Friedman T C, Gordon V M, Leppla S H, Klimpel K R, Birch N P, Loh Y P
Section on Cellular Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
Arch Biochem Biophys. 1995 Jan 10;316(1):5-13. doi: 10.1006/abbi.1995.1002.
Protective antigen (PA), an 83-kDa protein produced by Bacillus anthracis, requires proteolytic activation at a tetrabasic site (RKKR167) before it can combine with either edema factor or lethal factor on the cell surface. The complex is then endocytosed and the target cell intoxicated. Previous work has demonstrated that furin, a ubiquitously distributed, subtilisin-like protease, can perform this cleavage. In this study, another member of the furin family, PC1 (SPC3), was tested as a putative processing enzyme for PA. Recombinant PC1, partially purified from the medium of stably transfected L-cells, cleaved PA to a 63-kDa fragment (PA63) and a 20-kDa fragment (PA20). Amino-terminal sequence analysis of the 63 kDa product demonstrated that cleavage occurred between Arg167 and Ser168. The pH optimum for in vitro PA cleavage was 6.0 and the enzymatic activity was calcium-dependent. Medium from untransfected L-cells did not cleave PA. Site-directed mutagenesis of the tetrabasic cleavage site revealed that PC1 preferred to cleave sequences containing basic residues at positions -1 and -4 relative to the wild-type cleavage site, demonstrating that PC1 can cleave substrates at a monobasic residue site in vitro. Substrates having basic residues at the -1 and -2 positions were cleaved with approximately twofold less efficiency than wild-type PA. Mutants of PA containing basic residues in positions -1 and either -2 or -4 of the cleavage site were predicted to be substrates for PC1 and were more toxic to L-cells expressing PC1 than to untransfected L-cells. These results demonstrate that PA is cleaved by PC1 in vivo. Membranes from bovine intermediate lobe secretory vesicles which contain both prohormone convertases, PC1 and PC2, also cleaved PA to PA63 with a pH optimum of 5.5. Immunodepletion studies using antisera against PC1 and PC2 showed that these are the enzymes primarily responsible for the cleavage of PA in the membrane preparation. Thus, both recombinant PC1 and a membrane preparation containing endogenous PC1 can activate PA.
保护性抗原（PA）是炭疽芽孢杆菌产生的一种83 kDa蛋白，在与水肿因子或致死因子在细胞表面结合之前，需要在四碱基位点（RKKR167）进行蛋白水解激活。然后该复合物被内吞，靶细胞中毒。先前的研究表明，弗林蛋白酶（一种广泛分布的枯草杆菌蛋白酶样蛋白酶）可以进行这种切割。在本研究中，测试了弗林蛋白酶家族的另一个成员PC1（SPC3）作为PA的假定加工酶。从稳定转染的L细胞培养基中部分纯化的重组PC1将PA切割成一个63 kDa片段（PA63）和一个20 kDa片段（PA20）。对63 kDa产物的氨基末端序列分析表明，切割发生在Arg167和Ser168之间。体外PA切割的最适pH为6.0，酶活性依赖于钙。未转染L细胞的培养基不能切割PA。对四碱基切割位点进行定点诱变表明，PC1更倾向于切割相对于野生型切割位点在-1和-4位含有碱性残基的序列，这表明PC1可以在体外单碱基残基位点切割底物。在-1和-2位含有碱性残基的底物切割效率比野生型PA低约两倍。在切割位点的-1位以及-2或-4位含有碱性残基的PA突变体预计是PC1的底物，并且对表达PC1的L细胞比未转染的L细胞毒性更大。这些结果表明PA在体内被PC1切割。含有激素原转化酶PC1和PC2的牛中间叶分泌囊泡膜也将PA切割成PA63，最适pH为5.5。使用针对PC1和PC2的抗血清进行的免疫去除研究表明，这些是膜制剂中主要负责PA切割的酶。因此，重组PC1和含有内源性PC1的膜制剂都可以激活PA。
