Comparison of the bindin proteins of Strongylocentrotus franciscanus, S. purpuratus, and Lytechinus variegatus: sequences involved in the species specificity of fertilization.

Bindin is the sea urchin sperm acrosomal protein that is responsible for the species-specific adhesion of the sperm to the egg. Two new bindin cDNA sequences that contain the entire open reading frame for the binding precursor are reported: one for Strongylocentrotus franciscanus and one for Lytechinus variegatus. Both contain inverted repetitive sequences in their 3' untranslated regions, and the S. franciscanus cDNA contains an inverted repetitive sequence match between the 5' untranslated region and the coding region. The middle third of the mature bindin sequence is highly conserved in all three species, and the flanking sequences share short repeated sequences that vary in number between the species. Cross-fertilization data are reported for the species S. purpuratus, S. franciscanus, L. variegatus, and L. pictus. A barrier to cross-fertilization exists between the sympatric Strongylocentrotus species, but there is no barrier between the allopatric Lytechinus species.     

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.     

Species-specific inhibition of fertilization by a peptide derived from the sperm protein bindin.

The sperm protein bindin is responsible for the species-specific adhesion of the sperm to the egg. The regions of the bindin molecule responsible for forming the contact between the sperm and the egg were investigated by measuring the ability of peptides representing various regions of the bindin sequence to inhibit fertilization. Twenty-four peptides were studied: 7 based on the Strongylocentrotus purpuratus bindin sequence, 11 based on the S. franciscanus bindin sequence, and 6 control peptides. Values for the concentration of peptide required to inhibit 50% of the productive sperm contacts (IC50) were extracted from experimental measurements of the extent of fertilization in the presence of various concentrations. of these peptides. The IC50 value averaged 220 microM for the control peptides. Active peptides representing certain specific subregions of the bindin sequence displayed IC50 values < 10% of the average value for control peptides, and the IC50 for the most potent of the peptides tested was only approximately 1% of the control peptide value (IC50 = 2.2 microM). Furthermore, we found that a peptide representing a particular region of the S. franciscanus bindin sequence that differs from the S. purpuratus bindin sequence inhibits fertilization species specifically. For the reaction of S. purpuratus sperm and eggs, the IC50 of this peptide was approximately 120 microM, whereas for the reaction of S. franciscanus sperm and eggs it was only 8.6 microM. These results demonstrate that a few specific regions of the bindin molecule are involved in the sperm-egg contact and that certain of these regions mediate the species specificity of the interaction in a sequence-specific manner.     

Bindin,a multifunctional sperm ligand and the evolution of new species

Sea urchin sperm species-specifically adhere to the egg surface during fertilization. The protein which mediates this adhesion is known as bindin and cDNAs have recently been cloned and sequenced from several different species. Bindin proteins contain a highly conserved central domain flanked by much more highly divergent amino- and carboxyl-terminal domains. Investigations of the structure and function relationships indicate that the conserved domains may participate in membrane fusion and sulfated fucan binding activities, which may be conserved functions of bindin. The species-specific adhesion activity appears to be duplicated in both the amino- and carboxyl-terminal domain and may correspond to repeated sequence motifs found in these domains. The duplication of these sequence motifs and the redundancy of the adhesive domains may be important for the molecular mechanism of bindin evolution during speciation.     

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.     

Identification of a sperm receptor on the surface of the eggs of the sea urchin Arbacia punctulata

The possibility that the surface of the egg of the sea urchin Arbacia punctulata contains a species-specific receptor for sperm has been investigated. The extent of fertilization of eggs of A. punctulata, which is proportional to the number of sperm, is unaffected by the presence of either eggs or membranes prepared from eggs of Strongylocentrotus purpuratus. In marked contrast, membranes prepared from eggs of A. punctulata quantitatively inhibit fertilization of A. punctulata eggs by A. punctulata sperm. Several lines of evidence indicate that this inhibition is due to the presence of a membrane-associated glycoprotein that binds to the sperm, thus preventing them from interacting with receptor on the surface of the eggs. First, eggs treated with trypsin are incapable of being fertilized, although they can be activated with the Ca2+ ionophore A23187. Moreover, membranes prepared from eggs pretreated with trypsin do not inhibit fertilization of eggs. Second, receptor isolated in soluble form from surface membranes binds to sperm and thus prevents them from fertilizing eggs; the inhibition by soluble receptor is species-specific. Third, the soluble receptor binds to concanavalin A-Sepharose. Fourth, eggs are incapable of being fertilized if they are pretreated with concanavalin A. The specificity of inhibition, and the affect of trypsin and concanavalin A on intact eggs, suggest that the receptor is a species-specific macromolecule located on the surface of the eggs. The sensitivity of the receptor to trypsin, and its ability to bind to concanavalin A, indicate that it is a glycoprotein.     

Horseradish peroxidase. XXV. An electron spin resonance study of the low temperature photochemical reaction of compound I.

The insoluble acrosome granule content of sea urchin sperm consists of a single 30,500 dalton protein named bindin. Bindin mediates species-specific recognition and adhesion of sperm to the egg surface. Bindin from $\text{Strongylocentrotus purpuratus}$ (Sp) and $\text{Strongylocentrotus franciscanus}$ (Sf) have tyrosine as their single N-terminal amino acid. The pI of Sp bindin is 6.62 and of Sf 6.59. Amino acid analysis reveals almost identical composition between the two species for 16 amino acids. Only two (or three) amino acids, Pro and Asx, show large species differences. Tryptic peptide maps of the two species of bindin show very similar patterns with 24 spots of identical correspondence.     

Evaluation of Sequence Variation and Selection in the Bindin Locus of the Red Sea Urchin, Strongylocentrotus franciscanus

Recent evidence suggests that gamete recognition proteins may be subjected to directed evolutionary pressure that enhances sequence variability. We evaluated whether diversity enhancing selection is operating on a marine invertebrate fertilization protein by examining the intraspecific DNA sequence variation of a 273-base pair region located at the 5′ end of the sperm bindin locus in 134 adult red sea urchins (Strongylocentrotus franciscanus). Bindin is a sperm recognition protein that mediates species-specific gamete interactions in sea urchins. The region of the bindin locus examined was found to be polymorphic with 14 alleles. Mean pairwise comparison of the 14 alleles indicates moderate sequence diversity (p-distance = 1.06). No evidence of diversity enhancing selection was found. It was not possible to reject the null hypothesis that the sequence variation observed in S. franciscanus bindin is a result of neutral evolution. Statistical evaluation of expected proportions of replacement and silent nucleotide substitutions, observed versus expected proportions of radical replacement substitutions, and conformance to the McDonald and Kreitman test of neutral evolution all indicate that random mutation followed by genetic drift created the polymorphisms observed in bindin. Observed frequencies were also highly similar to results expected for a neutrally evolving locus, suggesting that the polymorphism observed in the 5′ region of S. franciscanus bindin is a result of neutral evolution. Received: 19 June 1998 / Accepted: 2 August 2000     

The molecular evolution of sperm bindin in six species of sea urchins (Echinoida: Strongylocentrotidae)

The acrosomal protein bindin attaches sperm to eggs during sea urchin fertilization. Complementary to ongoing functional biochemical studies, I take a comparative approach to explore the molecular evolution of bindin in a group of closely related free-spawning echinoid species. Two alleles of the mature bindin gene were sequenced for each of six species in the sea urchin family Strongylocentrotidae. The nucleotide sequences diverged by at least 1% per Myr at both silent and replacement sites. Two short sections flanking the conserved block show an excess of nonsynonymous substitutions. Each is homologous to a region that had been identified as a target of selection in other sea urchin comparisons. A large proportion of the bindin-coding sequence consists of a highly variable repeat region. Bindin sequences, even including the large intron, could not resolve the branching order among five of the species.      

Chemotaxis of Arbacia punctulata spermatozoa to resact, a peptide from the egg jelly layer

Resact, a peptide of known sequence isolated from the jelly layer of Arbacia punctulata eggs, is a potent chemoattractant for A. punctulata spermatozoa. The chemotactic response is concentration dependent, is abolished by pretreatment of the spermatozoa with resact, and shows an absolute requirement for millimolar external calcium. A. punctulata spermatozoa do not respond to speract, a peptide isolated from the jelly layer of Strongylocentrotus purpuratus eggs. This is the first report of animal sperm chemotaxis in response to a defined egg-derived molecule.     

Locale and level of bindin mRNA in maturing testis of the sea urchin, Strongylocentrotus purpuratus

This first study of the onset of spermatogenesis in the sea urchin, Strongylocentrotus purpuratus, was undertaken using individuals reared in the laboratory. Spermatogenesis commences about 11-12 months after metamorphosis in these animals. Bindin message accumulates in late spermatocytes and early spermatids which lie in the luminal germinal layer. Bindin message accumulates later than does the testis-specific histone, H2b-1, suggesting that different classes of genes are sequentially activated during the differentiation of sperm. We correlate the number of bindin mRNA molecules with morphological structure and with quantitative aspects of gonad maturation including the number of nuclei and of sperm. The results suggest that the bindin mRNA concentration in total RNA from testis at different stages of maturation reflects the change in the proportion of expressing cells in the total cell population of the testis.     

A peptide associated with eggs causes a mobility shift in a major plasma membrane protein of spermatozoa

A peptide (resact) associated with the eggs of the sea urchin, Arbacia punctulata, which stimulates sperm respiration rates by 5-10-fold, was purified and its amino acid sequence was determined. The sequence was found to be Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-Arg-Leu-NH2. The peptide was subsequently synthesized by solid phase methods, amidated at the carboxyl-terminal Leu, and shown to be identical to the isolated, native material. The peptide half-maximally stimulated A. punctulata spermatozoan respiration at 0.5 nM and half-maximally elevated cyclic GMP concentrations at 25 nM at an extracellular pH of 6.6. The increase in oxygen consumption was coupled with a stimulation of motility. However, at elevated extracellular pH (pH 8.0), resact failed to appreciably stimulate respiration while the elevations of cyclic GMP continued to occur. Resact did not cross-react with sperm cells obtained from Lytechinus pictus or Strongylocentrotus purpuratus; a peptide (speract) obtained from S. purpuratus eggs (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) which activates S. purpuratus sperm respiration did not stimulate A. punctulata spermatozoa. Resact caused a shift in the apparent molecular weight (160,000-150,000) of a major sperm plasma membrane protein; as with cyclic GMP elevations, this response was evident at extracellular pH values of both 6.6 and 8.0. The protein exists in the cell as a phosphoprotein and 32P is released coincident with the molecular weight change. Approximately 115 nM resact caused one-half-maximal conversion of the 160,000-dalton protein after 1 min of incubation. Resact caused the apparent molecular weight conversion of the protein within 5 s and appeared to do so in an irreversible manner. The molecular weight change of the protein was also observed after the addition of monensin A (25 microM) and NH4Cl (40 mM), two agents known to elevate intracellular pH and to increase sperm respiration rates. The membrane protein appears to be the enzyme guanylate cyclase, but since concentrations of resact causing one-half-maximal conversion of the Mr = 160,000 form of the enzyme are about 250 times higher than those causing one-half-maximal stimulation of respiration, the relationship of the apparent molecular weight conversion to a subsequent physiological event remains unclear.     

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.     

Positive selection and sequence rearrangements generate extensive polymorphism in the gamete recognition protein bindin

Bindin is a gamete recognition protein of sea urchins that mediates species-specific attachment of sperm to an egg-surface receptor during fertilization. Sequences of bindin from closely related urchins show fixed species-specific differences. Within species, highly polymorphic bindin alleles result from point substitution, insertion/deletion, and recombination. Since speciation, positive selection favoring allelic variants has generated diversity in bindin polypeptides. Intraspecific bindin variation can be tolerated by the egg receptor, which suggests functional parallels between this system and other flexible recognition systems, including immune recognition. These results show that polymorphism in mate recognition loci required for rapid evolution of sexual isolation can arise within natural populations.      

250 million years of bindin evolution

Bindin plays a central role in sperm-egg attachment and fusion in sea urchins (echinoids). Previous studies determined the DNA sequence of bindin in two orders of the class Echinoidea, representing 10% of all echinoid species. We report sequences of mature bindin from five additional genera, representing four new orders, including the distantly related sand dollars, heart urchins, and pencil urchins. The six orders in which bindin is now known include 70% of all echinoids, and indicate that bindin was present in the common ancestor of all extant sea urchins more than 250 million years ago. Over this span of evolutionary time there has been (1). remarkable conservation in the core region of bindin, particularly in a stretch of 29 amino acids that has not changed at all; (2). conservation of a motif of basic amino acids at the cleavage site between preprobindin and mature bindin; (3). more than a twofold change in length of mature bindin; and (4). emergence of high variation in the sequences outside the core, including the insertion of glycine-rich repeats in the bindins of some orders, but not others.     

Characterization of the Sperm Molecule Bindin in the Sea Urchin Genus <Emphasis Type="Italic">Paracentrotus</Emphasis>

Bindin is a sea urchin gamete-recognition protein that plays an essential role in the specificity of egg–sperm interactions and thus may be evolving under sexual selection and be related to speciation. Bindin has been found to evolve under strong selection in some sea urchin genera and neutrally in others. In this study, we characterized bindin in the two extant species of the genus Paracentrotus: P. lividus from the Atlanto-Mediterranean region and P. gaimardi from Brazil. The structure of the bindin molecule in Paracentrotus is similar to that of other genera studied thus far, consisting of a conserved core flanked by two variable regions and an intron of variable length located at the same conserved position as in other genera. Polymorphism in P. lividus is caused mainly by point substitutions and insertions/deletions, and length variations are caused mainly by the number of repeated motifs in the flanking regions. There is no evidence of recombination. Positive selection is acting on amino acid sites located in two regions flanking the conserved core.     

Purification and characterization of an extracellular fragment of the sea urchin egg receptor for sperm

Fertilization in the sea urchin involves species-specific interaction between the ligand bindin on the surface of acrosome-reacted sperm and a receptor of high molecular weight on the surface of the egg. Efforts to understand this interaction and the resultant signal transduction events leading to egg activation have been limited because of the large size and extreme insolubility of the intact receptor on the egg surface. Earlier work suggested that an alternative strategy would be to isolate proteolytic fragments of the extracellular domain of this receptor. Consequently, we have treated S. purpuratus eggs with a specific protease, lysylendoproteinase C. This enzyme treatment abolished the ability of eggs to bind sperm and resulted in the release of proteolytic fragments that bound to sperm and showed inhibitory activity in a fertilization bioassay. One of these fragments, presumed to be a fragment of the extracellular domain of the receptor, was purified to homogeneity by gel filtration and anion exchange chromatography and shown to be a 70-kD glycosylated protein. Several lines of evidence support the contention that this fragment is derived from the receptor. First, the fragment inhibited fertilization species specifically. Second, species specific binding of the 70-kD glycoprotein to acrosome-reacted sperm was directly demonstrated by using 125I-labeled receptor fragment. Third, the fragment exhibited the same species specificity in binding to isolated bindin particles. Species specificity was abolished by Pronase digestion of the fragment. This observation supports the hypothesis that although binding is mediated by the carbohydrate moieties, species specificity is dependent on the polypeptide backbone. The availability of a structurally defined fragment of the receptor will facilitate further studies of the molecular basis of gamete interaction.     

Purification of sea urchin sperm bindin by DEAE-cellulose chromatography

A procedure for purifying bindin from sperm of the sea urchin Strongylocentrotus purpuratus is presented in detail. The impure bindin, dissolved in 4 M urea, 50 mM sodium phosphate, pH 6.6, is adsorbed to DEAE-cellulose and eluted wit 4 M urea, 650 mM sodium phosphate, pH 6.6. The purified bindin is not contaminated with tubulin or histone HI. A precipitate of this DEAE-purified bindin, made by dialysis into Ca2+-free seawater and natural seawater, is a species-specific agglutinin of unfertilized eggs. This method of obtaining consistently pure preparations of bindin will aid in the analysis of its role in fertilization.     

The membrane form of guanylate cyclase. Homology with a subunit of the cytoplasmic form of the enzyme

A cDNA clone for the membrane form of guanylate cyclase has been isolated from the testis of the sea urchin Strongylocentrotus purpuratus. An open reading frame predicts a protein of 1125 amino acids including an apparent signal peptide of 21 residues; a single transmembrane domain of 25 amino acids divided the mature protein into an amino-terminal, extracellular domain of 485 amino acids and a carboxyl domain of 594 intracellular amino acids. Three potential Asn-linked glycosylation sites were present in the proposed extracellular domain. The deduced protein sequence was homologous to the protein kinase family and contained limited but significant regions of identity with a low molecular weight atrial natriuretic peptide receptor. The carboxyl region (202 amino acids) was 42% identical with a subunit of the cytoplasmic form of guanylate cyclase recently cloned from bovine lung (Koesling, D., Herz, J., Gausepohl, H., Niroomand, F., Hinsch, K.-D., Mulsch, A., Bohme, E., Schultz, G., and Frank, R. (1988) FEBS Lett. 239, 29-34). Therefore, the membrane form of guanylate cyclase is a member of an apparently large family of proteins that includes the low molecular weight atrial natriuretic peptide receptor, the soluble form of guanylate cyclase and protein kinases.