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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GC-D、retGC1(GC-E)和retGC2(GC-F)长约30个氨基酸。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的 STaR 突变体 CDELTA1015 和 CDELTA1023 的 GC 活性，它们分别缺乏 Gu^1^0^1^5-Phe^1^0^5^0 和 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 STaR 突变体细胞之间 cGMP 含量的基础水平没有差异。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的羧基末端区域可能对STa介导的GC激活具有抑制功能，因此羧基末端区域的缺失使得STa能够激活高GC活性。由Sf21昆虫细胞和重组杆状病毒构建的系统制备了高水平表达的STaR及其N末端胞外结构域。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。