Sadhukhan R, Sen G C, Ramchandran R, Sen I
Department of Molecular Cardiology, Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA.
Proc Natl Acad Sci U S A. 1998 Jan 6;95(1):138-43. doi: 10.1073/pnas.95.1.138.
Angiotensin-converting enzyme (ACE) is a type I ectoprotein that is cleaved off the cell surface by a plasma membrane-bound metalloprotease. However, CD4, another type I ectoprotein does not undergo such cleavage-secretion. In this study, we investigated the structural determinants of the ACE protein that regulate the cleavage-secretion process. Substitution and deletion mutations revealed that the cytoplasmic domain, the transmembrane domain, and the juxtamembrane region encompassing the major and the minor cleavage sites of ACE do not regulate its cleavage. Moreover, a chimeric protein containing the distal extracellular domain of CD4 and the juxtamembrane, transmembrane, and the cytoplasmic domains of ACE, although transported to the cell surface, was not cleavage-secreted. In contrast, the distal extracellular domain of ACE was shown to be the important determinant: a protein containing the distal extracellular domain of ACE and the juxtamembrane, transmembrane, and cytoplasmic domain of CD4 was efficiently cleaved off the cell surface. The chimeric protein was cleaved within the CD4 sequence and the responsible enzymatic activity was inhibited by Compound 3, a relatively specific inhibitor of the ACE secretase activity. These results demonstrate that, in a chimeric protein, the distal extracellular domain of a cleavable protein, such as ACE, can induce a proteolytic cleavage within the juxtamembrane domain of an uncleaved protein such as CD4.
血管紧张素转换酶（ACE）是一种I型细胞外蛋白，可被一种与质膜结合的金属蛋白酶从细胞表面切割下来。然而，另一种I型细胞外蛋白CD4却不会经历这种切割分泌过程。在本研究中，我们调查了调节ACE蛋白切割分泌过程的结构决定因素。替换和缺失突变表明，ACE的细胞质结构域、跨膜结构域以及包含主要和次要切割位点的近膜区域并不调节其切割过程。此外，一种包含CD4远端细胞外结构域以及ACE的近膜、跨膜和细胞质结构域的嵌合蛋白，尽管被转运到细胞表面，但并未被切割分泌。相反，ACE的远端细胞外结构域被证明是重要的决定因素：一种包含ACE远端细胞外结构域以及CD4的近膜、跨膜和细胞质结构域的蛋白能有效地从细胞表面被切割下来。嵌合蛋白在CD4序列内被切割，且其相关酶活性被化合物3（一种相对特异性的ACE分泌酶活性抑制剂）所抑制。这些结果表明，在嵌合蛋白中，可切割蛋白（如ACE）的远端细胞外结构域能够在未切割蛋白（如CD4）的近膜结构域内诱导蛋白水解切割。
