The oligomeric integrity of toposome is essential for its morphogenetic function

Sea urchin embryos are uniquely suitable for the study of morphogenetic cell interactions. Efforts to identify the molecules responsible for morphogenetic cell adhesion led to the isolation of a 22S glycoprotein complex from Paracentrotus lividus sea urchin embryo, that has been called toposome. The biological activity of toposome in mediating cellular adhesion has been fully documented. Its function in determining positional guidance during the development of the sea urchin embryo has been proposed. Here studies on the molecular structure of toposome are reported showing that, under non-reducing conditions, it is resolved in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in a major band with an apparent molecular weight of 260 kDa, a doublet of 180-160 kDa and a lower band of 80 kDa. Digestion with EndoH endoglycosidase reduced the molecular sizes of the bands of 10%, 20% and 40%, respectively. In order to establish if the oligomeric integrity of toposome was essential for its function, the biological activity of each subunit on cells dissociated from sea urchin blastula embryos was tested. The resulting swimming embryoids were lacking skeleton, while reaggregating cells supplemented with native toposome developed into pluteus-like structures with skeletal elements.     

Asymmetric inhibition of spicule formation in sea urchin embryos with low concentrations of gadolinium ion

As gastrulation proceeds during sea urchin embryogenesis, primary mesenchyme cells (PMCs) fuse to form syncytial cables, within which calcium is deposited as CaCO₃, and a pair of spicules is formed. Earlier studies suggested that calcium, previously sequestered by primary mesenchyme cells, is secreted and incorporated into growing spicules. We examined the effects of gadolinium ion (Gd³⁺), a Ca²⁺ channel blocker, on spicule formation. Gd³⁺ did not lead to a retardation of embryogenesis prior to the initiation of gastrulation and did not inhibit the ingression of PMCs from the blastula wall or their migration along the inner blastocoel surface. However, when embryos were raised in seawater containing submicromolar to a few micromolar Gd³⁺, of which levels are considered to be insufficient to block Ca²⁺ channels, a pair of triradiate spicules was formed asymmetrically. At 1–3 μmol/L Gd³⁺, many embryos formed only one spicule on either the left or right side, or embryos formed a very small second spicule. Induction of the spicule abnormality required the presence of Gd³⁺ specifically during late blastula stage prior to spicule formation. An accumulation or adsorption of Gd³⁺ was not detected anywhere in the embryos by X‐ray microanalysis, which suggests that Ca²⁺ channels were not inhibited. These results suggest that Gd³⁺ exerts an inhibitory effect on spicule formation through a mechanism that does not involve inhibition of Ca²⁺ channels.     

Evidence of a precursor-product relationship between vitellogenin and toposome,a glycoprotein complex mediating cell adhesion

Toposome, a large and oligomeric glycoprotein complex isolated from mesenchyme-blastula embryos, was defined as a cell-adhesion molecule expressing positional information specificities during sea urchin embryogenesis. This report describes the biochemical and functional characterization of the toposome precursor from sea urchin coelomic fluids of both male and female organisms. The molecule is isolated in the form of a 22S particle which has an apparent molecular mass of 200 kDa. An intermediate form is present in yolk granules of unfertilized eggs with a molecular mass of 180 kDa. The 200 kDa and 180 kDa polypeptides are defined as toposome precursors by Western blot and immunoprecipitation analyses using polyclonal and monoclonal toposome-specific antibodies. Comparison of the 200 kDa polypeptide and mesenchyme-blastula toposome by partial-proteolysis peptide-mapping shows that they are related in a precursor-product relationship. A morphogenetic cell-aggregation assay shows that toposome precursors promote cell adhesion of dissociated blastula cells, suggesting that processing is not required for the cell-adhesion function. The studies reported here present the first evidence that cell adhesion molecules first appear in the form of a 200 kDa polypeptide, previously named vitellogenin, and to which only a function as major-yolk-protein precursor has been ascribed.     

Studies on the Cellular Pathway Involved in Assembly of the Embryonic Sea Urchin Spicule

Micromeres from the 16-cell stage sea urchin embryo were isolated and cultured in vitro in seawater containing 3% horse serum. Under these conditions these cells differentiate into spicule-forming, primary mesenchyme cells. To obtain insight into the route traveled by Ca²⁺ to form the pseudocrystalline spicule composed of CaCO₃ and matrix proteins, studies with various inhibitors were undertaken. Experiments with members of several different classes of Ca²⁺ channel blockers established that the Ca²⁺ utilized for spiculogenesis must be taken up by the cells. Moreover, studies using two agents that disrupt the endomembrane system, monensin and brefeldin A, showed that both blocked spicule formation. Based on these experiments, we conclude that extracellular Ca²⁺ must enter the primary mesenchyme cells prior to being deposited extracellularly as CaCO₃ and that this ion and/or the matrix proteins found in the spicule are routed through the secretory pathway that has been established to exist in a wide variety of other cell types.     

High pH-induced acrosome reaction and Ca uptake in sea urchin sperm suspended in Na-free seawater

The egg jelly-induced acrosome reaction of sea urchin sperm requires the presence of Ca²⁺ and Na⁺ in seawater at its normal pH 8. Sperm suspended in seawater at pH 9 undergo the acrosome reaction in the absence of jelly. We have attempted to understand the role of external Na⁺ in this reaction. Sperm were suspended in Na⁺-free seawater and the percentage of acrosome reaction and the amount of Ca²⁺ uptake were determined as a function of external pH. High pH (9.0) in Na⁺-free medium without jelly triggered a high percentage (above 65%) of sperm acrosome reactions and a two to fourfold increase in Ca²⁺ uptake. Both the percentage of acrosome reactions and the amount of Ca²⁺ uptake were similar to those induced by either jelly or pH 9 in Na⁺-containing seawater. On the other hand, the absence of Na⁺ in seawater inhibits jelly from inducing Ca²⁺ uptake and acrosome reactions at pH 8.0 and even at pH 8.5. These results indicate that the Na⁺ requirement for the acrosome reaction induced by jelly is lost when triggering is by high pH. In contrast, Ca²⁺ was strictly required since sperm did not react in Ca²⁺-free seawater at pH 9. We also found that like the jelly-induced acrosome reaction the high-pH-induced acrosome reaction and Ca²⁺ uptake in complete and Na⁺-free seawater were inhibited by D600. This finding suggests that the same transport system for Ca²⁺ uptake associated with the acrosome reaction operates at both triggering conditions, i.e., jelly or pH 9. Although D600 is not now considered a specific blocker, its effect has suggested the involvement of Ca²⁺ channels in the acrosome reaction. This proposal is supported by our results with nisoldipine, a highly specific inhibitor of calcium channels. The drug inhibited both the sperm acrosome reaction and Ca²⁺ uptake induced by jelly or pH 9 in complete seawater.     

Sea urchin fertilization envelope: Uncoupling of cortical granule exocytosis from envelope assembly and isolation of an envelope intermediate from Strongylocentrotus purpuratus embryos

The sea urchin fertilization envelope (FE) is an extraembryonic coat which develops from the egg vitelline envelope (VE) and the secreted paracrystalline protein fraction of the cortical granules at fertilization. The FE undergoes further developmental changes postinsemination which are characterized by changes in envelope permeability, solubility in reducing and denaturing solvents, and morphology. We have developed a procedure to uncouple cortical granule exocytosis from assembly of the paracrystalline protein fraction onto the VE template. Egg suspensions were inseminated in normal seawater and diluted into Ca²⁺- and Mg²⁺-free seawater at 15 sec postinsemination. Phase-contrast and electron microscopic observations showed that the embryos formed a normally elevated, extremely thin envelope through which the cortical granule exudate permeated. Secretion studies showed that eggs which were diluted into divalent ion-free seawater postinsemination secreted as much protein into the surrounding seawater as eggs which had their VEs removed prior to the experiment. We have termed the envelope elevated in divalent ion-free seawater the VE1 and we believe that it is the VE structural component of the FE based on its thickness and morphology. VE1s were isolated by gentle physical means and the preparations appeared to be greater than 80% pure based on radioactive mixing experiments and on malate dehydrogenase and glucose-6-phosphate dehydrogenase marker studies. VE1s were at least 80% soluble based on extraction of radioiodinated preparations with reducing and denaturing solvents. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of VE1s showed eight major polypeptides which ranged from 30,500 to 270,000 in molecular weight.     

Cleavage stage mouse embryos share a common cell adhesion system with teratocarcinoma cells

We examined similarities in adhesive properties of mouse cleaving embryos at one- to eight-cell stages and of teratocarcinoma cells by aggregation studies. Teratocarcinoma cells and fibroblastic cells have a Ca²⁺-dependent cell-cell adhesion site (CDS), which is resistant to trypsin in the presence of Ca²⁺ but sensitive in the absence of Ca²⁺. When several embryos treated with trypsin in the presence of Ca²⁺ (TC) were kept in contact with each other, they fused into a single aggregate in the medium with Ca²⁺ but not without Ca²⁺. Embryos treated with trypsin in the absence of Ca²⁺ (TE) did not show such Ca²⁺-dependent aggregation. Aggregation of TC-treated embryos was inhibited by Fab fragments of antibody raised against TC-treated teratocarcinoma F9 cells. The aggregation-inhibitory effect of the Fab was removed by absorption with TC-treated teratocarcinoma cells, but not with TE-treated teratocarcinoma cells. This effect was not removed by absorption with fibroblasts and some other tissue cells. TC-treated embryos adhered to TC-treated teratocarcinoma cells, but not to TC-treated fibroblastic cells. These results suggest that early mouse embryos share a common CDS molecule with teratocarcinoma cells but not with fibroblastic cells.     

Purification and characteristics of Ca2+,Mg2+- and Ca2+,Mn2+-dependent and acid DNases from spermatozoa of the sea urchin Strongylocentrotus intermedius

Ca²⁺,Mg²⁺- and Ca²⁺,Mn²⁺-dependent and acid DNases were isolated from spermatozoa of the sea urchin Strongylocentrotus intermedius. The enzymes have been purified by successive chromatography on DEAE-cellulose, phenyl-Sepharose, Source 15Q, and by gel filtration, and the principal physicochemical and enzymatic properties of the purified enzymes were determined. Ca²⁺,Mg²⁺-dependent DNase (Ca,Mg-DNase) is a nuclear protein with molecular mass of 63 kD as the native form and its activity optimum is at pH 7.5. The enzyme activity in the presence of bivalent metal ions decreases in the series (Ca²⁺ + Mg²⁺) > Mn²⁺ = (Ca²⁺ + Mn²⁺) > (Mg²⁺ + EGTA) > Ca²⁺. Ca,Mg-DNase retains its maximal activity in sea water and is not inhibited by G-actin and N-ethylmaleimide, whereas Zn²⁺ inhibits the enzyme. The endogenous Ca,Mg-DNase is responsible for the internucleosomal cleavage of chromosomal DNA of spermatozoa. Ca²⁺,Mn²⁺-dependent DNase (Ca,Mn-DNase) has molecular mass of 25 kD as the native form and the activity optimum at pH 8.5. The enzyme activity in the presence of bivalent metal ions decreases in the series (Ca²⁺ + Mn²⁺) > (Ca²⁺ + Mg²⁺) > Mn²⁺ > (Mg²⁺ + EGTA). In seawater the enzyme is inactive. Zinc ions inhibit Ca,Mn-DNase. Acid DNase of spermatozoa (A-DNase) is not a nuclear protein, it has molecular mass of 37 kD as a native form and the activity optimum at pH 5.5, it is not activated by bivalent metal ions, and it is inhibited by N-ethylmaleimide and iodoacetic acid. Mechanisms of the endonuclease cleavage of double-stranded DNA have been established for the three enzymes. The possible involvement of DNases from sea urchin spermatozoa in programmed cell death is discussed.     

A sea urchin egg jelly peptide induces a cGMP-mediated decrease in sperm intracellular Ca(2+) before its increase

Speract, a sperm-activating peptide (SAP) from sea urchin eggs, increases the intracellular concentration of Ca²⁺ ([Ca²⁺]i) and modulates sperm motility. We measured the initial sperm response to speract using its caged analog and observed, for the first time, a small but significant decrease in sperm [Ca²⁺]i before the increase. Both directions of the [Ca²⁺]i change were completely blocked in high K⁺ seawater. Using membrane-permeant caged cyclic nucleotides (cNMP), only cGMP induced the decrease in [Ca²⁺]i although both cGMP and cAMP increased the [Ca²⁺]i. The decrease in the [Ca²⁺]i induced by cGMP was more notable following a second photolytic event, once [Ca²⁺]i had been elevated by an initial flash. This pattern of [Ca²⁺]i change was confirmed in individual sperm. These results together with pharmacological evidence suggest that the initial [Ca²⁺]i decrease is due to a Na⁺/Ca²⁺ exchanger activity, stimulated by hyperpolarization mediated by K⁺ efflux through cGMP-regulated K⁺ channels.     

Wheat germ agglutinin binding to the micromeres and primary mesenchyme cells of sea urchin embryos

Fluorescein isothiocyanate-conjugated wheat germ agglutinin (WGA-FITC) binds exclusively to the primary mesenchyme cells when the lectin is microinjected into the blastocoels of living Lytechinus pictus and Strongylocentrotus droebachiensis embryos. WGA-FITC binding increases throughout the period of primary mesenchyme cell migration and aggregation. Similar binding is observed in embryos cultured in sulfate-free seawater (SFSW) but not in seawater (ASW) containing tunicamycin. The temporal expression of WGA-FITC binding sites in vivo is also correlated with the pattern of binding observed in vitro. Sixteen-cell stage Arbacia punctulata embryos were dissociated in Ca²⁺ and Mg²⁺-free seawater (CMFSW) and the micromeres isolated using sucrose gradients. Arbacia micromeres, cultured in ASW containing calf serum, first bind WGA-FITC during the period when primary mesenchyme cell ingression occurs in control embryos. Micromeres cultured in the presence of tunicamycin do not develop WGA binding sites. The temporal expression of WGA-FITC binding in micromere cultures is unaffected by the absence of sulfate, but the size and morphology of aggregates cultured in SFSW differ from that of the controls.     

Characterization of a metalloproteinase: A late stage specific gelatinase activity in the sea urchin embryo

We have partially purified and characterized an 87 kDa gelatinase activity expressed in later stage sea urchin embryos. Cleavage activity was specific for gelatin and no cleavage of sea urchin peristome type I collagen, bovine serum albumin or casein was detected. Magnesium and Zn²⁺ inhibited the gelatinase and Ca²⁺ protected against inhibition. Ethylenediamine tetracetic acid, ethylenebisoxyethylenenitriol tetraacetic acid and 1,10-phenanthroline were inhibitory, suggesting that the gelatinase is a Ca²⁺- and Zn²⁺-dependent metalloproteinase. No inhibition was detected with serine or cysteine protease inhibitors and the vertebrate matrix metalloproteinase (MMP) inhibitor, Batimastat, was also ineffective. The vertebrate MMP activator p-aminophenylmercuric acetate was without effect. These results allow us to identify both similarities and differences between echinoderm and vertebrate gelatinases. J. Cell. Biochem. 66: 337–345, 1997. © 1997 Wiley-Liss, Inc.     

Tuning sperm chemotaxis by calcium burst timing

Marine invertebrate oocytes establish chemoattractant gradients that guide spermatozoa towards their source. In sea urchin spermatozoa, this relocation requires coordinated motility changes initiated by Ca²⁺-driven alterations in sperm flagellar curvature. We discovered that Lytechinus pictus spermatozoa undergo chemotaxis in response to speract, an egg-derived decapeptide previously noted to stimulate non-chemotactic motility alterations in Strongylocentrotus purpuratus spermatozoa. Sperm of both species responded to speract gradients with a sequence of turning episodes that correlate with transient flagellar Ca²⁺ increases, yet only L. pictus spermatozoa accumulated at the gradient source. Detailed analysis of sperm behavior revealed that L. pictus spermatozoa selectively undergo Ca²⁺ fluctuations while swimming along negative speract gradients while S. purpuratus sperm generate Ca²⁺ fluctuations in a spatially non-selective manner. This difference is attributed to the selective suppression of Ca²⁺ fluctuations of L. pictus spermatozoa as they swim towards the source of the chemoattractant gradient. This is the first study to compare and characterize the motility components that differ in chemotactic and non-chemotactic spermatozoa. Tuning of Ca²⁺ fluctuations and associated turning episodes to the chemoattractant gradient polarity is a central feature of sea urchin sperm chemotaxis and may be a feature of sperm chemotaxis in general.     

The synthesis and characterization of a clickable-photoactive NAADP analog active in human cells

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca²⁺ mobilizing second messenger which triggers Ca²⁺ release in both sea urchin egg homogenates and in mammalian cells. The NAADP binding protein has not been identified and the regulation of NAADP mediated Ca²⁺ release remains controversial. To address this issue, we have synthesized an NAADP analog in which 3-azido-5-azidomethylbenzoic acid is attached to the amino group of 5-(3-aminopropyl)-NAADP to produce an NAADP analog which is both a photoaffinity label and clickable. This ‘all-in-one-clickable’ NAADP (AIOC-NAADP) elicited Ca²⁺ release when microinjected into cultured human SKBR3 cells at low concentrations. In contrast, it displayed little activity in sea urchin egg homogenates where very high concentrations were required to elicit Ca²⁺ release. In mammalian cell homogenates, incubation with low concentrations of [³²P]AIOC-NAADP followed by irradiation with UV light resulted in labeling 23 kDa protein(s). Competition between [³²P]AIOC-NAADP and increasing concentrations of NAADP demonstrated that the labeling was selective. We show that this label recognizes and selectively photodervatizes the 23 kDa NAADP binding protein(s) in cultured human cells identified in previous studies using [³²P]5-N₃-NAADP.     

Requirement of extracellular calcium ions for various stages of fertilization and fertilization-related phenomena in the hamster

Using a semi-chemically defined medium, the requirement of extracellular Ca²⁺ for survival, capacitation, and acrosome reaction of spermatozoa as well as various stages of fertilization in the hamster was studied. A Ca²⁺-deficient environment is unfavorable for long-term survival of spermatozoa. Sperm capacitation may occur in Ca²⁺-deficient media, but not as efficiently as in normal media. The acrosome reaction definitely requires extracellular Ca²⁺. Other processes or phenomena that require extracellular Ca²⁺ are initiation and maintenance of hyperactivated motility of spermatozoa, penetration of acrosome-reacted spermatozoa into the zona pellucida, fusion of the spermatozoa with eggs, and the development of pronuclear eggs into two-cell embryos. Extracellular Ca²⁺ is apparently unnecessary for the attachment of spermatozoa to the zona and egg surfaces, decondensation of the sperm nucleus, and the development of sperm and egg pronuclei within the egg. These results were compared with data obtained in other species such as the sea urchin, mouse, rat and guinea pig.     

CHANGE IN THE ACTIVITY OF PYRUVATE DEHYDROGENASE COMPLEX IN SEA URCHIN EGGS FOLLOWING FERTILIZATION*

The activity of the pyruvate dehydrogenase complex in sea urchin eggs is localized in the crude mitochondrial fraction. The activity of the enzyme complex in the intact mitochondrial fraction of unfertilized eggs is too low to be estimated and is enhanced upon fertilization with a 5-min lag period. The activity of the enzyme complex in unfertilized eggs is enhanced by Ca²⁺at concentrations between 5 × 10^?5 M and 10^?3 M. The activity in fertilized eggs is blocked after incubation with 2 mM ATP, and the block of the activity is also released by Ca²⁺. The blockage of the enzyme complex activity is accompanied by phosphorylation of proteins, and release of the block by Ca²⁺ is concomitantly followed by the dephosphorylation of proteins in the mitochondrial fraction. The enzyme complex in unfertilized eggs will be assumed to be the one inhibited by phosphorylation. The enzyme complex will be activated upon fertilization as a consequence of the dephosphorylation, that is caused by the increase in intracellular concentration of Ca²⁺.     

A partial sequence of ionic changes associated with the acrosome reaction of Strongylocentrotus purpuratus

Relationships among several of the ion movements associated with the acrosome reaction of S. purpuratus were investigated. Egg jelly initiates ⁴⁵Ca²⁺ and ²²Na⁺ uptake, and K⁺ and H⁺ efflux. H⁺ efflux and ²²Na⁺ uptake occur with approximately equivalent stoichiometries as rapidly as the appearance of acrosomal rods, perhaps reflecting a linked process. Most K⁺ loss, as measured either by ⁴²K⁺ efflux or K⁺-ion-selective electrodes, occurs after the acrosome reaction is complete. Since an elevation of seawater K⁺ (from 10 to 15 mM) or the addition of 0.5 mM tetraethylammonium (TEA), an inhibitor of K⁺ channels, inhibits the acrosome reaction half-maximally, K⁺ movements or alterations of K⁺-dependent membrane potentials may regulate the triggering by jelly. Most, but not all, of the ⁴⁵Ca²⁺ influx is inhibited with a mixture of 10 μM FCCP, 1 mM CN^?, and 2 μg/ml oligomycin, suggesting that the mitochondria store most of the Ca²⁺. The extracellular Na⁺ concentration affects Ca²⁺ fluxes: sperm placed into 5 mM Na⁺ seawater have enhanced ⁴⁵Ca²⁺ uptake, but do not undergo the acrosome reaction, unless 30 mM Na⁺ is also added. Low Na⁺ concentrations lead to spontaneous triggering, by allowing for both Ca²⁺ influx and Na⁺-dependent H⁺ efflux. At least one early Ca²⁺ requirement precedes the Na⁺ and H⁺ movements, as inferred from attempts at reversing the inhibitors of jelly induction of the acrosome reaction. When sperm are incubated with jelly in the absence of Ca²⁺, then washed and incubated with jelly in the presence of Ca²⁺, the acrosome reaction is triggered only upon the second incubation. However, when sperm are mixed with jelly in the presence of the other inhibitors (verapamil, TEA, 5 mM Na⁺ seawater, low pH, or elevated K⁺), they are altered so that even upon subsequent washing, jelly-mediated triggering is no longer possible. This suggests the existence of an intermediate state in the reaction pathway, that follows an event for which Ca²⁺ is required, but that precedes the Na⁺ and H⁺ movements, which are inhibited by all inhibitors of the acrosome reaction. These data are used to develop a partial sequence of ionic changes associated with the triggering mechanism.     

The luminal Ca2+ chelator,TPEN, inhibits NAADP-induced Ca2+ release

The regulation of Ca²⁺ release by luminal Ca²⁺ has been well studied for the ryanodine and IP₃ receptors but has been less clear for the NAADP-regulated channel. In view of conflicting reports, we have re-examined the issue by manipulating luminal Ca²⁺ with the membrane-permeant, low affinity Ca²⁺ buffer, TPEN, and monitoring NAADP-induced Ca²⁺ release in sea urchin egg homogenate. NAADP-induced Ca²⁺ release was almost entirely blocked by TPEN (IC₅₀ 17–25 μM) which suppressed the maximal extent of Ca²⁺ release without altering NAADP sensitivity. In contrast, Ca²⁺ release via IP₃ receptors was 3- to 30-fold less sensitive to TPEN whereas that evoked by ionomycin was essentially unaffected. The effect of TPEN on NAADP-induced Ca²⁺ release was not due to an increase in the luminal pH or chelation of trace metals since it could not be mimicked by NH₄Cl or phenanthroline. The fact that TPEN had no effect upon ionophore-induced Ca²⁺ release also argued against a substantial reduction in the driving force for Ca²⁺ efflux. We propose that, in the sea urchin egg, luminal Ca²⁺ is important for gating native NAADP-regulated two-pore channels.     

The Effects of CapZ Peptide (TRTK-12) Binding to S100B-Ca as Examined by NMR and X-ray Crystallography

Structure-based drug design is underway to inhibit the S100B-p53 interaction as a strategy for treating malignant melanoma. X-ray crystallography was used here to characterize an interaction between Ca²⁺-S100B and TRTK-12, a target that binds to the p53-binding site on S100B. The structures of Ca²⁺-S100B (1.5-Å resolution) and S100B-Ca²⁺-TRTK-12 (2.0-Å resolution) determined here indicate that the S100B-Ca²⁺-TRTK-12 complex is dominated by an interaction between Trp7 of TRTK-12 and a hydrophobic binding pocket exposed on Ca²⁺-S100B involving residues in helices 2 and 3 and loop 2. As with an S100B-Ca²⁺-p53 peptide complex, TRTK-12 binding to Ca²⁺-S100B was found to increase the protein's Ca²⁺-binding affinity. One explanation for this effect was that peptide binding introduced a structural change that increased the number of Ca²⁺ ligands and/or improved the Ca²⁺ coordination geometry of S100B. This possibility was ruled out when the structures of S100B-Ca²⁺-TRTK-12 and S100B-Ca²⁺ were compared and calcium ion coordination by the protein was found to be nearly identical in both EF-hand calcium-binding domains (RMSD = 0.19). On the other hand, B-factors for residues in EF2 of Ca²⁺-S100B were found to be significantly lowered with TRTK-12 bound. This result is consistent with NMR ¹⁵N relaxation studies that showed that TRTK-12 binding eliminated dynamic properties observed in Ca²⁺-S100B. Such a loss of protein motion may also provide an explanation for how calcium-ion-binding affinity is increased upon binding a target. Lastly, it follows that any small-molecule inhibitor bound to Ca²⁺-S100B would also have to cause an increase in calcium-ion-binding affinity to be effective therapeutically inside a cell, so these data need to be considered in future drug design studies involving S100B.     

Ca-transport in sea urchin unfertilized eggs: Regulation by endogenous sulfated polysaccharides and K

Previous data from our laboratory showed that the reticulum of the sea cucumber smooth muscle body wall retains both a sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) and a sulfated polysaccharide. In this invertebrate, the transport of Ca²⁺ by the SERCA is naturally inhibited by these endogenous sulfated polysaccharides. The inhibition is reverted by K⁺ leading to an enhancement of the Ca²⁺ transport rate. We now show that vesicles derived from the endoplasmic reticulum of unfertilized eggs from the sea urchin Arbacia lixula retain a SERCA that is able to transport Ca²⁺ at the expense of ATP hydrolysis. As described for the sea cucumber SERCA isoform, the enzyme from the sea urchin is activated by K⁺ but not by Li⁺ and is inhibited by thapsigargin, a specific inhibitor of SERCA. A new sulfated polysaccharide was identified in the sea urchin eggs reticulum composed mainly by galactose, glucose, hexosamine and manose. After extraction and purification, this sulfated polysaccharide was able to inhibit the mammal SERCA isoform found in rabbit skeletal muscle and the inhibition is reversed by K⁺. These data suggest that the regulation of the SERCA pump by K⁺ and sulfated polysaccharides is not restricted to few marine invertebrates but is widespread.     

Alteration of Ca2+ homeostasis of sea urchin embryos by retinoid CD 367, dual effect on egg cleavage and embryonic development

The effect of a light stable retinoid (CD 367) was studied on sea urchin embryos. CD 367 did not affect sperm-egg interaction. In a range of concentrations between 10 and 100 μM, CD 367 delayed the first and the second cleavages. When added after fertilization, micromolar amounts of CD 367 delayed hatching and produced embryonic abnormalities in a dose-dependent manner. Mesodermal cells, primary (PMC) and secondary (SMC) mesenchyme cells migration was particularly disturbed, leading to exogastrulations and calcified spicules malformations. Concentrations of CD 367 higher than 8 μM were embryolethal. Micromolar amount of CD 367 increased plasmalemma Ca²⁺ permeability of fertilized eggs but not of unfertilized eggs. CD 367 inhibited ATP-dependent intra-cellular sequestration of Ca²⁺ in a range of concentrations similar to those affecting egg cleavage and embryonic structures. Since we were unable to detect nuclear receptors for CD 367 in sea urchin eggs and ovocytes, these effects probably are not related to interaction of the retinoid with members of the RAR family, to which CD 367 has a high affinity, but rather to its toxicity by the means of some unknown mechanisms.