Characterization of exogenous bacterial oligosaccharyltransferases in Escherichia coli reveals the potential for O-linked protein glycosylation in Vibrio cholerae and Burkholderia thailandensis

Abstract

Bacterial protein glycosylation systems from varying species have been functionally reconstituted in Escherichia coli. Both N- and O-linked glycosylation pathways, in which the glycans are first assembled onto lipid carriers and subsequently transferred to acceptor proteins by an oligosaccharyltransferase (OTase), have been documented in bacteria. The identification and characterization of novel OTases with different properties may provide new tools for engineering glycoproteins of biotechnological interest. In the case of OTases involved in O-glycosylation (O-OTases), there is very low sequence homology between those from different bacterial species. The Wzy_C signature domain common to these enzymes is also present in WaaL ligases; enzymes involved in lipopolysaccharide biosynthesis. Therefore, the identification of O-OTases using solely bioinformatic methods is problematic. The hypothetical proteins BTH_I0650 from Burkholderia thailandensis E264 and VC0393 from Vibrio cholerae N16961 contain the Wzy_C domain. In this work, we demonstrate that both proteins have O-OTase activity and renamed them PglL(Bt) and PglL(Vc), respectively, similar to the Neisseria meningitidis counterpart (PglL(Nm)). In E. coli, PglL(Bt) and PglL(Vc) display relaxed glycan and protein specificity. However, effective glycosylation depends upon a specific combination of the protein acceptor, glycan and O-OTase analyzed. This knowledge has important implications in the design of glycoconjugates and provides novel tools for use in glycoengineering applications. The codification of enzymatically active O-OTase in the genomes of members of the Vibrio and Burkholderia genera suggests the presence of still unknown O-glycoproteins in these organisms, which might have a role in bacterial physiology or pathogenesis.

Identification of a General O-linked Protein Glycosylation System in Acinetobacter baumannii and Its Role in Virulence and Biofilm Formation

“…Via a BLAST analysis, we identified a homolog to the N. meningitidis O- OTase PglL (A1S_3176; E-value 1e-9) that contained a Wzy_C motif. This motif is conserved in all O -OTases, but is also found in WaaL ligases, which catalyze the transfer of the O antigen to the Lipid A core [21], [22]. To date, only experimental determination allows the assignment of an ORF containing the Wzy_C motif as either an O -OTase or a ligase [23].…”

Identification of an O‐antigen chain length regulator, WzzP, in Porphyromonas gingivalis

“…), and Burkholderia thailandensis PglL Bt and V. cholera PglL Vc have an O‐linked oligosaccharyl transferase and contain a Wzy_C domain that is also present in WaaL (Gebhart et al. ). In P. gingivalis , the best‐matched Wzy_C domain was found in PGN_1302 (WaaL) (Rangarajan et al.…”

Identification of Bacterial Protein O-Oligosaccharyltransferases and Their Glycoprotein Substrates

“…This is consistent with Ye777/ waaL XS being a protein O -OTase, as previously speculated [29] and as predicted by our HMM analysis. During the preparation of this manuscript, PglL-like O -OTase BTH_I0650 of B. thailandensis [30], VC0393 of V. cholerae [30] and A1S_3176 of A. baumannii [31] were identified by homology with Neisseria PglL and the presence of the Wzy_C motif followed by experimentation to rule out a WaaL function. Our HMM analysis predicted that these genes were PglL homologs (Table 1), providing further support for our analysis.…”