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

Abstract

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 (PglB(Cj)). 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 (PglB(Dd)) 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 PglB(Cj), which adds two glycan chains to AcrA, a single oligosaccharide was attached to the protein by PglB(Dd). Site-directed mutagenesis of the five putative N-X-S/T glycosylation sites in AcrA and mass spectrometry analysis showed that PglB(Dd) does not recognize the "conventional bacterial glycosylation sequon" consisting of the sequence D/E-X(1)-N-X(2)-S/T (where X(1) and X(2) are any amino acid except proline), and instead used a different site for the attachment of the oligosaccharide than PglB(Cj.). Furthermore, PglB(Dd) 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.

Flagellar Glycosylation: Current Advances

“…However, recent reports on C. jejuni, Haemophilus influenzae, and Desulfovibrio desulfuricans N-glycosylations indicate that there is no conserved core structure in bacteria (Gross et al, 2008;Ielmini & Feldman, 2011;Young et al, 2002). Thus, in prokaryotes many different glycoprotein structures have been observed that display much more variation than those observed in eukaryotes.…”

Characterization of the Structurally Diverse N-Linked Glycans of Campylobacter Species

“…Recently released bacterial genome sequence data indicate that many members of the Deltaproteobacteria-Epsilonproteobacteria subdivision of the Proteobacteria contain pglB orthologues (13,15,25,37,44). Prominent genera within the Epsilonproteobacteria subdivision include Arcobacter, Campylobacter, and Helicobacter.…”

Recent developments in bacterial protein glycan coupling technology and glycoconjugate vaccine design

“…A PglB orthologue from the d-proteobacterium Desulfovibrio desulfuricans was also recently functionally characterized. DdPglB was shown to transfer the C. jejuni heptasaccharide to the acceptor protein AcrA in the recombinant E. coli system (Ielmini & Feldman, 2011). However, a distinct difference in acceptor site specificity was detected.…”

“…Only one asparagine residue was modified by DdPglB and mass spectrometry analysis of the acceptor site revealed the presence of a short, eukaryotic-like NXS/T sequon, demonstrating that DdPglB lacked the requirement for an acidic residue at the+2 position. Ielmini & Feldman (2011) also analysed the ability of DdPglB to transfer other glycans to AcrA and identified DATDH (2, 4-diacetamido-2,4,6-trideoxyhexose) as well as the E. coli O7 O-antigen polysaccharide, which contains an N-acetylglucosamine residue at the reducing end, as substrates for DdPglB. In addition to the CjPglB orthologues characterized to date, novel methods for high-throughput genome sequencing and an increased interest in micro-organisms from deepsea vent habitats have identified CjPglB orthologues in several novel species.…”

“…A surprising finding revealed that the high molecular mass adhesin proteins HMW1 and HMW2 of the Gram-negative c-Proteobacteria non-typeable H. influenzae are N-glycosylated (Grass et al, 2003), thus expanding the protein N-linked glycosylation beyond d-and e-Proteobacteria (Ielmini & Feldman, 2011;Nothaft & Szymanski, 2010). Around 75-80 % of non-typable H. influenzae express HMW1 and HMW2 proteins, which are essential in establishing the adherence of the bacteria to the epithelial cells of the respiratory tract of the human host, thus promoting and initiating colonization (St Geme et al, 1993, 1998.…”