Probing the Glycan Biosynthetic Machinery of Campylobacter Jejuni

N-linked protein glycosylation, the formation of a glycosylamide bond between an oligosaccharide and an asparagine side chain, is an essential protein modification required for the function, folding and routing of cell surface receptors, ion channels and lysosomal enzymes. N-linked glycosylation has been identified in eukaryotic and archaeal organisms, and has recently been discovered in the gram-negative enteropathogen Campylobacter jejuni (C. jejuni). <P> C. jejuni is a human-gut mucosal pathogen implicated in gastroenteritis, and is the leading cause of food-borne illness in North America. In addition, C. jejuni infection is associated with the development of peripheral neuropathies such as Guillain-Barre syndrome, an acute immune-mediated neuropathy. The enzymes PgIC, A, J and H are involved in oligosaccharide donor assembly for C. jejuni N-linked glycosylation. Deletion of the gene encoding PglH leads to either loss of C. jejuni viability, or a loss in human and chick cell colonization and hence pathogenicity. Therefore, understanding the activity of this enzyme may lead to potential treatments for C. jejuni infections. PglH is a polymerase enzyme, which catalyzes the transfer of three N-acetyl-galactosamine residues to an undecaprenyl diphosphate-linked glycan. <P> The studies outlined in this proposal focus on the characterization of the PglH reaction and the potential interactions of this enzyme with other proteins in the oligosaccharide donor biosynthesis (or Pgl) pathway. <P> 1) Based on sequence analysis and preliminary kinetic studies of PglH, we hypothesize that the PglH-catalyzed glycosyl transfer reactions occur at different catalytic sites on the enzyme, and that specific amino acid residues catalyze each transfer reaction. In order to test this model, we will utilize a series of mutagenesis and biophysical studies to investigate the mechanism by which this enzyme catalyzes multiple glycosyl transfer steps. <P> 2) The glycan assembling enzymes require a high degree of efficiency and fidelity. We hypothesize that some of this efficiency and fidelity is derived by direct interactions between sequential enzymes in the Pgl pathway. A series of co-immunoprecipitation, dual labeling and luminescence resonance energy transfer experiments will be utilized to investigate the ability of the Pgl proteins to interact with one another and the influence of such interactions on substrate/product flux through the PglJ and PglH reactions.