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.

热稳定的肠毒素受体（Star/GC-C）是膜相关的鸟叶基环酶（GC）家族的成员。与其他GC受体相比，遵循GC催化结构域，Star的羧基末端尾巴长约60个氨基酸，比NPR-A（CC-A）和NPR-B（CC-B）长约60个氨基酸，并且比GC-D，RETGCL（GC-E）和RetGC2（GC2（GC-F）长30个氨基酸。为了阐明其他羧基末端尾巴的功能作用，我们检查了两个缺乏GU^1^1^0^1^1^5-PHE^1^1^0^5^0的GC活性，分别是CDELTA1015和CDELTA1023，分别缺乏GU^1^1^1^1^1^5^0和Lys^1^1^0^0^2^3-PHE^1^1^0^0^0^5^5^0。与I Jim Sta孵育后，表达CDELTA1O15和CDELTA1023的细胞与表达野生型恒星的细胞相比，CGMP含量的基础水平与野生型恒星和恒星串扰的细胞之间的基础水平没有差异。此外，在蛋白质表达水平上未观察到CC活性的差异。此外，恒星的羧基末端尾部不仅引起了高最大CGMP产生水平，而且还引起了CGMP生产的高电位水平。这些结果表明，恒星的羧基末端区域可能具有STA介导的GC激活的抑制作用，因此，缺乏STA.STAR及其N末端的GC活性激活的羧基末端区域，以高水平的SF21昆虫细胞和Reculovirus构建的系统在高水平的表达中制备了高GC活性。重组恒星以6.2x10^ - ^1^0^0m的RD值保持其与Sta的结合活性，并以类似于Star表达的JN哺乳动物细胞系（例如COS-7）的水平，环酶催化活性。