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In the three domains of life, lipid-linked glycans contribute to various cellular processes ranging from protein glycosylation to glycosylphosphatidylinositol anchor biosynthesis to peptidoglycan assembly. In generating many of these glycoconjugates, phosphorylated polyprenol-based lipids are charged with single sugars by polyprenol phosphate glycosyltransferases. The resultant substrates serve as glycosyltransferase donors, complementing the more common nucleoside diphosphate sugars. It had been accepted that these polyprenol phosphate glycosyltransferases acted similarly, given their considerable sequence homology. Recent findings, however, suggest that matters may not be so simple. In this Opinion we propose that the stereochemistry of sugar addition by polyprenol phosphate glycosyltransferases is not conserved across evolution, even though the GT-A fold that characterizes such enzymes is omnipresent. Keywords: dolichol phosphate, dolichol phosphate glucose synthase, dolichol phosphate mannose synthase, polyprenol phosphate, protein glycosylation, stereochemistry, Israel Science Foundation (Grant 109/16), National Institutes of Health (U.S.) (Grant GM-039334), Israel Science Foundation (Grant 2253/15), National Natural Science Foundation (China) (Grant 2193/16), German-Israeli Foundation for Scientific Research and Development (Grant I-1290-416.13/2015)

Site selectivity of protein N-linked glycosylation is dependent on many factors, including accessibility of the modification site, amino acid composition of the glycosylation consensus sequence, and cellular localization of target proteins. Previous studies have shown that the bacterial oligosaccharyltransferase, PglB, of Campylobacter jejuni favors acceptor proteins with consensus sequences ((D/E)X[subscript 1]NX[subscript 2](S/T), where X[subscript 1,2] ≠ proline) in flexible, solvent-exposed motifs; however, several native glycoproteins are known to harbor consensus sequences within structured regions of the acceptor protein, suggesting that unfolding or partial unfolding is required for efficient N-linked glycosylation in the native environment. To derive insight into these observations, we generated structural homology models of the N-linked glycoproteome of C. jejuni. This evaluation highlights the potential diversity of secondary structural conformations of previously identified N-linked glycosylation sequons. Detailed assessment of PglB activity with a structurally characterized acceptor protein, PEB3, demonstrated that this natively folded substrate protein is not efficiently glycosylated in vitro, whereas structural destabilization increases glycosylation efficiency. Furthermore, in vivo glycosylation studies in both glyco-competent Escherichia coli and the native system, C. jejuni, revealed that efficient glycosylation of glycoproteins, AcrA and PEB3, depends on translocation to the periplasmic space via the general secretory pathway. Our studies provide quantitative evidence that many acceptor proteins are likely to be N-linked-glycosylated before complete folding and suggest that PglB activity is coupled to general secretion-mediated translocation to the periplasm. This work extends our understanding of the molecular mechanisms underlying N-linked glycosylation in bacteria. Keywords: N-linked glycosylation, oligosaccharyltransferase, protein folding, pr, National Institutes of Health (U.S.) (GM-039334), National Institutes of Health (U.S.) (GM-007270)

Post and co-translational protein processing reactions show a broad range of both specificity and reactivity. One such reaction, asparagine linked glycosylation, is specific for Asn-Xaa-Thr/Ser sequences and is interesting for the remarkable reactivity shown by the asparagine sidechain nitrogen. The effects of the substrate conformation are examined, and it is found that the ability to form an Asx-turn is important for the peptide to undergo glycosylation. The mechanism by which the reactivity of the asparagine is increased is investigated by examining the binding properties of a series of peptides containing asparagine analogs. A model mechanism, consistent with the observed results, is proposed.