Characterization of the first non-insect invertebrate functional angiotensin-converting enzyme (ACE): leech TtACE resembles the N-domain of mammalian ACE

Abstract

Angiotensin-converting enzyme (ACE) is a zinc metallopeptidase that plays a major role in blood homoeostasis and reproduction in mammals. In vertebrates, both transmembrane and soluble ACE, containing one or two homologous active sites, have been characterized. So far, several ACEs from invertebrates have been cloned, but only in insects. They are soluble and display a single active site. Using biochemical procedures, an ACE-like activity was detected in our model, the leech, Theromyzon tessulatum. Annelida is the most distant phylum in which an ACE activity has been observed. To gain more insight into the leech enzyme, we have developed a PCR approach to characterize its mRNA. The approx. 2 kb cDNA has been predicted to encode a 616-amino-acid soluble enzyme containing a single active site, named TtACE (T. tessulatum ACE). Surprisingly, its primary sequence shows greater similarity to vertebrates than to invertebrates. Stable in vitro expression of TtACE in transfected Chinese-hamster ovary cells revealed that the leech enzyme is a functional metalloprotease. As in mammals, this 79 kDa glycosylated enzyme functions as a dipeptidyl carboxypeptidase capable of hydrolysing angiotensin I to angiotensin II. However, a weak chloride inhibitory effect and acetylated N-acetyl-SDKP (Ac SDAcKP) hydrolysis reveal that TtACE activity resembles that of the N-domain of mammalian ACE. In situ hybridization shows that its cellular distribution is restricted to epithelial midgut cells. Although the precise roles and endogenous substrates of TtACE remain to be identified, characterization of this ancestral peptidase will help to clarify its physiological roles in non-insect invertebrate species.

A Crucial Role in Fertility for the Oyster Angiotensin-Converting Enzyme Orthologue CgACE

“…Indeed, drosophila ACEs intercalate between them, and Cg ACE appears close to the bacterial Xc ACE, whereas Tt ACE clusters with vertebrate isoforms. Because both leeches and oysters belong to the Lophotrochozoa, such an observation might reflect complex evolutionary relationships between annelids and molluscs, and may also involve the specific parasitic trait of leeches, as discussed elsewhere [30]. Besides, the finding of a unique catalytic region within Cg ACE, and more generally within all lophotrochozoan ACE-like enzymes described to date [30], seems consistent with the late occurrence of genome duplication during the course of animal evolution.…”

“…Because both leeches and oysters belong to the Lophotrochozoa, such an observation might reflect complex evolutionary relationships between annelids and molluscs, and may also involve the specific parasitic trait of leeches, as discussed elsewhere [30]. Besides, the finding of a unique catalytic region within Cg ACE, and more generally within all lophotrochozoan ACE-like enzymes described to date [30], seems consistent with the late occurrence of genome duplication during the course of animal evolution. Indeed, this duplication occurred far after the Protostomes-Deuterostomes divergence and is widely admitted to explain the presence of two active site ACE-coding sequences in vertebrate genomes, such as the human ace-1 gene [3].…”

“…Regardless of this point, the presence of ACE in oysters is consistent with the presence of ACE orthologues throughout the animal kingdom [30], [31]. However, the biological function(s) of such orthologues has never been addressed outside Vertebrates and Ecdysozoans, despite being of interest for the understanding of the evolution of enzyme/substrates systems.…”

A Modern Understanding of the Traditional and Nontraditional Biological Functions of Angiotensin-Converting Enzyme

“…Although the precise function of ACE in this location is not fully understood, the nonspecificity of ACE peptide cleavage suggests the hypothesis that it may play a role in the digestion of peptides (Rivière et al, 2004). Analysis has demonstrated that single-catalytic domain ACE-like enzymes are expressed in the gut of other species such as the moth (Spodoptera littoralis: ABW34729.1) or the leech (Theromyzon tessulatum: AAS57725.1) (Wijffels et al, 1996;Rivière et al, 2004;Lemeire et al, 2008). In the leech, the expression of ACE is restricted to intestinal epithelial cells.…”

Angiotensin-converting enzyme in Spodoptera littoralis: Molecular characterization, expression and activity profile during development

“…that Race is expressed in the embryonic, larval and pupal stages of D. melanogaster, as analyzed by Northern blotting. Also for the non-insect invertebrate T. tessulatum (leech), TtACE was present throughout the life cycle (Riviere et al, 2004). Fig.…”