1887

Abstract

Membrane-associated proteins of offer a challenge, as well as an opportunity, in the quest for better therapeutic and prophylactic interventions against tuberculosis. The authors have previously reported that extraction with the detergent Triton X-114 (TX-114) is a useful step in proteomic analysis of mycobacterial cell membranes, and detergent-soluble membrane proteins of mycobacteria are potent stimulators of human T cells. In this study 1-D and 2-D gel electrophoresis-based protocols were used for the analysis of proteins in the TX-114 extract of membranes. Peptide mass mapping (using MALDI-TOF-MS, matrix assisted laser desorption/ionization time of flight mass spectrometry) of 116 samples led to the identification of 105 proteins, 9 of which were new to the proteome. Functional orthologues of 73 of these proteins were also present in , suggesting their relative importance. Bioinformatics predicted that as many as 73 % of the proteins had a hydrophobic disposition. 1-D gel electrophoresis revealed more hydrophobic/transmembrane and basic proteins than 2-D gel electrophoresis. Identified proteins fell into the following major categories: protein synthesis, cell wall biogenesis/architecture and conserved hypotheticals/unknowns. To identify immunodominant proteins of the detergent phase (DP), 14 low-molecular-mass fractions prepared by continuous-elution gel electrophoresis were subjected to T cell activation assays using blood samples from BCG-vaccinated healthy donors from a tuberculosis endemic area. Analysis of the responses (cell proliferation and IFN- production) showed that the immunodominance of certain DP fractions was most probably due to ribosomal proteins, which is consistent with both their specificity for mycobacteria and their abundance. Other membrane-associated proteins, including transmembrane proteins/lipoproteins and ESAT-6, did not appear to contribute significantly to the observed T cell responses.

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2005-07-01
2019-08-21
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vol. , part 7, pp. 2411 - 2419

Protein identification data. Peptide-mass mapping of 116 samples [47 originating from detergent-phase (DP) and 69 from the aqueous-phase (AP) protein pools] led to the identification of 105 proteins. Typically, many proteins were present in >1 sample (mostly from 2-D gel electrophoresis) and many samples (especially from 1-D gel electrophoresis) contained >1 protein. Out of the identified proteins, 73 belonged to DP and 19 to AP protein pools. The remaining 13 proteins were common to both (DP/AP). A total of 9 proteins had no previous references in databases or publications pertaining to the proteome; hence they were considered as ‘new’ to the proteome. Bioinformatic analyses indicated that 77 (73 %) proteins (57 from DP, 12 from AP and 8 from DP/AP) could be considered as ‘membrane-associated’ since their GRAVY scores predicted a hydrophobic character. Similarly, 45 (43 %) proteins (34 from DP, 8 from AP and 3 from DP/AP) were considered as ‘membrane-bound’ as they were predicted to have one or more transmembrane regions with or without a lipid attachment site or signal sequence. Among the 9 newly described proteins, 8 (89 %) possessed transmembrane regions. According to the TubercuList database, the majority of the identified proteins belonged to the following five functional categories: category 2 (information pathways, 32 proteins), category 3 (cell wall and cell processes, 20 proteins), category 7 (intermediary metabolism and respiration, 22 proteins) and category [8+10] (unknowns+conserved hypotheticals, 20 proteins). Most (73 out of 105) proteins had a functional orthologue in .[ PDF file] (486kb)



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