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

Campylobacter jejuni has a general Winked glycosylation pathway (encoded by the pgl gene cluster), which culminates in the transfer of a heptasaccharide: GalNAc-alpha 1,4-GalNAc-alpha 1,4-(Glc beta 1,3)GalNAc-alpha 1,4-GalNAc-alpha 1,4-GalNAc-alpha 1,3-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 (PgIA, PgIH, PgII,and PgIJ) 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. PgIA and PgIJ add the first two GaINAc residues on to the isoprenoid-linked Bac carrier, respectively. Elongation of the trisaccharide with PgIH results in a hexasaccharicle revealing the polymerase activity of PgIH. The final branching glucose is then added by PgII, 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.