Bacterial N-Glycosylation Efficiency Is Dependent on the Structural Context of Target Sequons

• Published in: The Journal of biological chemistry Vol. 291; no. 42; pp. 22001 - 22010
• Main Authors: Silverman, Julie Michelle, Imperiali, Barbara
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 14.10.2016
• Subjects: Bacterial Secretion Systems - genetics; Bacterial Secretion Systems - metabolism; Campylobacter jejuni - genetics; Campylobacter jejuni - metabolism; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Glycobiology and Extracellular Matrices; Glycosylation; Hexosyltransferases - genetics; Hexosyltransferases - metabolism; Lipoproteins - genetics; Lipoproteins - metabolism; Membrane Proteins - genetics; Membrane Proteins - metabolism; Membrane Transport Proteins - genetics; Membrane Transport Proteins - metabolism; Periplasm - genetics; Periplasm - metabolism; oligosaccharyltransferase; protein folding; N-linked glycosylation; substrate specificity; protein translocation
• DOI: 10.1074/jbc.m116.747121
• ISSN: 0021-9258

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 NX (S/T), where X ≠ 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.