Desulfovibrio desulfuricans PglB homolog possesses oligosaccharyltransferase activity with relaxed glycan specificity and distinct protein acceptor sequence requirements

Oligosaccharyltransferases (OTases) are responsible for the transfer of carbohydrates from lipid carriers to acceptor proteins and are present in all domains of life. In bacteria, the most studied member of this family is PglB from Campylobacter jejuni (PglBCj). This enzyme is functional in Escherichia coli and, contrary to its eukaryotic counterparts, has the ability to transfer a variety of oligo- and polysaccharides to protein carriers in vivo. Phylogenetic analysis revealed that in the delta proteobacteria Desulfovibrio sp., the PglB homolog is more closely related to eukaryotic and archaeal OTases than to its Campylobacter counterparts. Genetic analysis revealed the presence of a putative operon that might encode all enzymes required for N-glycosylation in Desulfovibrio desulfuricans. D. desulfuricans PglB (PglBDd) was cloned and successfully expressed in E. coli, and its activity was confirmed by transferring the C. jejuni heptasaccharide onto the model protein acceptor AcrA. In contrast to PglBCj, which adds two glycan chains to AcrA, a single oligosaccharide was attached to the protein by PglBDd. Site-directed mutagenesis of the five putative N-X-S/T glycosylation sites in AcrA and mass spectrometry analysis showed that PglBDd does not recognize the "conventional bacterial glycosylation sequon" consisting of the sequence D/E-X1-N-X2-S/T (where X1 and X2 are any amino acid except proline), and instead used a different site for the attachment of the oligosaccharide than PglBCj. Furthermore, PglBDd exhibited relaxed glycan specificity, being able to transfer mono- and polysaccharides to AcrA. Our analysis constitutes the first characterization of an OTase from delta-proteobacteria involved in N-linked protein glycosylation.