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A 9.6 S protein is the third calcium-insoluble component of the sea urchin hyaline layer
ARCHIVBS
OF BIOCHEMISTRY
AND BIOPHYSICS
Vol. 294, No. 1, April, pp. 297-305, 1992
A 9.6 S Protein Is the Third Calcium-Insoluble Component of the Sea Urchin Hyaline Layer’ Robin W. Justice,* Glenn M. Nagel,tp2 Carl F. Gottschling,? and Edward J. Carroll, Jr.* *Department and Institute
Marina
F. Damis,?
of Biology, University of California, Riverside, California 92521, and tDepartment of Chemistry and Biochemistry for Molecular Biology and Nutrition, California State University, Fullerton, California 92634
Received October 11,1991, and in revised form December 9, 1991
A third major, calcium-insoluble component of the sea urchin (Strongylocentrotue purpuratue) hyaline layer has been purified and physically characterized. In the absence of divalent cations, the native, soluble protein has a sedimentation coe6lcient of 9.6 S and a molecular weight of 4.6 f 0.1 X 10’. These data indicate that this large protein assumes an elongated, nonspherical conformation in solution. Its sedimentation behavior and its mobility on nondenaturing electrophoretic gels distinguish the 9.6 S protein from the 11.6 S and 6.4 S hyalin proteins we have previously characterized. That the 6.4 S, 9.6 S, and 11.6 S proteins are the major calcium-insoluble structural components of the hyaline layer is supported by the fact that we have found them in a variety of hyalin protein fractions prepared by a number of standard approaches. All three proteins are precipitated by calcium ions, thus fitting the operational definition of hyalin. Evidence is presented that the 11.6 S protein may overlie the 9.6 S protein in the hyaline layer. QlS92AcademicPrem,Inc.
The sea urchin hyaline layer (l-3) is a proteinaceous extracellular coat that surrounds the early embryo. The blastomeres adhere to the hyaline layer (4) and its integrity is dependent on calcium ions in the seawater (5). The hyaline layer begins to appear a few seconds after insemination as the cortical reaction, a global exocytosis of secretory vesicles (the cortical granules) located just beneath the egg plasma membrane, commences. In transmission electron micrographs, material that appears to form the hyaline layer is seen emerging from the cortical granules 1 This research was supported, in part, by an award from Research Corporation to G.M.N. and a University of California, Riverside, Chancellor’s Patent Fund grant to R.W.J. ’ To whom correspondence should be addressed. ooo3-9&x/92 $3.00 Copyright 0 1992 by Academic Press, Inc. AI1 rights of reproduction in any form reserved.
during exocytosis (6,7). The early hyaline layer is overlain by a tough, impermeable fertilization envelope that also forms during the cortical reaction from the vitelline envelope and cortical granule-derived components. The major structural component of the hyaline layer, called hyalin (8), is operationally defined as a high-molecular-weight protein fraction soluble in the absence of divalent cations and rendered reversibly insoluble by calcium ions at concentrations comparable to that of seawater. Hyalin has been isolated from a number of sea urchin species and several distinct methods have been used for its isolation (these methods as applied in our laboratory are described, in turn, under Materials and Methods): (1) Hyalin has been dissolved from the surface of intact embryos after hatching (9) or after vitelline envelope disruption (10-13). (2) It has been collected from the cortical granules by activating eggs under conditions where formation of the fertilization envelope is blocked (14) or where the vitelline envelope is disrupted before egg activation (15). (3) Hyalin also has been obtained by isolating whole hyaline layers from embryos and dissolving the layers in 1 M urea (16) or Ca2+/Mg2+-free seawater (17). (4) In addition, hyalin has been precipitated from egg homogenates or extracts with Ca2+ (8, 18, 19). (5) Finally, hyalin has been isolated from intact cortical granules (20, 21). Polyclonal (22) and monoclonal (23) antibodies to hyalin localize to the cortical granules and some smaller vesicles before fertilization and to the hyaline layer after fertilization. It has been suggested that the hyaline layer has a role in the morphogenesis of the embryo because treatments that partially disrupt the hyaline layer also interfere with gastrulation (17, 24, 25). Evidence that this effect might be mediated by hyalin itself comes from work demonstrating that blastomeres adhere to hyalin in vitro, but those destined to migrate into the blastocoel lose their affinity for hyalin (11). Also, a monoclonal antibody di297
JUSTICE
rected against hyalin protein is able to block gastrulation and plutear arm formation (26). We have previously shown that when examined under nondenaturing conditions, hyalin actually contains up to three components by analytical ultracentrifugation analysis (12). The hyalin used in that study was prepared by treating acid-dejellied eggs with dithiothreitol to disrupt the vitelline envelopes (27), a treatment that prevents fertilization envelope formation, then dissolving the hyaline layers from intact 45-min embryos in a divalent cation-free medium and, finally, precipitating hyalin with 100 mM CaC12. Such preparations were enriched in one hyalin component, a 920-kDa, 11.6 S protein, when very mild (“less stringent”) dejellying conditions were employed. “More stringent” dejellying conditions resulted in additional components in the final Ca2+ precipitate. We subsequently described a 280-kDa, 6.4 S protein, isolated by allowing cortical granule exocytosis to take place into Ca2+/Mg2+-free seawater containing EGTA3 (28). This procedure leads to the formation of a thin, incompletely formed fertilization envelope permeable to the cortical granule contents (29). The Ca2+/Mg2+-insoluble fraction of this cortical granule exudate consists entirely of the 6.4 S and 11.6 S proteins. The 6.4 S component corresponds in electrophoretic and sedimentation behavior to the slowest sedimenting component of “more stringently dejellied” hyalin preparations. In the present study, we describe the isolation and physical characterization of the third, 9.6 S component from hyalin preparations. MATERIALS
AND METHODS
Gamete Preparation Gametes of Strongylmentrotus purpuratus (Alacrity, Inc., Redondo Beach, CA, and Pacific Bio-Marine, Venice, CA) were obtained by injection of 0.5 M KC1 into the perivisceral coelom of the adult. Sperm was collected “dry” from the aboral surface of shedding males and held on ice. Eggs were rinsed through 202~pm mesh Nitex to remove debris and washed with seawater at 11°C. Reagents, unless otherwise noted, were obtained from Sigma (St. Louis, MO).
Hyalin Preparations 1. Dissolved hyaline layers. Hyalin proteins were dissolved from the surface of intact embryos with disrupted vitelline envelopes by the method described in (12), with the following modifications. The washed eggs were not dejellied prior to dithiothreitol (DTT) treatment and the time used for the incubation with DTJ! to disrupt the vitelline envelopes was 3 min unless otherwise indicated. The hyaline layers were dissolved from the 45min embryo surfaces using a 2-min incubation in 475 mu NaCl, 25 mM KCl, 2 mM EGTA, 5 mM benzamidine, and the hyalin proteins were redissolved by dialysis at 4°C against 100 mM Tris-HCl,
3 Abbreviations used: DTT, dithiothreitol; EGTA, ethylene glycol bis(p-aminoethyl ether) N,N’-tetraacetic acid; BASW, artificial seawater (500 mM NaCl, 10 mM KCl, 10 mM Tris-HCl, 50 mM MgCl,, 10 mM CaCl,, pH 8.0) containing 5 mM benzamidine; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate.
ET AL. 5 mu benzamidine, 5 mu EDTA, pH 8.0, or 20 mM Tris, 5 mM benzamidine, 2 mM EGTA, pH 8.0.
2. Cortical granule exudate. The calcium-insoluble protein fraction of cortical granule exudate was prepared by a method previously described (28) with the following variations. Benzamidine (5 mM), to inhibit cortical granule protease activity (30), was added at times varying from 10 s before insemination to 5 min after insemination and the Caz’/ Mg*+-insoluble precipitate was dissolved as for the dissolved hyaline layers. 3. Whole hyaline luyers. Whole hyaline layers were isolated from prehatching embryos by a modification of the method described in (17). A suspension of 45-min S. purpuratua embryos in artificial seawater (500 mM NaCl, 10 mM KCl, 10 mM Tris-HCl, 50 mM MgClz, 10 mM CaClz, pH 8.0) containing 5 mu benzamidine (BASW) was homogenized for 3 min, ll”C, 14,000 rpm in a Waring blender to break the fertilization envelopes and remove the hyaline layers. The homogenate was then centrifuged 10 min, llOOg, 4°C giving a two-layered pellet with fertilization envelopes at the bottom and the whiter hyaline layers at the top, plus a frothy, floating mat enriched in hyaline layers. The top layer of the pellet was rinsed away from the lower layer by a gentle stream of BASW and then pooled with the floating material. The pooled material was homogenized in a Dounce apparatus (“A” pestle) in BASW on ice and centrifuged at llOOg, 10 min, 4’C. The homogenization and centrifugation were repeated, each time using only the floating material and the top fraction of the pellet, until all cytoplasmic debris was removed, as judged by light microscopy. At this point, only small fragments of fertilization envelopes remained and the hyaline layers were dissolved away from them by dialysis against 100 mM Tris-HCl, 5 mM EDTA, 5 mM benzamidine, pH 8.0, 4°C. Remaining fertilization envelopes were removed by centrifugation at ZO,OOOg,20 min, 4“C. Alternatively, the hyaline layers were physically separated from the fertilization envelope fragments using a sucrose step gradient by the method used in (17). To obtain whole hyaline layers from posthatching stages of S. purpuratm, embryos were allowed to develop at 11°C. The swimming embryos were concentrated by centrifugation at llOOg, 30 s, 4°C. The embryos were suspended in ice-cold BASW and homogenized with a Dounce apparatus (“A” pestle) to break the hyaline layers open. The hyaline layers were collected by centrifugation (llOOg, 10 min, 4°C) and homogenized in BASW. The homogenate was rinsed through 35-am mesh Nitex, which retains most of the hyaline layers. These processes were repeated until the filtrate was clear. The hyaline layers were then dissolved by dialysis against 100 mM Tris-HCl, 5 mu EDTA, 5 r&f benzamidine, pH 8.0,4”C. proteins 4. Whole egg homogenates. To prepare calcium-insoluble from whole egg homogenates unfertilized eggs were centrifuged 30 s at 3OOg, 4’C, the pelleted eggs were homogenized in a Dounce apparatus (“A” pestle) in 10 mM Tris-HCl, 5 mM benzamidine, pH 8.0, on ice, and 100 mM CaClz was added to the homogenate. After incubation for 30 min, on ice, the homogenate was centrifuged llOOg, 10 min, 4°C and 5 mM the pelleted material was dialyzed against 100 mM Tris-HCl, benzamidine, pH 8.0, 4°C. After dialysis, the insoluble material was removed by centrifugation at 2O,OOOg,20 min, 4”C, and 100 mM CaClz was added to the supernatant solution, which was incubated on ice, overnight. The calcium-insoluble material was collected by centrifugation at ZO,OOOg,20 min, 4°C. The precipitate was dissolved by dialysis at 4°C against 100 mM Tris-HCl, 5 mM EDTA, 5 mM benzamidine, 1 mM 3-amino-1,2,4,-triazole, pH 8.0.
5. Isolated cortical granules. Intact cortical granules were isolated from S. purpuratus by the procedure in (20) as modified in (21). The isolated cortical granules were lysed by homogenization in a Dounce apparatus (“A” pestle) in 100 mM Tris-HCl, 5 mM EDTA, 5 mM benzamidine, 1 mM 3-amino-1,2,4-triazole, pH 8.0, on ice. The crude lysate was centrifuged at ZO,OOOg,20 min, 4”C, 100 mu CaClz was added to the supernatant solution, and the mixture was incubated at 4”C, overnight. The calcium-insoluble material was collected by centrifugation at 2O,OOOg,20 min, 4°C. The precipitate was collected and dissolved as for the whole egg homogenate.
9.6 S HYALIN
Gel Electrophoresis
and Staining
Polyacrylamide gel electrophoresis (PAGE) was performed according to the method in (31) with the following modifications. Gels were 4% a&amide, 0.1% methylenebisacrylamide (A&age1 and bisAcrylage1, National Diagnostics, Highland Park, NJ) in 0.75 mM slab gels and 5 mM EDTA was included in the gels and all associated buffers as described in (28). Nondenaturing (native) gels omitted sodium dodecyl sulfate (SDS) from the gels and buffers, while all reducing gels included 4% j3mercaptoethanol in the sample buffer. The SDS-containing samples were incubated 2 h at 37°C prior to electrophoresis. Protein standards (Bio-Rad, Richmond, CA) for molecular weight estimation on reducing SDS gels were myosin (200 kDa), /3-galactosidase (116.25 kDa), phosphorylase B (92.5 kDa), bovine serum albumin (66.2 kDa), and ovalbumin (45 kDa). Native gels were stained with methylene blue (Matheson, Coleman and Bell, Norwood, OH) by a modification of the method in (32) for acidic proteins. Gels were washed in two changes of 200 ml 2:1, glassdistilled water:methanol, 5 mM sodium phosphate, pH 7.0 (gel fix), for 1 h each and then stained 10 min at room temperature in 200 ml 0.2% methylene blue, 5 mu sodium phosphate, pH 7.0. The gels were destained in 200 ml gel fix. For photography, gels were washed 5-10 min in 150 ml fresh gel fix (reserving the original destaining solution), moved to 50 ml fresh gel fix, and photographed immediately using Technical Pan film (Eastman Kodak Co., Rochester, NY). Gels were then returned to the reserved solution in which the stain remains stable indefinitely. Gels containing SDS were double-stained. First, after being rinsed in 40% methanol, 10% acetic acid, gels were stained in 0.1% Coomassie brilliant blue G-250,40% methanol, 10% acetic acid for 30 min at 37°C and then destained in several changes of 5% methanol, 7.5% acetic acid to reveal the molecular weight standards. Second, the gels were washed thoroughly with gel fix to neutralize the acid and then stained as above with methylene blue.
299
PROTEIN
tein, meniscus-depletion sedimentation equilibrium measurements (35) were made at 6800,7200, and 8000 rpm between 6 and 9°C. Run times varied from 43 to 134 h. The buffer employed in all experiments was 0.01 M Tris-HCl, 0.575 M NaCl, 0.01 M KCl, 5 mM EDTA, pH 8.0.
RESULTS
Hyalin was first prepared by dissolving hyaline layers from intact, 45min embryos in Ca2+/Mg2+-free seawater and subsequent Ca2+ precipitation. Before fertilization, 7-ml aliquots of eggs were treated with DTT to disrupt the vitelline layer (method 1 under Materials and Methods). The composition of hyalin prepared from these embryos was observed to vary with the length of DTT treatment (Fig. 1). A relatively brief treatment with the reductant (90 s) gave a hyalin fraction (1.7 mg total protein/aliquot) consisting mostly of the low-mobility, 11.6 S hyalin protein described in (12). Longer DTT treatment of other aliquots of the same batch of eggs gave hyalin fractions containing increased amounts of two additional components (5.5 f 0.8 mg total protein/aliquot). After very long DTT treatment (15 min) the hyalin fraction once again consisted mainly of the 11.6 S protein (2.9 mg total protein), though, unlike preparations using shorter DTT treatments, many of the eggs lysed on activation. The fastest migrating and least abundant material on
12345678
Purification
of Proteins by Rate-Zonal
Centrifugation
The 9.6 S protein was purified by rate-zonal centrifugation on 5-20% sucrose density gradients in 100 mM Tris-HCl, 5 mM EDTA, 5 mM benzamidine, pH 8.0, in 13.4-ml Beckman Ultraclear centrifuge tubes, 4“C. A dissolved hyalin or hyaline layer preparation (0.5 ml, 0.8 mg protein/ml) was layered on each gradient and the tubes were centrifuged in a Beckman SW41Ti swinging bucket rotor at 41,000 rpm, 25 h, 4”C, in a Beckman Model L5-65B preparative ultracentrifuge. The gradient was fractionated from the bottom of the tube, 0.4 ml/fraction at 4°C. The fractions were examined by native PAGE and fractions containing the 9.6 S component with a purity of 90% or more, as assessed after methylene blue staining, were pooled The pooled fractions were dialyzed against 100 mM Tris-HCl, 5 mM EDTA, 5 mM benzamidine, pH 8.0, to remove sucrose, concentrated by centrifugation in Centricon 10 microconcentrators (Amicon, Danvers, MA), and fractioned on a second set of 5-20% sucrose gradients (0.5 ml/gradient, 0.5 mg/ml). These gradients were treated as described above and the fractions were examined by native PAGE. The fractions containing only the 9.6 S component were pooled, dialyzed, and concentrated. The 11.6 S and 6.4 S proteins were obtained in pure form by a single round of 5-20% sucrose density gradient fractionation performed as for the 9.6 S protein except that the calcium-insoluble fraction of cortical granule exudate with benzamidine added 90 s after insemination (0.5 ml/gradient, 0.8 mg/ml) was used as the starting material. These preparations were virtually free of 9.6 S material. Protein concentration was determined calorimetrically (33) by using bovine serum albumin as a standard or from the ultraviolet absorbance (34).
Analytical
Ultracentrifugation
Sedimentation velocity and sedimentation equilibrium measurements were completed as previously described (12). For the purified 9.6 S pro-
-11.6 S -9.6s -6.4 5
FIG. 1. Native PAGE of hyalin proteins dissolved from the surface of intact, 45-min embryos with different degrees of vitelline envelope disruption. The length of time that the eggs were treated with dithiothreitol to disrupt the vitelline envelopes was varied as follows: 90-s treatment (lane 1, 1.7 pg protein); 3-min treatment (lane 2, 4.4 pg protein); 6-min treatment (lane 3, 6.1 pg protein); 9-min treatment (lane 4, 4.4 pg protein); 12-min treatment (lane 5, 5.5 pg protein); 15-min treatment (lane 6, 1.9 pg protein). Lane 7 contains 5.8 fig of Sephacryl S-500 column-purified 11.6 S protein. Lane 8 contains 10 pg of the calcium-insoluble fraction of cortical granule exudate with benzamidine added at 90 s after insemination consisting of the 11.6 S and 6.4 S components only. This figure and other figures depicting native PAGE are composites of lanes from two or more gels. To compare mobilities among gels a sample of hyalin proteins dissolved from intact, 45-min embryos was included on each gel and used to standardize the photographic prints, and the position of each of these standard components is marked on the right side of the gel.
300
JUSTICE
electrophoretic gels of the three-component mixtures corresponds to the 6.4 S protein isolated from the calciuminsoluble fraction of cortical granule exudate (Fig. 1) prepared as described in (28). Thus, by elimination, the intermediate electrophoretic hyalin component appeared to correspond to the protein having a sedimentation coefficient between the 6.4 S and 11.6 S proteins. This component previously has been observed in hyalin preparations from “more stringently dejellied” eggs (12). When the hyalin fraction was prepared from cortical granule exudate (method 2 under Materials and Methods), the presence of the intermediate component was dependent on the inclusion of benzamidine, a protease inhibitor, during cortical granule exocytosis. Cortical granule exudate is the material that leaks through the thin fertilization envelope that is formed when eggs are diluted into Ca2+/Mg2+-free seawater containing EGTA during the cortical reaction (28, 29). Analytical ultracentrifugal analysis of preparations treated with benzamidine before the cortical reaction revealed the presence of three components (Fig. 2). A number of similar analyses where benzamidine was added at times varying from 10 s before insemination to 300 s after insemination (Fig. 3) showed that the intermediate component was present only when 5 mM benzamidine was added before the start of the cortical reaction, a process beginning 28 s after insemination at 15°C. Electrophoretic gels of the calcium-insoluble fraction of cortical granule exudate with benzamidine added before the cortical reaction (10 s after insemination) showed the intermediate band, whereas when benzamidine was added after the cortical reaction, this band was absent (Fig. 4). These data also reinforce the conclusion that the intermediate electrophoretic and ultracentrifugal components are the same. To provide additional evidence that the three proteins we observed in hyalin fractions are actually hyaline layer components, whole hyaline layers were isolated from 45 min embryos or from hatched blastulas by a modification of the method in (17) (method 3 under Materials and Methods). In both preparations, fertilization envelopes were absent before solubilization of the hyaline layer.
FIG. 2. Schlieren profile of a three-component hyalin preparation. Hyalin protein prepared from the calcium-insoluble fraction of cortical granule exudate (benzamidine added 20 s after insemination) (see Materials and Methods) was examined in the analytical ultracentrifuge. The photograph was taken 72 mm after reaching a rotor velocity of 60,000 rpm at a rotor temperature of 6.2”C and a Schlieren diaphragm angle of 70’. Light path was 12 mm.
ET AL.
-20
0
20
40
60
60
100 300
lime (set)
FIG. 3.
Effect of the timing of benzamidine addition on the composition of the calcium-insoluble fraction of cortical granule exudate. The calcium-insoluble fraction of cortical granule exudate was prepared as described under Materials and Methods with the time of addition of benzamidine (to 5 mM final concentration) varied between 10 s before and 5 min after insemination. The composition of the calcium-insoluble fraction was determined by integration of Schlieren profiles after components were separated by analytical centrifugation (m, 6.4 S; A, 11.6 S; 0, 9.6 S). The arrow indicates the approximate beginning of the cortical reaction (CGE, 28 s after insemination), measured as the time when fertilization envelopes can first be seen elevating from egg surfaces by phase-contrast microscopy.
Three components with the same electrophoretic mobilities as those present in hyalin fractions dissolved from the surface of intact, 45min embryos were the only bands observed (Fig. 4). As noted above, hyalin has been isolated previously from egg homogenates and shown to originate within the cortical granules of the unfertilized egg. To determine whether all three native hyalin components were present within the egg before fertilization, a calcium precipitate of whole egg homogenates and the calcium-insoluble contents of isolated cortical granules (methods 4 and 5, respectively, under Materials and Methods) from unfertilized eggs were examined electrophoretically. Three components with the same mobilities as those in hyalin preparations were the only ones present on native gels (Fig. 4) of both preparations. The intermediate component, however, appeared somewhat diminished in proportion in the material prepared from isolated cortical granules. To purify the intermediate component, mixtures of hyalin proteins dissolved from the surface of intact, 45-min embryos or from dissolved, whole hyaline layers were subjected to rate-zonal centrifugation on 5-20% sucrose density gradients that included 5 mM benzamidine. Frac-
9.6 S HYALIN
-11.6s .9.6S .6.4 5
301
PROTEIN
Sedimentation velocity analysis of the purified protein gave the results shown in Fig. 6. The sedimentation coefficient, s20,w,was only moderately dependent on protein concentration, c. Fitting the data to the equation %!o,w = sL,& - kc)
FIG. 4. Native PAGE of preparations containing hyalin proteins. The following protein preparations are shown: hyalin proteins dissolved from the surface of intact, 45-min embryos (lane 1); calcium-insoluble protein fraction of cortical granule exudate with bsnxamidine added at 10 s after insemination (lane 2); calcium-insoluble protein fraction of cortical granule exudate with benxamidine added at 90 s after insemination (lane 3); proteins from whole hyaline layers isolated from 45-min embryos (lane 4); proteins from whole hyaline layers isolated from hatched blastulas (lane 5); calcium-insoluble proteins isolated from whole egg homogenates (lane 6); and calcium-insoluble proteins from a lysate of isolated cortical granules (lane 7). Approximately 10 fig of total protein was loaded on each lane.
tions were examined via electrophoresis. When relatively high concentrations of protein were loaded on the gradient (3.2 mg/tube; Fig. 5A), the separation of components was poor. This result is consistent with the observation that the sedimentation coefficient of the 11.6 S component is highly concentration dependent, being lower at higher protein concentrations (12). Thus the concentration of the protein mixture loaded on the gradient is critical to the success of the separation of the 11.6 S component from the intermediate component. At an eightfold lower protein concentration (0.4 mg/tube) the separation of components was greatly improved with a number of fractions containing >90% of the intermediate component by the gel assay (Fig. 5B). To achieve the final purification, fractions containing 90% or more of the intermediate component were pooled, concentrated as described under Materials and Methods, and fractionated on a second set of sucrose gradients. The second separation provided an excellent resolution of the intermediate component from the small amount of contaminating 11.6 S protein initially present (Fig. 5C). Fractions showing no detectable contamination with the 11.6 S protein by the gel assay were pooled and concentrated for further analysis. In addition, the 11.6 S and 6.4 S proteins were purified from the calcium-insoluble fraction of cortical granule exudate with benzamidine added 90 s after insemination (a preparation which lacks the intermediate component) by a single round of density gradient ultracentrifugation (data not shown).
gave a value of 9.6 S for s&~, the extrapolated value at infinite dilution, and a value for the constant It = 2 X 10e4ml/mg. Sedimentation equilibrium analysis yielded linear plots of In c vs ? (Fig. 7), indicating that the preparations were essentially monodisperse. Results from four determinations, at three different rotor velocities, gave a value of 447,000 + 5000 for the molecular weight. In combination, these data yield a value for the frictional ratio, f/to, of 2.3 and indicate a nonspherical shape for the 9.6 S protein. Assuming a shape approximating a prolate ellipsoid and an average hydration of 0.5 g H20/g protein, the calculated axial ratio, afb, is 14. Freshly purified 9.6 S material examined on a native polyacrylamide gel (Fig. 8) migrated as a single band with a mobility indistinguishable from that of the same component in the hyalin preparation from which it was isolated. This was also true of the 11.6 S and 6.4 S proteins (Fig. 8) purified by density-gradient sedimentation. This method of purification of the 6.4 S protein is thus more effective than the gel permeation chromatography used previously (28), where the column-purified protein exhibited a slight increase in mobility over the same component in the starting material. When the 9.6 S preparation was allowed to age at 4°C a faster-migrating band appeared on native gels of the preparation (Figure 8) with the same mobility as density gradient-purified 6.4 S protein. When purified 9.6 S material was examined under denaturing but not reducing conditions (in the presence of SDS but not P-mercaptoethanol) it resolved into four electrophoretic bands (Fig. 9). These four bands had the same relative mobilities as the four fastest-migrating bands resolved from hyalin containing all three native proteins (Fig. 9, compare lanes 3 and 4). The slow-migrating band in the hyalin mixture has the same mobility as the purified 11.6 S protein (Fig. 9, lane l), while, under the same conditions, the most rapidly migrating band in gels of both the mixture and the purified 9.6 S protein has the same mobility as the single band representing the sucrose gradient-purified 6.4 S component (Fig. 9, lane 2). These data suggest that the 6.4 S component may be derived from the 9.6 S component. Samples of the three native proteins purified on sucrose density gradients were also examined on reducing SDS gels (Fig. 9, lanes 5-8). The 11.6 S component migrates as a single band with an apparent size of 320 kDa. The 9.6 S component is not clearly resolved, but produces a smear of staining between 170 and 250 kDa. The 6.4 S
302
JUSTICE
ET AL.
C
B -11.6 S -9.6s -6.4 S
-11.6 S -9.6 S -6.4 S
FIG. 5. Native PAGE of fractions from 5-20% sucrose density gradients. Fractions were collected starting with the 20% end of each gradient. Samples of the fractions (16 pl of each) were loaded sequentially across the gel. (A) Dissolved whole hyaliie layers were used as the starting material and 3.2 mg of total protein was loaded on the gradient in A. Fractions 4 through 31 are shown. (B) Hyalin dissolved from intact, 45-min embryos was used as the starting material and 0.4 mg of total protein was loaded on the gradient in B. Fractions 5 through 16 are shown. (C) Fractions from six sucrose gradients identical to the gradient in B containing >90% 9.6 S protein, as assayed by native PAGE, were pooled, concentrated as described under Materials and Methods, and rerun on a 5-20% sucrose density gradient. Fractions 5 through 16 are shown; 0.25 mg of total protein was loaded on this gradient. The position at which each of the three native hyalin components runs is marked on the right side of each panel.
component migrates as a single band with a size of 180 kDa as reported in (28). Surprisingly, a three-component hyalin preparation run under reducing, denaturing conditions gives only one major band and several much fainter ones. While the major band has the mobility of the 320kDa, 11.6 S component, there is only a faint band at 250 kDa and no band at 180 kDa. DISCUSSION
We have now isolated and purified the three major protein components of the calcium-insoluble fraction of the embryonic extracellular matrix of the sea urchin generally referred to as the hyaline layer. Purification by rate-zonal centrifugation in the presence of the protease inhibitor benzamidine has allowed us to obtain homogeneous preparations of the previously described 11.6 S and 6.4 S components as well as a third, 9.6 S component which we had been unable to resolve by gel permeation chromatography. To achieve optimal separation of the 11.6 S and 9.6 S proteins by centrifugation, it was critical that low con-
centrations of protein be used, owing to the concentration dependence of the sedimentation coefficients of the proteins. The native 11.6 S component, which showed both a major and a minor band on reducing, denaturing gels when purified by gel-permeation chromatography (12,28), showed only the major, high-molecular-weight band (320 kDa) when purified in the ultracentrifuge. The 6.4 S protein had the same electrophoretic mobility before and after ultracentrifuge purification, while its mobility increased slightly after chromatographic purification (28), indicating that some degradation may have occurred in the latter case. The better preservation of 11.6 S and 6.4 S proteins likely is due to the continuous presence of protease inhibitor in the ultracentrifuge purification scheme. That the 9.6 S native protein is an authentic component of the hyaline layer was shown by the presence of 9.6 S protein in whole hyaline layers as well as in the calciuminsoluble fraction of material dissolved from the surface of embryos. The fact that no other proteins were observed as components of the five types of hyalin preparations we
9.6 S HYALIN
303
PROTEIN
-11.6s -9.65 -6.4 S
9.6 q
9.6 -
q q
9.4 -
9.2 -
9.0 9.0 -01 0.0 0.0
I 0.5
1 1.0
Protein Concentration
I 1.5
2.0
(wml)
FIG. 6. The dependence of the sedimentation coefficient of the purified 9.6 S protein on protein concentration. The value of sU),n(in Svedbergs) is plotted versus the protein concentration determined by integration of Schlieren boundaries. A value of sioW = 9.6 S was obtained after extrapolation to zero protein concentration.
examined indicates that these three are the only major calcium-insoluble proteins associated with the hyaline layer. All three hyalin proteins were present in whole hyaline layers from 45 min postfertilization until after hatching was complete; thus, these proteins apparently persist from the early embryo stage through the enzymatic hatching process. Analysis of the 9.6 S protein in the analytical ultracentrifuge revealed it to be a large, 450,000-molecularweight protein. Like the 11.6 S and 6.4 S proteins, the 9.6 S protein appears to have an elongated, nonspherical shape in solution. Our experiments showed only a modest
FIG. 8. Native polyacrylamide gel electrophoresis of 11.6 S, 9.6 S, and 6.4 S proteins purified by rate-zonal centrifugation. Purified 9.6 S protein was run on a native polyacrylamide gel just after the protein had been prepared (lane 1). The same sample was run under native conditions following a month of storage at 4’C (lane 2). Also illustrated are density gradient-purified 6.4 S (lane 3) and 11.6 S (lane 4) proteins. Approximately 5 pg of protein was loaded per well.
dependence of sZo,+., on protein concentration for the 9.6 S protein, in agreement with our previous findings (12). In this earlier work, we observed that szo,Wof the 11.6 S protein was remarkably concentration dependent such that the 11.6 S protein was not separable from the intermediate component (then termed “8.8 S”) at 2 mg of protein/ml, whereas two separate boundaries were readily
12
3
4
5
6
7
8
-0.4 B & ,c
-0.6
-1.2
-1.6 ! 49.6
I 50.0
I 50.2
I 50.4
I 50.6
rexp2 FIG. 7. Sedimentation equilibrium data for the purified 9.6 S protein. The results of a typical experiment are plotted as the natural logarithm of the Rayleigh fringe displacement versus the square of the radial position. The protein sample was centrifuged at a rotor velocity of 7200 rpm at a rotor temperature of 6°C.
FIG. 9. SDS-polyacrylamide gel electrophoresis of hyalin proteins. Protein preparations were denatured in the absence (lanes l-4) or presence (lanes 5-8) of fl-mercaptoethanol. The 11.6 S protein (lanes 1 and 5), 6.4 S protein (lanes 2 and 6), and 9.6 S protein (lanes 3 and 7) preparations were purified by rate-zonal centrifugation and approximately 10 pg of total protein was loaded per lane. Lanes 4 and 8 contain hyalin proteins dissolved from intact, 45min embryos; this preparation contained all three native components and is the same one shown under native conditions in Fig. 1, lane 2. Approximately 5 pg of total protein was loaded on lanes 4 and 8. For samples containing fl-mercaptoethanol, the chain sizes (in KDa)] indicated were obtained using standard proteins as described under Materials and Methods.
304
JUSTICE
ET AL.
observed at concentrations below 1 mg of protein/ml. The DTT may leave large amounts of the vitelline envelope higher sedimentation coefficient obtained for the inter- intact (37), although damaging it enough so that fertilmediate component in this study as compared to that ization envelope elevation does not occur (38). Thus, less previously reported may result from the fact that it was total protein was obtained from the 90-s treatment bemeasured previously only in mixtures composed predom- cause of the physical barrier of the partial envelope. By inantly of the 11.6 S protein. In addition, the difference this reasoning, the 9.6 S component, which appeared after may stem from the fact that previous preparations had longer treatments, must be less accessible to the dissolving suffered degradation. Data obtained from reducing SDS- medium than the 11.6 S component, perhaps because 9.6 gel electrophoresis (M, 170,000-250,000) in combination S underlies the 11.6 S protein in the hyaline layer. Imwith the sedimentation equilibrium results (M, 450,000) munocytochemical evidence has been obtained that supsuggest a dimeric structure for the 9.6 S protein. This ports this idea (39). A two-tiered structure for the hyaline conclusion must remain tentative, however, until more layer also agrees with electron microscopic observations information on the primary structure is available. Un- of the hyaline layer when fixed with ruthenium red (40) fortunately, attempts to obtain N-terminal sequence in- or alcian blue (41,42). The loss of 9.6 S protein from the formation for all three proteins failed, apparently due to hyalin fraction in the 15min treatment is probably exblocked termini. plained by proteolysis, since in this treatment group there The presence of all three hyalin proteins in a lysate of was some egg lysis on activation, at a time when no proisolated cortical granules is consistent with electron mi- teolytic inhibitors were present. Lysed eggs may release croscopic (6, 7) and immunological (22, 23) evidence for additional proteolytic enzymes to which the 9.6 S comthe intracellular origin of the hyaline layer. The same ponent is sensitive. It is notable that the composition of the 90-s DTT proteins were observed in whole egg homogenates and isolated cortical granules before fertilization and in the treatment hyalin fraction resembles that of the “less hyaline layer after fertilization. While there are differ- stringently dejellied” hyalin preparation described in (12) ences in the proportions of the hyaline layer components while the 3- to 12-min DTT treatment hyalin fractions in the three preparations, variations are likely to be a resemble “more stringently dejellied” preparations. We function of the extent to which the proteins were recov- feel these observations are explained by the fact that both ered and/or the success with which proteolysis was con- jelly and vitelline envelope material are reduced and disrupted by DTT and that the degree of vitelline envelope trolled in a given preparation. We have shown that when the cortical granule contents disruption is proportional to the amount of DTT exposure. were released into a Ca2+/Mg2-free medium so that the All else being equal, mild dejellying conditions reduce the hyaline layer material was unable to aggregate in normal effectiveness of DTT treatment compared to stringent fashion, the 9.6 S component was lost completely while dejellying. If the eggs are not dejellied, as in the current the 11.6 S and 6.4 S proteins remained. These data dem- study, this variable is eliminated. The three hyalin components are all quite acidic, based onstrate clearly the susceptibility of the 9.6 S protein to proteolysis because its presence was dependent on the on their strong staining with the basic dye methylene blue (32). The proteins stain very poorly with Coomassie blue. addition of protease inhibitor. Proteolysis of 9.6 S protein was concomitant with the cortical reaction, since the This acidity is consistent with the amino acid analysis for hyalin (8, 43; Justice and Carroll, unpublished data). presence of the 9.6 S protein was dependent on inhibitor being added before the cortical reaction began. It is known None of the three components react with silver stain unthat a large amount of proteolytic activity is released dur- less reduced. The negative charge conferred by the acidic ing cortical granule exocytosis (1, 36); however, under residues is likely to be important in the interaction of normal conditions these proteases must be inhibited from these proteins with calcium ions. We were unable to anacting on the 9.6 S protein, perhaps by the presence of alyze the interaction of the 9.6 S protein with Ca2+ and other divalent cations as has been done for the 11.6 S and divalent cations in the seawater. The number of hyaline layer components that could be 6.4 S proteins (12,28) because of the very limited amount dissolved from the surface of embryos varied with the of purified 9.6 S material that we were able to obtain. length of treatment with DTT before fertilization. Brief That the 9.6 S protein is functionally, at least, a calciumtreatment with DTT (e.g., 90 s) resulted in a hyalin frac- insoluble protein is shown by its appearance in a variety tion containing mostly 11.6 S protein. If the same con- of preparations which rely on this property. The structural relationship of the three hyalin comditions were used to dissolve the hyaline layers from the longer DTT treatment groups (3-12 min), over three times ponents to each other and to other proteins of the extracellular matrix is of major interest. Coffman and McClay as much total protein was obtained in the hyalin fraction and the 9.6 S component was present in large amounts. (44) have suggested that Ecto V, a hyaline layer compoThe 6.4 S protein was a relatively minor component in nent that is localized to the microvillar tips of the embryo, all of these hyalin fractions. These differences are con- could correspond to the 6.5 S (6.4 S) and/or 8.8 S (9.6 S) sistent with the observation that brief treatments with hyalin proteins as described in (12). The subunit molec-
9.6 S HYALIN
ular weight estimate (350 kDa), lack of aggregation with calcium ions, and glycosylation of Ecto V, however, make this unlikely. The subunit size of both the 6.4 S and 9.6 S proteins is fairly close to that of the 175kDa apical lamina protein (45); however, apical lamina proteins do not associate with Ca2+ (46). Evidence presented here supports the assertion that the 6.4 S component is derived from the 9.6 S protein by proteolysis. Amino acid sequence studies are in progress to address this issue directly. When mixtures containing all three hyalin components were examined on reducing SDS-gel electrophoresis, we observed only a single major band with a mobility comparable to that of the 320-kDa band expected for the 11.6 S component. A band with the mobility of the purified 6.4 S (180 kDa) protein was not apparent and only a very faint band with a mobility in the range of the 9.6 S protein was visible. It is possible that this behavior may be due to a combination of factors including (1) that the 11.6 S protein predominates in these preparations and produces an intense band, (2) that the 9.6 S protein is susceptible to degradation and is poorly resolved on the reducing SDS gel, and (3) that the 6.4 S component was a minor component of these hyalin preparations; however, it is also likely that the proteins are interacting anomalously so that resolution of the chains is not achieved. These facts may help to explain why hyalin has been traditionally regarded as a single protein with a chain size of approximately 300 kDa. While it now appears clear that the 300kDa band on reducing SDS-gels corresponds to the native 11.6 S protein (12), it is also clear that two additional proteins constitute hyalin prepared by a variety of procedures. Thus, the true composition of hyalin has been revealed more readily and reliably by analytical ultracentrifuge studies (47) than by SDS-gel electrophoresis. REFERENCES 1. Schuel, H. (1978) Gamete Res. 1, 299-382. 2. Giudice, G. (1986) The Sea Urchin Embryo. A Developmental logical System, Springer-Verlag, Berlin.
Bio-
3. Shapiro, B. M., Somers, C. E., and Weidman, J. J. (1989) in The Cell Biology of Fertilization (Schatten, H., and Schatten, G., Eds.), pp. 251-276, Academic Press, San Diego, CA. 4. Dan, K. (1960) Int. Rev. Cytol. 9,321-367. 5. Herbst, C. (1900) Wil/r&n ismen 9,424-463. 6. Afselius,
Roux’ Arch. Entwicklungsmech.
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B. A. (1956) Exp. Cell Res. 10, 257-285.
7. Endo, Y. (1961) Exp. Cell Res. 26, 383-397. 8. Stephens, R. E., and Kane, R. E. (1970) J. Cell Btil. 44,611-617. 9. Kane, R. E. (1973) Exp. Cell Res. 81,301-311. 10. McBlaine, 147.
P. J., and Carroll,
E. J., Jr. (1980) Deu. Bid. 75, 137-
PROTEIN
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11. McClay, D. R., and Fink, R. D. (1982) Deu. Biol. 92,285-293. 12. Gray, J., Justice, R., Nagel, G. M., and Carroll, E. J., Jr. (1986) J. Biol. Chem. 261,9282-9288. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.
Robinson, J. J. (1988) Biochem. J. 256,225-228. Kane, R. E. (1970) J. Cell Biol. 46,615-622. Bryan, J. (1970) J. Cell Biol. 44, 635-644. Vacquier, V. D. (1969) Ezp. Cell Res. 54, 140-142. Citkowitz, E. (1971) Deu. Bid. 24, 348-362. Kane, R. E., and Hersh, R. T. (1959) Exp. Cell Res. 16,59-69. Kane, R. E., and Stephens, R. E. (1969) J. Cell Bid. 41, 133-144. Vacquier, V. D., Tegner, M. J., and Epel, D. (1973) Exp. Cell Rex 80.111-119. Villacorta-Moeller, M. N., and Carroll, E. J., Jr. (1982) Deu. Biol. 94,415-424. Hylander, B. L., and Summers, R. G. (1982) Deu. Biol. 93, 368380. Vater, C. A., and Jackson, R. C. (1990) Mol. Reprod. Deu. 25, 215226. Moore, A. R. (1928) ProtopZusmu 3, 524-530. Vacquier, V. D., and Masia, D. (1968) Exp. Cell Res. 52,459-468. Adelson, D. L., and Humphreys, T. (1988) Development 104,391402. Epel, D., Weaver, A. M., and Mazia, D. (1970) Exp. Cell Res. 61, 64-68. Justice, R. W., Gottschling, C. F., Carroll, E. J., Jr., and Nagel, G. M. (1988) Arch. Biochem. Biophys. 266,136-145. Baginski, R. M., McBlaine, P. J., and Carroll, E. J., Jr. (1982) Gamete Res. 6, 39-52. Lois, A. L., Lackey, D. A., and Carroll, E. J., Jr. (1986) Gamete Res.
14,307-321. 31. Weber, K., Pringle, J. R., and Osborn, M. (1972) in Methods in Enzymology (Hirs, C. H. W., and Timasheff, S. N., Eds.), Vol. 26, pp. 3-27, Academic Press, New York. 32. Ruchel, R., Retief, A. E., and Richter-Landsberg, C. (1978) Anal. Biockem. 90,451-464. 33. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193,265-275. 34. Warburg, O., and Christian, W. (1941) Biockem. Z. 310, 384-421. 35. Yphantis, D. A. (1964) Biochemistry 3,297-317. 36. Vacquier, V. D., Epel, D., and Douglas, L. A. (1972) Nature (London) 237,34-36. 37. Chandler, D. E., and Heuser, J. (1980) J. Cell Biol. 84, 618-632. 38. Kubota, L. F., and Carroll, E. J., Jr. (1988) Gamete Res. 21,29-40. 39. Justice, R. W. (1989) Ph.D. dissertation, University of California, Riverside. 40. Lundgren, B. (1973) J. Submicrosc. Cytol. 5, 61-70. 41. Cameron, R. A., and Holland, N. D. (1985) Cell Tissue Res. 239, 455-458. 42. Spiegel, E., Howard, L., and Spiegel, M. (1989) J. Morphol. 199, 71-92. 43. Citkowitz, E. (1972) Deu. Bid. 27,494-503. 44. Coffman, J. A., and McClay, D. R. (1990) Deu. Bd 140,93-104. 45. Hall, H. G., and Vacquier, V. D. (1982) Deu. Bid. 89, 168-178. 46. Robinson, J. J. (1990) Biochem. Cell Bid. 68, 1083-1089. 47. Schachman, H. K. (1989) Nature (London) 341, 259-260.
OF BIOCHEMISTRY
AND BIOPHYSICS
Vol. 294, No. 1, April, pp. 297-305, 1992
A 9.6 S Protein Is the Third Calcium-Insoluble Component of the Sea Urchin Hyaline Layer’ Robin W. Justice,* Glenn M. Nagel,tp2 Carl F. Gottschling,? and Edward J. Carroll, Jr.* *Department and Institute
Marina
F. Damis,?
of Biology, University of California, Riverside, California 92521, and tDepartment of Chemistry and Biochemistry for Molecular Biology and Nutrition, California State University, Fullerton, California 92634
Received October 11,1991, and in revised form December 9, 1991
A third major, calcium-insoluble component of the sea urchin (Strongylocentrotue purpuratue) hyaline layer has been purified and physically characterized. In the absence of divalent cations, the native, soluble protein has a sedimentation coe6lcient of 9.6 S and a molecular weight of 4.6 f 0.1 X 10’. These data indicate that this large protein assumes an elongated, nonspherical conformation in solution. Its sedimentation behavior and its mobility on nondenaturing electrophoretic gels distinguish the 9.6 S protein from the 11.6 S and 6.4 S hyalin proteins we have previously characterized. That the 6.4 S, 9.6 S, and 11.6 S proteins are the major calcium-insoluble structural components of the hyaline layer is supported by the fact that we have found them in a variety of hyalin protein fractions prepared by a number of standard approaches. All three proteins are precipitated by calcium ions, thus fitting the operational definition of hyalin. Evidence is presented that the 11.6 S protein may overlie the 9.6 S protein in the hyaline layer. QlS92AcademicPrem,Inc.
The sea urchin hyaline layer (l-3) is a proteinaceous extracellular coat that surrounds the early embryo. The blastomeres adhere to the hyaline layer (4) and its integrity is dependent on calcium ions in the seawater (5). The hyaline layer begins to appear a few seconds after insemination as the cortical reaction, a global exocytosis of secretory vesicles (the cortical granules) located just beneath the egg plasma membrane, commences. In transmission electron micrographs, material that appears to form the hyaline layer is seen emerging from the cortical granules 1 This research was supported, in part, by an award from Research Corporation to G.M.N. and a University of California, Riverside, Chancellor’s Patent Fund grant to R.W.J. ’ To whom correspondence should be addressed. ooo3-9&x/92 $3.00 Copyright 0 1992 by Academic Press, Inc. AI1 rights of reproduction in any form reserved.
during exocytosis (6,7). The early hyaline layer is overlain by a tough, impermeable fertilization envelope that also forms during the cortical reaction from the vitelline envelope and cortical granule-derived components. The major structural component of the hyaline layer, called hyalin (8), is operationally defined as a high-molecular-weight protein fraction soluble in the absence of divalent cations and rendered reversibly insoluble by calcium ions at concentrations comparable to that of seawater. Hyalin has been isolated from a number of sea urchin species and several distinct methods have been used for its isolation (these methods as applied in our laboratory are described, in turn, under Materials and Methods): (1) Hyalin has been dissolved from the surface of intact embryos after hatching (9) or after vitelline envelope disruption (10-13). (2) It has been collected from the cortical granules by activating eggs under conditions where formation of the fertilization envelope is blocked (14) or where the vitelline envelope is disrupted before egg activation (15). (3) Hyalin also has been obtained by isolating whole hyaline layers from embryos and dissolving the layers in 1 M urea (16) or Ca2+/Mg2+-free seawater (17). (4) In addition, hyalin has been precipitated from egg homogenates or extracts with Ca2+ (8, 18, 19). (5) Finally, hyalin has been isolated from intact cortical granules (20, 21). Polyclonal (22) and monoclonal (23) antibodies to hyalin localize to the cortical granules and some smaller vesicles before fertilization and to the hyaline layer after fertilization. It has been suggested that the hyaline layer has a role in the morphogenesis of the embryo because treatments that partially disrupt the hyaline layer also interfere with gastrulation (17, 24, 25). Evidence that this effect might be mediated by hyalin itself comes from work demonstrating that blastomeres adhere to hyalin in vitro, but those destined to migrate into the blastocoel lose their affinity for hyalin (11). Also, a monoclonal antibody di297
JUSTICE
rected against hyalin protein is able to block gastrulation and plutear arm formation (26). We have previously shown that when examined under nondenaturing conditions, hyalin actually contains up to three components by analytical ultracentrifugation analysis (12). The hyalin used in that study was prepared by treating acid-dejellied eggs with dithiothreitol to disrupt the vitelline envelopes (27), a treatment that prevents fertilization envelope formation, then dissolving the hyaline layers from intact 45-min embryos in a divalent cation-free medium and, finally, precipitating hyalin with 100 mM CaC12. Such preparations were enriched in one hyalin component, a 920-kDa, 11.6 S protein, when very mild (“less stringent”) dejellying conditions were employed. “More stringent” dejellying conditions resulted in additional components in the final Ca2+ precipitate. We subsequently described a 280-kDa, 6.4 S protein, isolated by allowing cortical granule exocytosis to take place into Ca2+/Mg2+-free seawater containing EGTA3 (28). This procedure leads to the formation of a thin, incompletely formed fertilization envelope permeable to the cortical granule contents (29). The Ca2+/Mg2+-insoluble fraction of this cortical granule exudate consists entirely of the 6.4 S and 11.6 S proteins. The 6.4 S component corresponds in electrophoretic and sedimentation behavior to the slowest sedimenting component of “more stringently dejellied” hyalin preparations. In the present study, we describe the isolation and physical characterization of the third, 9.6 S component from hyalin preparations. MATERIALS
AND METHODS
Gamete Preparation Gametes of Strongylmentrotus purpuratus (Alacrity, Inc., Redondo Beach, CA, and Pacific Bio-Marine, Venice, CA) were obtained by injection of 0.5 M KC1 into the perivisceral coelom of the adult. Sperm was collected “dry” from the aboral surface of shedding males and held on ice. Eggs were rinsed through 202~pm mesh Nitex to remove debris and washed with seawater at 11°C. Reagents, unless otherwise noted, were obtained from Sigma (St. Louis, MO).
Hyalin Preparations 1. Dissolved hyaline layers. Hyalin proteins were dissolved from the surface of intact embryos with disrupted vitelline envelopes by the method described in (12), with the following modifications. The washed eggs were not dejellied prior to dithiothreitol (DTT) treatment and the time used for the incubation with DTJ! to disrupt the vitelline envelopes was 3 min unless otherwise indicated. The hyaline layers were dissolved from the 45min embryo surfaces using a 2-min incubation in 475 mu NaCl, 25 mM KCl, 2 mM EGTA, 5 mM benzamidine, and the hyalin proteins were redissolved by dialysis at 4°C against 100 mM Tris-HCl,
3 Abbreviations used: DTT, dithiothreitol; EGTA, ethylene glycol bis(p-aminoethyl ether) N,N’-tetraacetic acid; BASW, artificial seawater (500 mM NaCl, 10 mM KCl, 10 mM Tris-HCl, 50 mM MgCl,, 10 mM CaCl,, pH 8.0) containing 5 mM benzamidine; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate.
ET AL. 5 mu benzamidine, 5 mu EDTA, pH 8.0, or 20 mM Tris, 5 mM benzamidine, 2 mM EGTA, pH 8.0.
2. Cortical granule exudate. The calcium-insoluble protein fraction of cortical granule exudate was prepared by a method previously described (28) with the following variations. Benzamidine (5 mM), to inhibit cortical granule protease activity (30), was added at times varying from 10 s before insemination to 5 min after insemination and the Caz’/ Mg*+-insoluble precipitate was dissolved as for the dissolved hyaline layers. 3. Whole hyaline luyers. Whole hyaline layers were isolated from prehatching embryos by a modification of the method described in (17). A suspension of 45-min S. purpuratua embryos in artificial seawater (500 mM NaCl, 10 mM KCl, 10 mM Tris-HCl, 50 mM MgClz, 10 mM CaClz, pH 8.0) containing 5 mu benzamidine (BASW) was homogenized for 3 min, ll”C, 14,000 rpm in a Waring blender to break the fertilization envelopes and remove the hyaline layers. The homogenate was then centrifuged 10 min, llOOg, 4°C giving a two-layered pellet with fertilization envelopes at the bottom and the whiter hyaline layers at the top, plus a frothy, floating mat enriched in hyaline layers. The top layer of the pellet was rinsed away from the lower layer by a gentle stream of BASW and then pooled with the floating material. The pooled material was homogenized in a Dounce apparatus (“A” pestle) in BASW on ice and centrifuged at llOOg, 10 min, 4’C. The homogenization and centrifugation were repeated, each time using only the floating material and the top fraction of the pellet, until all cytoplasmic debris was removed, as judged by light microscopy. At this point, only small fragments of fertilization envelopes remained and the hyaline layers were dissolved away from them by dialysis against 100 mM Tris-HCl, 5 mM EDTA, 5 mM benzamidine, pH 8.0, 4°C. Remaining fertilization envelopes were removed by centrifugation at ZO,OOOg,20 min, 4“C. Alternatively, the hyaline layers were physically separated from the fertilization envelope fragments using a sucrose step gradient by the method used in (17). To obtain whole hyaline layers from posthatching stages of S. purpuratm, embryos were allowed to develop at 11°C. The swimming embryos were concentrated by centrifugation at llOOg, 30 s, 4°C. The embryos were suspended in ice-cold BASW and homogenized with a Dounce apparatus (“A” pestle) to break the hyaline layers open. The hyaline layers were collected by centrifugation (llOOg, 10 min, 4°C) and homogenized in BASW. The homogenate was rinsed through 35-am mesh Nitex, which retains most of the hyaline layers. These processes were repeated until the filtrate was clear. The hyaline layers were then dissolved by dialysis against 100 mM Tris-HCl, 5 mu EDTA, 5 r&f benzamidine, pH 8.0,4”C. proteins 4. Whole egg homogenates. To prepare calcium-insoluble from whole egg homogenates unfertilized eggs were centrifuged 30 s at 3OOg, 4’C, the pelleted eggs were homogenized in a Dounce apparatus (“A” pestle) in 10 mM Tris-HCl, 5 mM benzamidine, pH 8.0, on ice, and 100 mM CaClz was added to the homogenate. After incubation for 30 min, on ice, the homogenate was centrifuged llOOg, 10 min, 4°C and 5 mM the pelleted material was dialyzed against 100 mM Tris-HCl, benzamidine, pH 8.0, 4°C. After dialysis, the insoluble material was removed by centrifugation at 2O,OOOg,20 min, 4”C, and 100 mM CaClz was added to the supernatant solution, which was incubated on ice, overnight. The calcium-insoluble material was collected by centrifugation at ZO,OOOg,20 min, 4°C. The precipitate was dissolved by dialysis at 4°C against 100 mM Tris-HCl, 5 mM EDTA, 5 mM benzamidine, 1 mM 3-amino-1,2,4,-triazole, pH 8.0.
5. Isolated cortical granules. Intact cortical granules were isolated from S. purpuratus by the procedure in (20) as modified in (21). The isolated cortical granules were lysed by homogenization in a Dounce apparatus (“A” pestle) in 100 mM Tris-HCl, 5 mM EDTA, 5 mM benzamidine, 1 mM 3-amino-1,2,4-triazole, pH 8.0, on ice. The crude lysate was centrifuged at ZO,OOOg,20 min, 4”C, 100 mu CaClz was added to the supernatant solution, and the mixture was incubated at 4”C, overnight. The calcium-insoluble material was collected by centrifugation at 2O,OOOg,20 min, 4°C. The precipitate was collected and dissolved as for the whole egg homogenate.
9.6 S HYALIN
Gel Electrophoresis
and Staining
Polyacrylamide gel electrophoresis (PAGE) was performed according to the method in (31) with the following modifications. Gels were 4% a&amide, 0.1% methylenebisacrylamide (A&age1 and bisAcrylage1, National Diagnostics, Highland Park, NJ) in 0.75 mM slab gels and 5 mM EDTA was included in the gels and all associated buffers as described in (28). Nondenaturing (native) gels omitted sodium dodecyl sulfate (SDS) from the gels and buffers, while all reducing gels included 4% j3mercaptoethanol in the sample buffer. The SDS-containing samples were incubated 2 h at 37°C prior to electrophoresis. Protein standards (Bio-Rad, Richmond, CA) for molecular weight estimation on reducing SDS gels were myosin (200 kDa), /3-galactosidase (116.25 kDa), phosphorylase B (92.5 kDa), bovine serum albumin (66.2 kDa), and ovalbumin (45 kDa). Native gels were stained with methylene blue (Matheson, Coleman and Bell, Norwood, OH) by a modification of the method in (32) for acidic proteins. Gels were washed in two changes of 200 ml 2:1, glassdistilled water:methanol, 5 mM sodium phosphate, pH 7.0 (gel fix), for 1 h each and then stained 10 min at room temperature in 200 ml 0.2% methylene blue, 5 mu sodium phosphate, pH 7.0. The gels were destained in 200 ml gel fix. For photography, gels were washed 5-10 min in 150 ml fresh gel fix (reserving the original destaining solution), moved to 50 ml fresh gel fix, and photographed immediately using Technical Pan film (Eastman Kodak Co., Rochester, NY). Gels were then returned to the reserved solution in which the stain remains stable indefinitely. Gels containing SDS were double-stained. First, after being rinsed in 40% methanol, 10% acetic acid, gels were stained in 0.1% Coomassie brilliant blue G-250,40% methanol, 10% acetic acid for 30 min at 37°C and then destained in several changes of 5% methanol, 7.5% acetic acid to reveal the molecular weight standards. Second, the gels were washed thoroughly with gel fix to neutralize the acid and then stained as above with methylene blue.
299
PROTEIN
tein, meniscus-depletion sedimentation equilibrium measurements (35) were made at 6800,7200, and 8000 rpm between 6 and 9°C. Run times varied from 43 to 134 h. The buffer employed in all experiments was 0.01 M Tris-HCl, 0.575 M NaCl, 0.01 M KCl, 5 mM EDTA, pH 8.0.
RESULTS
Hyalin was first prepared by dissolving hyaline layers from intact, 45min embryos in Ca2+/Mg2+-free seawater and subsequent Ca2+ precipitation. Before fertilization, 7-ml aliquots of eggs were treated with DTT to disrupt the vitelline layer (method 1 under Materials and Methods). The composition of hyalin prepared from these embryos was observed to vary with the length of DTT treatment (Fig. 1). A relatively brief treatment with the reductant (90 s) gave a hyalin fraction (1.7 mg total protein/aliquot) consisting mostly of the low-mobility, 11.6 S hyalin protein described in (12). Longer DTT treatment of other aliquots of the same batch of eggs gave hyalin fractions containing increased amounts of two additional components (5.5 f 0.8 mg total protein/aliquot). After very long DTT treatment (15 min) the hyalin fraction once again consisted mainly of the 11.6 S protein (2.9 mg total protein), though, unlike preparations using shorter DTT treatments, many of the eggs lysed on activation. The fastest migrating and least abundant material on
12345678
Purification
of Proteins by Rate-Zonal
Centrifugation
The 9.6 S protein was purified by rate-zonal centrifugation on 5-20% sucrose density gradients in 100 mM Tris-HCl, 5 mM EDTA, 5 mM benzamidine, pH 8.0, in 13.4-ml Beckman Ultraclear centrifuge tubes, 4“C. A dissolved hyalin or hyaline layer preparation (0.5 ml, 0.8 mg protein/ml) was layered on each gradient and the tubes were centrifuged in a Beckman SW41Ti swinging bucket rotor at 41,000 rpm, 25 h, 4”C, in a Beckman Model L5-65B preparative ultracentrifuge. The gradient was fractionated from the bottom of the tube, 0.4 ml/fraction at 4°C. The fractions were examined by native PAGE and fractions containing the 9.6 S component with a purity of 90% or more, as assessed after methylene blue staining, were pooled The pooled fractions were dialyzed against 100 mM Tris-HCl, 5 mM EDTA, 5 mM benzamidine, pH 8.0, to remove sucrose, concentrated by centrifugation in Centricon 10 microconcentrators (Amicon, Danvers, MA), and fractioned on a second set of 5-20% sucrose gradients (0.5 ml/gradient, 0.5 mg/ml). These gradients were treated as described above and the fractions were examined by native PAGE. The fractions containing only the 9.6 S component were pooled, dialyzed, and concentrated. The 11.6 S and 6.4 S proteins were obtained in pure form by a single round of 5-20% sucrose density gradient fractionation performed as for the 9.6 S protein except that the calcium-insoluble fraction of cortical granule exudate with benzamidine added 90 s after insemination (0.5 ml/gradient, 0.8 mg/ml) was used as the starting material. These preparations were virtually free of 9.6 S material. Protein concentration was determined calorimetrically (33) by using bovine serum albumin as a standard or from the ultraviolet absorbance (34).
Analytical
Ultracentrifugation
Sedimentation velocity and sedimentation equilibrium measurements were completed as previously described (12). For the purified 9.6 S pro-
-11.6 S -9.6s -6.4 5
FIG. 1. Native PAGE of hyalin proteins dissolved from the surface of intact, 45-min embryos with different degrees of vitelline envelope disruption. The length of time that the eggs were treated with dithiothreitol to disrupt the vitelline envelopes was varied as follows: 90-s treatment (lane 1, 1.7 pg protein); 3-min treatment (lane 2, 4.4 pg protein); 6-min treatment (lane 3, 6.1 pg protein); 9-min treatment (lane 4, 4.4 pg protein); 12-min treatment (lane 5, 5.5 pg protein); 15-min treatment (lane 6, 1.9 pg protein). Lane 7 contains 5.8 fig of Sephacryl S-500 column-purified 11.6 S protein. Lane 8 contains 10 pg of the calcium-insoluble fraction of cortical granule exudate with benzamidine added at 90 s after insemination consisting of the 11.6 S and 6.4 S components only. This figure and other figures depicting native PAGE are composites of lanes from two or more gels. To compare mobilities among gels a sample of hyalin proteins dissolved from intact, 45-min embryos was included on each gel and used to standardize the photographic prints, and the position of each of these standard components is marked on the right side of the gel.
300
JUSTICE
electrophoretic gels of the three-component mixtures corresponds to the 6.4 S protein isolated from the calciuminsoluble fraction of cortical granule exudate (Fig. 1) prepared as described in (28). Thus, by elimination, the intermediate electrophoretic hyalin component appeared to correspond to the protein having a sedimentation coefficient between the 6.4 S and 11.6 S proteins. This component previously has been observed in hyalin preparations from “more stringently dejellied” eggs (12). When the hyalin fraction was prepared from cortical granule exudate (method 2 under Materials and Methods), the presence of the intermediate component was dependent on the inclusion of benzamidine, a protease inhibitor, during cortical granule exocytosis. Cortical granule exudate is the material that leaks through the thin fertilization envelope that is formed when eggs are diluted into Ca2+/Mg2+-free seawater containing EGTA during the cortical reaction (28, 29). Analytical ultracentrifugal analysis of preparations treated with benzamidine before the cortical reaction revealed the presence of three components (Fig. 2). A number of similar analyses where benzamidine was added at times varying from 10 s before insemination to 300 s after insemination (Fig. 3) showed that the intermediate component was present only when 5 mM benzamidine was added before the start of the cortical reaction, a process beginning 28 s after insemination at 15°C. Electrophoretic gels of the calcium-insoluble fraction of cortical granule exudate with benzamidine added before the cortical reaction (10 s after insemination) showed the intermediate band, whereas when benzamidine was added after the cortical reaction, this band was absent (Fig. 4). These data also reinforce the conclusion that the intermediate electrophoretic and ultracentrifugal components are the same. To provide additional evidence that the three proteins we observed in hyalin fractions are actually hyaline layer components, whole hyaline layers were isolated from 45 min embryos or from hatched blastulas by a modification of the method in (17) (method 3 under Materials and Methods). In both preparations, fertilization envelopes were absent before solubilization of the hyaline layer.
FIG. 2. Schlieren profile of a three-component hyalin preparation. Hyalin protein prepared from the calcium-insoluble fraction of cortical granule exudate (benzamidine added 20 s after insemination) (see Materials and Methods) was examined in the analytical ultracentrifuge. The photograph was taken 72 mm after reaching a rotor velocity of 60,000 rpm at a rotor temperature of 6.2”C and a Schlieren diaphragm angle of 70’. Light path was 12 mm.
ET AL.
-20
0
20
40
60
60
100 300
lime (set)
FIG. 3.
Effect of the timing of benzamidine addition on the composition of the calcium-insoluble fraction of cortical granule exudate. The calcium-insoluble fraction of cortical granule exudate was prepared as described under Materials and Methods with the time of addition of benzamidine (to 5 mM final concentration) varied between 10 s before and 5 min after insemination. The composition of the calcium-insoluble fraction was determined by integration of Schlieren profiles after components were separated by analytical centrifugation (m, 6.4 S; A, 11.6 S; 0, 9.6 S). The arrow indicates the approximate beginning of the cortical reaction (CGE, 28 s after insemination), measured as the time when fertilization envelopes can first be seen elevating from egg surfaces by phase-contrast microscopy.
Three components with the same electrophoretic mobilities as those present in hyalin fractions dissolved from the surface of intact, 45min embryos were the only bands observed (Fig. 4). As noted above, hyalin has been isolated previously from egg homogenates and shown to originate within the cortical granules of the unfertilized egg. To determine whether all three native hyalin components were present within the egg before fertilization, a calcium precipitate of whole egg homogenates and the calcium-insoluble contents of isolated cortical granules (methods 4 and 5, respectively, under Materials and Methods) from unfertilized eggs were examined electrophoretically. Three components with the same mobilities as those in hyalin preparations were the only ones present on native gels (Fig. 4) of both preparations. The intermediate component, however, appeared somewhat diminished in proportion in the material prepared from isolated cortical granules. To purify the intermediate component, mixtures of hyalin proteins dissolved from the surface of intact, 45-min embryos or from dissolved, whole hyaline layers were subjected to rate-zonal centrifugation on 5-20% sucrose density gradients that included 5 mM benzamidine. Frac-
9.6 S HYALIN
-11.6s .9.6S .6.4 5
301
PROTEIN
Sedimentation velocity analysis of the purified protein gave the results shown in Fig. 6. The sedimentation coefficient, s20,w,was only moderately dependent on protein concentration, c. Fitting the data to the equation %!o,w = sL,& - kc)
FIG. 4. Native PAGE of preparations containing hyalin proteins. The following protein preparations are shown: hyalin proteins dissolved from the surface of intact, 45-min embryos (lane 1); calcium-insoluble protein fraction of cortical granule exudate with bsnxamidine added at 10 s after insemination (lane 2); calcium-insoluble protein fraction of cortical granule exudate with benxamidine added at 90 s after insemination (lane 3); proteins from whole hyaline layers isolated from 45-min embryos (lane 4); proteins from whole hyaline layers isolated from hatched blastulas (lane 5); calcium-insoluble proteins isolated from whole egg homogenates (lane 6); and calcium-insoluble proteins from a lysate of isolated cortical granules (lane 7). Approximately 10 fig of total protein was loaded on each lane.
tions were examined via electrophoresis. When relatively high concentrations of protein were loaded on the gradient (3.2 mg/tube; Fig. 5A), the separation of components was poor. This result is consistent with the observation that the sedimentation coefficient of the 11.6 S component is highly concentration dependent, being lower at higher protein concentrations (12). Thus the concentration of the protein mixture loaded on the gradient is critical to the success of the separation of the 11.6 S component from the intermediate component. At an eightfold lower protein concentration (0.4 mg/tube) the separation of components was greatly improved with a number of fractions containing >90% of the intermediate component by the gel assay (Fig. 5B). To achieve the final purification, fractions containing 90% or more of the intermediate component were pooled, concentrated as described under Materials and Methods, and fractionated on a second set of sucrose gradients. The second separation provided an excellent resolution of the intermediate component from the small amount of contaminating 11.6 S protein initially present (Fig. 5C). Fractions showing no detectable contamination with the 11.6 S protein by the gel assay were pooled and concentrated for further analysis. In addition, the 11.6 S and 6.4 S proteins were purified from the calcium-insoluble fraction of cortical granule exudate with benzamidine added 90 s after insemination (a preparation which lacks the intermediate component) by a single round of density gradient ultracentrifugation (data not shown).
gave a value of 9.6 S for s&~, the extrapolated value at infinite dilution, and a value for the constant It = 2 X 10e4ml/mg. Sedimentation equilibrium analysis yielded linear plots of In c vs ? (Fig. 7), indicating that the preparations were essentially monodisperse. Results from four determinations, at three different rotor velocities, gave a value of 447,000 + 5000 for the molecular weight. In combination, these data yield a value for the frictional ratio, f/to, of 2.3 and indicate a nonspherical shape for the 9.6 S protein. Assuming a shape approximating a prolate ellipsoid and an average hydration of 0.5 g H20/g protein, the calculated axial ratio, afb, is 14. Freshly purified 9.6 S material examined on a native polyacrylamide gel (Fig. 8) migrated as a single band with a mobility indistinguishable from that of the same component in the hyalin preparation from which it was isolated. This was also true of the 11.6 S and 6.4 S proteins (Fig. 8) purified by density-gradient sedimentation. This method of purification of the 6.4 S protein is thus more effective than the gel permeation chromatography used previously (28), where the column-purified protein exhibited a slight increase in mobility over the same component in the starting material. When the 9.6 S preparation was allowed to age at 4°C a faster-migrating band appeared on native gels of the preparation (Figure 8) with the same mobility as density gradient-purified 6.4 S protein. When purified 9.6 S material was examined under denaturing but not reducing conditions (in the presence of SDS but not P-mercaptoethanol) it resolved into four electrophoretic bands (Fig. 9). These four bands had the same relative mobilities as the four fastest-migrating bands resolved from hyalin containing all three native proteins (Fig. 9, compare lanes 3 and 4). The slow-migrating band in the hyalin mixture has the same mobility as the purified 11.6 S protein (Fig. 9, lane l), while, under the same conditions, the most rapidly migrating band in gels of both the mixture and the purified 9.6 S protein has the same mobility as the single band representing the sucrose gradient-purified 6.4 S component (Fig. 9, lane 2). These data suggest that the 6.4 S component may be derived from the 9.6 S component. Samples of the three native proteins purified on sucrose density gradients were also examined on reducing SDS gels (Fig. 9, lanes 5-8). The 11.6 S component migrates as a single band with an apparent size of 320 kDa. The 9.6 S component is not clearly resolved, but produces a smear of staining between 170 and 250 kDa. The 6.4 S
302
JUSTICE
ET AL.
C
B -11.6 S -9.6s -6.4 S
-11.6 S -9.6 S -6.4 S
FIG. 5. Native PAGE of fractions from 5-20% sucrose density gradients. Fractions were collected starting with the 20% end of each gradient. Samples of the fractions (16 pl of each) were loaded sequentially across the gel. (A) Dissolved whole hyaliie layers were used as the starting material and 3.2 mg of total protein was loaded on the gradient in A. Fractions 4 through 31 are shown. (B) Hyalin dissolved from intact, 45-min embryos was used as the starting material and 0.4 mg of total protein was loaded on the gradient in B. Fractions 5 through 16 are shown. (C) Fractions from six sucrose gradients identical to the gradient in B containing >90% 9.6 S protein, as assayed by native PAGE, were pooled, concentrated as described under Materials and Methods, and rerun on a 5-20% sucrose density gradient. Fractions 5 through 16 are shown; 0.25 mg of total protein was loaded on this gradient. The position at which each of the three native hyalin components runs is marked on the right side of each panel.
component migrates as a single band with a size of 180 kDa as reported in (28). Surprisingly, a three-component hyalin preparation run under reducing, denaturing conditions gives only one major band and several much fainter ones. While the major band has the mobility of the 320kDa, 11.6 S component, there is only a faint band at 250 kDa and no band at 180 kDa. DISCUSSION
We have now isolated and purified the three major protein components of the calcium-insoluble fraction of the embryonic extracellular matrix of the sea urchin generally referred to as the hyaline layer. Purification by rate-zonal centrifugation in the presence of the protease inhibitor benzamidine has allowed us to obtain homogeneous preparations of the previously described 11.6 S and 6.4 S components as well as a third, 9.6 S component which we had been unable to resolve by gel permeation chromatography. To achieve optimal separation of the 11.6 S and 9.6 S proteins by centrifugation, it was critical that low con-
centrations of protein be used, owing to the concentration dependence of the sedimentation coefficients of the proteins. The native 11.6 S component, which showed both a major and a minor band on reducing, denaturing gels when purified by gel-permeation chromatography (12,28), showed only the major, high-molecular-weight band (320 kDa) when purified in the ultracentrifuge. The 6.4 S protein had the same electrophoretic mobility before and after ultracentrifuge purification, while its mobility increased slightly after chromatographic purification (28), indicating that some degradation may have occurred in the latter case. The better preservation of 11.6 S and 6.4 S proteins likely is due to the continuous presence of protease inhibitor in the ultracentrifuge purification scheme. That the 9.6 S native protein is an authentic component of the hyaline layer was shown by the presence of 9.6 S protein in whole hyaline layers as well as in the calciuminsoluble fraction of material dissolved from the surface of embryos. The fact that no other proteins were observed as components of the five types of hyalin preparations we
9.6 S HYALIN
303
PROTEIN
-11.6s -9.65 -6.4 S
9.6 q
9.6 -
q q
9.4 -
9.2 -
9.0 9.0 -01 0.0 0.0
I 0.5
1 1.0
Protein Concentration
I 1.5
2.0
(wml)
FIG. 6. The dependence of the sedimentation coefficient of the purified 9.6 S protein on protein concentration. The value of sU),n(in Svedbergs) is plotted versus the protein concentration determined by integration of Schlieren boundaries. A value of sioW = 9.6 S was obtained after extrapolation to zero protein concentration.
examined indicates that these three are the only major calcium-insoluble proteins associated with the hyaline layer. All three hyalin proteins were present in whole hyaline layers from 45 min postfertilization until after hatching was complete; thus, these proteins apparently persist from the early embryo stage through the enzymatic hatching process. Analysis of the 9.6 S protein in the analytical ultracentrifuge revealed it to be a large, 450,000-molecularweight protein. Like the 11.6 S and 6.4 S proteins, the 9.6 S protein appears to have an elongated, nonspherical shape in solution. Our experiments showed only a modest
FIG. 8. Native polyacrylamide gel electrophoresis of 11.6 S, 9.6 S, and 6.4 S proteins purified by rate-zonal centrifugation. Purified 9.6 S protein was run on a native polyacrylamide gel just after the protein had been prepared (lane 1). The same sample was run under native conditions following a month of storage at 4’C (lane 2). Also illustrated are density gradient-purified 6.4 S (lane 3) and 11.6 S (lane 4) proteins. Approximately 5 pg of protein was loaded per well.
dependence of sZo,+., on protein concentration for the 9.6 S protein, in agreement with our previous findings (12). In this earlier work, we observed that szo,Wof the 11.6 S protein was remarkably concentration dependent such that the 11.6 S protein was not separable from the intermediate component (then termed “8.8 S”) at 2 mg of protein/ml, whereas two separate boundaries were readily
12
3
4
5
6
7
8
-0.4 B & ,c
-0.6
-1.2
-1.6 ! 49.6
I 50.0
I 50.2
I 50.4
I 50.6
rexp2 FIG. 7. Sedimentation equilibrium data for the purified 9.6 S protein. The results of a typical experiment are plotted as the natural logarithm of the Rayleigh fringe displacement versus the square of the radial position. The protein sample was centrifuged at a rotor velocity of 7200 rpm at a rotor temperature of 6°C.
FIG. 9. SDS-polyacrylamide gel electrophoresis of hyalin proteins. Protein preparations were denatured in the absence (lanes l-4) or presence (lanes 5-8) of fl-mercaptoethanol. The 11.6 S protein (lanes 1 and 5), 6.4 S protein (lanes 2 and 6), and 9.6 S protein (lanes 3 and 7) preparations were purified by rate-zonal centrifugation and approximately 10 pg of total protein was loaded per lane. Lanes 4 and 8 contain hyalin proteins dissolved from intact, 45min embryos; this preparation contained all three native components and is the same one shown under native conditions in Fig. 1, lane 2. Approximately 5 pg of total protein was loaded on lanes 4 and 8. For samples containing fl-mercaptoethanol, the chain sizes (in KDa)] indicated were obtained using standard proteins as described under Materials and Methods.
304
JUSTICE
ET AL.
observed at concentrations below 1 mg of protein/ml. The DTT may leave large amounts of the vitelline envelope higher sedimentation coefficient obtained for the inter- intact (37), although damaging it enough so that fertilmediate component in this study as compared to that ization envelope elevation does not occur (38). Thus, less previously reported may result from the fact that it was total protein was obtained from the 90-s treatment bemeasured previously only in mixtures composed predom- cause of the physical barrier of the partial envelope. By inantly of the 11.6 S protein. In addition, the difference this reasoning, the 9.6 S component, which appeared after may stem from the fact that previous preparations had longer treatments, must be less accessible to the dissolving suffered degradation. Data obtained from reducing SDS- medium than the 11.6 S component, perhaps because 9.6 gel electrophoresis (M, 170,000-250,000) in combination S underlies the 11.6 S protein in the hyaline layer. Imwith the sedimentation equilibrium results (M, 450,000) munocytochemical evidence has been obtained that supsuggest a dimeric structure for the 9.6 S protein. This ports this idea (39). A two-tiered structure for the hyaline conclusion must remain tentative, however, until more layer also agrees with electron microscopic observations information on the primary structure is available. Un- of the hyaline layer when fixed with ruthenium red (40) fortunately, attempts to obtain N-terminal sequence in- or alcian blue (41,42). The loss of 9.6 S protein from the formation for all three proteins failed, apparently due to hyalin fraction in the 15min treatment is probably exblocked termini. plained by proteolysis, since in this treatment group there The presence of all three hyalin proteins in a lysate of was some egg lysis on activation, at a time when no proisolated cortical granules is consistent with electron mi- teolytic inhibitors were present. Lysed eggs may release croscopic (6, 7) and immunological (22, 23) evidence for additional proteolytic enzymes to which the 9.6 S comthe intracellular origin of the hyaline layer. The same ponent is sensitive. It is notable that the composition of the 90-s DTT proteins were observed in whole egg homogenates and isolated cortical granules before fertilization and in the treatment hyalin fraction resembles that of the “less hyaline layer after fertilization. While there are differ- stringently dejellied” hyalin preparation described in (12) ences in the proportions of the hyaline layer components while the 3- to 12-min DTT treatment hyalin fractions in the three preparations, variations are likely to be a resemble “more stringently dejellied” preparations. We function of the extent to which the proteins were recov- feel these observations are explained by the fact that both ered and/or the success with which proteolysis was con- jelly and vitelline envelope material are reduced and disrupted by DTT and that the degree of vitelline envelope trolled in a given preparation. We have shown that when the cortical granule contents disruption is proportional to the amount of DTT exposure. were released into a Ca2+/Mg2-free medium so that the All else being equal, mild dejellying conditions reduce the hyaline layer material was unable to aggregate in normal effectiveness of DTT treatment compared to stringent fashion, the 9.6 S component was lost completely while dejellying. If the eggs are not dejellied, as in the current the 11.6 S and 6.4 S proteins remained. These data dem- study, this variable is eliminated. The three hyalin components are all quite acidic, based onstrate clearly the susceptibility of the 9.6 S protein to proteolysis because its presence was dependent on the on their strong staining with the basic dye methylene blue (32). The proteins stain very poorly with Coomassie blue. addition of protease inhibitor. Proteolysis of 9.6 S protein was concomitant with the cortical reaction, since the This acidity is consistent with the amino acid analysis for hyalin (8, 43; Justice and Carroll, unpublished data). presence of the 9.6 S protein was dependent on inhibitor being added before the cortical reaction began. It is known None of the three components react with silver stain unthat a large amount of proteolytic activity is released dur- less reduced. The negative charge conferred by the acidic ing cortical granule exocytosis (1, 36); however, under residues is likely to be important in the interaction of normal conditions these proteases must be inhibited from these proteins with calcium ions. We were unable to anacting on the 9.6 S protein, perhaps by the presence of alyze the interaction of the 9.6 S protein with Ca2+ and other divalent cations as has been done for the 11.6 S and divalent cations in the seawater. The number of hyaline layer components that could be 6.4 S proteins (12,28) because of the very limited amount dissolved from the surface of embryos varied with the of purified 9.6 S material that we were able to obtain. length of treatment with DTT before fertilization. Brief That the 9.6 S protein is functionally, at least, a calciumtreatment with DTT (e.g., 90 s) resulted in a hyalin frac- insoluble protein is shown by its appearance in a variety tion containing mostly 11.6 S protein. If the same con- of preparations which rely on this property. The structural relationship of the three hyalin comditions were used to dissolve the hyaline layers from the longer DTT treatment groups (3-12 min), over three times ponents to each other and to other proteins of the extracellular matrix is of major interest. Coffman and McClay as much total protein was obtained in the hyalin fraction and the 9.6 S component was present in large amounts. (44) have suggested that Ecto V, a hyaline layer compoThe 6.4 S protein was a relatively minor component in nent that is localized to the microvillar tips of the embryo, all of these hyalin fractions. These differences are con- could correspond to the 6.5 S (6.4 S) and/or 8.8 S (9.6 S) sistent with the observation that brief treatments with hyalin proteins as described in (12). The subunit molec-
9.6 S HYALIN
ular weight estimate (350 kDa), lack of aggregation with calcium ions, and glycosylation of Ecto V, however, make this unlikely. The subunit size of both the 6.4 S and 9.6 S proteins is fairly close to that of the 175kDa apical lamina protein (45); however, apical lamina proteins do not associate with Ca2+ (46). Evidence presented here supports the assertion that the 6.4 S component is derived from the 9.6 S protein by proteolysis. Amino acid sequence studies are in progress to address this issue directly. When mixtures containing all three hyalin components were examined on reducing SDS-gel electrophoresis, we observed only a single major band with a mobility comparable to that of the 320-kDa band expected for the 11.6 S component. A band with the mobility of the purified 6.4 S (180 kDa) protein was not apparent and only a very faint band with a mobility in the range of the 9.6 S protein was visible. It is possible that this behavior may be due to a combination of factors including (1) that the 11.6 S protein predominates in these preparations and produces an intense band, (2) that the 9.6 S protein is susceptible to degradation and is poorly resolved on the reducing SDS gel, and (3) that the 6.4 S component was a minor component of these hyalin preparations; however, it is also likely that the proteins are interacting anomalously so that resolution of the chains is not achieved. These facts may help to explain why hyalin has been traditionally regarded as a single protein with a chain size of approximately 300 kDa. While it now appears clear that the 300kDa band on reducing SDS-gels corresponds to the native 11.6 S protein (12), it is also clear that two additional proteins constitute hyalin prepared by a variety of procedures. Thus, the true composition of hyalin has been revealed more readily and reliably by analytical ultracentrifuge studies (47) than by SDS-gel electrophoresis. REFERENCES 1. Schuel, H. (1978) Gamete Res. 1, 299-382. 2. Giudice, G. (1986) The Sea Urchin Embryo. A Developmental logical System, Springer-Verlag, Berlin.
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3. Shapiro, B. M., Somers, C. E., and Weidman, J. J. (1989) in The Cell Biology of Fertilization (Schatten, H., and Schatten, G., Eds.), pp. 251-276, Academic Press, San Diego, CA. 4. Dan, K. (1960) Int. Rev. Cytol. 9,321-367. 5. Herbst, C. (1900) Wil/r&n ismen 9,424-463. 6. Afselius,
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14,307-321. 31. Weber, K., Pringle, J. R., and Osborn, M. (1972) in Methods in Enzymology (Hirs, C. H. W., and Timasheff, S. N., Eds.), Vol. 26, pp. 3-27, Academic Press, New York. 32. Ruchel, R., Retief, A. E., and Richter-Landsberg, C. (1978) Anal. Biockem. 90,451-464. 33. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193,265-275. 34. Warburg, O., and Christian, W. (1941) Biockem. Z. 310, 384-421. 35. Yphantis, D. A. (1964) Biochemistry 3,297-317. 36. Vacquier, V. D., Epel, D., and Douglas, L. A. (1972) Nature (London) 237,34-36. 37. Chandler, D. E., and Heuser, J. (1980) J. Cell Biol. 84, 618-632. 38. Kubota, L. F., and Carroll, E. J., Jr. (1988) Gamete Res. 21,29-40. 39. Justice, R. W. (1989) Ph.D. dissertation, University of California, Riverside. 40. Lundgren, B. (1973) J. Submicrosc. Cytol. 5, 61-70. 41. Cameron, R. A., and Holland, N. D. (1985) Cell Tissue Res. 239, 455-458. 42. Spiegel, E., Howard, L., and Spiegel, M. (1989) J. Morphol. 199, 71-92. 43. Citkowitz, E. (1972) Deu. Bid. 27,494-503. 44. Coffman, J. A., and McClay, D. R. (1990) Deu. Bd 140,93-104. 45. Hall, H. G., and Vacquier, V. D. (1982) Deu. Bid. 89, 168-178. 46. Robinson, J. J. (1990) Biochem. Cell Bid. 68, 1083-1089. 47. Schachman, H. K. (1989) Nature (London) 341, 259-260.