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The proteolytic cleavage pathways of E-cadherin endogenously expressed in MDCK (Madin-Darby canine kidney) cells were characterized in cells treated with antimycin A and deoxyglucose to examine transmembrane protein processing under cellular stress. E-cadherin is a type I transmembrane protein which operates as the cell adhesion molecule component of the adherens junction, a complex of proteins involved in epithelial tissue development and integrity. We now demonstrate that treatment of MDCK cells with antimycin A and deoxyglucose activates caspase mediated pathways that cleave E-cadherin. E-cadherin is cleaved into two major fragments, with the sizes predicted by the location of a caspase-3 cleavage consensus sequence. Cleavage of E-cadherin and deposition of the C-terminal fragment into the cytoplasm are inhibited by the caspase inhibitor DEVD-CHO. Thus, a major mechanism for E-cadherin cleavage and dissolution of the adherens junction under antimycin/deoxyglucose treatment is caspase mediated, initiated by activation of an apoptosis pathway.

The solubilized jelly coat of Strongylocentrotus purpuratus eggs was boiled in SDS and mercaptoethanol, precipitated in ethanol, washed in 70% ethanol, lyophilized, and redissolved in seawater. The sample retained its activity to induce the sperm acrosome reaction (AR), showing that the AR inducer was resistant to harsh denaturing conditions. Egg jelly (EJ) was separated into its component macromolecules by denaturing polyacrylamide gel electrophoresis and the gel silver-stained. Several macromolecules ranging from approximately 30 to 380 kDa were consistently observed. These macromolecules were not seen by Coomassie blue staining. With the exception of the fucose sulfate polymer (FSP; 380 kDa) and the 300-kDa component, the other molecules were of minor abundance. Sephacryl-500 gel-filtration chromatography in detergents and disulfide reducing agents separated EJ into three fractions. The FSP and 300-kDa components were purified to homogeneity. A third fraction consisted of components of 30, 82, 116, and 138 kDa. The purified FSP and 300-kDa components had insignificant AR-inducing activity. Substantial AR activity was found only in the 30 to 138-kDa fraction. Ion-exchange chromatography and Centricon filtration separated the 82- and 138-kDa glycoproteins from the other components, but these two components could not be separated from each other. There may be other AR-inducing molecules in sea urchin EJ that were not visible after silver staining of gels. Also, some EJ components are so large that they did not enter the stacking gel. Of those macromolecules that were visible on gels after silver staining, only the fraction containing the 80- and 138-kDa proteins had significant AR-inducing activity.

Intact egg jelly (EJ) coats surrounding eggs of the sea urchin Strongylocentrotus purpuratus were visualized in stereo images of platinum replicas produced by the quick-freeze, deep-etch, rotary-shadowing technique. The hydrated EJ coat forms an extensive fibrous network that makes contact with the vitelline layer at the egg surface. Fibers are decorated along their length with particles, particle density being highest in the interior regions of the coat. The macromolecular components making up the EJ network were visualized by rotary-shadowing of mica-adsorbed EJ samples. Whole EJ coats solubilized in pH 5 sea-water and spread on the mica surface consist of complex networks of branching fibers decorated with large patches of amorphous material. As we have previously shown (Keller and Vacquier, 1994), EJ boiled in a dissolution buffer containing SDS and beta-mercaptoethanol and applied to a Sephacryl-500 gel filtration column can be separated into three fractions: a 380-kDa fucose sulfate polymer (FSP), which elutes in the void volume, and two column-included fractions consisting of intermediate (300 kDa) and low-molecular-weight (30- to 138-kDa) glycoproteins. Rotary-shadowing of the FSP fraction reveals branched fibrous components similar in appearance to that of solubilized whole EJ but devoid of any particulate decoration. In contrast, intermediate- and low-molecular-weight EJ components are strictly globular in appearance but are distinguishable on the basis of size. Ion-exchange purification of whole EJ yields two glycoproteins, of 82 and 138 kDa, having AR-inducing activity (Keller and Vacquier, 1994). Platinum replication shows these active components to be small spherical molecules about 8 nm in diameter. The above fractionation scheme requires harsh dissociation conditions. Indeed, if EJ is not boiled in SDS buffer before fractionation, the 300-kDa fraction and the FSP appear together in the void volume. Rotary-shadowing of this complex reveals a multistranded polymer, decorated with glycoproteins at specific kink points. Taken together, our data suggest that the EJ network is composed of a fucose sulfate polymer superstructure to which glycoproteins are bound.

When spermatozoa of Arbacia punctulata are labeled with 32P and treated with soluble egg jelly, radiolabel is incorporated into histone H3. The time course of labeling correlates with the period of chromatin decondensation of sperm pronuclei in eggs. Phosphorylation is on serine and may result from increased turnover of phosphate on H3. The macromolecular fraction of egg jelly (and not the peptide fraction) is the inducer of H3 phosphorylation. The reaction is dependent on external Ca2+ and is induced by monensin and A23187. H3 phosphorylation is not induced by the phosphodiesterase inhibitor IBMX and relatively high (250 microM) concentrations of the protein kinase inhibitor H8 are needed to block the reaction, suggesting that it is cAMP independent. A surprising finding is that merely diluting the cells into Na+ free media is the most effective method to induce the radiolabeling of H3. These results are in contrast to findings on the egg jelly induced phosphorylation of histone H1 in S. purpuratus spermatozoa. These species differences must reflect the great evolutionary divergence between these two sea urchin species in the mechanism of regulation of the phosphorylation of nuclear proteins during fertilization.