Publications

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

  Glover K.J., Weerapana E., Imperiali B.

  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- ... >> More

  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. << Less

  Proc. Natl. Acad. Sci. U.S.A. 102:14255-14259(2005) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Polyisoprenol specificity in the Campylobacter jejuni N-linked glycosylation pathway.

  Chen M.M., Weerapana E., Ciepichal E., Stupak J., Reid C.W., Swiezewska E., Imperiali B.

  Campylobacter jejuni contains a general N-linked glycosylation pathway in which a heptasaccharide is sequentially assembled onto a polyisoprenyl diphosphate carrier and subsequently transferred to the asparagine side chain of an acceptor protein. The enzymes in the pathway function at a membrane i ... >> More

  Campylobacter jejuni contains a general N-linked glycosylation pathway in which a heptasaccharide is sequentially assembled onto a polyisoprenyl diphosphate carrier and subsequently transferred to the asparagine side chain of an acceptor protein. The enzymes in the pathway function at a membrane interface and have in common amphiphilic membrane-bound polyisoprenyl-linked substrates. Herein, we examine the potential role of the polyisoprene component of the substrates by investigating the relative substrate efficiencies of polyisoprene-modified analogues in individual steps of the pathway. Chemically defined substrates for PglC, PglJ, and PglB are prepared via semisynthetic approaches. The substrates included polyisoprenols of varying length, double bond geometry, and degree of saturation for probing the role of the hydrophobic polyisoprene in substrate specificity. Kinetic analysis reveals that all three enzymes exhibit distinct preferences for the polyisoprenyl carrier whereby cis-double bond geometry and alpha-unsaturation of the native substrate are important features, while the precise polyisoprene length may be less critical. These findings suggest that the polyisoprenyl carrier plays a specific role in the function of these enzymes beyond a purely physical role as a membrane anchor. These studies underscore the potential of the C. jejuni N-linked glycosylation pathway as a system for investigating the biochemical and biophysical roles of polyisoprenyl carriers common to prokaryotic and eukaryotic glycosylation. << Less

  Biochemistry 46:14342-14348(2007) [PubMed] [EuropePMC]