Shedding of the germinal angiotensin I-converting enzyme (gACE) involves a serine protease and is activated by epididymal fluid.
The present report describes how the soluble germinal angiotensin I-converting enzyme (gACE) appears in the epididymal fluid, where it has been identified in some laboratory rodents and domestic ungulates. We showed that this gACE results from an active proteolytic process that releases the enzyme's extracellular domain from sperm in a precise spatiotemporal location during epididymal transit and that this process involves serine protease activity. Using polyclonal antibodies against the C-terminal intracellular sequence of ACE, a fragment of approximately 10 kDa was detected on the sperm extract only in the epididymal region, where the gACE release occurs. The fluid enzyme was purified, and the cleavage site was determined by mass spectrometry to be between Arg622 and Leu623 of the mature sheep gACE sequence (equivalent to Arg627 and Arg1203 of the human mature gACE and somatic ACE sequences, respectively). Thereafter, the C-terminal Arg was removed, leaving Ala621 as a C-terminal. Using an in vitro assay, gACE cleavage from sperm was strongly increased by the presence of epididymal fluid from the release zone, and this increase was inhibited specifically by the serine protease-inhibitor AEBSF but not by para-aminobenzamidine. None of the other inhibitors tested, such as metallo- or cystein-protease inhibitors, had a similar effect on release. It was also found that this process did not involve changes in gACE phosphorylation.
Thimon
Véronique
V
Gamètes Mâles et Fertilité, UMR 6175 INRA-CNRS-Université de Tours-Haras Nationaux, Nouzilly, France.
Métayer
Sonia
S
Belghazi
Maya
M
Dacheux
Françoise
F
Dacheux
Jean-Louis
JL
Gatti
Jean-Luc
JL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001550:Benzamidines / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D004789:Enzyme Activation; D004822:Epididymis / Q000378:metabolism*; D045604:Extracellular Fluid / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D007703:Peptidyl-Dipeptidase A / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D017421:Sequence Analysis; D012697:Serine Endopeptidases / Q000187:drug effects / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D012756:Sheep; D013094:Spermatozoa / Q000378:metabolism; D013450:Sulfones / Q000494:pharmacology; D013552:Swine
D001550:Benzamidines; D015842:Serine Proteinase Inhibitors; D013450:Sulfones; C002010:4-(2-aminoethyl)benzenesulfonylfluoride; C006500:4-aminobenzamidine; D007703:Peptidyl-Dipeptidase A; D012697:Serine Endopeptidases