Faust C, Ye B, Song K P
Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, 3601 Fourth Street, Lubbock, Texas, 79430, USA.
Biochem Biophys Res Commun. 1998 Oct 9;251(1):100-5. doi: 10.1006/bbrc.1998.9383.
Clostridium difficile, an anaerobic pathogen encountered in human enteric disease, produces two major virulence factors, toxins A and B, which are members of a clostridial family of large cytotoxins. These are glucosyltransferases, which use a UDP-sugar as co-substrate to glucosylate and inactivate small GTPases of the Rho or Ras families, culminating in cytotoxicity. Clinically, toxin A is perhaps the most important family member, because it causes major tissue damage in the course of disease, leading to a potentially lethal, pseudomembranous colitis. The location of the enzymatic domain of toxin A and mechanistic details of its action are not yet known, so we wished to localize this domain using gene deletion constructions from the full-length gene and by monitoring glucosylation activity of encoded protein products. Toxin A deletions were obtained by successively truncating the C-terminal coding region. These were transformed into E. coli, cell lysates were prepared and they were assayed for their ability to glucosylate Rho A protein, using an in vitro enzymatic assay. We report that the UDP-glucose binding site, the catalytic site for glucose transfer and the Rho A interaction site occur within the first 659 N-terminal amino acids of toxin A, i.e., less than 25% of the length of holotoxin A. Localization of the enzymatic domain of toxin A to these 659 N-terminal amino acids should greatly simplify studies on mechanistic details of this clinically important toxin.
艰难梭菌是一种在人类肠道疾病中发现的厌氧病原体，它产生两种主要的毒力因子，毒素A和毒素B，它们是梭菌属大细胞毒素家族的成员。这些是葡萄糖基转移酶，它们使用UDP-糖作为共底物来使Rho或Ras家族的小GTP酶糖基化并使其失活，最终导致细胞毒性。临床上，毒素A可能是最重要的家族成员，因为它在疾病过程中会导致主要组织损伤，引发潜在致命的伪膜性结肠炎。毒素A的酶结构域位置及其作用的机制细节尚不清楚，因此我们希望使用全长基因的基因缺失构建体并通过监测编码蛋白产物的糖基化活性来定位该结构域。通过连续截短C末端编码区获得毒素A缺失体。将这些缺失体转化到大肠杆菌中，制备细胞裂解物，并使用体外酶促测定法检测它们使Rho A蛋白糖基化的能力。我们报告UDP-葡萄糖结合位点、葡萄糖转移催化位点和Rho A相互作用位点位于毒素A的前659个N末端氨基酸内，即不到全毒素A长度的25%。将毒素A的酶结构域定位到这659个N末端氨基酸应该会大大简化对这种临床上重要毒素的机制细节的研究。
