Definition of the bacterial N-glycosylation site consensus sequence

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Abstract
The Campylobacter jejuni pgl locus encodes an N‐linked protein glycosylation machinery that can be functionally transferred into Escherichia coli. In this system, we analyzed the elements in the C. jejuni N‐glycoprotein AcrA required for accepting an N‐glycan. We found that the eukaryotic primary consensus sequence for N‐glycosylation is N terminally extended to D/E‐Y‐N‐X‐S/T (Y, X≠P) for recognition by the bacterial oligosaccharyltransferase (OST) PglB. However, not all consensus sequences were N‐glycosylated when they were either artificially introduced or when they were present in non‐C. jejuni proteins. We were able to produce recombinant glycoproteins with engineered N‐glycosylation sites and confirmed the requirement for a negatively charged side chain at position −2 in C. jejuni N‐glycoproteins. N‐glycosylation of AcrA by the eukaryotic OST in Saccharomyces cerevisiae occurred independent of the acidic residue at the −2 position. Thus, bacterial N‐glycosylation site selection is more specific than the eukaryotic equivalent with respect to the polypeptide acceptor sequence.