Abstract
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)X1NX2(S/T), where X1,2 ≠ 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.
Highlights
The heptasaccharide is transferred en bloc to an amide nitrogen of an asparagine located within a consensus sequence ((D/E)X1NX2(S/T), X1,2 proline) on an acceptor protein by a single integral membrane OTase, PglB, that is homologous to the catalytic subunit of eukaryotic OTases (STT3) [13, 18] (Fig. 1)
Ͼ60 glycoproteins have been identified from C. jejuni and Campylobacter lari, only four x-ray crystallographic structures (CjaA, PEB3, JlpA, and PglB) and one NMR structure of truncated AcrA have been determined (24, 25, 29 –32)
Through the analysis of native N-glycosylation sites of C. jejuni glycoproteins in vitro and in vivo, we show that PglB substrate selectivity is dependent on the structure and accessibility of the target asparagine residue
Summary
In Silico Analysis of Glycosylation Sites—Previously, a survey of eukaryotic N-linked glycoproteins structures (x-ray crystallographic data) revealed that N-glycosylation sequons adopt diverse secondary structure conformations [26]. Analysis of the four known x-ray crystal structures shows that the bacterial glycosylation consensus sequence adopts various conformational states (Fig. 2A). These four protein structures include five solventexposed glycosylation sites: one in an ␣-helix, one in a structured turn between a -strand and ␣-helix, and three in non-structured loops. PEB3, a C. jejuni homodimeric surfaceassociated protein with immunogenic properties and predicted transporter functions [25, 34], was selected for these studies because it is a reported soluble glycoprotein, and the x-ray crystallographic structure was previously determined, which shows the native glycosylation site in a structurally defined region of the protein [24, 25] (Fig. 2A). Determined using PEB3 x-ray crystallographic structure (PDB code 2HXW)
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