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Molecular cloning of human testicular angiotensin-converting enzyme: the testis isozyme is identical to the C-terminal half of endothelial angiotensin-converting enzyme.

ABSTRACT: Angiotensin-converting enzyme (ACE; EC 3.4.15.1) is a zinc-containing dipeptidyl carboxypeptidase widely distributed in mammalian tissues and is thought to play a critical role in blood pressure regulation. Testis contains a unique, androgen-dependent ACE isozyme of unknown function. We have determined the cDNA sequence for human testicular ACE; it encodes a protein that is identical, from residue 37 to its C terminus, to the second half or C-terminal domain of the endothelial ACE sequence [Soubrier, F., Alhenc-Gelas, F., Hubert, C., Allegrini, J., John, M., Tregear, G. & Corvol, P. (1988) Proc. Natl. Acad. Sci. USA 85, 9386-9390]. The full-length human testis ACE cDNA was constructed from a composite of cloned cDNAs, obtained by a combination of (i) immunoscreening and hybridization screening of a human testicular cDNA library in lambda gt11 and (ii) hybridization screening of human testis cDNAs constructed with ACE-specific primers and amplified by the polymerase chain reaction. The protein sequence inferred consists of a 732-residue preprotein including a 31-residue signal peptide. The mature polypeptide has a molecular weight of 80,073. The testis enzyme contains the second of the two putative metal-binding sites (His-Glu-Met-Gly-His) identified in endothelial ACE. This indicates that the functionally active catalytic site is within the C-terminal domain of the endothelial enzyme, accounting for the previous finding that these two structurally dissimilar isozymes are virtually identical catalytically. Of 22 testis ACE cDNAs cloned and sequenced, 3 have unique 5' regions, consisting of inserted, deleted, or substituted sequences up to 328 base pairs long, which have apparently arisen by alternative pre-mRNA splicing.

SUBMITTER: Ehlers MR

PROVIDER: S-EPMC298146 | biostudies-other | 1989 Oct

REPOSITORIES: biostudies-other

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Molecular cloning of human testicular angiotensin-converting enzyme: the testis isozyme is identical to the C-terminal half of endothelial angiotensin-converting enzyme.

Ehlers M R MR Fox E A EA Strydom D J DJ Riordan J F JF

Proceedings of the National Academy of Sciences of the United States of America 19891001 20

Angiotensin-converting enzyme (ACE; EC 3.4.15.1) is a zinc-containing dipeptidyl carboxypeptidase widely distributed in mammalian tissues and is thought to play a critical role in blood pressure regulation. Testis contains a unique, androgen-dependent ACE isozyme of unknown function. We have determined the cDNA sequence for human testicular ACE; it encodes a protein that is identical, from residue 37 to its C terminus, to the second half or C-terminal domain of the endothelial ACE sequence [Soub ...[more]

PMID: 2554286

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Project description:Diminished release and function of endothelium-derived nitric oxide coupled with increases in reactive oxygen species production is critical in endothelial dysfunction. Recent evidences have shown that activation of the protective axis of the renin-angiotensin system composed by angiotensin-converting enzyme 2, angiotensin-(1-7), and Mas receptor promotes many beneficial vascular effects. This has led us to postulate that activation of intrinsic angiotensin-converting enzyme 2 would improve endothelial function by decreasing the reactive oxygen species production. In the present study, we tested 1-[[2-(dimetilamino)etil]amino]-4-(hidroximetil)-7-[[(4-metilfenil)sulfonil]oxi]-9H-xantona-9 (XNT), a small molecule angiotensin-converting enzyme 2 activator, on endothelial function to validate this hypothesis. In vivo treatment with XNT (1 mg/kg per day for 4 weeks) improved the endothelial function of spontaneously hypertensive rats and of streptozotocin-induced diabetic rats when evaluated through the vasorelaxant responses to acetylcholine/sodium nitroprusside. Acute in vitro incubation with XNT caused endothelial-dependent vasorelaxation in aortic rings of rats. This vasorelaxation effect was attenuated by the Mas antagonist D-pro7-Ang-(1-7), and it was reduced in Mas knockout mice. These effects were associated with reduction in reactive oxygen species production. In addition, Ang II-induced reactive oxygen species production in human aortic endothelial cells was attenuated by preincubation with XNT. These results showed that chronic XNT administration improves the endothelial function of hypertensive and diabetic rat vessels by attenuation of the oxidative stress. Moreover, XNT elicits an endothelial-dependent vasorelaxation response, which was mediated by Mas. Thus, this study indicated that angiotensin-converting enzyme 2 activation promotes beneficial effects on the endothelial function and it is a potential target for treating cardiovascular disease.

Project description:The somatic and testis isoforms of angiotensin-converting enzyme (ACE) are both C-terminally anchored ectoproteins that are shed by an unidentified secretase. Although testis and somatic ACE both share the same stalk and membrane domains the latter was reported to be shed inefficiently compared with testis ACE, and this was ascribed to cleavage at an alternative site [Beldent, Michaud, Bonnefoy, Chauvet and Corvol (1995) J. Biol. Chem. 270, 28962-28969]. These differences constitute a useful model system of the regulation and substrate preferences of the ACE secretase, and hence we investigated this further. In transfected Chinese hamster ovary cells, human somatic ACE (hsACE) was indeed shed less efficiently than human testis ACE, and shedding of somatic ACE responded poorly to phorbol ester activation. However, using several analytical techniques, we found no evidence that the somatic ACE cleavage site differed from that characterized in testis ACE. First, anti-peptide antibodies raised to specific sequences on either side of the reported cleavage site (Arg(1137)/Leu(1138)) clearly recognized soluble porcine somatic ACE, indicating that cleavage was C-terminal to Arg(1137). Second, a competitive ELISA gave superimposable curves for porcine plasma ACE, secretase-cleaved porcine somatic ACE (eACE), and trypsin-cleaved ACE, suggesting similar C-terminal sequences. Third, mass-spectral analyses of digests of released soluble hsACE or of eACE enabled precise assignments of the C-termini, in each case to Arg(1203). These data indicated that soluble human and porcine somatic ACE, whether generated in vivo or in vitro, have C-termini consistent with cleavage at a single site, the Arg(1203)/Ser(1204) bond, identical with the Arg(627)/Ser(628) site in testis ACE. In conclusion, the inefficient release of somatic ACE is not due to cleavage at an alternative stalk site, but instead supports the hypothesis that the testis ACE ectodomain contains a motif that activates shedding, which is occluded by the additional domain found in somatic ACE.

Project description:The emergence of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutants and breakthrough infections despite available coronavirus disease 2019 (COVID-19) vaccines calls for antiviral therapeutics. The application of soluble angiotensin converting enzyme 2 (ACE2) as a SARS-CoV-2 decoy and reduce cell bound ACE2-mediated virus entry is limited by a short plasma half-life. This work presents a recombinant human albumin ACE2 genetic fusion (rHA-ACE2) to increase the plasma half-life by a FcRn-driven cellular recycling mechanism, investigated using a wild type (WT) albumin sequence and sequence engineered with null FcRn binding (NB). Binding of rHA-ACE2 fusions to SARS-CoV-2 spike protein subdomain 1 (S1) was demonstrated (WT-ACE2 KD = 3.28E-8 M and NB-ACE2 KD = 3.17E-8 M) using Bio-Layer Interferometry and dose-dependent in vitro inhibition of host cell infection of pseudotyped viruses displaying surface SARS-CoV-2 spike (S) protein. FcRn-mediated in vitro recycling was translated to a five times greater plasma half-life of WT-ACE2 (t½ β=13.5 h) than soluble ACE2 (t½ β=2.8 h) in humanised FcRn/albumin double transgenic mice. The rHA-ACE2-based SARS-CoV-2 decoy system exhibiting FcRn-driven circulatory half-life extension introduced in this work offers the potential to expand and improve the anti-COVID-19 anti-viral drug armoury. STATEMENT OF SIGNIFICANCE: The COVID-19 pandemic has highlighted the need for rapid development of efficient antiviral therapeutics to combat SARS-CoV-2 and new mutants to lower morbidity and mortality in severe cases, and for people that are unable to receive a vaccine. Here we report a therapeutic albumin ACE2 fusion protein (rHAACE2) that can bind SARS-CoV-2 S protein decorated virus-like particles to inhibit viral infection, and exhibits extended in vivo half-life compared to ACE2 alone. Employing ACE2 as a binding decoy for the virus is expected to efficiently inhibit all SARS-CoV-2 mutants as they all rely on binding with endogenous ACE2 for viral cell entry and, therefore, rHA-ACE2 constitutes a versatile addition to the therapeutic arsenal for combatting COVID-19.

Dataset Information

Molecular cloning of human testicular angiotensin-converting enzyme: the testis isozyme is identical to the C-terminal half of endothelial angiotensin-converting enzyme.

Publications

Molecular cloning of human testicular angiotensin-converting enzyme: the testis isozyme is identical to the C-terminal half of endothelial angiotensin-converting enzyme.

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