Mycobacterium tuberculosis glycoproteomics based on ConA-lectin affinity capture of mannosylated proteins.
ABSTRACT A Mycobacterium tuberculosis culture filtrate enriched with mannose-containing proteins was resolved by 2-DE gel. After ConA ligand blotting, 41 proteins were identified by mass spectrometry as putative glycoproteins with 34 of them new probably mannosylated proteins. These results contribute to the construction of the ConA affinity glycoprotein database of M. tuberculosis, and provide useful information for understanding the biological role of glycoproteins in mycobacteria.
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ABSTRACT: Mycobacterium tuberculosis (Mtb) causes tuberculosis, one of the leading causes of fatal infectious diseases worldwide. Cell-cell recognition between the pathogen Mtb and its host are mediated in part by glycosylated proteins. So far, glycoproteins in Mtb are understudied and for only very few glycoproteins glycosylation sites have been described, e.g., alanine and proline rich secreted protein apa, superoxide dismutase SODC, lipoprotein lpqH and MPB83/MPT83. In this study, glycosylated proteins in Mtb culture filtrate were investigated using liquid chromatography-mass spectrometry approaches and bioinformatic analyses. To validate the presence of glycoproteins, several strategies were pursued including collision induced dissociation, high energy collision dissociation and electron transfer dissociation techniques, and bioinformatics analyses involving a neutral loss search for glycosylated moieties. After extensive data curation, we report glycosylation sites for thirteen Mtb glycoproteins using a combination of mass spectrometry techniques on a dataset collected from culture filtrate proteins. This is the first glycoproteomics study identifying glycosylation sites on mycobacterial culture filtrate proteins (CFP) on a global scale. This article is part of a Special Issue entitled:Trends in Microbial Proteomics.Journal of proteomics 05/2013; · 5.07 Impact Factor
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ABSTRACT: Virulence of the gastric pathogen Helicobacter pylori (Hp) is directly linked to the pathogens ability to glycosylate proteins; for example, Hp flagellin proteins are heavily glycosylated with the unusual nine-carbon sugar pseudaminic acid, and this modification is absolutely essential for Hp to synthesize functional flagella and colonize the host stomach. Although Hp glycans are linked to pathogenesis, the Hp glycome remains poorly understood; only the two flagellin glycoproteins have been firmly characterized in Hp. Evidence from our laboratory suggests that Hp synthesizes a large number of as-yet unidentified glycoproteins. Here we set out to discover Hp glycoproteins by coupling glycan metabolic labeling with mass spectrometry analysis. An assessment of the subcellular distribution of azide-labeled proteins by Western blot analysis indicated that glycoproteins are present throughout Hp and may therefore serve diverse functions. To identify these species, azide-labeled glycoproteins were tagged via Staudinger ligation, enriched by tandem affinity chromatography, and analyzed by multidimensional protein identification technology. Direct comparison of enriched azide-labeled glycoproteins with a mock-enriched control by both SDS-PAGE and mass spectrometry-based analyses confirmed the selective enrichment of azide-labeled glycoproteins. We identified 125 candidate glycoproteins with diverse biological functions, including those linked with pathogenesis. Mass spectrometry analyses of enriched azide-labeled glycoproteins before and after cleavage of O-linked glycans revealed the presence of Staudinger ligation-glycan adducts in samples only after beta-elimination, confirming the synthesis of O-linked glycoproteins in Hp. Finally, the secreted colonization factors urease alpha and urease beta were biochemically validated as glycosylated proteins via Western blot analysis as well as by mass spectrometry analysis of cleaved glycan products. These data set the stage for the development of glycosylation-based therapeutic strategies, such as new vaccines based on natively glycosylated Hp proteins, to eradicate Hp infection. Broadly, this report validates metabolic labeling as an effective and efficient approach for the identification of bacterial glycoproteins.Molecular & Cellular Proteomics 06/2013; · 7.25 Impact Factor
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ABSTRACT: The fungal and plant plasma membrane H(+)-ATPases play critical roles in the physiology of yeast, plant and protozoa cells. We identified two genes encoding two plasma membrane H(+)-ATPases in the basidiomycete Ustilago maydis, one protein with higher identity to fungal (um02581) and the other to plant (um01205) H(+)-ATPases. Proton pumping activity was 5-fold higher when cells were grown in minimal medium with ethanol compared to cells cultured in rich YPD medium, but total vanadate-sensitive ATPase activity was the same in both conditions. In contrast, the activity in cells cultured in minimal medium with glucose was 2-fold higher than in YPD or ethanol, implicating mechanisms for the regulation of the plasma membrane ATPase activity in U. maydis. Analysis of gene expression of the H(+)-ATPases from cells grown under different conditions, showed that the transcript expression of um01205 (plant-type) was higher than that of um02581 (fungal-type). The translation of the two proteins was confirmed by mass spectrometry analysis. Unlike baker's yeast and plant H(+)-ATPases, where the activity is increased by a short incubation with glucose or sucrose, respectively, U. maydis H(+)-ATPase activity did not change in response to these sugars. Sequence analysis of the two U. maydis H(+)-ATPases revealed the lack of canonical threonine and serine residues which are targets of protein kinases in Saccharomyces cerevisiae and Arabidopsis thaliana plasma membrane H(+)-ATPases, suggesting that phosphorylation of the U. maydis enzymes occurs at different amino acid residues.Journal of Bioenergetics 07/2013; · 1.60 Impact Factor