scholarly journals Regulated Proteolysis by Cortical Granule Serine Protease 1 at Fertilization

2004 ◽  
Vol 15 (5) ◽  
pp. 2084-2092 ◽  
Author(s):  
Sheila A. Haley ◽  
Gary M. Wessel

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 β-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.

Development ◽  
1994 ◽  
Vol 120 (5) ◽  
pp. 1325-1333 ◽  
Author(s):  
M. Laidlaw ◽  
G.M. Wessel

Cortical granules are secretory vesicles formed in the eggs of most animals and are essential for the prevention of polyspermy in these organisms. We have studied the biogenesis of cortical granules in sea urchin oocytes by identifying cDNA clones that encode proteins targeted selectively to the cortical granules. These cDNA clones were identified by an immunoscreen of a cDNA library using antibodies to proteins of the fertilization envelope. Four different mRNAs were identified, ranging from 4 kb to 13 kb in length, that encoded proteins targeted specifically to cortical granules. Accumulation of these mRNAs began very early in oogenesis, in oocytes approximately 10–15 microns in diameter, and continued throughout oogenesis. The mRNAs reached peak abundance (on a per cell basis) in germinal vesicle stage oocytes, and the accumulation of each mRNA was linear with respect to oocyte growth. During breakdown of the germinal vesicle these mRNAs were degraded so that in eggs the mRNA signals were at background levels. Antibodies generated to recombinant proteins made from each of these cDNA clones showed that in the oocyte each cognate protein appeared early in oogenesis. These proteins accumulated only in cortical granules: no accumulation was seen in the cytoplasm, in Golgi, or in other vesicles, and no heterogeneity of the contents was seen within the population of cortical granules. Using these antibodies we show that cortical granules accumulated linearly throughout oogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)


2006 ◽  
Vol 17 (12) ◽  
pp. 5241-5252 ◽  
Author(s):  
Julian L. Wong ◽  
Gary M. Wessel

Preventing polyspermy during animal fertilization relies on modifications to the egg's extracellular matrix. On fertilization in sea urchins, the contents of cortical granules are secreted and rapidly assemble into the egg's extracellular vitelline layer, forming the fertilization envelope, a proteinaceous structure that protects the zygote from subsequent sperm. Here, we document rendezvin, a gene whose transcript is differentially spliced to yield proteins destined for either cortical granules or the vitelline layer. These distinctly trafficked variants reunite after cortical granule secretion at fertilization. Together, they help coordinate assembly of the functional fertilization envelope, whose proteome is now defined in full.


Blood ◽  
2004 ◽  
Vol 103 (6) ◽  
pp. 2299-2307 ◽  
Author(s):  
Masayuki Okada ◽  
Souichi Adachi ◽  
Tsuyoshi Imai ◽  
Ken-ichiro Watanabe ◽  
Shin-ya Toyokuni ◽  
...  

Abstract Caspase-independent programmed cell death can exhibit either an apoptosis-like or a necrosis-like morphology. The ABL kinase inhibitor, imatinib mesylate, has been reported to induce apoptosis of BCR-ABL–positive cells in a caspase-dependent fashion. We investigated whether caspases alone were the mediators of imatinib mesylate–induced cell death. In contrast to previous reports, we found that a broad caspase inhibitor, zVAD-fmk, failed to prevent the death of imatinib mesylate–treated BCR-ABL–positive human leukemic cells. Moreover, zVAD-fmk–preincubated, imatinib mesylate–treated cells exhibited a necrosis-like morphology characterized by cellular pyknosis, cytoplasmic vacuolization, and the absence of nuclear signs of apoptosis. These cells manifested a loss of the mitochondrial transmembrane potential, indicating the mitochondrial involvement in this caspase-independent necrosis. We excluded the participation of several mitochondrial factors possibly involved in caspase-independent cell death such as apoptosis-inducing factor, endonuclease G, and reactive oxygen species. However, we observed the mitochondrial release of the serine protease Omi/HtrA2 into the cytosol of the cells treated with imatinib mesylate or zVAD-fmk plus imatinib mesylate. Furthermore, serine protease inhibitors prevented the caspase-independent necrosis. Taken together, our results suggest that imatinib mesylate induces a caspase-independent, necrosis-like programmed cell death mediated by the serine protease activity of Omi/HtrA2.


2011 ◽  
Vol 47 (3) ◽  
pp. 345-353 ◽  
Author(s):  
P. Mäntylä ◽  
E. Buduneli ◽  
G. Emingil ◽  
T. Tervahartiala ◽  
P. J. Pussinen ◽  
...  

1961 ◽  
Vol 113 (2) ◽  
pp. 359-380 ◽  
Author(s):  
Georges Ungar ◽  
Takuso Yamura ◽  
Jacqueline B. Isola ◽  
Sidney Kobrin

Protease activity was measured through the hydrolysis of synthetic amino acid esters in body fluids and tissues of guinea pigs, rats, mice, and humans. Significant in vitro activation was observed in serum and lung slices of sensitized guinea pigs on addition of the specific antigen. Increased proteolytic activity was also seen in reverse anaphylaxis. More marked activation occurred when guinea pig serum was treated with peptone and guinea pig or rat serum was treated with agar. Protease activation was demonstrated in specimens of human skin under the influence of a poison ivy extract or croton oil added in vitro. Urinary protease activity of guinea pigs increased significantly during the first hours of anaphylactic shock and very markedly in peptone shock. Peptone shock, elicited in mice pretreated with H. pertussis, was accompanied by a considerable increase in protease activity in the peritoneal fluid as compared with non-pretreated mice which were insensitive to peptone. Proteolytic activity resulting from the activation procedures was due to a number of proteases. The dominant substrate affinity and inhibition patterns suggest that serum and urine proteases are similar to but not identical with plasmin. Anaphylactic activation exhibited patterns different from those resulting from the action of anaphylactoid agents. Tissue enzymes are either of cathepsin- or chymotrypsin-type or mixtures of both. Some of the activated enzymes, although remarkably effective in hydrolyzing amino acid esters, show no activity on protein substrates. This does not justify, however, their designation as "esterases." They probably belong to the class of specific proteases acting only on a single or a small number of functionally significant protein substrates. There is at present sufficient evidence to prove not only that protease activation does occur in anaphylaxis and anaphylactoid conditions but also that it is an important component of the chain of reactions leading to the allergic response.


2001 ◽  
Vol 285 (4) ◽  
pp. 863-872 ◽  
Author(s):  
Marie-Thérèse Château ◽  
Véronique Robert-Hebmann ◽  
Christian Devaux ◽  
Jean-Bernard Lazaro ◽  
Bruno Canard ◽  
...  

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