The Angiotensin-converting enzyme (Ance) gene of Drosophila melanogaster is a homologue of mammalian angiotensin-converting enzyme (ACE), a peptidyl dipeptidase implicated in regulation of blood pressure and male fertility. In Drosophila, Ance protein is present in vesicular structures within spermatocytes and immature spermatids. It is also present within the lumen of the testis and the waste bag, and is associated with the surface of elongated spermatid bundles. Ance mRNA is found mainly in large primary spermatocytes and is not detectable in cyst cells. Testes lacking germ cells have reduced levels of ACE activity, and no Ance protein is detectable by immunocytochemistry, indicating that the germ cells are the major site of Ance synthesis. Ance mutant testes lack individualised sperm and have very few actin-based individualisation complexes. Spermatid nuclei undergo scattering along the cyst and have abnormal morphology, similar to other individualisation mutants. Mutant spermatids also have abnormal ultrastructure with grossly defective mitochondrial derivatives. The failure of Ance mutant testes to form individualisation complexes may be due to a failure in correct spermatid differentiation. Taken together, the expression pattern and mutant phenotype suggest that Ance is required for spermatid differentiation, probably through the processing of a regulatory peptide synthesised within the developing cyst.

SUMMARY Angiotensin I-converting enzyme (ACE) expressed on the surface of endothelial cells is responsible for the last step in the synthesis of circulating angiotensin II and the inactivation of bradykinin. Mammalian ACE is also expressed in the prostate with other components of the renin–angiotensin system, and in developing spermatids, where the peptidase activity is known to be critical for normal sperm function. The importance of an ACE gene to male fertility has also been demonstrated in Drosophila melanogaster, where Ance is expressed in spermatids, and hypomorphic alleles of Ance cause a defect in spermiogenesis. Here we show that ANCE, which shares many enzymatic properties with mammalian ACE, is also a product of the male accessory gland of D. melanogaster. It is expressed in the secondary cells and is associated with the electron dense granule within the large vesicles of these cells. ACE proteolytic activity is lost from the accessory glands during mating,consistent with transfer to the mated female in the seminal fluid. The accessory gland ACE-like activity might have an evolutionarily conserved function processing biologically active peptides with a role in male fertility.

Background:In Drosophila melanogaster, the male seminal fluid contains proteins that are important for reproductive success. Many of these proteins are synthesised by the male accessory glands and are secreted into the accessory gland lumen, where they are stored until required. Previous studies on the identification of Drosophila accessory gland products have largely focused on characterisation of male-specific accessory gland cDNAs from D. melanogaster and, more recently, Drosophila simulans. In the present study, we have used a proteomics approach without any sex bias to identify proteins in D. melanogaster accessory gland secretions.Results: Thirteen secreted accessory gland proteins, including seven new accessory gland proteins, were identified by 2D-gel electrophoresis combined with mass spectrometry of tryptic fragments. They included protein-folding and stress-response proteins, a hormone, a lipase, a serpin, a cysteine-rich protein and two peptidases, a pro-enzyme form of a cathepsin K-like cysteine peptidase and a γ-glutamyl transpeptidase. Enzymatic studies established that accessory gland secretions contain a cysteine peptidase zymogen that can be activated at low pH. This peptidase may have a role in the processing of female and other male-derived proteins, but is unlikely to be involved in the processing of the sex peptide. γ-Glutamyl transpeptidases are type II integral membrane proteins; however, the identified AG γ-glutamyl transpeptidase (GGT-1) is unusual in that it is predicted to be a soluble secreted protein, a prediction that is supported by biochemical evidence. GGT-1 is possibly involved in maintaining a protective redox environment for sperm. The strong γ-glutamyl transpeptidase activity found in the secretions provides an explanation for the observation that glutamic acid is the most abundant free amino acid in accessory gland secretions of D. melanogaster. Conclusion:We have applied biochemical approaches, not used previously, to characterise prominent D. melanogaster accessory gland products. Of the thirteen accessory gland secreted proteins reported in this study, six were represented in a D. simulans male accessory gland EST library that was biased for male-specific genes. Therefore, the present study has identified seven new secreted accessory gland proteins, including GGT-1, which was not recognised previously as a secreted accessory gland product.

The mammalian neprilysin (NEP) family members are typically type II membrane endopeptidases responsible for the activation/inactivation of neuropeptides and peptide hormones. Differences in substrate specificity and subcellular localization of the seven mammalian NEPs contribute to their functional diversity. The sequencing of the Drosophila melanogaster genome has revealed a large expansion of this gene family, resulting in over 20 fly NEP-like genes, suggesting even greater diversity in structure and function than seen in mammals. We now report that one of these genes (Nep2) codes for a secreted endopeptidase with a highly restricted pattern of expression. D. melanogaster NEP2 is expressed in the specialized stellate cells of the renal tubules and in the cyst cells that surround the elongating spermatid bundles in adult testis, suggesting roles for the peptidase in renal function and in spermatogenesis. D. melanogaster NEP2 was found in vesicle-like structures in the syncytial cytoplasm of the spermatid bundles, suggesting that the protein was acquired by endocytosis of protein secreted from the cyst cells. Expression of NEP2 cDNA in D. melanogaster S2 cells confirmed that the peptidase is secreted and is only weakly inhibited by thiorphan, a potent inhibitor of human NEP. D. melanogaster NEP2 also differs from human NEP in the manner in which the peptidase cleaves the tachykinin, GPSGFYGVR-amide. Molecular modelling suggests that there are important structural differences between D. melanogaster NEP2 and human NEP in the S1' and S2' ligand-binding subsites, which might explain the observed differences in inhibitor and substrate specificities. A soluble isoform of a mouse NEP-like peptidase is strongly expressed in spermatids, suggesting an evolutionarily conserved role for a soluble endopeptidase in spermatogenesis.