Abrami L, Fivaz M, Decroly E, Seidah N G, Jean F, Thomas G, Leppla S H, Buckley J T, van der Goot F G
Department of Biochemistry, University of Geneva, 30 Quai E. Ansermet, 1211 Geneva, Switzerland.
J Biol Chem. 1998 Dec 4;273(49):32656-61. doi: 10.1074/jbc.273.49.32656.
Aerolysin is secreted as an inactive dimeric precursor by the bacterium Aeromonas hydrophila. Proteolytic cleavage within a mobile loop near the C terminus of the protoxin is required for oligomerization and channel formation. This loop contains the sequence KVRRAR432, which should be recognized by mammalian proprotein convertases such as furin, PACE4, and PC5/6A. Here we show that these three proteases cleave proaerolysin after Arg-432 in vitro, yielding active toxin. We also investigated the potential role of these enzymes in the in vivo activation of the protoxin. We found that Chinese hamster ovary cells were able to convert the protoxin to aerolysin in the absence of exogenous proteases and that activation did not require internalization of the toxin. The furin inhibitor alpha1-antitrypsin Portland reduced the rate of proaerolysin activation in vivo, and proaerolysin processing was even further reduced in furin-deficient FD11 Chinese hamster ovary cells. The cells were also less sensitive to proaerolysin than wild type cells; however, transient transfection of FD11 cells with the cDNA encoding furin conferred normal sensitivity to the protoxin. Together these findings argue that furin catalyzes the cell-surface activation of proaerolysin in vivo.
气单胞菌溶素由嗜水气单胞菌作为无活性的二聚体前体分泌。原毒素C末端附近的一个可移动环内的蛋白水解切割是寡聚化和通道形成所必需的。这个环包含序列KVRRAR432，它应该被诸如弗林蛋白酶、PACE4和PC5/6A等哺乳动物前蛋白转化酶识别。在这里，我们表明这三种蛋白酶在体外切割原气单胞菌溶素的432位精氨酸后，产生活性毒素。我们还研究了这些酶在原毒素体内激活中的潜在作用。我们发现中国仓鼠卵巢细胞能够在没有外源性蛋白酶的情况下将原毒素转化为气单胞菌溶素，并且激活不需要毒素内化。弗林蛋白酶抑制剂α1-抗胰蛋白酶波特兰降低了原气单胞菌溶素在体内的激活速率，并且在缺乏弗林蛋白酶的FD11中国仓鼠卵巢细胞中原气单胞菌溶素的加工甚至进一步减少。这些细胞对原气单胞菌溶素的敏感性也低于野生型细胞；然而，用编码弗林蛋白酶的cDNA瞬时转染FD11细胞赋予了对原毒素正常的敏感性。这些发现共同表明弗林蛋白酶在体内催化原气单胞菌溶素的细胞表面激活。
