Enhancing O-linking oligosaccharyltransferase functionality through directed evolution

Abstract

Polysaccharide protein conjugate vaccines consist of bacterial polysaccharides covalently linked to carrier proteins. Bioconjugate vaccines are a type of polysaccharide protein conjugate vaccine produced by oligosaccharyltransferases, which catalyze the en bloc transfer of polysaccharides to specific amino acid motifs, called sequons, engineered into carrier proteins. The O-linking oligosaccharyltransferase PglS has been shown to have the broadest substrate repertoire, transferring virtually any saccharide to engineered carrier proteins, making it an attractive bioconjugation tool for next generation vaccine development. Yet, the efficiency of glycan transfer varies depending on the polysaccharide substrate. Successful bioconjugation hinges upon the ability of an oligosaccharyltransferase to efficiently transfer a polysaccharide to the engineered carrier protein. Therefore, enhancing glycosylation efficiency to produce carrier proteins that are highly glycosylated is a key aspect of developing scalable processes. Using directed evolution and the group B Streptococcal serotype V capsular polysaccharide as substrate, we identified single amino acid substitutions in PglS that improved enzymatic transfer of the group B Streptococcal serotype V polysaccharide to carrier proteins. Combinatorial amino acid substitutions and the incorporation of multiple sequons in the carrier protein further increased production and quality of the bioconjugate as determined by enzyme-linked immunosorbant assay and mass spectrometry (MS). Unexpectedly, the PglS variants were found to glycosylate two independent serine residues located within the sequon, a phenomenon not observed for the wildtype enzyme, resulting in significantly enhanced glycosylation activity. Such engineered PglS oligosaccharyltransferases, which increase the ratio of polysaccharide to carrier protein, are expected to improve large scale bioconjugation processes.

Keywords: OTase; bioconjugation; directed evolution; oligosaccharyltransferase; protein glycosylation.