ENTEROTOXICITY OF HEAT-STABLE ENTEROTOXIN PRODUCED FROM ENTEROTOXIGENIC ESCHERICHIA COLI

产肠毒素大肠杆菌产生的热稳定肠毒素的肠毒性

基本信息

批准号：
09670289
负责人：
HIRAYAMA Toshiya
金额：
$ 1.98万
依托单位：
NAGASAKI UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

Heat-stable enterotoxin receptor (STaR/GC-C) is a member of membrane-associated guanylyl cyclase (GC) family. As compared with other GC receptors, following GC catalytic domain, STaR has prolonged carboxy-terminal tail which is about 60 amino acids longer than NPR-A (CC-A) and NPR-B (CC-B), and about 30 amino acids longer than GC-D, retGCl (GC-E), and retGC2 (GC-F). To elucidate the functional role of the additional carboxy-terminal tail, we examined the GC activities of two truncate STaR mutants, CDELTA1015 and CDELTA1023, which lack Gu^1^0^1^5-Phe^1^0^5^0 and Lys^1^0^2^3-Phe^1^0^5^0, respectively. After incubation with I jiM STa, the cells expressing CDELTA1O15 and CDELTA1023 accumulated about 20- and 10-folds higher cGMP than the cells expressing wild-typc STaR.The basal level of cGMP contents were not different between the cells with wild-type STaR and STaR mutants. Furthermore, the difference of CC activity was not observed at protein expression level. In addition, removal of carboxy-terminal tail of STaR induced not only high maximum level of cGMP-production but also high potential level of cGMP-produciion. These results suggest that the carboxy-terminal region of STaR might have an inhibitory function of STa-mediated GC activation, and therefore the lack of the carboxy-terminal region allowed to be activated high GC activity by STa.STaR and its N-terminal extraceltular domain were prepared at a high level of expression from a system constructed of Sf21 insect cells and recombinant baculovirus. The recombinant STaR retained its binding activity to STa with a Rd value of 6.2x10^-^1^0^0M and cyclase catalytic activity at a level similar to those of STaR expressed jn mammalian cell lines, such as COS-7.

热稳定肠毒素受体 (STaR/GC-C) 是膜相关鸟苷酸环化酶 (GC) 家族的成员。与其他GC受体相比，在GC催化结构域之后，STaR具有延长的羧基末端尾部，比NPR-A（CC-A）和NPR-B（CC-B）长约60个氨基酸，比NPR-A（CC-A）和NPR-B（CC-B）长约30个氨基酸比 GC-D、retGC1 (GC-E) 和 retGC2 (GC-F) 更长。为了阐明额外的羧基末端尾部的功能作用，我们检查了两个截短 STaR 突变体 CDELTA1015 和 CDELTA1023 的 GC 活性，它们缺乏 Gu^1^0^1^5-Phe^1^0^5^0 和分别为 Lys^1^0^2^3-Phe^1^0^5^0。与 I jiM STa 孵育后，表达 CDELTA1O15 和 CDELTA1023 的细胞积累的 cGMP 比表达野生型 STaR 的细胞高约 20 倍和 10 倍。野生型 STaR 和野生型 STaR 细胞之间 cGMP 含量的基础水平没有差异。 STAR 突变体。此外，在蛋白质表达水平上没有观察到CC活性的差异。此外，去除 STaR 的羧基末端尾部不仅诱导高最大水平的 cGMP 产生，而且诱导高潜在水平的 cGMP 产生。这些结果表明 STaR 的羧基端区域可能对 STa 介导的 GC 激活具有抑制功能，因此羧基端区域的缺失使得 STa 能够激活高 GC 活性。 STaR 及其 N 端细胞外结构域是从由 Sf21 昆虫细胞和重组杆状病毒构建的系统中以高水平表达制备的。重组 STaR 保留了其与 STa 的结合活性，Rd 值为 6.2x10^-^1^0^0M，环化酶催化活性水平与在哺乳动物细胞系（如 COS-7）中表达的 STaR 相似。