ENTEROTOXICITY OF HEAT-STABLE ENTEROTOXIN PRODUCED FROM ENTEROTOXIGENIC ESCHERICHIA COLI

耐热肠毒素受体(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催化区之后，STAR的羧基末端长度比NPR-A(CC-A)和NPR-B(CC-B)长约60个氨基酸，比GC-D、retGCl(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巴的功能作用，我们检测了两个截短的星形突变体CDELTA1015和CDELTA1023的GC活性，它们分别缺少Gu^1^1^1^5-Phe^1^0^5^0和Lys^1^0^2^3-Phe^1^0^5^0。用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，cGMP含量的基础水平在野生型和野生型STAR突变体细胞中没有差异。此外，在蛋白表达水平上没有观察到CC活性的差异。此外，去除STAR的羧基末端不仅能产生高水平的cGMP-产生，而且还能产生高水平的cGMP-产生。这些结果表明，STAR的羧基末端区域可能具有抑制STA介导的GC激活的功能，因此缺少STAR能够激活高GC活性的羧基末端区域。STAR及其N末端胞外结构域在由Sf21昆虫细胞和重组杆状病毒构建的系统中得到高效表达。重组STAR与STAR的结合活性保持在6.2×10~(-1)×10~(-1)M之间，环化酶催化活性与表达STAR的哺乳动物细胞株COS-7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.