Kessler E, Safrin M, Gustin J K, Ohman D E
Maurice and Gabriela Goldschleger Eye Research Institute, Tel-Aviv University Sackler Faculty of Medicine, Sheba Medical Center, Tel-Hashomer 52621, Israel.
J Biol Chem. 1998 Nov 13;273(46):30225-31. doi: 10.1074/jbc.273.46.30225.
Pseudomonas aeruginosa elastase and the LasA protease are synthesized as preproenzymes with long amino-terminal propeptides. The elastase propeptide is cleaved autocatalytically in the periplasm to form a transient, inactive elastase-propeptide complex. In contrast, the processing of proLasA does not involve autoproteolysis. In this study, we analyzed short-term P. aeruginosa cultures under conditions that minimize proteolysis and found that an elastase-propeptide complex is secreted, and then the propeptide is degraded extracellularly, apparently by elastase itself. LasA protease, on the other hand, was found to be secreted in its unprocessed 42-kDa proenzyme form. The processing of proLasA occurred extracellularly, and it involved the transient appearance of a 28-kDa intermediate and the respective 14-kDa LasA propeptide fragment. The processing of proLasA in P. aeruginosa strain FRD740, which does not express elastase, also proceeded via the 28-kDa intermediate, but the rate of processing was greatly reduced. This low rate of proLasA processing was further reduced when the activity of a secreted lysine-specific protease was blocked. Purified secreted proteases of P. aeruginosa (i.e. elastase, the lysine-specific protease, and alkaline proteinase) converted proLasA to the active enzyme. Processing by elastase and the lysine-specific enzyme, but not by alkaline proteinase, proceeded via the 28-kDa intermediate, and both were far more effective than alkaline proteinase in converting proLasA to the mature enzyme. We conclude that LasA protease and elastase are secreted with their propeptides, which are then degraded by secreted proteases of P. aeruginosa. In addition to their other functions, the propeptides may play a role in targeting their respective enzymes across the outer membrane.
铜绿假单胞菌弹性蛋白酶和LasA蛋白酶以具有长氨基末端前肽的前体酶形式合成。弹性蛋白酶前肽在周质中自催化裂解，形成一种短暂的、无活性的弹性蛋白酶-前肽复合物。相比之下，前LasA的加工不涉及自蛋白酶解。在本研究中，我们在使蛋白水解最小化的条件下分析了铜绿假单胞菌的短期培养物，发现弹性蛋白酶-前肽复合物被分泌，然后前肽在细胞外被降解，显然是被弹性蛋白酶自身降解。另一方面，发现LasA蛋白酶以未加工的42 kDa前体酶形式被分泌。前LasA的加工发生在细胞外，它涉及一个28 kDa中间体和相应的14 kDa LasA前肽片段的短暂出现。在不表达弹性蛋白酶的铜绿假单胞菌菌株FRD740中，前LasA的加工也通过28 kDa中间体进行，但加工速率大大降低。当一种分泌的赖氨酸特异性蛋白酶的活性被阻断时，前LasA的这种低加工速率进一步降低。纯化的铜绿假单胞菌分泌蛋白酶（即弹性蛋白酶、赖氨酸特异性蛋白酶和碱性蛋白酶）将前LasA转化为活性酶。弹性蛋白酶和赖氨酸特异性酶的加工通过28 kDa中间体进行，但碱性蛋白酶不行，并且在将前LasA转化为成熟酶方面，两者都比碱性蛋白酶有效得多。我们得出结论，LasA蛋白酶和弹性蛋白酶与其前肽一起被分泌，然后被铜绿假单胞菌的分泌蛋白酶降解。除了它们的其他功能外，前肽可能在将它们各自的酶靶向穿过外膜方面发挥作用。
