Engineered oligosaccharyltransferases with greatly relaxed acceptor-site specificity

Nat Chem Biol. 2014 Oct;10(10):816-22. doi: 10.1038/nchembio.1609. Epub 2014 Aug 17.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Asparagine / chemistry*
  • Asparagine / metabolism
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Campylobacter jejuni / chemistry*
  • Campylobacter jejuni / enzymology
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Gene Expression
  • Glycosylation
  • Hexosyltransferases / chemistry*
  • Hexosyltransferases / genetics
  • Hexosyltransferases / metabolism
  • Membrane Proteins / chemistry*
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism
  • Models, Molecular
  • Molecular Sequence Data
  • Protein Engineering*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Sequence Alignment
  • Substrate Specificity


  • Bacterial Proteins
  • Membrane Proteins
  • Recombinant Proteins
  • Asparagine
  • Hexosyltransferases
  • dolichyl-diphosphooligosaccharide - protein glycotransferase