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V D Vacquier

Publications and source records attributed to V D Vacquier.

At least 19 recordsLinked to original sources
PubMed2001

The crystal structure of a fusagenic sperm protein reveals extreme surface properties.

Sp18 is an 18 kDa protein that is released from abalone sperm during the acrosome reaction. It coats the acrosomal process where it is thought to mediate fusion between sperm and egg cell membranes. Sp18 is evolutionarily related to lysin, a 16 kDa abalone sperm protein that dissolves the vitelline envelope surrounding the egg. The two proteins were generated by gene duplication followed by rapid divergence by positive selection. Here, we present the crystal structure of green abalone sp18 resolved to 1.86 A. Sp18 is composed of a bundle of five alpha-helices with surface clusters of basic and hydrophobic residues, giving it a large dipole moment and making it extremely amphipathic. The large clusters of hydrophobic surface residues and domains of high positive electrostatic surface charge explain sp18's ability as a potent fusagen of liposomes. The overall fold of sp18 is similar to that of green abalone lysin; however, the surface features of the proteins are quite different, accounting for their different roles in fertilization. This is the first crystal structure of a protein implicated in sperm-egg fusion during animal fertilization.

Acrosome↗
PubMed2001

Abalone lysin: the dissolving and evolving sperm protein.

Abalone sperm lysin is a non-enzymatic protein that creates a hole for sperm passage in the envelope surrounding the egg. Lysin exhibits species-specificity in making the hole and it evolves rapidly by positive selection. Our studies have focused on combining structural, biochemical, and evolutionary data to understand the mechanism of action and evolution of this remarkable protein. Currently, more is known about lysin than about any other protein involved in animal fertilization. We present an hypothesis to explain lysin's rapid evolution and the evolution of species-specific fertilization in this order of mollusks. We also propose a two-step model for lysin's action in which a dimer of lysin binds species-specifically to its glycoprotein receptor, and then monomerizes and binds the receptor in a non-species-specific manner. This experimental system yields data relevant to the general problem of molecular recognition between cell surfaces, and is also important to our thinking about how new species arise in the sea. BioEssays 23:95-103, 2001.

Acrosome↗
PubMed2001

Glycobiology of sperm-egg interactions in deuterostomes.

The process of fertilization begins when sperm contact the outermost egg investment and ends with fusion of the two haploid pronuclei in the egg cytoplasm. Many steps in fertilization involve carbohydrate-based molecular recognition between sperm and egg. Although there is conservation of gamete recognition molecules within vertebrates, their homologues have not yet been discovered in echinoderms and ascidians (the invertebrate deuterostomes). In echinoderms, long sulfated polysaccharides act as ligands for sperm receptors. Ascidians employ egg coat glycosides that are recognized by sperm surface glycosidases. Vertebrate egg coats contain zona pellucida (ZP) family glycoproteins, whose carbohydrates bind to sperm receptors. Several candidate sperm receptors for vertebrate ZP proteins have been identified and are discussed here. This brief review focuses on new information concerning fertilization in deuterostomes (the phylogenetic group including echinoderms, ascidians, and vertebrates) and highlights protein-carbohydrate interactions involved in this process.

Animals↗
PubMed2001

Embryos of the sea urchin Strongylocentrotus purpuratus synthesize a dermatan sulfate enriched in 4-O- and 6-O-disulfated galactosamine units.

Unfertilized eggs of the sea urchin Strongylocentrotus purpuratus are surrounded by a gelatinous layer rich in sulfated fucan. Shortly after fertilization this polysaccharide disappears, but 24 h later the embryos synthesize high amounts of dermatan sulfate concomitantly with the mesenchyme blastula-early gastrula stage when the larval gut is forming. This glycosaminoglycan has the same backbone structure [4-alpha-L-IdoA-1-->3-beta-D-GalNAc-1](n) as the mammalian counterpart but possesses a different sulfation pattern. It has a high content of 4-O- and 6-O-disulfated galactosamine units. In addition, chains of this dermatan sulfate are considerable longer than those of vertebrate tissues. Adult sea urchin tissues contain high concentrations of sulfated polysaccharides, but dermatan sulfate is restricted to the adult body wall where it accounts for approximately 20% of the total sulfated polysaccharides. In addition, sulfation at the 4-O-position decreases markedly in the dermatan sulfate from adult sea urchin when compared with the glycan from larvae. Overall, these results demonstrate the occurrence of dermatan sulfates with unique sulfation patterns in this marine invertebrate. The physiological implication of these oversulfated dermatan sulfates is unclear. One hypothesis is that interactions between components of the extracellular matrix in marine invertebrates occur at higher salt concentrations than in vertebrates and therefore require glycosaminoglycans with increased charge density.

Acetylgalactosamine↗
PubMed2001

Polymorphism in abalone fertilization proteins is consistent with the neutral evolution of the egg's receptor for lysin (VERL) and positive darwinian selection of sperm lysin.

The evolution of species-specific fertilization in free-spawning marine invertebrates is important for reproductive isolation and may contribute to speciation. The biochemistry and evolution of proteins mediating species-specific fertilization have been extensively studied in the abalone (genus Haliotis). The nonenzymatic sperm protein lysin creates a hole in the egg vitelline envelope by species-specifically binding to its egg receptor, VERL. The divergence of lysin is promoted by positive Darwinian selection. In contrast, the evolution of VERL does not depart from neutrality. Here, we cloned a novel nonrepetitive region of VERL and performed an intraspecific polymorphism survey for red (Haliotis rufescens) and pink (Haliotis corrugata) abalones to explore the evolutionary forces affecting VERL. Six statistical tests showed that the evolution of VERL did not depart from neutrality. Interestingly, there was a subdivision in the VERL sequences in the pink abalone and a lack of heterozygous individuals between groups, suggesting that the evolution of assortative mating may be in progress. These results are consistent with a model which posits that egg VERL is neutrally evolving, perhaps due to its repetitive structure, while sperm lysin is subjected to positive Darwinian selection to maintain efficient interaction of the two proteins during sperm competition.

Amino Acid Sequence↗
PubMed2000

The high resolution crystal structure of green abalone sperm lysin: implications for species-specific binding of the egg receptor.

Abalone sperm lysin is a 16 kDa acrosomal protein used by sperm to create a hole in the egg vitelline envelope. Lysins from seven California abalone exhibit species-specificity in binding to their egg receptor, and range in sequence identity from 63 % to 90 %. The crystal structure of the sperm lysin dimer from Haliotis fulgens (green abalone) has been determined to 1.71 A by multiple isomorphous replacement. Comparisons with the structure of the lysin dimer from Haliotis rufescens (red abalone) reveal a similar overall fold and conservation of features contributing to lysin's amphipathic character. The two structures do, however, exhibit differences in surface residues and electrostatics. A large clustering of non-conserved surface residues around the waist and clefts of the dimer, and differences in charged residues around these regions, indicate areas of the molecule which may be involved in species-specific egg recognition.

Acrosome↗
PubMed2000

Maximum-likelihood analysis of molecular adaptation in abalone sperm lysin reveals variable selective pressures among lineages and sites.

Maximum-likelihood models of codon substitution were used to analyze sperm lysin genes of 25 abalone (HALIOTIS:) species to identify lineages and amino acid sites under diversifying selection. The models used the nonsynonymous/synonymous rate ratio (omega = d(N)/d(S)) as an indicator of selective pressure and allowed the ratio to vary among lineages or sites. Likelihood ratio tests suggested significant variation in selective pressure among lineages. The variable selective pressure provided an explanation for the previous observation that the omega ratio is >1 in comparisons of closely related species and <1 in comparisons of distantly related species. Computer simulations demonstrated that saturation of nonsynonymous substitutions and constraint on lysin structure were unlikely to account for the observed pattern. Lineages linking closely related sympatric species appeared to be under diversifying selection, while lineages separating distantly related species from different geographic locations were associated with low evolutionary rates. The selective pressure indicated by the omega ratio was found to vary greatly among amino acid sites in lysin. Sites under potential diversifying selection were identified. Ancestral lysins were inferred to trace the route of evolution at individual sites and to provide lysin sequences for future laboratory studies.

Adaptation, Biological↗
PubMed2000

Positive selection and propeptide repeats promote rapid interspecific divergence of a gastropod sperm protein.

Male-specific proteins have increasingly been reported as targets of positive selection and are of special interest because of the role they may play in the evolution of reproductive isolation. We report the rapid interspecific divergence of cDNA encoding a major acrosomal protein of unknown function (TMAP) of sperm from five species of teguline gastropods. A mitochondrial DNA clock (calibrated by congeneric species divided by the Isthmus of Panama) estimates that these five species diverged 2-10 MYA. Inferred amino acid sequences reveal a propeptide that has diverged rapidly between species. The mature protein has diverged faster still due to high nonsynonymous substitution rates (> 25 nonsynonymous substitutions per site per 10(9) years). cDNA encoding the mature protein (89-100 residues) shows evidence of positive selection (Dn/Ds > 1) for 4 of 10 pairwise species comparisons. cDNA and predicted secondary-structure comparisons suggest that TMAP is neither orthologous nor paralogous to abalone lysin, and thus marks a second, phylogenetically independent, protein subject to strong positive selection in free-spawning marine gastropods. In addition, an internal repeat in one species (Tegula aureotincta) produces a duplicated cleavage site which results in two alternatively processed mature proteins differing by nine amino acid residues. Such alternative processing may provide a mechanism for introducing novel amino acid sequence variation at the amino-termini of proteins. Highly divergent TMAP N-termini from two other tegulines (Tegula regina and Norrisia norrisii) may have originated by such a mechanism.

Acrosome↗
PubMed2000

1.35 and 2.07 A resolution structures of the red abalone sperm lysin monomer and dimer reveal features involved in receptor binding.

Abalone sperm use lysin to make a hole in the egg's protective vitelline envelope (VE). When released from sperm, lysin first binds to the VE receptor for lysin (VERL) then dissolves the VE by a non-enzymatic mechanism. The structures of the monomeric and dimeric forms of Haliotis rufescens (red abalone) lysin, previously solved at 1.90 and 2.75 A, respectively, have now been refined to 1.35 and 2.07 A, respectively. The monomeric form of lysin was refined using previously obtained crystallization conditions, while the dimer was solved in a new crystal form with four molecules (two dimers) per asymmetric unit. These high-resolution structures reveal alternate residue conformations, enabling a thorough analysis of the conserved residues contributing to the amphipathic nature of lysin. The availability of five independent high-resolution copies of lysin permits comparisons leading to insights on the local flexibility of lysin and alternative conformations of the hypervariable N-terminus, thought to be involved in species-specific receptor recognition. The new analysis led to the discovery of the basic nature of a cleft formed upon dimerization and a patch of basic residues in the dimer interface. Identification of these features was not possible at lower resolution. In light of this new information, a model explaining the binding of sperm lysin to egg VERL and the subsequent dissolution of the egg VE is proposed.

Animals↗
PubMed1999

Interspecies chimeric sperm lysins identify regions mediating species-specific recognition of the abalone egg vitelline envelope.

Abalone sperm lysin is a nonenzymatic, 16-kDa protein that creates a hole in the egg vitelline envelope (VE) through which the sperm swims to fuse with the egg. The dissolution of isolated VE by lysin is species specific. Interspecies comparisons show that the most divergent region of lysin is the N-terminal segment of residues 1-12 which is always species-unique. The C-terminus and three internal segments are moderately variable between species, but not species unique. Analysis of nucleotide substitutions shows that lysin evolves rapidly by positive Darwinian selection, suggesting that there is adaptive value in altering its amino acid sequence. The results reported here, in which segments of lysin were exchanged between two species, prove by direct experimentation that the interspecies variable termini play major roles in the species-specific recognition between sperm lysin and the egg VE.

Acrosome↗
PubMed1999

Structure of the sulfated alpha-L-fucan from the egg jelly coat of the sea urchin Strongylocentrotus franciscanus: patterns of preferential 2-O- and 4-O-sulfation determine sperm cell recognition.

The egg jelly coats of sea urchins contains sulfated polysaccharides responsible for inducing the sperm acrosome reaction which is an obligatory event for sperm binding to, and fusion with, the egg. Here, we extend our study to the sea urchin Strongylocentrotus franciscanus. The egg jelly of this species contains a homofucan composed of 2- O -sulfated, 3-linked units which is the simplest structure ever reported for a sulfated fucan. This polysaccharide was compared with other sulfated alpha-L-fucans as inducers of acrosome reaction in conspecific and heterospecific sperm. Although all these fucans are linear polymers composed of 3-linked alpha-L-fucopyranosyl units, they differ in the proportions of 2-O- and 4-O-sulfation. The reactivity of the sperm of each species is more sensitive to the egg jelly sulfated fucan found in their own species. The reactivity of the sperm does not correlate with the charge density of the fucan, but with the proportion of 2-O- and 4-O-sulfation. The pattern of sulfation may be an important feature for recognition of fucans by the sperm receptor contributing to the species-specificity of fertilization.

Acrosome Reaction↗
PubMed1999

Rapid evolution of fertilization selectivity and lysin cDNA sequences in teguline gastropods.

Proteins mediating intercellular recognition face opposing selective forces as they evolve: purifying selection to maintain function, and diversifying selection to alter specificity. Lysin is a 16-kDa protein which enables sperm of free-spawning marine snails to make a hole in the vitelline layer (VE) surrounding conspecific eggs. Previous work on abalone (Haliotis spp.) has shown that positive selection promotes rapid interspecific divergence of lysin. Here, we present data on the specificity of VE dissolution by four species of teguline gastropods, along with lysin cDNA sequences. The teguline and abalone lineages diverged over 250 MYA. As in abalone, VE dissolution by lysin in tegulines is species-selective, and positive selection promotes rapid interspecific divergence over the entire mature protein. Nonsynonymous substitution rates, calculated using a mtCOI molecular clock calibrated by two Tegula species separated by the Isthmus of Panama, are high (> 25 substitutions per site per 10(9) years). However, the extensive replacements in teguline lysins are overwhelmingly conservative with respect to type, charge, and polarity of residues. Predictions of secondary structure suggest that the size and position of alpha-helices are also conserved, even through pairwise amino acid identities between Haliotis rufescens and the different tegulines are less than 15%.

Amino Acid Sequence↗
PubMed1998

Evolution of gamete recognition proteins.

REVIEW Although fertilization has been studied for more than a century, the cell surface proteins mediating the process are only now becoming known. Gamete interaction in animals appears to be molecularly complex. Although it is difficult to generalize at present, diversity of structure may be a recurring theme in the evolution of fertilization proteins. Examples of rapid evolution of fertilization proteins by positive selection are known, and concerted evolution can influence the differentiation of gamete recognition proteins between closely related species.

Animals↗
PubMed1998

Increased association of synaptosome-associated protein of 25 kDa with syntaxin and vesicle-associated membrane protein following acrosomal exocytosis of sea urchin sperm.

Synaptosomal-associated protein of 25 kDa (SNAP-25) is a palmitoylated integral membrane protein expressed almost exclusively in neuronal and neuroendocrine tissues. This protein forms a ternary complex with vesicle-associated membrane protein (VAMP) and syntaxin, which is thought to regulate the fusion of plasma and vesicle membranes during exocytosis. We report the identification of SNAP-25 expressed in sea urchin sperm. Sea urchin SNAP-25 shares greater identity with mammalian SNAP-25 than with mammalian SNAP-23, a ubiquitously expressed homologue believed to regulate membrane fusion in non-neuronal tissues. Sea urchin sperm contain a single exocytotic vesicle, the acrosomal vesicle, whose contents are exposed during the acrosome reaction. Fusion of the plasma membrane with the acrosomal vesicle membrane at multiple points (vesiculation) results in the release of SNAP-25 with the shed acrosome reaction vesicles. A complex containing SNAP-25, syntaxin, and VAMP is present in sperm, as detected by affinity chromatography and immunoprecipitation. Although this complex is present prior to the acrosome reaction, the amount of complex increases over 4-fold following acrosomal exocytosis. These findings support the involvement of SNAP-25 in the invertebrate sperm acrosome reaction, possibly through increased association with VAMP and syntaxin driving the fusion of plasma and acrosomal membranes.

Acrosome↗
PubMed1998

Nonsynonymous substitution in abalone sperm fertilization genes exceeds substitution in introns and mitochondrial DNA.

Strong positive Darwinian selection acts on two sperm fertilization proteins, lysin and 18-kDa protein, from abalone (Haliotis). To understand the phylogenetic context for this dramatic molecular evolution, we obtained sequences of mitochondrial cytochrome c oxidase subunit I (mtCOI), and genomic sequences of lysin, 18-kDa, and a G protein subunit. Based on mtDNA differentiation, four north Pacific abalone species diverged within the past 2 million years (Myr), and remaining north Pacific species diverged over a period of 4-20 Myr. Between-species nonsynonymous differences in lysin and 18-kDa exons exceed nucleotide differences in introns by 3.5- to 24-fold. Remarkably, in some comparisons nonsynonymous substitutions in lysin and 18-kDa genes exceed synonymous substitutions in mtCOI. Lysin and 18-kDa intron/exon segments were sequenced from multiple red abalone individuals collected over a 1,200-km range. Only two nucleotide changes and two sites of slippage variation were detected in a total of >29,000 nucleotides surveyed. However, polymorphism in mtCOI and a G protein intron was found in this species. This finding suggests that positive selection swept one lysin allele and one 18-kDa allele to fixation. Similarities between mtCOI and lysin gene trees indicate that rapid adaptive evolution of lysin has occurred consistently through the history of the group. Comparisons with mtCOI molecular clock calibrations suggest that nonsynonymous substitutions accumulate 2-50 times faster in lysin and 18-kDa genes than in rapidly evolving mammalian genes.

Animals↗
PubMed1998

Concerted evolution in an egg receptor for a rapidly evolving abalone sperm protein.

Gamete interactions during fertilization exhibit species specificity. In abalone, the sperm protein lysin species-specifically creates a hole in the egg envelope. Lysin evolves rapidly by positive Darwinian selection. Evolution of the egg receptor for lysin provides the selective pressure for lysin's divergence. The egg receptor for lysin is a tandemly repeated sequence that evolves by concerted evolution. Concerted evolution in the egg receptor could explain the rapid, adaptive evolution in sperm lysin and may provide an underlying molecular mechanism that gives rise to species-specific fertilization.

Amino Acid Sequence↗