Biomedical subjects

V D Vacquier

Publications and source records attributed to V D Vacquier.

At least 37 records · Page 2Linked to original sources

PubMed1998

Females of the sea urchin Strongylocentrotus purpuratus differ in the structures of their egg jelly sulfated fucans.

The egg jelly coats of sea urchins contain sulfated fucans which bind to a sperm surface receptor glycoprotein to initiate the signal transduction events resulting in the sperm acrosome reaction. The acrosome reaction is an ion channel regulated exocytosis which is an obligatory event for sperm binding to, and fusion with, the egg. Approximately 90% of individual females of the sea urchin Strongylocentrotus purpuratus spawned eggs having only one of two possible sulfated fucan electrophoretic isotypes, a slow migrating (sulfated fucan I), or a fast migrating (sulfated fucan II) isotype. The remaining 10% of females spawned eggs having both sulfated fucan isotypes. The two sulfated fucan isotypes were purified from egg jelly coats and their structures determined by NMR spectroscopy and methylation analysis. Both sulfated fucans are linear polysaccharides composed of 1-->3-linked alpha-L-fucopyranosyl units. Sulfated fucan I is entirely sulfated at the O -2 position but with a heterogeneous sulfation pattern at O -4 position. Sulfated fucan II is composed of a regular repeating sequence of 3 residues, as follows: [3-alpha-L-Fuc p -2,4(OSO3)-1-->3-alpha-L-Fuc p -4(OSO3)-1-->3-alpha-L-Fuc p -4(OSO3)-1]n. Both purified sulfated fucans have approximately equal potency in inducing the sperm acrosome reaction. The significance of two structurally different sulfated fucans in the egg jelly coat of this species could relate to the finding that the sperm receptor protein which binds sulfated fucan contains two carbohydrate recognition modules of the C-type lectin variety which differ by 50% in their primary structure.

Acrosome↗

PubMed1998

Mitochondrial DNA and bindin gene sequence evolution among allopatric species of the sea urchin genus Arbacia.

Sea urchins of the genus Arbacia (order Stirodonta) have discontinuous allopatric distributions ranging over thousands of kilometers. Mitochondrial DNA (mtDNA) sequences were used to reconstruct phylogenetic relationships of four Arbacia species and their geographic populations. There is little evidence of genetic structuring of populations within species, except in two cases at range extremes. The mtDNA sequence differentiation between species suggests that divergence occurred about 4-9 MYA. Gene sequences encoding the sperm protein bindin and its intron were obtained and compared with the mtDNA phylogeny. Sea urchins among the well-studied echinoid order Camarodonta, with degrees of mtDNA divergence similar to those of Arbacia species, are known to have remarkable variation in bindin. However, in Arbacia, little variation in deduced amino acid sequences of bindin was found, indicating that purifying selection acts on the protein. In contrast, bindin intron sequences showed much differentiation, including numerous insertion/deletions. Fertilization experiments performed between a divergent pair of Arbacia species from the Atlantic and Pacific Oceans revealed no evidence of blocks to gamete recognition. In Arbacia, fertilization specificities may have evolved relatively slowly as a result of extensive gene flow within species, greater functional constraint on the bindin polypeptide, or reduced selective pressure for species recognition in singly occurring species.

Amino Acid Sequence↗

PubMed1998

Direct sequencing of genomic DNA for characterization of a satellite DNA in five species of eastern Pacific abalone.

A tandemly repeated satellite DNA of 290-291 base pairs (bp) was identified by SalI digestion of genomic DNA of five species of Eastern Pacific (California) abalone (genus, Haliotis). Following cloning and sequencing of one repeat unit from one species, the consensus sequences of this satellite were determined for five species by directly sequencing genomic DNA using satellite-specific primers. Phylogenetic trees of the consensus satellite sequences had the same topology as trees constructed for two abalone sperm acrosomal proteins. In 12 randomly picked clones of the Red abalone (H. rufescens) SalI satellite, 16 positions varied, the variation being spread throughout the sequence. GenBank database searches found no significant similarities between this satellite and known sequences. Southern analysis showed that all 290-bp SalI repeats were excised from genomic DNA by Sau3A1 digestion. The tandem arrangement of satellite repeats was confirmed by sequencing through the SalI site into the next repeat using genomic DNA as template, time-dependent appearance of DNA ladders with an approximate 300-bp spacing in SalI digests of genomic DNA, and ladders of bands with an approximate 300-bp spacing generated by polymerase chain reaction (PCR) using genomic DNA as template. In the Red abalone, the 290-bp SalI satellite represents approximately 0.5% of total DNA, equivalent to approximately 28,000 copies per haploid genome. The species-specific consensus sequence of this satellite, obtained directly using genomic DNA as the sequencing template, provides a molecular marker that could be used for identification of hybrid parentage, taxonomy, population identification, and forensic studies.

Animals↗

PubMed1997

The fucose sulfate polymer of egg jelly binds to sperm REJ and is the inducer of the sea urchin sperm acrosome reaction.

The sea urchin sperm cell is an advantageous model for studying ligand-mediated exocytosis. Sperm can be obtained in vast quantities and induced to undergo exocytosis of the acrosomal vesicle with great synchrony. During sea urchin fertilization, egg jelly (EJ) triggers the sperm acrosome reaction (AR) which is required for sperm binding and fusion with the egg. Uncertainty exists as to the exact biochemical nature of the AR inducer. The following study was performed in an attempt to clarify the nature of the inducer. EJ from individual females (Strongylocentrotus purpuratus) was analyzed on SDS-PAGE gels. Each female had a unique composition of EJ macromolecules, but all females possessed the previously described fucose sulfate polymer (FSP). Two electrophoretic isotypes of FSP were discovered; 87% of females had only one isotype and 13% had both. Both FSP isotypes bound to the REJ protein (receptor for egg jelly) purified from sperm. The two FSP isotypes had almost equal potency in inducing the AR. EJ was fractionated by DEAE chromatography in 6 M urea/4% beta-mercaptoethanol. All AR-inducing activity coeluted with FSP. FSP, purified by trypsin digestion followed by dialysis, was twice as active as the non-trypsin-digested control at inducing the AR. EJ was digested with proteinase K, boiled in detergent and beta-mercaptoethanol, and subjected to sucrose density gradient sedimentation. The FSP and AR activity had superimposable sedimentation patterns. Purified FSP had no associated peptide component. Sperm from individual males differed in the concentration dependency of purified FSP to induce the AR. The data indicate that the 138/82 kDa EJ glycoproteins, previously thought to act as AR inducers, do not appear to be involved in triggering the AR. The data are consistent with the hypothesis that FSP is the only inducer of the AR of this sea urchin species.

Acrosome↗

PubMed1997

The exocytosis regulatory proteins syntaxin and VAMP are shed from sea urchin sperm during the acrosome reaction.

Syntaxin is a cytoplasmically oriented plasma membrane protein and VAMP (vesicle-associated membrane protein; synaptobrevin) is a protein associated with the secretory vesicle membrane. These two proteins form part of a complex which is thought to mediate the fusion of plasma and vesicle membranes during exocytosis. This paper reports the identification of syntaxin and VAMP homologues in sea urchin sperm. During fertilization, sea urchin sperm release the contents of a single vesicle, the acrosomal vesicle, exposing the membrane destined to fuse with the egg. During acrosomal exocytosis, the plasma membrane over the acrosomal vesicle fuses at multiple points with the acrosomal membrane (vesiculation) and syntaxin and VAMP are shed with the resulting membrane vesicles. Sea urchin sperm syntaxin and VAMP are associated in a complex as detected by immunoprecipitation. Following acrosomal exocytosis, syntaxin and VAMP cosediment to denser fractions on sucrose gradients showing that they have undergone associative changes during or after the acrosome reaction. Syntaxin and VAMP localization and loss during acrosomal exocytosis support a role for these proteins in regulating the acrosome reaction.

Acrosome↗

PubMed1997

The abalone egg vitelline envelope receptor for sperm lysin is a giant multivalent molecule.

Abalone sperm lysin is a 16-kDa acrosomal protein, which nonenzymatically and species selectively creates a hole in the egg vitelline envelope (VE) through which the sperm passes to reach the egg cell membrane. The crystal structures of both monomeric and dimeric lysins have been solved and the sequences of lysins from 20 abalone species have been determined. As a first step in understanding the molecular mechanism by which lysin creates a hole in the VE, its VE receptor was isolated. The VE receptor for lysin (VERL) is an unbranched, rod-like molecule with an approximate relative molecular mass of 2 million; half the mass being carbohydrate. Fluorescence polarization studies showed positive cooperativity in the binding of lysin to VERL (EC50 approximately 9 nM) and were consistent with the species selectivity of lysin in dissolving VEs. Each molecule of VERL bound between 126 and 142 molecules of monomeric lysin (two independent assays), showing that VERL possesses repetitive lysin-binding motifs.

Animals↗

PubMed1997

Positive Darwinian selection on two homologous fertilization proteins: what is the selective pressure driving their divergence?

Most examples of positive selection inferred from nucleotide sequence data involve hostpathogen interactions. However, positive selection also promotes the divergence of proteins mediating sperm-egg recognition in marine invertebrates. The abalone spermatozoon has a large acrosomal vesicle containing two proteins of 16 kDa and 18 kDa. Lysin, the 16-kDa protein, exhibits species-specificity in dissolving a hole in the egg vitelline envelope through which the sperm swims to reach the egg plasma membrane. The 18-kDa protein coats the sperm acrosomal process and probably mediates fusion of the two gametes. In this review, we compare sequences of both proteins from five species of California abalones. Both proteins show extensive divergence which has been promoted by positive Darwinian selection. The ratios of nonsynonymous to synonymous nucleotide substitutions may be the highest yet discovered for full-length sequences. Although extensive divergence has occurred, there is conservation of the shape and polarity of residues in both proteins. The two acrosomal proteins arose by a gene duplication followed by their extensive divergence. Five hypotheses are presented which attempt to explain the nature of the unknown selective force responsible for the robust positive selection. The positive selection may, in some unknown way, be related to the establishment of prezygotic barriers to reproduction. Because positive selection promotes the divergence of unrelated, species-specific gamete recognition proteins in both abalones and sea urchins, we predict that positive selection may be a general phenomenon in the evolution of gamete recognition systems in marine invertebrates.

Amino Acid Sequence↗

PubMed1996

The sea urchin sperm receptor for egg jelly is a modular protein with extensive homology to the human polycystic kidney disease protein, PKD1.

During fertilization, the sea urchin sperm acrosome reaction (AR), an ion channel-regulated event, is triggered by glycoproteins in egg jelly (EJ). A 210-kD sperm membrane glycoprotein is the receptor for EJ (REJ). This conclusion is based on the following data: purified REJ binds species specifically to EJ dotted onto nitrocellulose, an mAb to REJ induces the sperm AR, antibody induction is blocked by purified REJ, and purified REJ absorbs the AR-inducing activity of EJ. Overlapping fragments of REJ cDNA were cloned (total length, 5,596 bp). The sequence was confirmed by microsequencing six peptides of mature REJ and by Western blotting with antibody to a synthetic peptide designed from the sequence. Complete deglycosylation of REJ followed by Western blotting yielded a size estimate in agreement with that of the mature amino acid sequence. REJ is modular in design; it contains one EGF module and two C-type lectin carbohydrate-recognition modules. Most importantly, it contains a novel module, herein named the REJ module (700 residues), which shares extensive homology with the human polycystic kidney disease protein (PKD1). Mutations in PKD1 cause autosomal dominant polycystic kidney disease, one of the most frequent genetic disease of humans. The lesion in cellular physiology resulting from mutations in the PKD1 protein remains unknown. The homology between REJ modules of the sea urchin REJ and human PKD1 suggests that PKD1 could be involved in ionic regulation.

Amino Acid Sequence↗

PubMed1995

Liposome fusion induced by a M(r) 18,000 protein localized to the acrosomal region of acrosome-reacted abalone spermatozoa.

A M(r) 18,000 protein is secreted by abalone spermatozoa during the acrosome reaction. Immunofluorescence of acrosome-reacted sperm localizes the protein as a coating on the spent acrosomal granule hull and on the surface of the acrosomal process. The membrane of the acrosomal process fuses with the egg plasma membrane at fertilization. The M(r) 18,000 acrosomal protein aggregates negatively charged (but not neutral) large unilamellar liposomes and renders them permeable to internal probe. The M(r) 18,000 proteins from two abalone species are potent inducers of intervesicular lipid mixing in the resonance energy transfer assay, suggesting that they mediate the fusion of lipid bilayers. Predicted secondary structures of these proteins show the presence of strongly amphipathic alpha-helices that may be active in the perturbation of phospholipid bilayers. The M(r) 18,000 protein may mediate sperm-egg fusion during fertilization.

Acrosome↗

PubMed1995

Extraordinary divergence and positive Darwinian selection in a fusagenic protein coating the acrosomal process of abalone spermatozoa.

During fertilization in marine invertebrates, fusion between sperm and egg cell membranes occurs at the tip of the sperm acrosomal process. In abalone sperm the acrosomal process is coated with an 18-kDa protein. In situ, this protein has no effect on the egg vitelline envelope, but in vitro it is a potent fusagen of liposomes. Thus, the 18-kDa protein may mediate membrane fusion between the gametes, a step in gamete recognition known to restrict heterospecific fertilization in other species. The cDNA and deduced amino acid sequences of the 18-kDa protein were determined for five species of California abalone. The deduced amino acid sequences exhibit extraordinary divergence; the percent identity varies from 27% to 87%. Analysis of nucleotide substitution shows extremely high frequencies of amino acid-altering substitution compared to silent substitution, demonstrating that positive Darwinian selection promotes the divergence of this protein. However, amino acid replacement is conservative with respect to size and polarity of residue. The data support the developing idea that in free-spawning marine invertebrates, the proteins mediating fertilization may be subjected to intense, and as yet unknown, selective forces. The extraordinary divergence of fertilization proteins may be related to the establishment of barriers to heterospecific fertilization.

Acrosome↗

PubMed1995

Structural features of the abalone egg extracellular matrix and its role in gamete interaction during fertilization.

Abalone eggs are surrounded by a complex extracellular coat that contains three distinct elements: the jelly layer, the vitelline envelope, and the egg surface coat. In this study we used light and electron microscopy to describe these three elements in the red abalone (Haliotis rufescens) and ascribe function to each based on their interactions with sperm. The jelly coat is a spongy matrix that lies at the outermost margin of the egg and consists of variably sized fibers. Sperm pass through this layer with their acrosomes intact and then go on to bind to the vitelline envelope. The vitelline envelope is a multilamellar fibrous layer that appears to trigger the acrosome reaction after the sperm binding. Next, sperm release lysin from their acrosomal granules, a nonenzymatic protein that dissolves a hole in the vitelline envelope through which the sperm swims. Sperm then contact the egg surface coat, a network of uniformly sized filaments lying directly above the egg plasma membrane. This layer mediates attachment of sperm, via their acrosomal process, to the egg surface.

Animals↗

PubMed1995

Crystal structure and subunit dynamics of the abalone sperm lysin dimer: egg envelopes dissociate dimers, the monomer is the active species.

Lysin is a 16-kD acrosomal protein used by abalone spermatozoa to create a hole in the egg vitelline envelope (VE) by a nonenzymatic mechanism. The crystal structure of the lysin monomer is known at 1.9 A resolution. The surface of the molecule reveals two tracks of basic residues running the length of one surface of the molecule and a patch of solvent-exposed hydrophobic residues on the opposite surface. Here we report that lysin dimerizes via interaction of the hydrophobic patches of monomers. Triton X-100 dissociates the dimer. The crystal structure of the dimer is described at 2.75 A resolution. Fluorescence energy transfer experiments show that the dimer has an approximate KD of 1 microM and that monomers exchange rapidly between dimers. Addition of isolated egg VE dissociates dimers, implicating monomers as the active species in the dissolution reaction. This work represents the first step in the elucidation of the mechanism by which lysin enables abalone spermatozoa to create a hole in the egg envelope during fertilization.

Animals↗

PubMed1995

Positive selection is a general phenomenon in the evolution of abalone sperm lysin.

Lysin is a 16kDa acrosomal protein used by abalone sperm to create a hole in the egg vitelline envelope (VE). The interaction of lysin with the VE is species-selective and is one step in the multistep fertilization process that restricts heterospecific (cross-species) fertilization. For this reason, the evolution of lysin could play a role in establishing prezygotic reproductive isolation between species. Previously, we sequenced sperm lysin cDNAs from seven California abalone species and showed that positive Darwinian selection promotes their divergence. In this paper an additional 13 lysin sequences are presented representing species from Japan, Taiwan, Australia, New Zealand, South Africa, and Europe. The total of 20 sequences represents the most extensive analysis of a fertilization protein to date. The phylogenetic analysis divides the sequences into two major clades, one composed of species from the northern Pacific (California and Japan) and the other composed of species from other parts of the world. Analysis of nucleotide substitution demonstrates that positive selection is a general process in the evolution of this fertilization protein. Analysis of nucleotide and codon usage bias shows that neither parameter can account for the robust data supporting positive selection. The selection pressure responsible for the positive selection on lysin remains unknown.

Amino Acid Sequence↗

PubMed1994

Acid pH crystallization of the basic protein lysin from the spermatozoa of red abalone (Haliotis rufescens).

A new crystal form of dimeric red lysin, a distinctly basic protein (M(r) = 16 070) from the red abalone (Haliotis rufescens), has been obtained using ammonium sulfate as precipitant with a sodium citrate-boric acid-citric acid buffer at pH 4.5. The acid pH crystal form resulted from a study aimed at developing conditions favorable to the sitting-drop vapor-diffusion crystallization of other abalone lysins which do not crystallize at neutral or basic pH conditions. The space group is P222(1) with cell dimensions a = 51.2, b = 47.0, c = 123.8 A and two molecules per asymmetric unit.

Journal Article↗

PubMed1994

The isolation of acrosome-reaction-inducing glycoproteins from sea urchin egg jelly.

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

Acrosome↗

PubMed1994

The sea urchin egg jelly coat consists of globular glycoproteins bound to a fibrous fucan superstructure.

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

Animals↗

PubMed1994

A unique expression pattern for a sperm membrane protein during sea urchin spermatogenesis.

Specific mRNAs coding for a 63 kDa sperm membrane protein (63-SMP) were localised in Strongylocentrotus purpuratus testis sections using in situ hybridisation techniques. 35S-labelled antisense RNA probes transcribed from a 766 base pair fragment of the gene coding for the 63-SMP hybridised to all spermatogenic cells in the basal germinal epithelia of testicular acini, except the most peripherally located (least differentiated) spermatogonia. No hybridisation to the luminally located mature spermatozoa or somatic cells of the testis was observed. Using monoclonal antibody J17/30 and indirect immunofluorescence techniques, the 63-SMP was localised to the same subset of spermatogenic cells that contain the 63-SMP mRNA, suggesting that expression of this gene is transcriptionally controlled. In combination with previous studies on the expression of sperm histones and sperm binding, these results show that multiple, perhaps sequential, classes of gene activity contribute to the differentiation of sea urchin sperm.

Animals↗

PubMed1993

The crystal structure of lysin, a fertilization protein.

Lysin, a protein from abalone sperm, creates a hole in the envelope of the egg, permitting the sperm to pass through the envelope and fuse with the egg. The structure of lysin, refined at 1.9 angstroms resolution, reveals an alpha-helical, amphipathic molecule. The surface of the protein exhibits three features: two tracks of basic residues that span the length of the molecule, a solvent-exposed cluster of aromatic and aliphatic amino acids, and an extended amino-terminal hypervariable domain that is species-specific. The structure suggests possible mechanisms of action.

Amino Acid Sequence↗