The distal ectodomain of angiotensin-converting enzyme regulates its cleavage-secretion from the cell surface

Abstract

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 cleavagesecreted. 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.

Shedding of somatic angiotensin-converting enzyme (ACE) is inefficient compared with testis ACE despite cleavage at identical stalk sites

“…Second, Sadhukhan et al [26] showed that when the extracellular domain of testis ACE was fused to the stalk, TM and cytoplasmic domains of CD4 (a membrane protein that is usually resistant to shedding), the construct was efficiently cleaved and released. These data suggest, but do not prove conclusively, that the testis ACE extracellular domain contains a recognition motif that stimulates the ACE secretase.…”

“…Moreover, somatic ACE was shown to be shed much less efficiently than testis ACE, which has been interpreted to mean that the second, N-terminal domain of somatic ACE modulates the activity of the secretase and directs it to an alternative cleavage site [23]. Ectodomain-mediated modulation of secretase activity is supported by data indicating that the ACE secret-ase has a remarkably low stalk-sequence specificity [24,25] and that the ACE ectodomain promotes cleavage of stalks from shedding-resistant proteins [26]. However, the reported somatic ACE cleavage site [22] is not consistent with data indicating that the size of the residual anchoring domain is 6.9 kDa [15], or that deletion of 47 residues proximal to the TM domain in testis ACE renders it catalytically inactive [21].…”

Maintenance of Normal Blood Pressure and Renal Functions Are Independent Effects of Angiotensin-converting Enzyme

“…The slides were blocked for 2 h at 25°C in PBS ϩ 10% horse serum ϩ 0.3% Triton X-100 (blocking buffer). The anti-rabbit sACE antibody (20), which weakly binds with mouse ACE in this procedure, diluted 1:1000 in blocking buffer was applied to the slides in a humid chamber for 16 h at 4 C. Following washes in PBS ϩ 0.3% Triton X-100 (PBST), anti-goat-fluorescein isothiocyanate (Santa Cruz Biotechnology, Inc.) was applied to each section for 2 h in the dark at 25°C. Following washes in PBST, VectaShield (Vector Laboratories) diluted 1:1 in PBS was applied.…”

Increased Shedding of Angiotensin-converting Enzyme by a Mutation Identified in the Stalk Region

“…Cleavage secretion of ACE is topologically constrained to an accessible stalk region of at least 11 residues in length and requires a minimum distance of 3 residues from the proximal extracellular domain and 8 residues from the membrane (10). Furthermore, mutational analysis of the stalk region has shown that conservation of the amino acid sequence was not essential for shedding, even though the secretase seems to have a weak preference for cleavage after Arg or Lys residues (10,13,14). Thus, the critical parameter, for cleavage efficiency seems to be the conformation of the stalk more than the amino acid sequence.…”