Cleavage of arginyl-arginine and lysyl-arginine from the C-terminus of pro-hormone peptides by human germinal angiotensin I-converting enzyme (ACE) and the C-domain of human somatic ACE.
Mammalian germinal angiotensin I-converting enzyme (gACE) is a single-domain dipeptidyl carboxypeptidase found exclusively in male germ cells, which has almost identical sequence and enzymic properties with the C-domain of the two-domain somatic ACE. Mutant mice that do not express gACE are infertile, suggesting a role for the enzyme in the processing of undefined peptides involved in fertilization. A number of spermatid peptides [e.g. cholecystokinin (CCK) and gastrin] are processed from pro-hormones by endo- and exo-proteolytic cleavages which might generate substrates for gACE. We have shown that peptide hormone intermediates with Lys/Arg-Arg at the C-terminus are high-affinity substrates for human gACE. gACE from human sperm cleaved Arg-Arg from the C-terminus of the CCK5-GRR (GWMDFGRR), a peptide corresponding to the C-terminus of a CCK-gastrin prohormone intermediate. Hydrolysis of CCK5-GRR by recombinant human C-domain ACE was Cl- dependent, with maximal activity achieved in 5-10 mM NaCl at pH 6.4. C-Domain ACE cleaved Lys/Arg-Arg from the C-terminus of dynorphin-(1-7), a pro-TRH peptide KRQHPGKR, and two insect peptides FSPRLGKR and FSPRLGRR. C-Domain ACE displayed high affinity towards all these substrates with Vmax/Km values between 14 and 113 times greater than the Vmax/Km for the conversion of the best known ACE substrate, angiotensin I, into angiotensin II. In conclusion, we have identified a new class of substrates for human gACE, and we suggest that gACE might be an alternative to carboxypeptidase E for the trimming of basic dipeptides from the C-terminus of intermediates generated from pro-hormones by subtilisin-like convertases in human male germ cells.
Isaac
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RE
School of Biology, University of Leeds, Leeds LS2 9JT, U.K.
Williams
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Sajid
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Corvol
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P
Coates
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D002712:Chlorides / Q000494:pharmacology; D002766:Cholecystokinin / Q000378:metabolism*; D004151:Dipeptides / Q000378:metabolism; D005755:Gastrins / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008297:Male; D010446:Peptide Fragments / Q000378:metabolism; D007703:Peptidyl-Dipeptidase A / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism; D013094:Spermatozoa / Q000201:enzymology*
D002712:Chlorides; D004151:Dipeptides; D005755:Gastrins; D010446:Peptide Fragments; D011498:Protein Precursors; D011994:Recombinant Proteins; C076685:arginylarginine; C042873:preprogastrin; C042082:preprocholecystokinin; D002766:Cholecystokinin; D007703:Peptidyl-Dipeptidase A