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Ovoperoxidase is a cortical granule-derived enzyme that hardens the sea urchin fertilization envelope by catalyzing the formation of dityrosine residues. Ovoperoxidase works in concert with a second protein, proteoliaisin, which anchors ovoperoxidase to the nascent fertilization envelope in a divalent cation-dependent manner. In this study, we examined the Ca(2+)-dependent interaction of proteoliaisin with ovoperoxidase in rotary-shadowed Pt replicas. Ovoperoxidase, a uniformly sized globular molecule, binds to a distal portion of rod-shaped proteoliaisin when low concentrations of Ca2+ are present. Higher Ca2+ concentrations lead to the formation of extended proteoliaisin strands that are decorated along their lengths with ovoperoxidase. Using immunogold labeling, we also examined the assimilation of these two proteins into the fertilization envelope in quick-frozen, deeply etched samples. Both proteins are abundant in the fertilization envelope as early as one minute after fertilization. Coincident with paracrystalline coating of the envelope, the labeling density is markedly reduced, suggesting that antigenic sites may be masked by the paracrystalline coat. This suggests that the ovoperoxidase-proteoliaisin complex resides within the central portion of the fertilization envelope, rather than in the paracrystalline coat.

Fertilization of the sea urchin egg leads to the assembly of an extracellular matrix, the fertilization envelope. Ovoperoxidase, the enzyme implicated in hardening the fertilization envelope, is inserted into the assembling structure via a Ca2+-dependent interaction with the protein proteoliasin (P. Weidman and B. M. Shapiro, 1987, J. Cell Biol. 105, 561-567). In the present report, polyclonal antisera were raised to ovoperoxidase and proteoliasin (purified from eggs of Strongylocentrotus purpuratus) and characterized by Western blot analysis and an enzyme-linked immunoabsorbent assay (ELISA). By indirect immunofluorescence microscopy all cortical granules of unfertilized eggs, as well as the fertilization envelope, contained both proteoliasin and ovoperoxidase. At the ultrastructural level both proteins are localized to the electron-dense spiral lamellae of the cortical granules. Western blot analysis revealed that ovoperoxidase and proteoliasin persist in early embryos until hatching, but are absent from later developmental stages. Homogenates of eggs of several other echinoderm species (Strongylocentrotus droebachiensis, Strongylocentrotus franciscanus, Pisaster ochraceus, Dendraster excentricus, and Lytechinus pictus) also contain proteins antigenically similar to ovoperoxidase and proteoliaisin, indicating that many echinoderms utilize a similar strategy for assembly of the fertilization envelope. The results underline the need for postsecretory controls in the extracellular matrix modifications that accompany the cortical reaction.