BackgroundAngiotensin-converting enzyme (ACE), which metabolizes many peptides and plays a key role in blood pressure regulation and vascular remodeling, as well as in reproductive functions, is expressed as a type-1 membrane glycoprotein on the surface of endothelial and epithelial cells. ACE also presents as a soluble form in biological fluids, among which seminal fluid being the richest in ACE content - 50-fold more than that in blood.Methods/Principal FindingsWe performed conformational fingerprinting of lung and seminal fluid ACEs using a set of monoclonal antibodies (mAbs) to 17 epitopes of human ACE and determined the effects of potential ACE-binding partners on mAbs binding to these two different ACEs. Patterns of mAbs binding to ACEs from lung and from seminal fluid dramatically differed, which reflects difference in the local conformations of these ACEs, likely due to different patterns of ACE glycosylation in the lung endothelial cells and epithelial cells of epididymis/prostate (source of seminal fluid ACE), confirmed by mass-spectrometry of ACEs tryptic digests.ConclusionsDramatic differences in the local conformations of seminal fluid and lung ACEs, as well as the effects of ACE-binding partners on mAbs binding to these ACEs, suggest different regulation of ACE functions and shedding from epithelial cells in epididymis and prostate and endothelial cells of lung capillaries. The differences in local conformation of ACE could be the base for the generation of mAbs distingushing tissue-specific ACEs.

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The effect of low concentrations of hydrogen peroxide (10-100 µM) on sperm motility and on the activity of the sperm enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDS) was investigated. Incubation of semen samples with 10 and 100 µM hydrogen peroxide increased the content of spermatozoa with progressive motility by 20 and 18%, respectively, and enhanced the activity of GAPDS in the sperm cells by 27 and 20% compared to a semen sample incubated without additions. It was also found that incubation with 10 µM hydrogen peroxide increased the content of reduced glutathione (GSH) in sperm cells by 50% on average compared to that in the control samples. It is supposed that low concentrations of hydrogen peroxide activate the pentose phosphate pathway, resulting in NADPH synthesis and the reduction of the oxidized glutathione by glutathione reductase yielding GSH. The formed GSH reduces the oxidized cysteine residues of the GAPDS active site, increasing the activity of the enzyme, which in turn enhances the content of sperm cells with progressive motility. Thus, the increase in motile spermatozoa in the presence of low concentrations of hydrogen peroxide can serve as an indicator of normal functioning of the antioxidant defense system in sperm cells.

Epithelial cells of prostate express significant level of ACE and, as a result, seminal fluid has 50-fold more ACE than plasma. The substitution of highly specialized prostate epithelial cells by tumor cells results in dramatic decrease in ACE production in prostate tissues. We performed detailed characterization of ACE status in prostate tissues from patients with benign prostate hyperplasia (BPH) and prostate cancer (PC) using new approach- ACE phenotyping, that includes evaluation of: 1) ACE activity with two substrates (HHL and ZPHL); 2) the ratio of the rates of their hydrolysis (ZPHL/HHL ratio); 3) the ratio of immunoreactive ACE protein to ACE activity; 4) the pattern of mAbs binding to different epitopes on ACE – ACE conformational fingerprint - to reveal conformational changes in prostate ACE due to prostate pathology. ACE activity dramatically decreased and the ratio of immunoreactive ACE protein to ACE activity increased in PC tissues. The catalytic parameter, ZPHL/HHL ratio, increased in prostate tissues from all patients with PC, but was did not change for most |BPH patients. Nevertheless, prostate tissues of several patients diagnosed with BPH based on histology, also demonstrated decreased ACE activity and increased immunoreactive ACE protein/ACE activity and ZPHL/HHL ratios, that could be considered as more early indicators of prostate cancer development than routine histology. Thus, ACE phenotyping of prostate biopsies has a potential to be an effective approach for early diagnostics of prostate cancer or at least for differential diagnostics of BPH and PC.

Angiotensin I‐converting enzyme (ACE) is a glycoprotein, consisting of two homologous domains within a single polypeptide chain. ACE concentration in biological fluids is an important parameter of clinical observation; its increase or decrease may accompany various pathologies. Currently, the exact crystal structure of the two‐domain ACE form is still unknown because of microheterogeneity and intensity of the enzyme glycosylation. Raman spectroscopy provides the qualitative and quantitative analysis of many compounds, including proteins. For the first time, surface‐enhanced Raman scattering (SERS) spectra of native and thermo‐denatured human ACE have been demonstrated with full assignment. Denaturation leads to SERS intensity increase and bands shifting. Detailed band assignment and discussion are included to elucidate the potential site of ACE interaction with the silver surface. Based on SERS spectra, we characterized the region on the ACE molecule in contact with the substrate and demonstrated the model of the two‐domain ACE adsorbed on a silver matrix.