Regulated proteolysis by cortical granule serine protease 1 at fertilization

Abstract

Cortical granules are specialized organelles whose contents interact with the extracellular matrix of the fertilized egg to form the block to polyspermy. In sea urchins, the granule contents form a fertilization envelope (FE), and this construction is critically dependent upon protease activity. An autocatalytic serine protease, cortical granule serine protease 1 (CGSP1), has been identified in the cortical granules of Strongylocentrotus purpuratus eggs, and here we examined the regulation of the protease activity and tested potential target substrates of CGSP1. We found that CGSP1 is stored in its full-length, enzymatically quiescent form in the granule, and is inactive at pH 6.5 or below. We determined the pH of the cortical granule by fluorescent indicators and micro-pH probe measurements and found the granules to be pH 5.5, a condition inhibitory to CGSP1 activity. Exposure of the protease to the pH of seawater (pH 8.0) at exocytosis immediately activates the protease. Activation of eggs at pH 6.5 or lower blocks activation of the protease and the resultant FE phenotypes are indistinguishable from a protease-null phenotype. We find that native cortical granule targets of the protease are beta-1,3 glucanase, ovoperoxidase, and the protease itself, but the structural proteins of the granule are not proteolyzed by CGSP1. Whole mount immunolocalization experiments demonstrate that inhibition of CGSP1 activity affects the localization of ovoperoxidase but does not alter targeting of structural proteins to the FE. The mistargeting of ovoperoxidase may lead to spurious peroxidative cross-linking activity and contribute to the lethality observed in protease-null cells. Thus, CGSP1 is proteolytically active only when secreted, due to the low pH of the cortical granules, and it has a small population of targets for cleavage within the cortical granules.

Figure 1.

Spectral profile of LysoSensor Yellow/Blue DND-160 was determined in situ by a Zeiss 510 equipped for META spectral imaging (A–C) and by a fluorimeter (D). After standard recordings at various pHs, the LysoSensor spectrum was determined in situ in whole eggs slightly compressed to reveal the cortical granules at the surface of the cell (A), or in isolated cell surface complex consisting of cortical granules attached to the plasma membrane (B and C). Pseudocolor was assigned to pixels based on an overlay with standards. (D) Isolated cell surface complex was also examined in a fluorimeter after treatment with LysoSensor. In comparison with standards of defined pH, the cortical granules closely overlay the pH 5.5 standard.

Figure 2.

CGSP1 is inhibited at low pH. (A) Column-purified CGSP1 was assayed in vitro at different pH values by using BAEE as a substrate. (B) Brightfield images of live eggs activated with A23187 in low pH sea water form abnormal fertilization envelopes, similar to the phenotype displayed when eggs are activated in the presence of SBTI. Bar, 40 μm.

Figure 5.

Enzymatic contents of the cortical granule are cleaved at fertilization. Immunoblot analysis of cortical granule exudate using antibodies generated against the enzymes of the granule. The enzymes are cleaved in a protease-dependent manner after fertilization. SBTI concentration 100 μg/ml.

Figure 3.

Immunoblot analysis of cortical granule exudate in the presence and absence of protease inhibitors indicates that these proteins are not cleaved by CGSP1. SBTI concentration 100 μg/ml.

Figure 4.

Fertilization envelope forms but does not detach in protease-null cells. However, the targeting of the structural proteins of the envelope is not affected by the absence of protease activity. Arrows indicate the FE on fixed cells. Bar, 40 μm.

Figure 7.

Protease activity has opposing effects on ovoperoxidase and β-1,3 glucanase. (A) Egg β-1,3 glucanase is more active when CGSP1 is active. In vitro glucanase activity was detected by measuring laminarin hydrolysis by β-1,3 glucanase in the presence or absence of the protease inhibitor benzamidine. (B) Protease activity suppresses ovoperoxidase activity initially after fertilization. In vivo ovoperoxidase was measured by an Amplex Red-based fluorescence assay.

Figure 6.

Protease activity is necessary for targeting ovoperoxidase to the fertilization envelope. Ovoperoxidase localizes to the fertilization envelope after cortical granule exocytosis in fixed embryos (A and B). However, when CGSP1 is inhibited, the ovoperoxidase is mistargeted to the egg plasma membrane (C and D). (E) Quantification of ovoperoxidase in the fertilization versus the plasma membrane. Bar, 40 μm.