Export of recombinant Mycobacterium tuberculosis superoxide dismutase is dependent upon both information in the protein and mycobacterial export machinery -: A model for studying export of leaderless proteins by pathogenic mycobacteria
Export of recombinant Mycobacterium tuberculosis superoxide dismutase is dependent upon both information in the protein and mycobacterial export machinery -: A model for studying export of leaderless proteins by pathogenic mycobacteria
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DOI：
10.1074/jbc.274.7.4281
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发表时间：
1999-02-12
影响因子：
4.8
通讯作者：
Horwitz, MA
中科院分区：
文献类型：
--
作者：
Harth, G;Horwitz, MA
我们研究了结核分枝杆菌10种主要胞外蛋白之一的酶活性超氧化物歧化酶在其天然宿主和异源宿主耻垢分枝杆菌中的表达和胞外释放。我们发现，结核分枝杆菌超氧化物歧化酶基因编码类似于23，000的M-r的无前导多肽，代表该酶的四个相同亚基之一，在正常生长条件下组成型表达，在过氧化氢应激条件下以B倍增加的水平表达。高致病性分枝杆菌M.结核分枝杆菌表达的超氧化物歧化酶是非致病性分枝杆菌M的93倍。结核分枝杆菌和结核分枝杆菌的表达量分别为21%和76%，结核分枝杆菌和结核分枝杆菌的表达量分别为21%和76%，结核分枝杆菌和结核分枝杆菌的表达量是结核分枝杆菌和结核分枝杆菌的350倍。smeiglitazone、重组M.结核菌超氧化物歧化酶的表达水平是内源酶的8.4倍，输出比例（66%）接近同源宿主; smeglycoprotein输出的重组酶比内源酶多26倍。有趣的是，M.结核和M.当酶在重组宿主中表达时和当纯化的酶一起孵育时，smeglycerides酶容易地和化学计量地彼此交换，形成四个亚基的五种不同复合物;然而，每个亚基保留其特征金属离子，即IM的铁。结核和锰对M.与细胞相关酶相比，重组IM，smegalloy的上清液酶富集了M，结核杆菌酶亚基，与M的优先输出一致。结核酶重组耻垢分枝结核超氧化物歧化酶反式补充了超氧化物歧化酶缺陷型大肠杆菌，导致菌株对氧化应激的敏感性降低，但该酶不从该非分枝杆菌宿主输出。我们的研究结果表明，结核分枝杆菌超氧化物歧化酶的出口信息包含在蛋白质中，但出口还需要特定于分枝杆菌的出口机制。
We have investigated the expression and extracellular release of enzymatically active superoxide dismutase, one of the 10 major extracellular proteins of Mycobacterium tuberculosis, both in its native host and in the heterologous host Mycobacterium smegmatis, We found that the M, tuberculosis superoxide dismutase gene, encoding a leaderless polypeptide of M-r similar to 23,000 representing one of the four identical subunits of the enzyme, is expressed constitutively under normal growth conditions and at a B-fold increased level under conditions of hydrogen peroxide stress. The highly pathogenic mycobacterium M. tuberculosis expresses 93-fold more superoxide dismutase than the nonpathogenic mycobacterium M. smegmatis, and it exports a much higher proportion of expressed enzyme (76 versus 21%); taking both expression and export into consideration, IM, tuberculosis exports similar to 350-fold more enzyme than M, smegmatis, In M. smegmatis, recombinant M. tuberculosis superoxide dismutase is expressed at 8.4 times the level of the endogenous enzyme and the proportion exported (66%) approaches that in the homologous host; hence M. smegmatis exports up to 26-fold more of the recombinant than endogenous enzyme. Interestingly, subunits of the M. tuberculosis and M. smegmatis enzymes readily and stoichiometrically exchange with each other, forming five different complexes of four subunits, both when the enzymes are expressed in the recombinant host and when the purified enzymes are incubated together; however, each subunit retains its characteristic metal ion, iron for IM. tuberculosis and manganese for M.. smegmatis, Compared with the cell-associated enzyme, the supernatant enzyme of recombinant IM, smegmatis is enriched for M, tuberculosis enzyme subunits, consistent with preferential export of the M. tuberculosis enzyme. Recombinant M. tuberculosis superoxide dismutase transcomplements a superoxide dismutase-deficient Escherichia coli, resulting in a reduction of sensitivity of the strain to oxidative stress, but the enzyme is not exported from this nonmycobacterial host. Our findings indicate that the information for export of the M, tuberculosis superoxide dismutase is contained within the protein but that export additionally requires export machinery specific to mycobacteria.