Scheving L A, Chong K M
Department of Pathology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2576, USA.
Am J Physiol. 1997 Jun;272(6 Pt 1):C1995-2004. doi: 10.1152/ajpcell.1997.272.6.C1995.
Many strains of enterotoxigenic Escherichia coli produce a heat-stable peptide enterotoxin (STa) that binds to the intestinal receptor guanylyl cyclase C (GC-C). STa receptors are structurally heterogeneous, but the molecular events causing this heterogeneity remain obscure. We examined the influence of cell position along the villus-crypt axis on STa receptor heterogeneity by fractionating EDTA-dissociated cells that detached in a villus-to-crypt direction. STa affinity labeling experiments revealed that the initially released villus "tip" fraction had four major STa binding proteins (STBPs), with relative molecular weight (M(r)) of 150,000, 135,000, 125,000, and 95,000, that did not react with a GC-C carboxy-terminal antibody. Yet succeeding villus cell fractions had major immunoreactive STBPs with M(r) of 275,000 and 250,000. Limited proteolysis of these larger GC-C isoforms produced 1) smaller STBPs that had M(r) similar to those in the initial villus fraction, 2) a 65,000 M(r) protein GC-C isoform that did not bind STa, and 3) elevated basal and STa-induced cyclase activity. Our data show that STBP structural heterogeneity in the intact intestine arises largely from multisite proteolytic processing of GC-C.
许多产肠毒素大肠杆菌菌株会产生一种热稳定肽肠毒素（STa），它能与肠道受体鸟苷酸环化酶C（GC-C）结合。STa受体在结构上具有异质性，但导致这种异质性的分子事件仍不清楚。我们通过分离沿绒毛-隐窝轴方向解离的EDTA解离细胞，研究了细胞沿绒毛-隐窝轴的位置对STa受体异质性的影响。STa亲和标记实验表明，最初释放的绒毛“顶端”部分有四种主要的STa结合蛋白（STBPs），相对分子质量（M(r)）分别为150,000、135,000、125,000和95,000，它们不与GC-C羧基末端抗体发生反应。然而，随后的绒毛细胞部分有主要的免疫反应性STBPs，其M(r)为275,000和250,000。对这些较大的GC-C异构体进行有限的蛋白酶解产生了：1）较小的STBPs，其M(r)与初始绒毛部分的相似；2）一种65,000 M(r)的蛋白质GC-C异构体，它不结合STa；3）基础和STa诱导的环化酶活性升高。我们的数据表明，完整肠道中STBP的结构异质性很大程度上源于GC-C的多位点蛋白水解加工。
