title: Enzymatic activity of sperm proprotein convertase is important for mammalian fertilization.
abstract: Proprotein convertase subtilisin/kexin 4 (PCSK4) is implicated for sperm fertilizing ability, based on studies using Pcsk4-null mice. Herein we demonstrated proprotein convertase (PC) activity in intact sperm and acrosomal vesicles. To determine whether this activity was important for sperm fertilizing ability, a peptide inhibitor was designed based on PCSK4 prodomain sequence (proPC4(75-90)), which contains its primary autocatalytic cleavage site. ProPC4(75-90) inhibited recombinant PCSK4's activity with a K(i) value of 5.4 µM, and at 500 µM, it inhibited sperm PC activity almost completely. Treatment of sperm with proPC4(75-90) inhibited their egg fertilizing ability in a dose dependent manner. Correlation between sperm PC activity and fertilizing ability showed a high co-efficient value (>0.9), indicating the importance of sperm PC activity in fertilization. In particular, sperm PC activity was important for capacitation and zona pellucida (ZP)-induced acrosome reaction, since proPC4(75-90) -treated sperm showed markedly decreased rates in these two events. These results were opposite to those observed in Pcsk4-null sperm, which contained higher PC activity than wild type sperm, possibly due to overcompensation by PCSK7, the other PCSK enzyme found in sperm. ADAM2 (45 kDa), a sperm plasma membrane protein, involved in sperm-egg plasma membrane interaction, was also processed into a smaller form (27 kDa) during capacitation at a much reduced level in proPC4(75-90) -treated sperm. This result suggested that ADAM2 may be a natural substrate of sperm PCSK4 and its cleavage by the enzyme during acrosome reaction may be relevant to the fertilization process.
authors.affiliation: Chronic Disease, Ottawa Hospital Research Institute, University of Ottawa, Ottawa, Ontario, Canada.
authors.forename: Sitthichai
authors.initials: S
authors.lastname: Iamsaard
authors.forename: Rapeepun
authors.initials: R
authors.lastname: Vanichviriyakit
authors.forename: Greanggrai
authors.initials: G
authors.lastname: Hommalai
authors.forename: Arpornrad
authors.initials: A
authors.lastname: Saewu
authors.forename: Nopparat
authors.initials: N
authors.lastname: Srakaew
authors.forename: Boonsirm
authors.initials: B
authors.lastname: Withyachumnarnkul
authors.forename: Ajoy
authors.initials: A
authors.lastname: Basak
authors.forename: Nongnuj
authors.initials: N
authors.lastname: Tanphaichitr
mesh_terms: D051722:ADAM Proteins / Q000378:metabolism*; D000177:Acrosome / Q000187:drug effects / Q000201:enzymology; D020101:Acrosome Reaction / Q000187:drug effects; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D000072196:Fertilins; D005306:Fertilization; D008297:Male; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D043484:Proprotein Convertases; D012697:Serine Endopeptidases / Q000378:metabolism*; D013075:Sperm Capacitation / Q000187:drug effects; D013094:Spermatozoa / Q000187:drug effects / Q000201:enzymology*; D013379:Substrate Specificity; D013381:Subtilisins; D015044:Zona Pellucida / Q000187:drug effects / Q000201:enzymology
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D004791:Enzyme Inhibitors; D008562:Membrane Glycoproteins; C460143:decanoylRVKRchloromethylketone; C489717:Pcsk4 protein, mouse; D043484:Proprotein Convertases; D012697:Serine Endopeptidases; D013381:Subtilisins; D051722:ADAM Proteins; C000606458:Adam2 protein, mouse; D000072196:Fertilins