Characterization of a proteinase that cleaves zona pellucida glycoprotein ZP2 following activation of mouse eggs.
Here, we describe an in vitro assay that has permitted further characterization of a proteinase (called "ZP2-proteinase") that is released upon activation of ovulated mouse eggs and cleaves ZP2, one of three glycoproteins present in mouse zonae pellucidae. Results presented suggest that ZP2-proteinase readily diffuses through the zona pellucida within 5 min of activation of eggs by ionophore A23187 and carries out limited proteolysis of ZP2. Appearance of ZP2-proteinase is completely dependent upon activation of eggs, consistent with it being present in cortical granule exudate. The proteinase is insensitive to a wide variety of proteinase inhibitors, but is inhibited when either an anti-ZP2 monoclonal antibody or an Fab fragment of the antibody is bound to ZP2. Proteolysis occurs near the amino- or carboxy-terminus of ZP2, producing a 23,000 Mr glycopeptide(s) that remains attached to ZP2 by intramolecular disulfide bonds. HPLC fractionation of activated egg exudate suggests that ZP2-proteinase has an apparent Mr between 21,000 and 34,000. Proteolysis of ZP2 correlates with "hardening" of the zona pellucida following egg activation and, thus, may be responsible for one aspect of the zona reaction.
Moller
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Department of Cell and Developmental Biology, Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110.
Wassarman
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D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D004527:Egg Proteins / Q000378:metabolism*; D005306:Fertilization; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008562:Membrane Glycoproteins; D051379:Mice; D010063:Ovum / Q000378:metabolism* / Q000502:physiology*; D010447:Peptide Hydrolases / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D011956:Receptors, Cell Surface; D015044:Zona Pellucida / Q000378:metabolism*; D000072216:Zona Pellucida Glycoproteins
D004527:Egg Proteins; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D011480:Protease Inhibitors; D011956:Receptors, Cell Surface; C000606515:ZP2 protein, human; D000072216:Zona Pellucida Glycoproteins; C000606520:Zp2 protein, mouse; D000001:Calcimycin; D010447:Peptide Hydrolases