Abstract
Conjugate vaccines belong to the most efficient preventive measures against life-threatening bacterial infections. Functional expression of N-oligosaccharyltransferase (N-OST) PglB of Campylobacter jejuni in Escherichia coli enables a simplified production of glycoconjugate vaccines in prokaryotic cells. Polysaccharide antigens of pathogenic bacteria can be covalently coupled to immunogenic acceptor proteins bearing engineered glycosylation sites. Transfer efficiency of PglBCj is low for certain heterologous polysaccharide substrates. In this study, we increased glycosylation rates for Salmonella enterica sv. Typhimurium LT2 O antigen (which lacks N-acetyl sugars) and Staphylococcus aureus CP5 polysaccharides by structure-guided engineering of PglB. A three-dimensional homology model of membrane-associated PglBCj, docked to the natural C. jejuni N-glycan attached to the acceptor peptide, was used to identify potential sugar-interacting residues as targets for mutagenesis. Saturation mutagenesis of an active site residue yielded the enhancing mutation N311V, which facilitated fivefold to 11-fold increased in vivo glycosylation rates as determined by glycoprotein-specific ELISA. Further rounds of in vitro evolution led to a triple mutant S80R-Q287P-N311V enabling a yield improvement of S. enterica LT2 glycoconjugates by a factor of 16. Our results demonstrate that bacterial N-OST can be tailored to specific polysaccharide substrates by structure-guided protein engineering.
Highlights
N-linked glycosylation is a key posttranslational protein modification in eukaryotic cells
To select potential OS interacting residues of PglBCj as targets for mutagenesis, we constructed a structural model of PglBCj
Using previously established techniques for the screening of N-OSTs mutant libraries [33], we were able to demonstrate that PglB of C. jejuni can be evolved for enhanced transfer of heterologous polysaccharide substrates
Summary
N-linked glycosylation is a key posttranslational protein modification in eukaryotic cells. PglBCj has an extraordinarily relaxed specificity for the lipid-linked polysaccharide substrate. Does it transfer the natural C. jejuni oligosaccharides (OSs) (figure 1a), and O antigen lipopolysaccharide structures of numerous Gram-negative bacteria and capsular antigen polysaccharides of Gram-positive bacteria [7 –11]. It is possible to combine this relaxed glycan specificity with the introduction of glycosylation site consensus sequences at specific positions of any desired protein for the production of custom glycoproteins in E. coli [12,13]. This technology is of particular value for the production of conjugate vaccines where surface polysaccharide antigens
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