In vitro assembly of the undecaprenylpyrophosphate-linked heptasaccharide for prokaryotic N-linked glycosylation

Abstract

Campylobacter jejuni has a general N-linked glycosylation pathway (encoded by the pgl gene cluster), which culminates in the transfer of a heptasaccharide: GalNAc-alpha1,4-GalNAc-alpha1,4-(Glcbeta1,3)-GalNAc-alpha1,4-GalNAc-alpha1,4-GalNAc-alpha1,3-Bac [where Bac is bacillosamine (2,4-diacetamido-2,4,6-trideoxyglucose)] from a membrane-anchored undecaprenylpyrophosphate (Und-PP)-linked donor to the asparagine side chain of proteins at the Asn-X-Ser/Thr motif. Herein we report, the cloning, overexpression, and purification of four of the glycosyltransferases (PglA, PglH, PglI, and PglJ) responsible for the biosynthesis of the Und-PP-linked heptasaccharide. Starting with chemically synthesized Und-PP-linked Bac and various combinations of enzymes, we have deduced the precise functions of these glycosyltransferases. PglA and PglJ add the first two GalNAc residues on to the isoprenoid-linked Bac carrier, respectively. Elongation of the trisaccharide with PglH results in a hexasaccharide revealing the polymerase activity of PglH. The final branching glucose is then added by PglI, which prefers native lipids for optimal activity. The sequential activities of the glycosyl transferases in the pathway can be reconstituted in vitro. This pathway represents an ideal venue for investigating the integrated functions of a series of enzymatic processes that occur at a membrane interface.

Fig. 1.

Chemical structure of the Und-PP-linked heptasaccharide [GalNAc-α1,4-GalNAc-α1,4- (Glcβ1,3)-GalNAc-α1,4-GalNAc-α1,4-GalNAc-α1,3-Bac- α-1-PP-Und, where Bac is bacillosamine (2,4-diacetamido-2,4,6-trideoxyglucose)].

Fig. 2.

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

Fig. 3.

Current model of the C. jejuni pgl N-glycosylation pathway.

Fig. 4.

Ni-NTA purified glycosyl transferases. (A) Coomassie-stained polyacrylamide gel. (B) Anti-T7-Tag Western blot analysis. M_r markers (lane 1), PglA 43 kDa (lane 2), PglH 41 kDa (lane 3), PglI 36 kDa (lane 4), and PglJ 41 kDa (lane 5). The higher M_r band observed in lane 4 is due to protein oligomerization. The higher M_r band in lane 3 does not react with the anti-T7 Ab and represents an impurity brought through the purification.

Fig. 5.

HPLC traces of the 2AB-labeled saccharide products. (A) PglA. (B) PglA + PglJ. (C) PglA + PglJ +PglH. (D) PglA + PglJ + PglH first then extracted followed by addition of PglI (bacterial membrane fraction). Asterisks denote major saccharide peaks. The two peaks at 25 and 28 min are present in each trace and represent unknown byproducts of the 2AB-labeling process.

Fig. 6.

MALDI-MS of 2AB-labeled saccharide products. (A) PglA. (B) PglA + PglJ. (C) PglA + PglJ +PglH. (D) PglA + PglJ + PglH first then extracted followed by addition of PglI (bacterial membrane fraction). Major peaks represent sodium ion adducts. Additional peaks seen in B and C are due to potassium ion adducts.

Fig. 7.

Plot of Und-PP-disaccharide product formation vs. time by using PglA, UDP-GalNAc, and the three Und-PP-monosaccharides shown; dotted line, Und-PP-GlcNAc; dashed line, Und-PP-6-hydroxybacillosamine; solid line, Und-PP-Bac. Results represent a typical experiment.