ENTEROTOXICITY OF HEAT-STABLE ENTEROTOXIN PRODUCED FROM ENTEROTOXIGENIC ESCHERICHIA COLI

热稳定型肠毒素受体（STaR/GC- c）是膜相关观酰基环化酶（GC）家族的一员。与其他GC受体相比，在GC催化结构域之后，STaR具有较长的羧基末端尾部，比NPR-A （CC-A）和NPR-B （CC-B）长约60个氨基酸，比GC- d、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部的功能作用，我们检测了两个截断的STaR突变体CDELTA1015和CDELTA1023的GC活性，它们分别缺乏Gu^1^0^1^ 1^5- phe ^1^0^5^0和Lys^1^0^2^3-Phe^1^0^5^0。与I jiM STa孵养后，表达cdelta1015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基础含量在STaR野生型和STaR突变体细胞间无显著差异。此外，在蛋白表达水平上未观察到CC活性的差异。此外，去除STaR的羧基末端尾部，不仅能提高cgmp的最大产量，而且还能提高cgmp的潜在产量。这些结果表明，STaR的羧基末端区域可能对STa介导的GC激活具有抑制作用，因此缺乏羧基末端区域允许STa激活高GC活性。在Sf21昆虫细胞和重组杆状病毒构建的系统中，高水平表达了STaR及其n端胞外结构域。重组STaR与STa的结合活性为6.2x10^-^1^0^0 m，环化酶催化活性与COS-7等哺乳动物细胞系中表达的STaR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.