Hewlett E L, Gray M C, Ehrmann I E, Maloney N J, Otero A S, Gray L, Allietta M, Szabo G, Weiss A A, Barry E M
Department of Medicine, University of Virginia School of Medicine, Charlottesville 22908.
J Biol Chem. 1993 Apr 15;268(11):7842-8.
Bordetella pertussis adenylate cyclase (AC) toxin has the abilities to 1) enter target cells where it catalyzes cyclic AMP production and 2) lyse sheep erythrocytes, and these abilities require post-translational modification by the product of an accessory gene cyaC (Barry, E. M., Weiss, A. A., Ehrmann, E. E., Gray, M. C., Hewlett, E. L., and Goodwin, M. St. M. (1991) J. Bacteriol. 173, 720-726). In the present study, AC toxin has been purified from an organism with a mutation in cyaC, BPDE386, and evaluated for its physical and functional properties in order to determine the basis for its lack of toxin and hemolytic activities. AC toxin from BPDE386 is indistinguishable from wild-type toxin in enzymatic activity, migration on SDS-polyacrylamide gel electrophoresis, ability to bind calcium, and calcium-dependent conformational change. Although unable to elicit cAMP accumulation, AC toxin from BPDE386 exhibits binding to the surface of Jurkat cells which is comparable to that of wild-type toxin. This target cell interaction is qualitatively different, however, in that 99% of the mutant toxin remains sensitive to trypsin, whereas approximately 20% of cell-associated wild-type toxin enters a trypsin-resistant compartment. To evaluate the ability of this mutant AC toxin to function at its intracellular site of action, the cAMP-stimulated L-type calcium current in frog atrial myocytes was used. Extracellular addition of wild-type toxin results in cAMP-dependent events that include activation of calcium channels and enhancement of calcium current. In contrast, there is no response to externally applied toxin from BPDE386. When injected into the cell interior, however, the AC toxin from BPDE386 is able to produce increases in the calcium current comparable to those observed with wild-type toxin. Although AC toxin from BPDE386 is unaffected in its enzymatic activity, calcium binding, and calcium-dependent conformational change, the mutation in cyaC does result in a toxin which is able to bind to target cells but unable to elicit cAMP accumulation. In that AC toxin from BPDE386 is able to function normally when injected artificially to an intracellular site, we conclude that the disruption of cyaC produces a defect in insertion and transmembrane delivery of the catalytic domain.
百日咳博德特氏菌腺苷酸环化酶（AC）毒素具有以下能力：1）进入靶细胞并在其中催化环磷酸腺苷（cAMP）的产生；2）裂解绵羊红细胞，而这些能力需要由辅助基因cyaC的产物进行翻译后修饰（巴里，E.M.，魏斯，A.A.，埃尔曼，E.E.，格雷，M.C.，休利特，E.L.，以及古德温，M.圣M.（1991年）《细菌学杂志》173卷，720 - 726页）。在本研究中，从cyaC发生突变的菌株BPDE386中纯化出了AC毒素，并对其物理和功能特性进行了评估，以确定其缺乏毒素活性和溶血活性的原因。来自BPDE386的AC毒素在酶活性、在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳上的迁移、结合钙的能力以及钙依赖性构象变化方面与野生型毒素没有区别。尽管无法引发cAMP积累，但来自BPDE386的AC毒素与野生型毒素一样，能与Jurkat细胞表面结合。然而，这种与靶细胞的相互作用在性质上有所不同，因为99%的突变毒素对胰蛋白酶仍敏感，而约20%与细胞结合的野生型毒素进入了对胰蛋白酶有抗性的区室。为了评估这种突变AC毒素在其细胞内作用位点发挥功能的能力，使用了蛙心房肌细胞中cAMP刺激的L型钙电流。在细胞外添加野生型毒素会导致依赖cAMP的事件，包括钙通道的激活和钙电流的增强。相比之下，对来自BPDE386的外部施加毒素没有反应。然而，当注入细胞内部时，来自BPDE386的AC毒素能够使钙电流增加，与野生型毒素所观察到的情况相当。尽管来自BPDE386的AC毒素在酶活性、钙结合和钙依赖性构象变化方面未受影响，但cyaC中的突变确实导致产生了一种能够与靶细胞结合但无法引发cAMP积累的毒素。鉴于来自BPDE386的AC毒素在人工注入细胞内位点时能够正常发挥功能，我们得出结论，cyaC的破坏在催化结构域的插入和跨膜转运方面产生了缺陷。
