Engineering N-linked protein glycosylation with diverse O antigen lipopolysaccharide structures in Escherichia coli

Abstract

Campylobacter jejuni has a general N-linked protein glycosylation system that can be functionally transferred to Escherichia coli. In this study, we engineered E. coli cells in a way that two different pathways, protein N-glycosylation and lipopolysaccharide (LPS) biosynthesis, converge at the step in which PglB, the key enzyme of the C. jejuni N-glycosylation system, transfers O polysaccharide from a lipid carrier (undecaprenyl pyrophosphate) to an acceptor protein. PglB was the only protein of the bacterial N-glycosylation machinery both necessary and sufficient for the transfer. The relaxed specificity of the PglB oligosaccharyltransferase toward the glycan structure was exploited to create novel N-glycan structures containing two distinct E. coli or Pseudomonas aeruginosa O antigens. PglB-mediated transfer of polysaccharides might be valuable for in vivo production of O polysaccharides-protein conjugates for use as antibacterial vaccines.conjugate vaccines ͉ oligosaccharyltransferase ͉ STT3 ͉ glycoengineering

Recombinant Glycans on an S‐Layer Self‐Assembly Protein: A New Dimension for Nanopatterned Biomaterials

“…Inspection of the S-layer protein sequence revealed the presence of one putative Nglycosylation site at position N 879 (DVNQT; the glycosylated asparagine residue is underlined), conforming with the amino acid sequence requirement of the oligosaccharyl:protein transferase PglB of C. jejuni, which is the key enzyme for protein Nglycosylation. [22,28,35] Thus, in a first glycosylation approach, the potential of PglB to glycosylate native SgsE with its endogenous heptasaccharide Glc(GalNAc) 5 Bac substrate in E. coli was investigated by expression of ssPelB-A_SgsE from plasmid pET22b-A_SgsE in E. coli BL21 Star (DE3) harboring pACYCpgl. [21] However, no glycosylated protein was visible on a Western immunoblot, implying that the putative N-glycosylation site of SgsE at position N 879 was not recognized by PglB (not shown).…”

“…This approach is based on the finding of Feldman and coworkers, [22,35] who discovered that the oligosaccharyl:protein transferase PglB of C. jejuni is able to transfer glycan chains with different 2-acetamido sugars at the reducing end to the N-glycosylation sites of proteins. [22,29,35] In our study, we investigated the possibility that PglB can also transfer glycan chains with GlcNAc at the reducing end (as present in O7) onto the engineered Thr 620 N-glycosylation site on SgsE.…”

“…This strain should favor the PglB-mediated transfer of O-antigen from its carrier to the protein acceptor, because of accumulation of undecaprenylpyrophosphate-linked polysaccharide. [22] PglB was expressed under the control of the arabinose-inducible BAD promoter from plasmid pMAF10. [22] The two modified S-layer protein forms A_SgsE_T12 and G_SgsE_T12 with the PelB signal peptide, the extended N-glycosylation site from C. jejuni replacing the O-glycosylation site T 620 and a C-terminal hexahistidine-tag were cloned from pET22b into pBAD24 vector, which is arabinose inducible.…”

“…[11] Recent demonstrations of the functional transfer of protein glycosylation pathways into the experimental model organism Escherichia coli at the genetic level opens new avenues for carbohydrate engineering of S-layer proteins. [21,22] Based on the availability of molecular tools, the Pgl protein N-glycosylation system from Campylobacter jejuni [23] and the E. coli O7 antigen biosynthesis system [24] were used for the design of SgsE-neoglycoproteins. The C. jejuni Pgl enzymes synthesize a heptasaccharide with the structure D-GalNAc-α1,4-D-GalNAc-α1,4-(D-Glc-β1,3)-D-GalNAc-α1,4-D-GalNAc-α1,4-D-GalNAc-α1,3-D-Bac, where Bac is 2,4-diacetamido-2,4,6-trideoxy-D-Glc, [25] involving the lipid carrier undecaprenylpyrophosphate.…”

“…[27,28] Using E. coli as a host, it was shown that PglB has relaxed substrate specificity, transferring several O-antigen polysaccharides, carrying a 2-acetamido modification at the reducing-end sugar of the glycan, to distinct protein glycosylation sites. [22] Among the glycans tested in the course of this previous study was also the E. coli O7 antigen with the repeating unit…”