Chemoenzymatic synthesis of glycopeptides with PglB, a bacterial oligosaccharyl transferase from Campylobacter jejuni

Abstract

The gram-negative bacterium Campylobacter jejuni has a general N-linked glycosylation pathway encoded by the pgl gene cluster. One of the proteins in this cluster, PgIB, is thought to be the oligosaccharyl transferase due to its significant homology to Stt3p, a subunit of the yeast oligosaccharyl transferase complex. PgIB has been shown to be involved in catalyzing the transfer of an undecaprenyl-linked heptasaccharide to the asparagine side chain of proteins at the Asn-X-Ser/Thr motif. Using a synthetic disaccharide glycan donor (GaINAc-alpha1,3-bacillosamine-pyrophosphate-undecaprenyl) and a peptide acceptor substrate (KDFNVSKA), we can observe the oligosaccharyl transferase activity of PgIB in vitro. Furthermore, the preparation of additional undecaprenyl-linked glycan variants reveals the ability of PgIB to transfer a wide variety of saccharides. With the demonstration of PgIB activity in vitro, fundamental questions surrounding the mechanism of N-linked glycosylation can now be addressed.

Figure 1

Schematic representation of the C. jejuni pgl N-glycosylation process

Figure 2. Design of peptide substrates and overview of PglA and PglB reactions

(A) Sequence alignment of residues flanking the glycosylation sites of PEB3 andAcrA and resulting consensus peptide. (B) Synthesis of radiolabeled undecaprenyl-linked disaccharide using PglA, synthetic undecaprenyl-pyrophosphate bacillosamine, and tritiated UDP-GalNAc. Bold highlights position of the tritium label. (C) Overview of desired PglB reaction using lipid-linked saccharide highlighted in (B) and the consensus peptide shown in (A).

Figure 3. In vitro activity of wild type and mutant PglB

(A) Sequence alignment of the highly conserved OT hexa-amino acid motif. The PglB mutant has two alanine substitutions within this motif. (B) Anti-T7 Tag Western blot of bacterial membranes overexpressing PglB and the mutant counterpart. Lane 1, Wild type ; Lane 2, Mutant (C) Plot of glycopeptide product formation as a function of time. Solid line, Wild type; Dashed line, Mutant. This plot is a representation of product formation and should not be interpreted as kinetic data.

Figure 4. Reverse-phase HPLC traces of peptide and glycopeptide products. (These experiments were performed with the more synthetically accessible 6-hydroxybacillosamine derivative)

(A) HPLC trace of peptide after PglB wild type reaction. (B) MALDI-MS of glycopeptide product (C) Radioactive HPLC trace of wild type PglB reaction. (D) HPLC trace of peptide after PglB mutant reaction. (E) MALDI-MS of unglycosylated peptide product. (F) Radioactive HPLC trace of mutant PglB reaction.

Figure 5

Specificity of PglB for undecaprenyl-pyrophosphate linked disaccharides. (³H-GalNAc-X-PP-Und). Solid line, X= bacillosamine; Dashed line, X= GlcNAc; Dotted line, X= 6-hydroxybacillosamine. This plot is a representation of product formation, it is not a representation of kinetic analysis, and strong conclusions about acceptance rates should not be made.

Figure 6. Utilization of various undecaprenyl-pyrophosphate linked saccharide intermediates. The more readily available 6-hydroxybacillosamine substrate was used for this study in place of the native bacillosamine. The variation in the levels of DPM incorporation reflects the different amounts of lipid-linked sugar substrate in each reaction; Therefore conclusions about the relative reaction rates cannot be made

(A) ³H-GalNAc-Bac-PP-Und. (B) GalNAc-³H-GalNAc-Bac-PP-Und. (C) (GalNAc)₄-³H-GalNAc-Bac-PP-Und. (D) (Glc)-(GalNAc)₄-³H-GalNAc-Bac-PP-Und.

Figure 7. Peptide substrate specificity of PglB

(A) Radioactive Assay of product formation. Solid line, octapeptide consensus sequence (KDFNVSKA); Dashed line, tripeptide consensus for yeast OT (Bz-NLT-NHMe). 6-hydroxybacillosamine was used in this study in place of the native bacillosamine substrate. Plot is not a representation of kinetic parameters. (B) HPLC trace of Bz-NLT-NHMe reaction. (C) MALDI-MS of Bz-NLT-NHMe glycopeptide (peak at T_R = 27.0 min).