Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Chen MM

PROVIDER: S-EPMC2585822 | biostudies-literature | 2007 Dec

REPOSITORIES: biostudies-literature

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

Chen Mark M MM Weerapana Eranthie E Ciepichal Ewa E Stupak Jacek J Reid Christopher W CW Swiezewska Ewa E Imperiali Barbara B

Biochemistry 20071123 50

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 relativ ...[more]

PMID: 18034500

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Project description:Campylobacter jejuni is a major gastrointestinal pathogen generally acquired via consumption of poorly prepared poultry. N-linked protein glycosylation encoded by the pgl gene cluster targets >80 membrane proteins and is required for both nonsymptomatic chicken colonization and full human virulence. Despite this, the biological functions of N-glycosylation remain unknown. We examined the effects of pgl gene deletion on the C. jejuni proteome using label-based liquid chromatography/tandem mass spectrometry (LC-MS/MS) and validation using data independent acquisition (DIA-SWATH-MS). We quantified 1359 proteins corresponding to ∼84% of the C. jejuni NCTC 11168 genome, and 1080 of these were validated by DIA-SWATH-MS. Deletion of the pglB oligosaccharyltransferase (ΔpglB) resulted in a significant change in abundance of 185 proteins, 137 of which were restored to their wild-type levels by reintroduction of pglB (Δaaz.batpglB::ΔpglB). Deletion of pglB was associated with significantly reduced abundances of pgl targets and increased stress-related proteins, including ClpB, GroEL, GroES, GrpE and DnaK. pglB mutants demonstrated reduced survival following temperature (4 °C and 46 °C) and osmotic (150 mm NaCl) shock and altered biofilm phenotypes compared with wild-type C. jejuni Targeted metabolomics established that pgl negative C. jejuni switched from aspartate (Asp) to proline (Pro) uptake and accumulated intracellular succinate related to proteome changes including elevated PutP/PutA (proline transport and utilization), and reduced DctA/DcuB (aspartate import and succinate export, respectively). ΔpglB chemotaxis to some substrates (Asp, glutamate, succinate and α-ketoglutarate) was reduced and associated with altered abundance of transducer-like (Tlp) proteins. Glycosylation negative C. jejuni were depleted of all respiration-associated proteins that allow the use of alternative electron acceptors under low oxygen. We demonstrate for the first time that N-glycosylation is required for a specific enzyme activity (Nap nitrate reductase) that is associated with reduced abundance of the NapAB glycoproteins. These data indicate a multifactorial role for N-glycosylation in C. jejuni physiology.

Dataset Information

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

Publications

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

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