摘要：The central enzyme in the Campylobacter jejuni asparagine-linked glycosylation pathway is the oligosaccharyltransferase (OST), PglB, which transfers preassembled glycans to specific asparagine residues in target proteins. While C. jejuni PglB ( Cj PglB) can transfer many diverse glycan structures, the acceptor sites that it recognizes are restricted predominantly to those having a negatively charged residue in the −2 position relative to the asparagine. Here, we investigated the acceptor-site preferences for 23 homologs with natural sequence variation compared to Cj PglB. Using an ectopic trans-complementation assay for Cj PglB function in glycosylation-competent Escherichia coli , we demonstrated in vivo activity for 16 of the candidate OSTs. Interestingly, the OSTs from Campylobacter coli, Campylobacter upsaliensis , Desulfovibrio desulfuricans , Desulfovibrio gigas , and Desulfovibrio vulgaris , exhibited significantly relaxed specificity towards the −2 position compared to Cj PglB. These enzymes glycosylated minimal N-X-T motifs in multiple targets and each followed unique, as yet unknown, rules governing acceptor-site preferences. One notable example is D. gigas PglB, which was the only bacterial OST to glycosylate the Fc domain of human immunoglobulin G at its native ‘QYNST’ sequon. Overall, we find that a subset of bacterial OSTs follow their own rules for acceptor-site specificity, thereby expanding the glycoengineering toolbox with previously unavailable biocatalytic diversity.
