Abstract
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 (CjPglB) 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 CjPglB. Using an ectopic trans-complementation assay for CjPglB 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 CjPglB. 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.
Highlights
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
A glycan comprised of three N-acetylhexosamine (HexNAc) molecules was covalently bound to N261 of HmcA in the motif 259TANGT26318, which lacks a negatively charged amino acid in the − 2 position. This would suggest that D. gigas PglB (DgPglB) may not follow the minus two rule seen for Campylobacter jejuni PglB (CjPglB); the substrate specificity of DgPglB remains to be unequivocally determined
This panel spanned four classes of bacteria, with 19 OSTs from species belonging to the epsilonproteobacteria, 4 OSTs from deltaproteobacteria (Geobacter and Desulfovibrio sp.), and 1 OST from aquificae (Hydrogenivirga sp.) (Fig. 1a)
Summary
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. The OSTs from Campylobacter coli, Campylobacter upsaliensis, Desulfovibrio desulfuricans, Desulfovibrio gigas, and Desulfovibrio vulgaris, exhibited significantly relaxed specificity towards the −2 position compared to CjPglB These enzymes glycosylated minimal N-X-T motifs in multiple targets and each followed unique, as yet unknown, rules governing acceptor-site preferences. Two notable exceptions were reported recently whereby homologous PglBs from Campylobacter lari (ClPglB) and Desulfovibrio desulfuricans (DdPglB) were evaluated using reconstituted protein glycosylation pathways in Escherichia coli cells[16] Both ClPglB and DdPglB catalyzed a low level of glycosylation at a nonconsensus sequon, NNN274ST, in AcrA13,17, a naturally occurring N-linked glycoprotein from C. jejuni that serves as a model glycosylation target. D. gigas PglB (DgPglB) exhibited the most relaxed specificity based on its unique ability to tolerate nearly any residue in the − 2 position and glycosylate the Fc domain of human immunoglobulin G (IgG), the first example of native Fc glycosylation in E. coli
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