ENTEROTOXICITY OF HEAT-STABLE ENTEROTOXIN PRODUCED FROM ENTEROTOXIGENIC ESCHERICHIA COLI

热稳定肠毒素受体（星星/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，星星在GC催化结构域之后具有延长的羧基末端尾，其比NPR-A（CC-A）和NPR-B（CC-B）长约60个氨基酸，并且比GC-D、retGC 1（GC-E）和retGC 2（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，我们检测了两个截短的星星突变体CDELTA 1015和CDELTA 1023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1 μ M STa孵育后，表达CDELTA 1015和CDELTA 1023的细胞比表达野生型星星的细胞积累约20倍和10倍的cGMP。此外，在蛋白表达水平上没有观察到CC活性的差异。此外，星星的羧基末端尾部的去除不仅诱导高的cGMP-产生的最大水平，而且诱导高的cGMP-产生的潜在水平。这些结果表明星星的羧基端区域可能具有抑制STa介导的GC激活的功能，因此，羧基端区域的缺失允许STa激活高GC活性。星星及其N端胞外结构域在Sf 21昆虫细胞和重组杆状病毒构建的系统中以高水平表达。重组星星保留了其与STa的结合活性，Rd值为6.2 × 10 ^-^1^0^0 M，环化酶催化活性水平与哺乳动物细胞系（如COS-7）中表达的星星相似。

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.