Characterization of posttranslational modification of 19 kDa protein expressed by Mycobacterium avium subspecies paratuberculosis
Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne's disease, a chronic enteritis in ruminants, and has recently been linked to Crohn's disease in humans. To generate an effective vaccine against MAP, it is necessary to identify MAP antigens that trigger protective immunity. Unfortunately, not much is known about MAP proteins despite decades of research. We have previously shown that a 4.8 kb insert from MAP will produce a 16 kDa recombinant protein when expressed in Escherichia coli and 19 kDa recombinant protein when expressed in M smegmatis ( smeg 19K). The difference of 3 kDa in size of these expressed proteins may be related to posttranslational modificatjons that occur in Mycobacterium species. We hypothesized that smeg19K is a lipoglycoprotein since blast analysis revealed approximately 76 % amino acid identity between the MAP 19 kDa protein and a known lipoglycoprotein, the 19 kDa protein of M tuberculosis. This prediction was confirmed following positive staining of smeg19K with Sudan Black 4B, a postelectrophoresis dye used to stain for lipids. Smeg 19K has also stained positively for glycosylation with the lectin concavalin A, a highly specific stain for mannose residues. As expected, treatment with tunicamycin (an antibiotic known to inhibit N-glycosylation) and treatment with deglycosylation assay (non-specific for mannose ), showed no reduction in size of 19 kDa glycolipoproteins. Since covalent modification of proteins with acyl or glycosyl moieties alter immunogenicity and/or pathogenicity, the study here provides foundation for future experiments regarding the antigenicity of MAP 19 kDa lipoglycoprotein and its role in disease pathogenicity.
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Naser, Saleh A.
Bachelor of Science (B.S.)
Burnett College of Biomedical Sciences
Molecular Biology and Microbiology
Arts and Sciences -- Dissertations, Academic;Dissertations, Academic -- Arts and Sciences
Length of Campus-only Access
Honors in the Major Thesis
Spinelli, Natalia, "Characterization of posttranslational modification of 19 kDa protein expressed by Mycobacterium avium subspecies paratuberculosis" (2008). HIM 1990-2015. 756.