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 ␤-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. INTRODUCTIONPolyspermy is lethal to the eggs of most species. If more than one sperm fertilizes the egg, then the newly formed zygote will cleave abnormally and die. One effective way to block polyspermy is for the egg to rapidly modify its extracellular surface after sperm activation so that subsequent sperm cannot reach the egg cell surface. A conserved mechanism for this modification is for the fertilizing sperm to induce the egg to exocytose contents from specialized organelles called cortical granules. The contents of the granule serve to modify the extracellular matrix of the egg, making it impenetrable to additional sperm fusions. The cortical granules have a specific and singular role during development: these organelles are present only in eggs and oocytes, and most of the known content proteins are expressed selectively in oocytes. Although the molecular mechanisms used to block polyspermy are different between animals, all mammals, most vertebrates, and many invertebrates, including the sea urchin share this fundamental process Wessel et al., 2001).Cortical granules of the sea urchin contain a diverse population of proteins, including structural molecules, enzym...

Fertilization and Nicotinic Acid Adenine Dinucleotide Phosphate Induce pH Changes in Acidic Ca2+ Stores in Sea Urchin Eggs

“…upon vesicle fusion with the plasma membrane, the alkaline extracellular medium exchanges with the acidic lumen of cortical granules, the primary egg exocytotic vesicles (12,14). Therefore, we simultaneously recorded pH L changes and exocytosis to see whether there was any spatial or temporal overlap.…”

“…The pH L of acidic vesicles in unfertilized eggs has been reported to be approximately pH 5.5 for both cortical granules (14) and yolk platelets (18), and it has been proposed that the pH within these vesicles serves to regulate luminal enzyme activity (14, 19 -21) and/or vesicular integrity (18). That the pH L within egg acidic vesicles can slowly acidify has been suggested during oogenesis (11,20,21) and post-fertilization (10), but with the exception of the rapid alkalinization of cortical granules upon fusion and exocytosis (12,14), rapid increases in the pH L of acidic vesicles have not been reported.…”

“…That the pH L within egg acidic vesicles can slowly acidify has been suggested during oogenesis (11,20,21) and post-fertilization (10), but with the exception of the rapid alkalinization of cortical granules upon fusion and exocytosis (12,14), rapid increases in the pH L of acidic vesicles have not been reported.…”

CO2-induced fertilization impairment in Strongylocentrotus droebachiensis collected in the Arctic

“…Eggs classified as UF included no lifting-off of the FE (NLO), unfertilized eggs (UnF) and fertilization failure (FF). Impairment in the FE formation may be involved in polyspermy events as documented in the literature (Vacquier et al 1973;LaFleur et al 1998;Haley and Wessel 2004b;Levitan et al 2007;Runnström and Manelli 1964;Longo 1978;Longo and Schuel 1973;Hiramoto 1962). For this reason, eggs in the highest lesion scale exhibited one or more pathological changes, such as irregular divisions, protrusion and bulges formation or cytoplasmic degeneration, together with abnormalities in the FE and/or HL.…”

“…Posterior studies identified and characterized the proteins involved in the process of cortical granules exocytosis Wessel 2004a, b, 1999;Wong and Wessel 2008) and observed fertilized eggs with similar ''blebby'' envelopes, after treatment in artificial seawater adjusted to pH 8, 7, 6, 5 and 4 as in our study. Haley and Wessel (2004b) showed that the cortical granules serine protease 1 (CGSP1), a protein secreted during the cortical reaction, is inhibited at lowered pH, decreasing its activity down to 25 % at pH 7. CGSP1 is activated at fertilization, mediates the protease responses that follow the cortical reaction and contributes to the block to polyspermy.…”