Maturation, Aging and Death in Reproductive Tissues
(M604) IN VIVO SHEDDING OF THE GERMINAL ANGIOTENSIN-CONVERTING ENZYME (gACE) INVOLVES A SERINE PROTEASE AND IS CONTROLLED BY EPIDIDYMAL FACTOR(S).
Thimon, Veronique1, Metayer, Sonia1, Belghazi, Maya1, Dacheux, Francoise1, Dacheux, Jean-Louis1, Gatti, Jean-Luc1, 1 Institut National de la Recherche Agronomique, Nouzilly, France
ABSTRACT- A number of different cell-surface proteins are targets of specific proteolytic processing that releases their extracellular domain and may function as a post-translational switch for their biological activity. Among these proteins, the membrane angiotensin-converting enzyme (ACE), a key factor in blood-pressure control, has been shown in vitro to be shed from cells, a process also suggested to occur in vivo and allowing this protein to become a blood-circulating enzyme. We have previously reported that the specific germinal ACE isoform (gACE) is found in the epididymal fluid of different mammals and that this gACE originates from the sperm surface. Here, we demonstrate that the extracellular domain of gACE is liberated from spermatozoa by an active proteolytic process that occurs in a precise spatio-temporal location during epididymal transit. Using polyclonal antibodies against the C-terminal intracellular sequence of ACE, a fragment of about 10 kDa was detected on ram and boar sperm membrane only in the epididymal region where in vivo the gACE liberation occurs. Using an in vitro assay, we showed that the gACE cleavage from ram sperm is specifically inceased by the presence of the epididymal fluid from the liberation zone and that this process involves a protease inhibited by the serine protease inhibitor AEBSF but not by para-aminobenzamidine. The involvement of a serine protease is also supported nature of the cleavage site: the fluid liberated enzyme was purified and by a mass spectrometry this site was situated after Arg650 of the gACE sequence. We also demonstrate that the gACE release did not involved tyrosine or serine phosphorylations of the gACE intra- or extra-cellular domains. These results indicated that the male epididymis provides a good model to investigate the mechanism by which the ACE could be released from cells and also to explore the nature of the ACE-sheddase involved in this important physiological regulatory mechanism. This data may also help to understand the infertility phenotype resulting from ACE knockout in mouse, showing the importance of the gACE for male reproduction.
KEY WORDS: epididyme, sheddase, angiotensin-I converting enzyme, fertility