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

Abstract

Angiotensin-converting enzyme (ACE) is a highly conserved metallopeptidase. In mammals, the somatic isoform governs blood pressure whereas the germinal isoform (tACE) is required for fertility. In Ecdysozoans, ACE-like enzymes are implicated in reproduction. Despite ACE orthologues being present from bacteria to humans, their function(s) remain(s) unknown in distant organisms such as Lophotrochozoans. In silico analysis of an oyster (Crassostrea gigas) EST library suggested the presence of an ACE orthologue in molluscs. Primer walking and 5′-RACE revealed that the 1.9 kb cDNA encodes CgACE, a 632 amino acid protein displaying a conserved single active site and a putative C-terminal transmembrane anchor, thus resembling human tACE, as supported by molecular modelling. FRET activity assays and Maldi-TOF spectrometry indicated that CgACE is a functional dipeptidyl-carboxypeptidase which is active on Angiotensin I and sensitive to ACE inhibitors and chloride ion concentration. Immunocytochemistry revealed that, as its human counterpart, recombinant CgACE is synthesised as a transmembrane enzyme. RT-qPCR, in-situ hybridization and immunohistochemistry shed light on a tissue, and development stage, specific expression pattern for CgACE, which is increased in the gonad during spermatogenesis. The use of ACE inhibitors in vivo indicates that the dipeptidase activity of CgACE is crucial for the oyster fertilization. Our study demonstrates that a transmembrane active ACE is present in the oyster Crassostrea gigas, and for the first time ascribes a functional role for ACE in Lophotrochozoans. Its biological function in reproduction is conserved from molluscs to humans, a finding of particular evolutionary interest especially since oysters represent the most important aquaculture resource worldwide.

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

“…Also, chicken (Gallus gallus) lacks testis ACE expression so that the physiologic requirement for testis ACE does not appear absolute (Esther et al, 1994). ACE, however, has been implicated in playing an important role in the fertility of insects and oysters (Ekbote et al, 2003;Hurst et al, 2003;Riviere et al, 2011). Thus, the role of ACE in reproduction may have played an important role in maintaining the structure of at least one of the two domains.…”

GigaTON: an extensive publicly searchable database providing a new reference transcriptome in the pacific oyster Crassostrea gigas

“…This indicates a noticeable difference in protein sets between the Pacific oyster and other invertebrates. Such a discrepancy between invertebrates seems surprising, except if one considers the growing number of documented lophotrochozoan features, such as DNA methylation patterns [ 24 , 30 ], angiotensin-converting enzyme characteristics [ 31 , 32 ] or BMP cDNA sequences [ 33 ], that display a closer resemblance with vertebrates than with ecdysozoans. Besides, in a similar fashion than mapping long 454 reads helped assembling genomic scaffolds [ 13 ], the GigaTON database provides even longer contigs that we think could importantly help improve the current assembly of the oyster genome.…”

DNA methylation in adults and during development of the self‐fertilizing mangrove rivulus, Kryptolebias marmoratus

“…RT-qPCR was performed as previously described (Riviere, Fellous, Franco, Bernay, & Favrel, 2011). Briefly, samples were extracted using affinity chromatography (Nucleospin RNA II kit, Macherey-Nagel).…”