Direct Biochemical Evidence for the Utilization of UDP-bacillosamine by PglC, an Essential Glycosyl-1-phosphate Transferase in the Campylobacter jejuni N-Linked Glycosylation Pathway ² Kerney Jebrell Glover, ‡ Eranthie Weerapana, ‡ Mark M. Chen, and Barbara Imperiali* Departments of Chemistry and Biology, Massachusetts Institute of Technology, 77 Massachusetts AVenue, Cambridge, Massachusetts 02139 ReceiVed January 31, 2006 ABSTRACT: Campylobacter jejuni has a general N-linked glycosylation pathway, encoded by the pgl gene cluster. In C. jejuni, a heptasaccharide is transferred from an undecaprenyl pyrophosphate donor [GalNAc- R1,4-GalNAc-R1,4-(Glc1,3)-GalNAc-R1,4-GalNAc-R1,4-GalNAc-R1,3-Bac-R1-PP-undecaprenyl, where Bac is bacillosamine (2,4-diacetamido-2,4,6-trideoxyglucose)] to the asparagine side chain of target proteins at the Asn-X-Ser/Thr motif. In this study, we have cloned, overexpressed in Escherichia coli, and purified PglC, the glycosyl-1-phosphate transferase responsible for the first step in the biosynthesis of the undecaprenyl-linked heptasaccharide donor. In addition, we report the first synthetic route to uridine 5′- diphosphobacillosamine. Using the uridine 5′-diphosphobacillosamine and undecaprenyl phosphate, we demonstrate the ability of PglC to produce undecaprenyl pyrophosphate bacillosamine using radiolabeled HPLC and mass spectral analysis. In addition, we revealed that PglC does not accept uridine 5′-diphospho- N-acetylglucosamine or uridine 5′-diphospho-N-acetylgalactosamine as substrates but will accept uridine 5′-diphospho-6-hydroxybacillosamine, an analogue of bacillosamine that retains the C-6 hydroxyl functionality from the biosynthetic precursor. The in vitro characterization of PglC as a bacillosamine 1-phosphoryl transferase provides direct evidence for the early steps in the C. jejuni N-linked glycosylation pathway, and the coupling of PglC with the latter glycosyltransferases (PglA, PglJ, PglH, and PglI) allows for the “one-pot” chemoenzymatic synthesis of the undecaprenyl pyrophosphate heptasaccharide donor. Recent studies have revealed the presence of N-linked glycosylation in the bacterial domain. Specifically, the Gram- negative bacterium Campylobacter jejuni has a general N-linked glycosylation pathway encoded by the pgl gene locus (1-4). C. jejuni 1 has been implicated in a variety of gastroenterological disorders, and evidence suggests that these N-linked glycans play a major role in host adherence, invasion, and colonization (5). Similar to the analogous process in eukaryotes, N-linked glycosylation involves the transfer of a preassembled glycan from a polyisoprenyl pyrophosphate-linked donor onto the asparagine side chain at an Asn-Xaa-Ser/Thr motif. In C. jejuni, the glycan donor is an undecaprenyl pyrophosphate (Und-PP) linked hep- tasaccharide, GalNAc-R1,4-GalNAc-R1,4-(Glc1,3)-GalNAc- R1,4-GalNAc-R1,4-GalNAc-R1,3-Bac-R1-PP-Und, where Bac is the unusual sugar, bacillosamine (2,4-diacetamido-2,4,6- trideoxyglucose) (6) (Figure 1). In the pgl gene locus, bioinformatic analysis has identified five glycosyltransferases (PglC, PglA, PglJ, PglH, and PglI) (3, 7). Mutational and in vitro analyses of these glycosyl- transferases have established their involvement in the as- sembly of the undecaprenyl pyrophosphate-linked heptasac- charide. PglA adds a GalNAc residue to undecaprenyl pyrophosphate bacillosamine (Und-PP-Bac) to form the disaccharide, PglJ adds the second GalNAc residue to form the trisaccharide, PglH adds the next three GalNAc residues to afford the hexasaccharide, and finally PglI adds the single branching glucose residue (8, 9). However, the role of PglC, a putative glycosyl-1-phosphate transferase, which is pro- posed to be involved in the synthesis of the first Und-PP- glycan intermediate, has not been fully determined. ² This work was supported in full by the NIH: Grant GM039334 to B.I. and postdoctoral fellowship (Grant GM65699) to K.J.G. * To whom correspondence should be addressed. E-mail: imper@mit.edu. ‡ These authors contributed equally to this work. 1 Abbreviations: C. jejuni, Campylobacter jejuni; UDP-Bac, uridine 5′-diphosphobacillosamine; UDP-GlcNAc, uridine 5′-diphospho-N- acetylglucosamine; UDP-GalNAc, uridine 5′-diphospho-N-acetylga- lactosamine; Und-P, undecaprenyl phosphate; Glc, glucose; HexNAc, N-acetylhexosamine; CHAPSO, 3-[(3-cholamidopropyl)dimethylam- monio]-2-hydroxy-1-propanesulfonate; CHAPS, 3-[(3-cholamidopro- pyl)dimethylammonio]-1-propanesulfonate. FIGURE 1: Chemical structure of the undecaprenyl pyrophosphate- linked heptasaccharide. 5343 Biochemistry 2006, 45, 5343-5350 10.1021/bi0602056 CCC: $33.50 © 2006 American Chemical Society Published on Web 04/05/2006