Identification of a cysteine protease responsible for degradation of sperm histones during male pronucleus remodeling in sea urchins.
We have identified a 60-kDa cysteine protease that is associated with chromatin in sea urchin zygotes. This enzyme was found to be present as a proenzyme in unfertilized eggs and was activated shortly after fertilization. At a pH of 7.8-8.0, found after fertilization, the enzyme degraded the five sperm-specific histones (SpH), while the native cleavage-stage (CS) histone variants remained unaffected. Based on its requirements for reducing agents, its inhibition by sulfhydryl blocking compounds and its sensitivity to the cysteine-type protease inhibitors (2S,3S)-trans-epoxysuccinyl-L-leucyl-amido-3-methylbutane-ethyl-es ter (E-64 d), cystatin and leupeptin, this protease can be defined as a cysteine protease. Consistently, this protease was not affected by serine-type protease inhibitors phenylmethylsulfonyl fluoride (PMSF) and pepstatin. The substrate selectivity and pH modulation of the protease activity strongly suggest its role in the removal of sperm-specific histones, which determines sperm chromatin remodeling after fertilization. This suggestion was further substantiated by the inhibition of sperm histones degradation in vivo by E-64 d. Based on these three lines of evidence, we postulate that this cysteine protease is responsible for the degradation of sperm-specific histones which occurs during male pronucleus formation.
Imschenetzky
M
M
Department of Molecular Biology, Universidad de Conceptión, Chile.
Díaz
F
F
Montecino
M
M
Sierra
F
F
Puchi
M
M
D000818:Animals; D002467:Cell Nucleus / Q000201:enzymology / Q000378:metabolism*; D002843:Chromatin / Q000201:enzymology; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004527:Egg Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D004792:Enzyme Precursors / Q000737:chemistry; D005260:Female; D006657:Histones / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008247:Lysosomes / Q000201:enzymology; D008297:Male; D008970:Molecular Weight; D019163:Reducing Agents / Q000494:pharmacology; D012617:Sea Urchins; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013084:Sperm-Ovum Interactions; D013094:Spermatozoa / Q000378:metabolism*; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D015053:Zygote / Q000201:enzymology
D002843:Chromatin; D015853:Cysteine Proteinase Inhibitors; D004527:Egg Proteins; D004792:Enzyme Precursors; D006657:Histones; D019163:Reducing Agents; D015842:Serine Proteinase Inhibitors; D013439:Sulfhydryl Reagents; D003546:Cysteine Endopeptidases