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Cleavage of arginyl-arginine and lysyl-arginine from the C-terminus of pro-hormone peptides by human germinal angiotensin I-converting enzyme (ACE) and the C-domain of human somatic ACE.

ABSTRACT: Mammalian germinal angiotensin I-converting enzyme (gACE) is a single-domain dipeptidyl carboxypeptidase found exclusively in male germ cells, which has almost identical sequence and enzymic properties with the C-domain of the two-domain somatic ACE. Mutant mice that do not express gACE are infertile, suggesting a role for the enzyme in the processing of undefined peptides involved in fertilization. A number of spermatid peptides [e.g. cholecystokinin (CCK) and gastrin] are processed from pro-hormones by endo- and exo-proteolytic cleavages which might generate substrates for gACE. We have shown that peptide hormone intermediates with Lys/Arg-Arg at the C-terminus are high-affinity substrates for human gACE. gACE from human sperm cleaved Arg-Arg from the C-terminus of the CCK5-GRR (GWMDFGRR), a peptide corresponding to the C-terminus of a CCK-gastrin prohormone intermediate. Hydrolysis of CCK5-GRR by recombinant human C-domain ACE was Cl- dependent, with maximal activity achieved in 5-10 mM NaCl at pH 6.4. C-Domain ACE cleaved Lys/Arg-Arg from the C-terminus of dynorphin-(1-7), a pro-TRH peptide KRQHPGKR, and two insect peptides FSPRLGKR and FSPRLGRR. C-Domain ACE displayed high affinity towards all these substrates with Vmax/Km values between 14 and 113 times greater than the Vmax/Km for the conversion of the best known ACE substrate, angiotensin I, into angiotensin II. In conclusion, we have identified a new class of substrates for human gACE, and we suggest that gACE might be an alternative to carboxypeptidase E for the trimming of basic dipeptides from the C-terminus of intermediates generated from pro-hormones by subtilisin-like convertases in human male germ cells.

SUBMITTER: Isaac RE

PROVIDER: S-EPMC1218959 | biostudies-other | 1997 Dec

REPOSITORIES: biostudies-other

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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:Animal aging is characterized by progressive, degenerative changes in many organ systems. Because age-related degeneration is a major contributor to disability and death in humans, treatments that delay age-related degeneration are desirable. However, no drugs that delay normal human aging are currently available. To identify drugs that delay age-related degeneration, we used the powerful Caenorhabditis elegans model system to screen for FDA-approved drugs that can extend the adult lifespan of worms. Here we show that captopril extended mean lifespan. Captopril is an angiotensin-converting enzyme (ACE) inhibitor used to treat high blood pressure in humans. To explore the mechanism of captopril, we analyzed the acn-1 gene that encodes the C. elegans homolog of ACE. Reducing the activity of acn-1 extended the mean life span. Furthermore, reducing the activity of acn-1 delayed age-related degenerative changes and increased stress resistance, indicating that acn-1 influences aging. Captopril could not further extend the lifespan of animals with reduced acn-1, suggesting they function in the same pathway; we propose that captopril inhibits acn-1 to extend lifespan. To define the relationship with previously characterized longevity pathways, we analyzed mutant animals. The lifespan extension caused by reducing the activity of acn-1 was additive with caloric restriction and mitochondrial insufficiency, and did not require sir-2.1, hsf-1 or rict-1, suggesting that acn-1 functions by a distinct mechanism. The interactions with the insulin/IGF-1 pathway were complex, since the lifespan extensions caused by captopril and reducing acn-1 activity were additive with daf-2 and age-1 but required daf-16. Captopril treatment and reducing acn-1 activity caused similar effects in a wide range of genetic backgrounds, consistent with the model that they act by the same mechanism. These results identify a new drug and a new gene that can extend the lifespan of worms and suggest new therapeutic strategies for addressing age-related degenerative changes.

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Cleavage of arginyl-arginine and lysyl-arginine from the C-terminus of pro-hormone peptides by human germinal angiotensin I-converting enzyme (ACE) and the C-domain of human somatic ACE.

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