The sequence of the Arbacia punctulata bindin cDNA and implications for the structural basis of species-specific sperm adhesion and fertilization

Bindin is the major protein component of the acrosome granule of sea urchin sperm which mediates the species-specific adhesion of sperm to the egg surface during fertilization. Bindin isolated from both Arbacia punctulata and Strongylocentrotus purpuratus sperm demonstrate a distinct adhesive preference for eggs of the same species although a significant amount of cross-species reactivity is observed. Here we describe the isolation and sequence of A. punctulata bindin cDNA clones and a comparison of the predicted protein sequence with the sequence previously reported for S. purpuratus bindin (Gao et al., 1986, Proc. Natl. Acad. Sci., USA 83, 8634-8638). Bindins from these genera show substantial sequence similarity in both the mature bindin domain and the probindin precursor region. The most striking identity is a region of 42 conserved amino acids in the central part of the mature bindins. This conserved domain may be responsible for conserved functions of bindin. Regions flanking this conserved element on both the amino and carboxyl side are more highly divergent, suggesting that they are responsible for the species-specific properties of bindin. The mature A. punctulata sequence contains a putative transmembrane segment between residues 431 and 451 that is absent from S. purpuratus bindin. This structural element may account for the previous observation that isolated A. punctulata bindin uniquely forms multilamellar structures reminiscent of lipid bilayers and binds significant amounts of phospholipid and detergent. The structure of this hydrophobic segment also displays a number of similarities to viral fusion peptides.     

Poly(A)-containing messenger ribonucleoprotein complexes from sea urchin eggs and embryos: polypeptides associated with native and UV-crosslinked mRNPs

Fertilization of sea urchin eggs results in the rapid recruitment of stored messages into polyribosomes. Whether translational control in sea urchin eggs is mediated by macromolecules associated with the stored messages remains unknown, since preparations of messenger ribonucleoprotein complexes (mRNPs) were active in protein synthesis in a rabbit reticulocyte lysate. To facilitate the study of mRNPs, chromatography on oligo(dT)-cellulose was used to purify poly(A)-containing mRNPs from eggs and embryos of the sea urchin Strongylocentrotus purpuratus. Nonpolyribosomal mRNPs purified from eggs had a similar sedimentation in sucrose to unpurified mRNPs, a peak buoyant density in metrizamide of 1.22 g/cm3, and peak buoyant densities in Cs2SO4 in 1.42 g/cm3 after fixation with glutaraldehyde and 1.46 g/cm3 without fixation. Nonpolyribosomal mRNPs from eggs and zygotes contained 5-10 major proteins on sodium dodecylsulfate (SDS) polyacrylamide gels, and numerous minor bands. UV-irradiation of living eggs of the sea urchin Arbacia punctulata produced cross-linked mRNPs which contained a similar pattern of polypeptides to noncross-linked mRNPs. The polypeptides associated with embryonic polyribosomal mRNPs were also qualitatively similar to those present in nonpolyribosomal mRNPs, although stoichiometric differences may exist.     

Isolation and characterization of proteolytic fragments of the sea urchin sperm receptor that retain species specificity

The sea urchin sperm receptor isolated from the eggs of Strongylocentrotus purpuratus is a high molecular weight proteoglycan-like molecule. Previous studies in our laboratory suggested that the sperm receptor has two functional components, glycosaminoglycan chains that are responsible for sperm binding and polypeptide chains that control species specificity in the binding process. We have investigated this idea further by generating fragments of the receptor by limited proteolytic digestion of the egg cell surface. The results of experiments with these receptor preparations support the hypothesis that the species specificity of inhibition of fertilization observed in a competitive bioassay is conferred by the polypeptide portion of the receptor molecule. Studies with various receptor preparations reveal that the presence of at least 30% of the polypeptide by weight is required to inhibit fertilization species specifically. Receptor preparations containing less than 10% protein lack species specificity and inhibit fertilization in both S. purpuratus and Arbacia punctulata.     

Poly(A)-Containing Messenger Ribonucleoprotein Complexes from Sea Urchin Eggs and Embryos: Polypeptides Associated with Native and UV-Crosslinked mRNPs

Abstract. Fertilization of sea urchin eggs results in the rapid recruitment of stored messages into polyribosomes. Whether translational control in sea urchin eggs is mediated by macromolecules associated with the stored messages remains unknown, since preparations of messenger ribonucleoprotein complexes (mRNPs) were active in protein synthesis in a rabbit reticulocyte lysate. To facilitate the study of mRNPs, chromatography on oligo(dT)-cellulose was used to purify poly(A)-containing mRNPs from eggs and embryos of the sea urchin Strongylocentrotus purpuratus . Nonpolyribosomal mRNPs purified from eggs had a similar sedimentation in sucrose to unpurified mRNPs, a peak buoyant density in metrizamide of 1.22 g/cm³, and peak buoyant densities in Cs₂SO₄ in 1.42 g/cm³ after fixation with glutaraldehyde and 1.46 g/cm³ without fixation. Nonpolyribosomal mRNPs from eggs and zygotes contained 5–10 major proteins on sodium dodecylsulfate (SDS) polyacrylamide gels, and numerous minor bands. UV-irradiation of living eggs of the sea urchin Arbacia punctulata produced cross-linked mRNPs which contained a similar pattern of polypeptides to noncross-linked mRNPs. The polypeptides associated with embyronic polyribosomal mRNPs were also qualitatively similar to those present in nonpolyribosomal mRNPs, although stoichiometric differences may exist.     

Conserved pattern of embryonic actin gene expression in several sea urchins and a sand dollar

An examination of the size and relative abundance of actin-coding RNA in embryos of four sea urchins (Strongylocentrotus purpuratus, Strongylocentrotus droebachiensis, Arbacia punctulata, Lytechinus variegatus) and one sand dollar (Echinarachnius parma) reveals a generally conserved program of expression. In each species the relative abundance of these sequences is low in early embryos and begins to rise during late cleavage or blastula stages. In the four sea urchins, actin-coding RNAs increase between approximately 9- and 35-fold by pluteus or an earlier stage, and in the sand dollar about 5.5-fold by blastula. A major actin-coding RNA class of 2.0-2.2 kilobases (kb) is found in each species. A smaller actin-coding RNA class, which accumulates during embryogenesis, is also present in S. purpuratus (1.8 kb), S. droebachiensis (1.9 kb), and A. punctulata (1.6 kb), but apparently absent in L. variegatus and E. parma. In S. droebachiensis, actin-coding RNA is relatively abundant in unfertilized eggs and drops sharply by the 16-cell stage. This is in contrast to the other sea urchins where the actin message content is relatively low in eggs and does not change substantially in the embryos throughout early cleavage. The observations in this study suggest that the pattern of embryonic expression of at least some members of this gene family is ancient and conserved.     

Purification and properties of soluble actin from sea urchin eggs

Unfertilized eggs of the sea urchin, Strongylocentrotus purpuratus, were homogenized in a buffer containing 0.1 M KCl and 2 mM MgCl2 at pH 6.85. About 50% of the actin was recovered in the high-speed supernate of the homogenate. More than 80% of the actin in this supernate was found to be monomeric upon gel filtration chromatography through a Sephadex G-150 column or by a DNase I inhibition assay. The critical concentration for polymerization of this actin prior to further purification was 0.3-0.9 mg/ml under various conditions. Actin was purified to near homogeneity from the Sephadex G-150 pool with high yield. The purified actin had a critical concentration for polymerization of 0.02-0.03 mg/ml. The isoelectric point of the crude actin and the purified actin was the same. Indeed, we found that there is only one isoelectric focusing species of actin in the sea urchin egg, and it has an isoelectric point more basic than rabbit skeletal muscle actin. The discrepancy between the polymerizability of the crude and purified actin may be due to the presence of factors in the crude fraction which inhibit the polymerization of actin.     

Phenotypic switching to long cilia effected by various proteases: results with Dendraster excentricus and Stronglyocentrotus purpuratus blastulae

Incubation in trypsin effects a phenotypic switch from short to long cilia (greater than 30 micron) in hatched blastulae of the sea urchin, Arbacia punctulata. To determine how trypsin causes such a switch we tested whether the phenomenon was unique to the species, Arbacia, and to the protease, trypsin. With two other echinoderm species, the sand dollar, Dendraster excentricus, and the sea urchin, Strongylocentrotus purpuratus, trypsin incubation increased the percentage of long cilia. During incubation of D. excentricus in trypsin, the percentage of long cilia increased progressively from the normal 10% long cilia of the apical tuft to 45-50% long cilia covering 1/2-3/4 of the embryo. With S. purpuratus blastulae, however, the percentage of long cilia was lower (32-40%) and the results were more variable. Of the additional proteases tested with D. excentricus, elastase was more effective than trypsin in terms of the percentage of long cilia (58%), the mean length, and the broad distribution of lengths formed. Thermolysin was about as effective as trypsin but chymotrypsin was much less so. Thus, increases in ciliary length were not unique to a particular echinoderm species or to incubation in trypsin. The magnitude of the change in length distribution, however, was species- and enzyme-dependent. An extracellular or membrane component with differential susceptibility to various proteases may, therefore, be involved in altering ciliary length.     

The onset of collagen synthesis in sea urchin embryos

In Arbacia punctulata and Strongylocentrotus purpuratus, two species of sea urchins, collagen synthesis begins during gastrulation and increases many-fold before reaching a plateau in the late pluteus stage. A collagen extraction method involving treatment with 0.1 M NaOH and hot 10% trichloroacetic acid provided the basis for a sensitive assay of collagen synthesis.     

Species-specific sperm adhesion in sea urchins. A quantitative investigation of bindin- mediated egg agglutination

Bindin, a protein component of the acrosomal vesicle of sea urchin sperm, has been isolated from Arbacia punctulata and strongylocentrous purpuratus. Using this isolated bindin, we have devised a quantitative assay for bindin-mediated egg agglutination and compared the agglutination of bindin eggs from A. puntulata and S. purpuratus. Bindin- mediated agglutination is species –specific in both species, although a measurable degree of heterotypic interaction is observed. Homotypic bindin-egg interactions differ significantly from heterotypic interactions both in the extent of agglutination and the size of the resulting aggregates. We also provide direct evidence that bindin particles agglutinate eggs by adhering to the surfaces of adjacent eggs. Although the A. punctulata bindin preparation displays the same functional properties and consists of one major polypeptide of the same apparent molecular weight as S. purpuratus bindin, its morphology is very different. Unlike the spherical aggregates observed with S. purpuratus bindin, A punctulata bindin exists as lamellar vesicles and binds significant amounts of phospholipids and Triton X-100, suggesting that it may be tightly associated with the acrosomal membrane. Having defined a number of the basic parameters of bindin-mediated agglutination, we examined the effect of a number of saccharides and glycopeptides on bindin-mediated egg agglutination. Carbohydrate-containing components derived from the egg cell surface by proteolysis were found to inhibit bindin-mediated egg agglutination at low concentrations, but this inhibition is not species specific.     

Association of ribosomes with in vitro assembled microtubules

Microtubules were purified from unfertilized eggs of the sea urchins Arbacia punctulata, Lytechinus pictus, Lytechinus variegatus, and Strongylocentrotus purpuratus. Numerous densely stained particles (24 x 26 nm) are associated with microtubules isolated from each of these sea urchins. The most striking aspect of this structure is an extended, slightly curved arm that appears to attach the particles to the microtubule. Morphologically similar particles are associated with microtubules of the isolated first cleavage mitotic apparatus. The particles are attached to the microtubules by ionic interactions and contain large amounts of extractable RNA. Based upon their size and density, RNA and protein composition, and sedimentation in sucrose gradients, the microtubule-associated particles are identified as ribosomes.     

The increased phosphorylation of ribosomal protein S6 in Arbacia punctulata is not a universal event in the activation of sea urchin eggs

Eggs of the sea urchins Arbacia punctulata (Ap), Lytechinus pictus (Lp), and Strongylocentrotus purpuratus (Sp) were labeled to equilibrium with 32PO3-4. Approximately 65-70% of the label in extractable adenine nucleotides comigrates chromatographically with ATP. Autoradiograms of one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) slab gels show that each species possesses a distinct complement of phosphate-exchangeable phosphoproteins. No changes in the phosphoprotein composition are detected in Lp and Sp eggs as a result of fertilization or development for 2.5 hr (with the possible exception of a 43,000 Mr protein in Lp). In Ap, increases in the phosphorylation of bands at Mr's 30,000, 55,000, and 105,000 are seen during the first 10 min postinsemination. The 30,000 Mr band in Ap eggs has previously been identified as ribosomal protein S6 and the hypothesis presented that its increased phosphorylation may be an important step in the activation of protein synthesis at fertilization (D. G. Ballinger and T. Hunt, 1981, Dev. Biol. 87, 277-285). In Lp and Sp eggs S6 (identified by two-dimensional PAGE) is heavily phosphorylated in the unfertilized state and the extent of labeling does not increase after fertilization. If the increased phosphorylation of S6 seen in Ap is indeed related to translational activation, then these results suggest that different sea urchin species may rely on different mechanisms for the activation of protein synthesis.     

Individual histone messenger RNAs: Identification by template activity

Newly synthesized polysomal messenger RNAs from cleavage stage embryos of the sea urchin Arbacia punctulata and Lytechinus pictus that contain putative histone mRNAs have been fractionated on 6% polyacrylamide slab gels. At least 8 RNA species with unique electrophoretic mobilities have been recognized. The complex of RNAs has been eluted from the gels in three groups, A, B, and C, in increasing order of mobility. The template activity of the three fractions and the unfractionated starting material was examined in the mouse Krebs II ascites tumor cell-free protein synthesizing system. The unfractionated messenger complex programs the synthesis of proteins that coelectrophorese exclusively with sea urchin histones in both sodium dodecyl sulfate and acid urea gel systems. The products of in vitro protein synthesis stimulated by the individual polyacrylamide gel RNA fractions were similarly examined. Each stimulated protein synthesis and was enriched for specific histone templates. We conclude that RNA fraction A is template for histone f1, C is template for histone f2a1, and B serves as template for f2b, f2a2, and f3 histones. A minor degree of contamination of the A and B RNA fractions was obvious from the production of other histones by each template. The co-electrophoresis of specific template activity with specific radiolabeled RNAs supports the concept that most or all of the labeled RNAs are indeed themselves the histone mRNAs.     

Sperm–egg binding: Identification of a species-specific sperm receptor from eggs of strongylocentrotus purpuratus

We have attempted to identify a surface component of echinoderm eggs that is involved in the species-specific binding of sperm. Cell surface membranes from eggs of the sea urchins Strongylocentrotus purpuratus or Arbacia punctulata were radioiodinated, detergent-treated, and subjected to density-gradient centrifugation. In the presence of bindin, the complementary binding protein isolated from sperm, one component of the membranes sedimented to a different density. This membrane component bound-species specifically to sperm that had undergone the acrosome reaction. This binding led to an inhibition of the ability of treated sperm to fertilize eggs. Exhaustive proteolytic digestion of this receptor fraction yields a high molecular weight glycopeptide that can also bind to bindin. It therefore appears that this egg surface membrane fraction contains a functionally intact, species-specific receptor for sperm.     

Sea urchin morphogenesis and cell-hyalin adhesion are perturbed by a monoclonal antibody specific for hyalin

We have generated and characterized a monoclonal antibody (McA Tg-HYL) that recognizes sea urchin hyalin as evidenced by immunofluorescence staining of the hyaline layer (HL) and immunoblot staining of the hyalin protein band. On immunoblots of HL extracts only the hyalin protein reacted with McA Tg-HYL. Immunoprecipitates of radioactive proteins from embryos incubated with [35S]methionine yielded radioactive hyalin and 190, 140 and 105 x 10(3) Mr proteins associated with hyalin. McA Tg-HYL was generated against Tripneustes gratilla embryos but reacts with hyalin from the distantly related sea urchin species, Colobocentrotus atratus, Strongylocentrotus purpuratus, Arbacia punctulata, Lytechinus variegatus and Lytechinus pictus. Developing embryos of the above-mentioned six species were treated with McA Tg-HYL and did not gastrulate or form arms. Observations of treated embryos revealed areas of separation of the hyaline layer from the underlying embryonic cells, suggesting that McA Tg-HYL was interfering with binding of the cells to the HL. Using the centrifugation-based adhesion assay of McClay et al. (Proc. natn. Acad. Sci. U.S.A. 78, 4975-4979, 1981), Fab' fragments of McA Tg-HYL were found to inhibit cell-hyalin binding. McA Tg-HYL did not inhibit hyalin gelation in vitro or the reaggregation of dissociated blastula cells. We postulate that McA Tg-HYL recognizes an evolutionarily conserved hyalin domain involved in cell-hyalin binding and required for normal epithelial folding.     

Temperature induced isozyme variants in individuals of the sea urchin, Arbacia punctulata.

1. The technique of microacrylamide slab gel electrophoresis was used to repeatedly monitor the qualitative isozyme composition in the tube feet of the sea urchin, Arbacia punctulata exposed to different temperature regimes. 2. Four enzyme systems were assayed. Three (MDH, HK, and ACPH) were monomorphic for each animal studied and no change in band migration pattern was observed. Banding patterns for the fourth system (EST) were observed to vary in the same individual. 3. The reversible induction of esterase variants in the tube feet of the same individual is reported. 4. The change was temperature dependent, and a period of 7-14 days acclimation was required to induce the pattern alteration.