Maresso Anthony W, Riese Matthew J, Barbieri Joseph T
Microbiology and Molecular Genetics, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, Wisconsin 53226, USA.
Biochemistry. 2003 Dec 9;42(48):14249-57. doi: 10.1021/bi035053i.
Pseudomonas aeruginosa ExoS is a bifunctional type III cytotoxin. The N-terminus (residues 1-232) is a Rho GTPase activating protein (GAP) domain, while the C-terminus (residues 233-453) is a FAS-dependent ADP-ribosyltransferase domain that targets Ras and Ras-like GTPases. A membrane localization domain (residues 51-72) localizes ExoS to a perinuclear region within eukaryotic cells. Recent studies observed that ExoS is auto-ADP-ribosylated upon delivery into eukaryotic cells. Auto-ADP-ribosylated ExoS analyzed from eukaryotic cells displayed pI heterogeneity and prompted an analysis of this heterogeneity. Bacterial-associated ExoS and ExoS that had been secreted by P. aeruginosa also showed pI heterogeneity with five charge forms ranging in pI from 5.1 to 5.9. The pI heterogeneity of ExoS was independent of a mass change and thus represented molecular charge conformers. Urea was not required to observe the pI conformers of ExoS; it enhanced the resolution and formation of pI conformers during the focusing component of the analysis. ExoS(E381D), a mutant deficient in ADP-ribosyltransferase activity, isolated from cultured cells showed charge forms that migrated to a more acidic pI than type III secreted ExoS but more basic than auto-ADP-ribosylated ExoS. Incubation of cell lysates with Mn(2+) shifted the pI of ExoS(E381D) to a pI identical to secreted ExoS. This indicates that within the mammalian cells ExoS undergoes a negatively charged modification, in addition to auto-ADP-ribosylation observed for wild-type ExoS. ExoT, ExoU, and YopE also focus into multiple pI forms, suggesting that this is a common property of type III cytotoxins.
铜绿假单胞菌外毒素S是一种双功能III型细胞毒素。其N端（第1 - 232位氨基酸残基）是一个Rho GTP酶激活蛋白（GAP）结构域，而C端（第233 - 453位氨基酸残基）是一个依赖FAS的ADP核糖基转移酶结构域，作用于Ras和Ras样GTP酶。一个膜定位结构域（第51 - 72位氨基酸残基）将外毒素S定位于真核细胞内的核周区域。最近的研究发现，外毒素S在被递送至真核细胞后会发生自身ADP核糖基化。从真核细胞中分析得到的自身ADP核糖基化外毒素S显示出pI异质性，并促使对这种异质性进行分析。与细菌相关的外毒素S以及由铜绿假单胞菌分泌的外毒素S也表现出pI异质性，有五种电荷形式，pI范围从5.1到5.9。外毒素S的pI异质性与质量变化无关，因此代表分子电荷构象体。观察外毒素S的pI构象体不需要尿素；它在分析的聚焦阶段提高了pI构象体的分辨率和形成。从培养细胞中分离出的缺乏ADP核糖基转移酶活性的突变体外毒素S（E381D），其电荷形式迁移到比III型分泌外毒素S更酸性的pI，但比自身ADP核糖基化外毒素S更碱性。用Mn(2+)孵育细胞裂解物会使外毒素S（E381D）的pI转变为与分泌外毒素S相同的pI。这表明在哺乳动物细胞内，外毒素S除了发生野生型外毒素S所观察到的自身ADP核糖基化外，还会经历带负电荷的修饰。外毒素T、外毒素U和YopE也聚焦为多种pI形式，表明这是III型细胞毒素的共同特性。
