Mycobacterium tuberculosis Glycoproteomics Based on ConA-Lectin Affinity Capture of Mannosylated Proteins

Departamento de Inmunologia, Instituto de Investigaciones Biomedicas, Universidad Nacional Autonoma de Mexico, Mexico.
Journal of Proteome Research (Impact Factor: 4.25). 03/2009; 8(2):721-33. DOI: 10.1021/pr800756a
Source: PubMed


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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Available from: Antonio J. Vallecillo, Jun 22, 2015

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Article: Mycobacterium tuberculosis Glycoproteomics Based on ConA-Lectin Affinity Capture of Mannosylated Proteins

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    • "LprG is an antigenic lipoprotein with a potential role in bacterial cell wall assembly. This protein was previously described as P27 in the Mycobacterium tuberculosis complex [2] [3] [4] [5] and the lprG gene has been annotated as Rv1411c in the "
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    ABSTRACT: The lprG-p55 operon of Mycobacterium tuberculosis and Mycobacterium bovis is involved in the transport of toxic compounds. P55 is an efflux pump that provides resistance to several drugs, while LprG is a lipoprotein that modulates the host's immune response against mycobacteria. The knockout mutation of this operon severely reduces the replication of both mycobacterial species during infection in mice and increases susceptibility to toxic compounds. In order to gain insight into the function of LprG in the Mycobacterium avium complex, in this study, we assayed the effect of the deletion of lprG gene in the D4ER strain of Mycobacterium avium subsp. avium. The replacement of lprG gene with a hygromycin cassette caused a polar effect on the expression of p55. Also, a twofold decrease in ethidium bromide susceptibility was observed and the resistance to the antibiotics rifampicin, amikacin, linezolid, and rifabutin was impaired in the mutant strain. In addition, the mutation decreased the virulence of the bacteria in macrophages in vitro and in a mice model in vivo. These findings clearly indicate that functional LprG and P55 are necessary for the correct transport of toxic compounds and for the survival of MAA in vitro and in vivo.
    Full-text · Article · May 2014 · BioMed Research International
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    • "For example, the unusual structures of bacterial glycans, which often contain amino-and deoxy-carbohydrates exclusively found in bacteria (12, 23–25), hampers their identification using existing tools. Though methods such as the use of glycan-binding reagents (20,24,26,27) and periodic acid/hydrazide glycan labeling (15) have successfully detected glycoproteins in a range of bacteria, they present limitations. Glycan bindingbased methods are often limited because of the unavailability of lectins or antibodies with binding specificity for glycosylated proteins in the bacteria of interest (14, 22). "
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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.
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    • "The cloning, expression and purification of the M. tuberculosis Rv1411c (p27) protein was performed as previously described [45]. The full length of Rv1818c (PE_PGRS33) gene cloned into PET15b fused to a histidine tag was a kind gift from Dr. M.J. Brennan (CBER, FDA, Bethesda, MD, USA). "
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