CAMPYLOBACTER jejuni; BACTERIAL proteins; GLYCOSYLATION; ASPARAGINE
- Publication
Nature Chemical Biology, 2014, Vol 10, Issue 10, p816
- ISSN
1552-4450
- Publication type
Article
- DOI
10.1038/nchembio.1609