Functional Characterization of Bacterial Oligosaccharyltransferases Involved in O-Linked Protein Glycosylation

ABSTRACTProtein glycosylation is an important posttranslational modification that occurs in all domains of life. Pilins, the structural components of type IV pili, are O glycosylated inNeisseria meningitidis,Neisseria gonorrhoeae, and some strains ofPseudomonas aeruginosa. In this work, we characterized theP. aeruginosa1244 andN. meningitidisMC58 O glycosylation systems inEscherichia coli. In both cases, sugars are transferred en bloc by an oligosaccharyltransferase (OTase) named PglL inN. meningitidisand PilO inP. aeruginosa. We show that, like PilO, PglL has relaxed glycan specificity. Both OTases are sufficient for glycosylation, but they require translocation of the undecaprenol-pyrophosphate-linked oligosaccharide substrates into the periplasm for activity. Whereas PilO activity is restricted to short oligosaccharides, PglL is able to transfer diverse oligo- and polysaccharides. This functional characterization supports the concept that despite their low sequence similarity, PilO and PglL belong to a new family of “O-OTases” that transfer oligosaccharides from lipid carriers to hydroxylated amino acids in proteins. To date, such activity has not been identified for eukaryotes. To our knowledge, this is the first report describing recombinant O glycoproteins synthesized inE. coli.