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In the present study, an isoform of angiotensin-converting enzyme was characterized from the testis of a decapod crustacean, the crayfish Astacus leptodactylus. Angiotensin-converting enzyme cDNA, obtained by 3'- to 5' RACE of testis RNAs, codes for a predicted one-domain protein similar to the mammalian germinal isoform of angiotensin-converting enzyme. All amino acid residues involved in enzyme activity are highly conserved, and a potential C-terminus transmembrane anchor may be predicted from the sequence. Comparison of this testicular isoform with angiotensin-converting enzyme from other crustaceans, namely Carcinus maenas, Homarus americanus (both reconstituted for this study from expressed-sequence tag data) and Daphnia pulex, suggests that membrane-bound angiotensin-converting enzyme occurs widely in crustaceans, conversely to other invertebrate groups where angiotensin-converting enzyme is predominantly a soluble protein. In situ hybridization and immunohistochemistry performed on testis sections show that angiotensin-converting enzyme mRNA is mainly localized in spermatogonias, whereas protein is present in spermatozoids. By contrast, in vas deferens, immunoreactivity is detected in the seminal fluid rather than in germ cells. Accordingly, angiotensin-converting enzyme activity assays of testis and vas deferens extracts demonstrate that the enzyme is present in the membrane fraction in testis, but in the soluble fraction in vas deferens. Taken together, the results obtained in the present study suggest that, during the migration of spermatozoids from testis to vas deferens, the enzyme is cleaved from the membrane of the germ cells and released into the seminal fluid. To our knowledge, this present study is the first to report such a maturation process for angiotensin-converting enzyme outside of mammals.

The present study was initiated to characterize angiotensin-converting enzyme (ACE) in Crustaceans. Using degenerate DNA primers deduced from consensus sequences located upward and downward from the active site of ACEs from different arthropod species, several tissues from the crayfish Astacus leptodactylus were screened by RT-PCR. Amplicons were obtained from hepatopancreas, testis and hemocytes. Analysis of the predicted protein sequences after cloning and Northern blot experiments revealed an original and complex polymorphism of the ACE-like active site. Two variants were obtained in the hepatopancreas, one displaying a 6.4 kb size transcript, probably corresponding to a double domain ACE, with an unusual active site structure while the other had a transcript size of 2.5 kb, close to the size of the transcript obtained in testis and hemocytes (2 and 3kb, respectively), likely representing single domain enzymes. Functional assays using a synthetic substrate were performed from the different tissues and showed a maximal ACE-like activity associated to membrane fraction from testis and hepatopancreas.