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Ca2+-transport in sea urchin unfertilized eggs: Regulation by endogenous sulfated polysaccharides and K+
Biochimica et Biophysica Acta 1760 (2006) 1529 – 1535 www.elsevier.com/locate/bbagen
Ca 2+ -transport in sea urchin unfertilized eggs: Regulation by endogenous sulfated polysaccharides and K + Ana M. Landeira-Fernandez a,⁎, Rafael S. Aquino a,b , Paulo A.S. Mourão a,b , Leopoldo de Meis a a
b
Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Caixa Postal 68041, Rio de Janeiro, RJ, 21941-590, Brazil Laboratorio de Tecido Conjuntivo, Hospital Universitario Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, Caixa Postal 68041, Rio de Janeiro, RJ, 21941-590, Brazil Received 5 April 2006; received in revised form 26 May 2006; accepted 2 June 2006 Available online 7 June 2006
Abstract 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. © 2006 Elsevier B.V. All rights reserved. Keywords: Ca2+-transport; Endoplasmic reticulum; K+ activation; SERCA; Sea urchin egg; Sulfated polysaccharide
1. Introduction The Ca2+ release from intracellular stores is involved in the regulation of a variety of physiological events such as: muscle contraction, platelet and neuronal activation, fertilization, etc [1–7]. In sea urchins, the immediate consequence of the sperm– egg fusion is the generation of a single large transient elevation in cytosolic free [Ca2+], which is absolutely required for egg activation e.g. cortical granule exocytosis, the respiratory burst, the increase in intracellular pH, increases in DNA and protein synthesis [8–10]. Sea urchin eggs contain both Inositol 1,4,5-trisphosphate (InsP3) and ryanodine receptor (RyR) endoplasmic reticulum (ER) Ca2+ release channels. The microinjection of either InsP3, ryanodine or the cyclic adenosine diphosphate ribose (cADPR) increases the cytoplasmatic Ca2+ concentrations and activates ⁎ Corresponding author. Tel.: +55 21 2562 6787; fax: +55 21 2270 8647. E-mail address: [email protected] (A.M. Landeira-Fernandez). 0304-4165/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2006.06.001
eggs [11–13]. In addition, earlier studies demonstrated a significantly ATP-dependent and oxalate supported rate of Ca2+-uptake using the microssomal fraction from sea urchin unfertilized eggs derived from Hemicentrotus pulcherrimus and Strongylocentrotus droebachiensis [14,15]. More recently, the gene encoding the SERCA pump from the sea urchin S. purpuratus has been cloned and found to be similar to the mammalian SERCA2 isoform [16]. These results demonstrated the existence of intracellular ER compartments responsible for the maintenance of Ca2+ stores in the sea urchin eggs. The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) is responsible for the maintenance of low cytoplasmatic Ca2+concentrations (∼ 0.1 μM), pumping Ca2+ from the cytosol into the ER at the expense of ATP hydrolysis. The SERCA pump is a cation-transport ATPase that belongs to the P-type or E1/E2 ATPases family. This family of enzymes is characterized by the formation of an acylphosphate during its catalytic cycle and the inhibition by low concentrations of vanadate. This family of enzymes also includes the Na + /K + -ATPase, the plasma
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membrane Ca2+ -ATPase and the plant and yeast plasma membrane H+-ATPase [17,18]. We have previously shown that heparin and other highly sulfated polysaccharides were able to modulate in vitro the activity of several P-type ATPases. Curiously, the inhibition of P-type ATPases by sulfated polysaccharides is completely reverted in the presence of K+, a monovalent cation that usually has no effect on the ATPases [19–23]. More recently, we show that the invertebrate SERCA isoform found in the smooth muscle from the sea cucumber Ludwigothurea grisea was naturally dependent of K+ to reach maximal activity due to the presence of endogenous sulfated polysaccharides, which regulate its activity [23 24]. In the present work we demonstrated, that the microsomal fraction derived from the unfertilized eggs of the sea urchin Arbacia lixula also retains a SERCA-like pump that was able to transport Ca2+ at the expense of ATP hydrolysis. The Ca2+-uptake was activated several times by K+ but not Li+. Even more significantly, we also found that vesicles derived from the sea urchin unfertilized eggs retain a sulfated polysaccharide, which is able to inhibit the rabbit SERCA Ca2+-transport. We hypothesized that, similar to the SERCA pump found on the smooth muscle of the sea cucumber, SERCA isoform from the sea urchin unfertilized eggs can be intrinsically inhibited by the presence of a sulfated polysaccharide, which might be conferring the K+ dependence. 2. Materials and methods 2.1. Gametes extraction Mature species of A. lixula sea urchin were collected in Praia Vermelha (Rio de Janeiro, Brazil) and gametes were isolated by intracelomic injection of 0.5 M KCl (∼5 ml/specimen). Eggs were collected in artificial seawater (450 mM NaCl, 9 mM KCl, 48 mM MgSO4·7H2O, 10 mM CaCl2, and 6 mM NaHCO3).
2.2. Preparation of microsomes After collecting the eggs, the egg jelly was removed by pH shock, as described by SeGall and Lennarz [25]. The suspension of eggs, without the egg jelly, was centrifuged for 3 min at 3,000 rpm and the pellet (approximately 5 ml) was resuspended in 50 ml of an ice-cold solution, containing 10 mM MOPSTRIS buffer (pH 7.0), 10% sucrose, 1 mM EDTA, 30 mM NaSO4, 450 mM NaCl, 10 mM KCl, and 1 mM phenylmethylsulfonyl fluoride and homogenized in a warping blender. The egg homogenate was centrifuged at 14,000×g for 20 min at 4 °C. The supernatant was filtered through cheesecloth and centrifuged twice at 100,000×g for 40 min at 4 °C. The pellet (total microsomes) was collected, suspended in 1 ml of ice-cold buffer, containing 50 mM MOPS-TRIS (pH 7.0), 800 mM sucrose, 5 mM NaN3 and 1 mM EDTA, and stored in liquid nitrogen until use. The microsomes derived from rabbit muscle were isolated as described by Eletr and Inesi [26].
2.4. Extraction and purification of sulfated polysaccharides from microsome preparation The sulfated polysaccharides were extracted from microsome preparation (∼ 5 ml) by papain digestion and partially purified by ethanol precipitation [28]. After precipitation, the crude polysaccharide (∼ 50 mg) were applied to a Mono Q-FPLC column (HR 5/5, Amersham Pharmacia Biotech), equilibrated with 20 mM Tris/HCl (pH 8.0) and 5 mM EDTA. The column was developed by a linear gradient of 0–3 M NaCl in the same buffer. The flow rate of the column was 0.5 ml/min, and fractions of 0.5 ml were collected and assayed by metachromasia using 1,9-dimethyl-methylene blue [29]. The fractions were pooled, dialyzed against distilled water and lyophilized. The Mono-Q purified sulfated polysaccharide fractions (20 mg of each) were chromatographed separately on a Sepharose 12 column. The column was equilibrated with 0.2 M sodium bicarbonate (pH 6.0), and eluted with the same solution. The flow rate of the column was 0.5 ml/min and fractions of 0.5 ml were collected in regular intervals. Fractions checked by a metacromatic assay were pooled, dialyzed against distilled water and lyophilized.
2.5. Chemical analysis After acid hydrolysis (6.0 M trifluoroacetic acid, 100 °C for 5 h) of the sulfated polysaccharide (∼ 200 μg), the sugars were identified by paper chromatography in n-butanol: pyridine:water (3:2:1,v/v) for 48 h on Whatman No. 1 paper, followed by silver nitrate staining.
2.6. Polyacrylamide gel electrophoresis Sulfate polysaccharides (∼ 10 μg) were applied to a 1-mm-tick 6% polyacrylamide slab gel. After electrophoresis at 100 V for ∼ 1 h in 0.06 M sodium barbital (pH 8.6), the gel was stained with 0.1% toluidine blue in 1% acetic acid and washed for ∼ 6 h in 1% acetic acid.
3. Results and discussion 3.1. Sea urchin unfertilized eggs retain a SERCA-like isoform Vesicles derived from the sea urchin unfertilized eggs retain a Ca2+-ATPase, that is able to transport Ca2+ in the presence of ATP and 10 mM Pi or 5 mM oxalate (as Ca2+-precipitant agents). The rate of Ca2+-transport by the microsomal fraction from the sea urchin unfertilized eggs was totally inhibited by the addition of 1 μM of thapsigargin or 100 μM of ciclopiazonic acid, which are specific inhibitors of SERCA isoforms, without any effect in the plasma membrane Ca2+-ATPase. Also, the addition of a Ca2+ ionophore (A23187) to the reaction medium prevents the formation of the Ca2+ gradient (Fig. 1). These results indicate that the Ca2+-gradient measured in our assays was achieved by the activity of a SERCA pump and that the contribution of a possible contaminant plasma membrane Ca2+ATPase or some K+ dependent transporter, such as Na+/Ca2+ exchanger, for the total Ca2+-transport is insignificant. 3.2. Activation of the SERCA pump by Ca2+ -precipitant agents
2.3. Ca2+-uptake 2+
Ca -uptake was measured by liquid scintillation counting [27]. After filtration, the filters were washed five times with 5 ml of 3 mM LaNO3 and the radioactivity remaining on filters was counted on a liquid scintillation counter. Unless otherwise specified, the reactions were performed at 25 °C for sea urchin and 35 °C for rabbit and were started by the addition of 0.020 mg/ ml of vesicles.
The time courses for Ca2+-transport, catalyzed by the SERCA pump from the sea urchin unfertilized eggs, in the presence of two different Ca2+-precipitant agents, are shown in Fig. 2A and B. The vesicles were able to accumulate Ca2+ when incubated in media containing ATP, MgCl2 and either 10 mM Pi or 5 mM oxalate (open circles in Fig. 2). The amount of Ca2+
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Fig. 1. Effect of different SERCA inhibitors on time dependence Ca2+-uptake catalyzed by the sea urchin microsomes. The reaction medium was composed of 50 mM Mops/Tris (pH 7.0), 1 mM MgCl2, 100 mM KCl, 1 mM ATP, 10 μM CaCl2 and 10 mM Pi in the absence (●) or in the presence of either 0.1 mM ciclopiazonic acid (○), 5 μM thapsigargin (▴) or 5 μM A23187 (■). Ca2+ uptake was measured as described under Materials and methods.
retained by the vesicles, in the absence of oxalate and Pi, was small and difficult to measure with the method used (data not shown). These two anions are known to increase the Ca2+ loading capacity of microsomes isolated from different tissues, including skeletal muscle, blood platelets and brain [30–32]. During Ca 2+ transport, the cation diffuses through the membrane and forms Ca2+ phosphate and Ca2+ oxalate crystals in the vesicles lumen [30]. In the presence of oxalate, the rate of Ca2+-uptake was ∼ 2 times faster and the amount of Ca2+ retained during the steady-state by the vesicles was twice higher than that measured in the presence of Pi (Fig. 2A and B). 3.3. Activation of Ca2+ transport promoted by K+ Even though the increase of Ca2+ transport observed in the presence of oxalate as a precipitant agent (open circles in Fig.
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2B), the sea urchin SERCA activity was significant lower than that observed in vesicles obtained from mammals. A similar low activity was also observed for the SERCA sea cucumber muscle [24]. In that case, we have already demonstrated that this low Ca2+ transport activity is due to an inhibitory effect of an endogenous sulfated polysaccharide [24]. Possibly, a similar event may occur with the SERCA from the sea urchin eggs. To examine this possibility we tested the effect of K+, an antagonist of the sulfated polysaccharide inhibition, on Ca2+ transport activity by the sea urchin vesicles. Curiously, the rate of Ca2+-transport catalyzed by the SERCA pump from the sea urchin unfertilized eggs was activated 3–4 times after the addition of 100 mM K+ to the reaction medium (closed circles in Fig. 2), while Li+ had no effect (closed triangles). This activation was independent of the Ca2+ precipitant agent used, being the same either in the presence of oxalate or Pi. In control experiments, the rate of Ca2+-uptake was 22.0 ± 4.8 (n = 6) and 14.8 ± 2.5 (n = 6) nmol Ca2+/mg/20 min in the presence of 5 mM oxalate or 10 mM Pi, respectively. But, after the addition of K+, the activity increased up to 90.0 ± 9.5 (n = 6) and 46.8 ± 5.0 (n = 6) nmol Ca2+/mg/20 min, respectively. The physiological dependence of K+ to reach maximal activity seems to be a specific feature of the marine invertebrates SERCA isoforms, found in the sea urchin unfertilized eggs and also in the smooth muscle of sea cucumber [24]. The dependence of K+ is not observed in microsomes preparations obtained from other animal tissues [33]. Like the sea urchin SERCA, the smooth muscle SERCA isoform from the sea cucumber presents a similar degree of activation (3–4 times) after the addition of 100 mM of K+ but not Li+ [33]. On the other hand, this distinct monovalent cation dependence was previously observed in vitro with different well studied P-type ATPases, upon addition of exogenous sulfated polysaccharide. In the presence of low concentrations of heparin or other highly sulfated polysaccharides, the activity of the rabbit skeletal muscle vesicles SERCA, human blood platelets SERCA [19], rat brain SERCA [21] and plasma membrane H + -ATPase from corn roots [20] was
Fig. 2. Effect of Ca2+-precipitant agents and of different cations on time dependence Ca2+-uptake catalyzed by the sea urchin microsomes. The reaction medium was composed of 50 mM Mops–Tris (pH 7.0), 1 mM MgCl2, 1 mM ATP, 10 μM 45CaCl2 and in the presence of either 5 mM oxalate–Tris (Panel A) or 10 mM Pi (Panel B). The reactions were carried out in the absence (○) or in the presence of 100 mM of either KCl (●) or LiCl (▴). The values represent the average ± SE of six experiments made with at least four different preparations.
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completely inhibited and K+, but not Li+, was able to revert this inhibition. In the case of the SERCA isoform from sea cucumber smooth muscle, the natural dependence by K+ to reach maximal activity was related to the presence of an endogenous sulfated polysaccharide [23,24]. 3.4. Occurrence of sulfated polysaccharides in the sea urchin vesicles The K+ dependence on Ca2+-transport to reach maximal activity by vesicles derived from the sea urchin unfertilized eggs suggests the presence of a sulfated polysaccharide in the sea urchin vesicles, modulating the SERCA activity. In order to test this possibility we attempted to identify sulfated polysaccharides in vesicles derived from ER of the sea urchin unfertilized eggs depleted of their jelly coat. The vesicles were submitted to a protease digestion, ethanol precipitation and fractionation of the polysaccharides on an anion-exchange chromatography (Mono-Q-FPLC). The chromatography displayed two distinct sulfated polysaccharide fractions eluted at ∼ 1.6 M NaCl (F1) and ∼ 2.3 M NaCl (F2) (Fig. 3). Polyacrylamide gel electrophoresis (Fig. 4, lane 6) suggests that fraction F2 is the sulfated polysaccharide originated from the jelly coat since both possess a high molecular mass (compare lanes 3 and 6, Fig. 5). It has been described that the jelly coats from different species of sea urchins possess a high content of sulfated polysaccharides that are involved mainly in the fertilization, as inducers of sperm the acrosome reaction. For the A. lixula species it has been shown that the egg jelly coat contains a linear sulfated α-L-fucans, with regular tetrasaccharide repeating units [34–36]. The fraction eluted with lower salt concentration (F1) is a distinct sulfated polysaccharide, with a single, narrow and well defined band on polyacrylamide gel electrophoresis (Fig. 4, lane 5), representing a low-molecular-weight sulfated polysacchar-
Fig. 3. Purification of the sulfated polysaccharides from the vesicles of the unfertilized eggs of A. lixula on Mono Q-FPLC. The sulfated polysaccharides extracted from the sea urchin vesicles were applied to a Mono Q-FPLC and purified as described under Materials and methods. Fractions were assayed for metachromasia (○) and NaCl concentration (dashed line). The fractions indicated by the horizontal bars in the panel were pooled, dialyzed against distilled water and lyophilized.
Fig. 4. Polyacrylamide gel electrophoresis of the sulfated polysaccharides extracted from the sea urchin unfertilized eggs. Samples of sulfated polysaccharide (∼ 10 μg of each) were applied to 6% 1 mm thick polyacrylamide gel slabs in 0.02 M sodium barbital (pH 8.6), and run for 30 min at 100 V. After electrophoresis, the sulfated polysaccharides were stained with 0.1% toluidine blue in 1% acetic acid and washed for about 4 h in 1% acetic acid. The numbers on the top of the gel indicates: standard dextran sulfates with molecular masses of 8 and 500 kDa (1and 10); sulfated polysaccharides extracted from either the sea urchin eggs without their egg jellies (2), the purified egg jelly (3) or the ER vesicles (4); fractions F1 (5) and F2 (6) obtained from the Mono Q chromatography (see Fig. 3); fractions F1-a (7) and F1-b (8) obtained from chromatography on Superose 12 (see Fig. 5) and the sialoglycoconjugate purified from the sea urchin egg jelly (9) (see Alves et al., 1997).
ide. A further purification of the fraction containing this new sulfated polysaccharide (F1, from Fig. 3) was achieved by gel filtration chromatography on Superose 12. Fig. 5 shows the elution pattern and revealed that fraction F1 is contaminated with small amount of the sulfated fucan from jelly coat (Fig. 5, fraction F1a) as indicated by the polyacrylamide electrophoresis (Fig. 4, lane 7) and an agarose gel electrophoresis (data not shown), while an other fraction (Fig. 5, fraction F1-b) contains
Fig. 5. Chromatography of the sulfated polysaccharides from the vesicles of A. lixula on Superose 12. The sulfated polysaccharide from the sea urchin vesicles (∼ 18 mg), partially purified on the Mono Q column (fraction F1 from Fig. 3) (●) and (∼ 1 mg) of the sulfated polysaccharides extracted from the egg jelly (○) were chromatographed on Superose 12. Fractions were eluted as described under Materials and methods, checked for their metacromasia and pooled as indicated by the horizontal bars in the panel. These fractions were dialyzed against distilled water and lyophilized.
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mainly the new sulfated polysaccharide with a molecular mass of approximately 8 kDa (Fig. 5, lane 8). Chemical analysis of the purified sulfated polysaccharide reveals a heterogeneous content of sugars, with galactose, glucose hexosamine and small amounts of manose. In order to determinate its chemical structure, the purified sulfated polysaccharide was analyzed using high field nuclear magnetic resonance. However, due to its high heterogeneity, the NMR analysis revealed a very complex spectrum, avoiding structural determination (data not shown). The attempt to characterize the purified sulfated polysaccharide using mass spectrometry was also unsuccessful, possibly due to the high molecular size and complexity of this polysaccharide (data not shown). Further structural characterization of this sulfated polysaccharide was not possible due to scarcity of material. The possibility of the studied sample to be a glycoconjugate is refuted due to the extraction procedure, exhaustive digestion with papain, and the absence of significant amounts of amino acids in the 1H-NMR spectrum. Therefore, we believe this is a polysaccharide. Overall, these results showed that vesicles from ER from the sea urchin unfertilized eggs contain a sulfated polysaccharide that presents a clear difference from the high-molecular-weight sulfated polysaccharide previous described in the sea urchin egg jelly. While egg jelly sulfated polysaccharides present simple and repetitive sequences, composed of fucose units, the sulfated polysaccharide associated to the endoplasmic reticulum vesicles (ERV) display a highly heterogeneous sugar composition but a low and homogeneous molecular size. Interestingly, the unique sulfated polysaccharides associated with the Ca2+-ATPases found in sea urchins and sea cucumbers [24] have a complex sugar composition but present a sharp electrophoretic migration on polyacrylamide gel, denoting a more homogeneous molecular size. In the case of the sea urchin we observed a single electrophoretic band while the sea cucumber showed diverse sharps bands. These results suggest that, although highly complex, those sulfated polysaccharide
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may have a regular structure, which indicates a complex physiological function. 3.5. Effect of sulfated polyssacharide on Ca2+ -transport by rabbit muscle vesicles We tested the effect of the sea urchin sulfated polysaccharide (fraction F1-b) on the Ca2+-transport catalyzed by the rabbit SERCA. Fig. 6A shows that increasing concentrations of the purified sulfated polysaccharide from the ERV (fraction F1-b) was able to inhibit the rabbit Ca2+-transport. This inhibition was totally antagonized by 100 mM KCl, as observed in Fig. 6B. In the absence of the sulfated polysaccharide, K+ has no effect on rabbit Ca2+-transport. However, in the presence of 100 μg/ml of ERV sulfated polysaccharide, the activity was totally inhibited and the rabbit SERCA became dependent on K+ to reach maximal activity (Fig. 6B). Increasing concentrations of the purified sulfated fucan extracted from the jelly coat from A. lixula was also able to inhibit the rabbit Ca 2+ -transport, but it required higher concentrations than the ERV polysaccharide (Fig. 6A and Table 1). We expressed the inhibitory effect of the polysaccharides on weight rather than on molar basis because the repeating structure of these compounds is capable of multiple interactions. An expression of our results on molar basis could reflect a “false” difference between the high and low-molecular weight polysaccharides isolated from the sea urchin egg. 3.6. Specificity of the inhibitory effect of the sulfated polysaccharide on ATPase The in vitro experiments showed that the inhibitory effect of the sulfated polysaccharides on different P-types ATPases can be related to the sulfated content of the polysaccharide [19–21]. For the P-types ATPases, the inhibitory effects of low and highmolecular-weight dextran sulfates and of heparin were about the
Fig. 6. Effect of sulfated polysaccharides extracted from sea urchin unfertilized eggs (A) and of K+ (B) on Ca2+-uptake catalyzed by rabbit muscle vesicles. The reaction medium was composed of 50 mM Mops–Tris (pH 7.0), 1 mM MgCl2, 1 mM ATP, 10 μM CaCl2 and 10 mM Pi. On Panel A symbols represent increasing concentrations of (○) crude egg jelly, (▴) unfractionated and (●) superose 12-purified (fraction F1-b) sulfated polysaccharides. On panel B symbols represent incubations in the absence (○) or in the presence of either 100 mM KCl (Δ), 100 μg/ml of sulfated polysaccharide (fraction F1-b) (●) or the mixture of 100 μg/ml the purified sulfated polysaccharide plus 100 mM KCl (■).
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Table 1 Concentration required for half-maximal (K0.5) inhibition promoted by sulfated polysaccharide on Ca2+-uptake catalyzed by rabbit muscle vesicles Sulfated Polysaccharide
K0.5 (μg/ml)
Heparin
4.0
Chondroitin 6-sulfate
1.0
Sulfated polysaccharide from sea cucumber vesicles Egg Jelly Crude sulfated polysaccharide from the sea urchin unfertilized eggs Purified sulfated polysaccharide from the sea urchin unfertilized eggs
0.1 40.0 40.0 4.0
References de Meis and Suzano (1994) de Meis and Suzano (1994) Landeira-Fernandez et al. (2000a) This work This work This work
same, but it required significantly higher concentrations of dermatan sulfate to achieve the same inhibitory effect. However, this does not apply to the sulfated polysaccharides extracted from sea urchin. The sulfated fucan derived from the egg jelly seems to have higher sulfate content than the ERV sulfated polysaccharide, since it is eluted from the anion exchange column with higher NaCl concentration than the ERV polysaccharide (Fig. 3). However, the concentration necessary to achieve the half-maximum inhibition is approximately 10 times higher for the egg jelly sulfated polysaccharide than for the ERV polysaccharide (Fig. 6A and Table 1). Furthermore, the sulfated fucan from the egg jelly has a significant higher molecular size than the polysaccharide from the ERV. The inhibitory effect of the ERV sulfated polysaccharide from sea urchin on the mammal SERCA activity is about the same as heparin, low-molecular-weight dextran sulfate and fucosylated chondroitin sulfate from the sea cucumber (Table 1). The sulfated polysaccharide with more potent inhibitory effect is the one extracted from the smooth muscle of sea cucumber [24]. In a previous work we showed that the inhibitory effect of the sulfated polysaccharide is independent of its molecular size [24]. Together these data indicate that the inhibitory effect of the ERV sulfated polysaccharide from the sea urchin unfertilized eggs on Ca2+-uptake is not a single consequence of its sulfated content or molecular size but depends on the particular structure of the molecule. 3.7. “Physiological importance of SERCA regulation by K+ and sulfated polysaccharides” Early studies about the cytoplasmic K+ concentration in sea urchin eggs have shown that basal cytoplasmic [K+] is in the range of 200–250 mM [37] and only a modest increases in [K+]i have been reported to occur during fertilization [38]. Thus, apparently at physiological conditions the SERCA pump from sea urchin eggs would be always working at maximal activity. However, different authors have shown that a substantial decrease in cytoplasmic [K+] can occur during cell apoptosis [39,40]. Barbiero et al. [41] used the K+-sensitive fluorescent dye PBFI to estimate that the [K+]i in apoptotic mouse L fibro-
blast cells falls below 50 mM. Also, measurements performed on lymphoma cells during dexamethasone-induced apoptosis also confirmed early K+ loss [42], which may account to 50–75% of the initial [K+]i [43,44]. These results raise the possibility that during programmed cell death the activity of the sea urchin SERCA is inhibited leading to an increase in the cytoplasmic [Ca2+]. Apoptosis was also demonstrable in sea urchin oocytes, eggs and early embryos [45,46], and is a key event during oocyte maturation in mammals [47]. Thus, maybe the K+ regulation on sea urchin SERCA activity can be involved in the programmed cell death pathway. Nevertheless, more studies in sea urchin eggs must be done and the regulation of SERCA by K+ and sulfated polysaccharides still remains an open question. In conclusion, echinoderms are the only group, so far studied, that have sulfated polysaccharides in the membrane of the ER or SR, conferring natural SERCA K+ dependence. Further studies with other organisms are necessary to elucidate the evolutionary and physiological significance of this observation. Nevertheless, our results emphasize a particular mechanism for regulation of the Ca2+ transport found in marine organisms based on the expression of a sulfated polysaccharide together with the Ca2+ ATPase. Acknowledgments We are grateful to Valdecir Suzano and Antonio Carlos Miranda for the technical assistance. We are also grateful to Antonio Galina for snorkeling and helpful to get the gametes from sea urchins. This work was supported with grants from Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). Rafael S. Aquino is a PhD fellow from CNPq. References [1] M.J. Berridge, A tale of two messengers, Nature 365 (1993) 388–389. [2] A. Galione, A. McDougall, W.B. Busa, N. Willmott, J. Gillot, M. Whitaker, Redundant mechanisms of calcium induced calcium release underlying calcium waves during fertilization of sea-urchin eggs, Science 261 (1993) 348–352. [3] A. Galione, S. Patel, G.C. Churchill, NAADP-induced calcium release in sea urchin eggs, Biol. Cell 92 (2000) 197–204. [4] T.P. Dousa, E.N. Chini, K.W. Beers, Adenine nucleotide diphosphate: emerging second messengers acting via intracellular Ca2+ release, Am. J. Phys. 271 (1996) C1007–C1024. [5] J.M. Cancela, G.C. Churchill, A. Galione, Coordination of agonist-induced Ca2+-signalling patterns by NAADP in pancreatic acinar cells, Nature 398 (1999) 74–76. [6] A.N.K. Yusufi, J. Cheng, M.A. Thompson, E.N. Chini, I.P. Grande, NAADP elicits specific microsomal Ca2+ release from mammalian cells, Biochem. J. 353 (2001) 531–536. [7] E.N. Chini, Interactions between intracellular Ca2+ stores: Ca2+ released from the NAADP pool potentiates cADPRinduced Ca2+ release, Brazil. J. Med. Biol. Res. 35 (2002) 543–547. [8] V.D. Vacquier, The isolation of intact cortical granules from sea urchin eggs: Calcium ions trigger granule discharge, Dev. Biol. 43 (1975) 62–74. [9] R.S. Zucker, R.A. Steinhardt, Prevention of the cortical reaction in fertilized sea urchin eggs by injection of calcium-chelating ligands, Biochim. Biophys. Acta 541 (1978) 459–466.
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Ca 2+ -transport in sea urchin unfertilized eggs: Regulation by endogenous sulfated polysaccharides and K + Ana M. Landeira-Fernandez a,⁎, Rafael S. Aquino a,b , Paulo A.S. Mourão a,b , Leopoldo de Meis a a
b
Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Caixa Postal 68041, Rio de Janeiro, RJ, 21941-590, Brazil Laboratorio de Tecido Conjuntivo, Hospital Universitario Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, Caixa Postal 68041, Rio de Janeiro, RJ, 21941-590, Brazil Received 5 April 2006; received in revised form 26 May 2006; accepted 2 June 2006 Available online 7 June 2006
Abstract 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. © 2006 Elsevier B.V. All rights reserved. Keywords: Ca2+-transport; Endoplasmic reticulum; K+ activation; SERCA; Sea urchin egg; Sulfated polysaccharide
1. Introduction The Ca2+ release from intracellular stores is involved in the regulation of a variety of physiological events such as: muscle contraction, platelet and neuronal activation, fertilization, etc [1–7]. In sea urchins, the immediate consequence of the sperm– egg fusion is the generation of a single large transient elevation in cytosolic free [Ca2+], which is absolutely required for egg activation e.g. cortical granule exocytosis, the respiratory burst, the increase in intracellular pH, increases in DNA and protein synthesis [8–10]. Sea urchin eggs contain both Inositol 1,4,5-trisphosphate (InsP3) and ryanodine receptor (RyR) endoplasmic reticulum (ER) Ca2+ release channels. The microinjection of either InsP3, ryanodine or the cyclic adenosine diphosphate ribose (cADPR) increases the cytoplasmatic Ca2+ concentrations and activates ⁎ Corresponding author. Tel.: +55 21 2562 6787; fax: +55 21 2270 8647. E-mail address: [email protected] (A.M. Landeira-Fernandez). 0304-4165/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2006.06.001
eggs [11–13]. In addition, earlier studies demonstrated a significantly ATP-dependent and oxalate supported rate of Ca2+-uptake using the microssomal fraction from sea urchin unfertilized eggs derived from Hemicentrotus pulcherrimus and Strongylocentrotus droebachiensis [14,15]. More recently, the gene encoding the SERCA pump from the sea urchin S. purpuratus has been cloned and found to be similar to the mammalian SERCA2 isoform [16]. These results demonstrated the existence of intracellular ER compartments responsible for the maintenance of Ca2+ stores in the sea urchin eggs. The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) is responsible for the maintenance of low cytoplasmatic Ca2+concentrations (∼ 0.1 μM), pumping Ca2+ from the cytosol into the ER at the expense of ATP hydrolysis. The SERCA pump is a cation-transport ATPase that belongs to the P-type or E1/E2 ATPases family. This family of enzymes is characterized by the formation of an acylphosphate during its catalytic cycle and the inhibition by low concentrations of vanadate. This family of enzymes also includes the Na + /K + -ATPase, the plasma
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membrane Ca2+ -ATPase and the plant and yeast plasma membrane H+-ATPase [17,18]. We have previously shown that heparin and other highly sulfated polysaccharides were able to modulate in vitro the activity of several P-type ATPases. Curiously, the inhibition of P-type ATPases by sulfated polysaccharides is completely reverted in the presence of K+, a monovalent cation that usually has no effect on the ATPases [19–23]. More recently, we show that the invertebrate SERCA isoform found in the smooth muscle from the sea cucumber Ludwigothurea grisea was naturally dependent of K+ to reach maximal activity due to the presence of endogenous sulfated polysaccharides, which regulate its activity [23 24]. In the present work we demonstrated, that the microsomal fraction derived from the unfertilized eggs of the sea urchin Arbacia lixula also retains a SERCA-like pump that was able to transport Ca2+ at the expense of ATP hydrolysis. The Ca2+-uptake was activated several times by K+ but not Li+. Even more significantly, we also found that vesicles derived from the sea urchin unfertilized eggs retain a sulfated polysaccharide, which is able to inhibit the rabbit SERCA Ca2+-transport. We hypothesized that, similar to the SERCA pump found on the smooth muscle of the sea cucumber, SERCA isoform from the sea urchin unfertilized eggs can be intrinsically inhibited by the presence of a sulfated polysaccharide, which might be conferring the K+ dependence. 2. Materials and methods 2.1. Gametes extraction Mature species of A. lixula sea urchin were collected in Praia Vermelha (Rio de Janeiro, Brazil) and gametes were isolated by intracelomic injection of 0.5 M KCl (∼5 ml/specimen). Eggs were collected in artificial seawater (450 mM NaCl, 9 mM KCl, 48 mM MgSO4·7H2O, 10 mM CaCl2, and 6 mM NaHCO3).
2.2. Preparation of microsomes After collecting the eggs, the egg jelly was removed by pH shock, as described by SeGall and Lennarz [25]. The suspension of eggs, without the egg jelly, was centrifuged for 3 min at 3,000 rpm and the pellet (approximately 5 ml) was resuspended in 50 ml of an ice-cold solution, containing 10 mM MOPSTRIS buffer (pH 7.0), 10% sucrose, 1 mM EDTA, 30 mM NaSO4, 450 mM NaCl, 10 mM KCl, and 1 mM phenylmethylsulfonyl fluoride and homogenized in a warping blender. The egg homogenate was centrifuged at 14,000×g for 20 min at 4 °C. The supernatant was filtered through cheesecloth and centrifuged twice at 100,000×g for 40 min at 4 °C. The pellet (total microsomes) was collected, suspended in 1 ml of ice-cold buffer, containing 50 mM MOPS-TRIS (pH 7.0), 800 mM sucrose, 5 mM NaN3 and 1 mM EDTA, and stored in liquid nitrogen until use. The microsomes derived from rabbit muscle were isolated as described by Eletr and Inesi [26].
2.4. Extraction and purification of sulfated polysaccharides from microsome preparation The sulfated polysaccharides were extracted from microsome preparation (∼ 5 ml) by papain digestion and partially purified by ethanol precipitation [28]. After precipitation, the crude polysaccharide (∼ 50 mg) were applied to a Mono Q-FPLC column (HR 5/5, Amersham Pharmacia Biotech), equilibrated with 20 mM Tris/HCl (pH 8.0) and 5 mM EDTA. The column was developed by a linear gradient of 0–3 M NaCl in the same buffer. The flow rate of the column was 0.5 ml/min, and fractions of 0.5 ml were collected and assayed by metachromasia using 1,9-dimethyl-methylene blue [29]. The fractions were pooled, dialyzed against distilled water and lyophilized. The Mono-Q purified sulfated polysaccharide fractions (20 mg of each) were chromatographed separately on a Sepharose 12 column. The column was equilibrated with 0.2 M sodium bicarbonate (pH 6.0), and eluted with the same solution. The flow rate of the column was 0.5 ml/min and fractions of 0.5 ml were collected in regular intervals. Fractions checked by a metacromatic assay were pooled, dialyzed against distilled water and lyophilized.
2.5. Chemical analysis After acid hydrolysis (6.0 M trifluoroacetic acid, 100 °C for 5 h) of the sulfated polysaccharide (∼ 200 μg), the sugars were identified by paper chromatography in n-butanol: pyridine:water (3:2:1,v/v) for 48 h on Whatman No. 1 paper, followed by silver nitrate staining.
2.6. Polyacrylamide gel electrophoresis Sulfate polysaccharides (∼ 10 μg) were applied to a 1-mm-tick 6% polyacrylamide slab gel. After electrophoresis at 100 V for ∼ 1 h in 0.06 M sodium barbital (pH 8.6), the gel was stained with 0.1% toluidine blue in 1% acetic acid and washed for ∼ 6 h in 1% acetic acid.
3. Results and discussion 3.1. Sea urchin unfertilized eggs retain a SERCA-like isoform Vesicles derived from the sea urchin unfertilized eggs retain a Ca2+-ATPase, that is able to transport Ca2+ in the presence of ATP and 10 mM Pi or 5 mM oxalate (as Ca2+-precipitant agents). The rate of Ca2+-transport by the microsomal fraction from the sea urchin unfertilized eggs was totally inhibited by the addition of 1 μM of thapsigargin or 100 μM of ciclopiazonic acid, which are specific inhibitors of SERCA isoforms, without any effect in the plasma membrane Ca2+-ATPase. Also, the addition of a Ca2+ ionophore (A23187) to the reaction medium prevents the formation of the Ca2+ gradient (Fig. 1). These results indicate that the Ca2+-gradient measured in our assays was achieved by the activity of a SERCA pump and that the contribution of a possible contaminant plasma membrane Ca2+ATPase or some K+ dependent transporter, such as Na+/Ca2+ exchanger, for the total Ca2+-transport is insignificant. 3.2. Activation of the SERCA pump by Ca2+ -precipitant agents
2.3. Ca2+-uptake 2+
Ca -uptake was measured by liquid scintillation counting [27]. After filtration, the filters were washed five times with 5 ml of 3 mM LaNO3 and the radioactivity remaining on filters was counted on a liquid scintillation counter. Unless otherwise specified, the reactions were performed at 25 °C for sea urchin and 35 °C for rabbit and were started by the addition of 0.020 mg/ ml of vesicles.
The time courses for Ca2+-transport, catalyzed by the SERCA pump from the sea urchin unfertilized eggs, in the presence of two different Ca2+-precipitant agents, are shown in Fig. 2A and B. The vesicles were able to accumulate Ca2+ when incubated in media containing ATP, MgCl2 and either 10 mM Pi or 5 mM oxalate (open circles in Fig. 2). The amount of Ca2+
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Fig. 1. Effect of different SERCA inhibitors on time dependence Ca2+-uptake catalyzed by the sea urchin microsomes. The reaction medium was composed of 50 mM Mops/Tris (pH 7.0), 1 mM MgCl2, 100 mM KCl, 1 mM ATP, 10 μM CaCl2 and 10 mM Pi in the absence (●) or in the presence of either 0.1 mM ciclopiazonic acid (○), 5 μM thapsigargin (▴) or 5 μM A23187 (■). Ca2+ uptake was measured as described under Materials and methods.
retained by the vesicles, in the absence of oxalate and Pi, was small and difficult to measure with the method used (data not shown). These two anions are known to increase the Ca2+ loading capacity of microsomes isolated from different tissues, including skeletal muscle, blood platelets and brain [30–32]. During Ca 2+ transport, the cation diffuses through the membrane and forms Ca2+ phosphate and Ca2+ oxalate crystals in the vesicles lumen [30]. In the presence of oxalate, the rate of Ca2+-uptake was ∼ 2 times faster and the amount of Ca2+ retained during the steady-state by the vesicles was twice higher than that measured in the presence of Pi (Fig. 2A and B). 3.3. Activation of Ca2+ transport promoted by K+ Even though the increase of Ca2+ transport observed in the presence of oxalate as a precipitant agent (open circles in Fig.
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2B), the sea urchin SERCA activity was significant lower than that observed in vesicles obtained from mammals. A similar low activity was also observed for the SERCA sea cucumber muscle [24]. In that case, we have already demonstrated that this low Ca2+ transport activity is due to an inhibitory effect of an endogenous sulfated polysaccharide [24]. Possibly, a similar event may occur with the SERCA from the sea urchin eggs. To examine this possibility we tested the effect of K+, an antagonist of the sulfated polysaccharide inhibition, on Ca2+ transport activity by the sea urchin vesicles. Curiously, the rate of Ca2+-transport catalyzed by the SERCA pump from the sea urchin unfertilized eggs was activated 3–4 times after the addition of 100 mM K+ to the reaction medium (closed circles in Fig. 2), while Li+ had no effect (closed triangles). This activation was independent of the Ca2+ precipitant agent used, being the same either in the presence of oxalate or Pi. In control experiments, the rate of Ca2+-uptake was 22.0 ± 4.8 (n = 6) and 14.8 ± 2.5 (n = 6) nmol Ca2+/mg/20 min in the presence of 5 mM oxalate or 10 mM Pi, respectively. But, after the addition of K+, the activity increased up to 90.0 ± 9.5 (n = 6) and 46.8 ± 5.0 (n = 6) nmol Ca2+/mg/20 min, respectively. The physiological dependence of K+ to reach maximal activity seems to be a specific feature of the marine invertebrates SERCA isoforms, found in the sea urchin unfertilized eggs and also in the smooth muscle of sea cucumber [24]. The dependence of K+ is not observed in microsomes preparations obtained from other animal tissues [33]. Like the sea urchin SERCA, the smooth muscle SERCA isoform from the sea cucumber presents a similar degree of activation (3–4 times) after the addition of 100 mM of K+ but not Li+ [33]. On the other hand, this distinct monovalent cation dependence was previously observed in vitro with different well studied P-type ATPases, upon addition of exogenous sulfated polysaccharide. In the presence of low concentrations of heparin or other highly sulfated polysaccharides, the activity of the rabbit skeletal muscle vesicles SERCA, human blood platelets SERCA [19], rat brain SERCA [21] and plasma membrane H + -ATPase from corn roots [20] was
Fig. 2. Effect of Ca2+-precipitant agents and of different cations on time dependence Ca2+-uptake catalyzed by the sea urchin microsomes. The reaction medium was composed of 50 mM Mops–Tris (pH 7.0), 1 mM MgCl2, 1 mM ATP, 10 μM 45CaCl2 and in the presence of either 5 mM oxalate–Tris (Panel A) or 10 mM Pi (Panel B). The reactions were carried out in the absence (○) or in the presence of 100 mM of either KCl (●) or LiCl (▴). The values represent the average ± SE of six experiments made with at least four different preparations.
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completely inhibited and K+, but not Li+, was able to revert this inhibition. In the case of the SERCA isoform from sea cucumber smooth muscle, the natural dependence by K+ to reach maximal activity was related to the presence of an endogenous sulfated polysaccharide [23,24]. 3.4. Occurrence of sulfated polysaccharides in the sea urchin vesicles The K+ dependence on Ca2+-transport to reach maximal activity by vesicles derived from the sea urchin unfertilized eggs suggests the presence of a sulfated polysaccharide in the sea urchin vesicles, modulating the SERCA activity. In order to test this possibility we attempted to identify sulfated polysaccharides in vesicles derived from ER of the sea urchin unfertilized eggs depleted of their jelly coat. The vesicles were submitted to a protease digestion, ethanol precipitation and fractionation of the polysaccharides on an anion-exchange chromatography (Mono-Q-FPLC). The chromatography displayed two distinct sulfated polysaccharide fractions eluted at ∼ 1.6 M NaCl (F1) and ∼ 2.3 M NaCl (F2) (Fig. 3). Polyacrylamide gel electrophoresis (Fig. 4, lane 6) suggests that fraction F2 is the sulfated polysaccharide originated from the jelly coat since both possess a high molecular mass (compare lanes 3 and 6, Fig. 5). It has been described that the jelly coats from different species of sea urchins possess a high content of sulfated polysaccharides that are involved mainly in the fertilization, as inducers of sperm the acrosome reaction. For the A. lixula species it has been shown that the egg jelly coat contains a linear sulfated α-L-fucans, with regular tetrasaccharide repeating units [34–36]. The fraction eluted with lower salt concentration (F1) is a distinct sulfated polysaccharide, with a single, narrow and well defined band on polyacrylamide gel electrophoresis (Fig. 4, lane 5), representing a low-molecular-weight sulfated polysacchar-
Fig. 3. Purification of the sulfated polysaccharides from the vesicles of the unfertilized eggs of A. lixula on Mono Q-FPLC. The sulfated polysaccharides extracted from the sea urchin vesicles were applied to a Mono Q-FPLC and purified as described under Materials and methods. Fractions were assayed for metachromasia (○) and NaCl concentration (dashed line). The fractions indicated by the horizontal bars in the panel were pooled, dialyzed against distilled water and lyophilized.
Fig. 4. Polyacrylamide gel electrophoresis of the sulfated polysaccharides extracted from the sea urchin unfertilized eggs. Samples of sulfated polysaccharide (∼ 10 μg of each) were applied to 6% 1 mm thick polyacrylamide gel slabs in 0.02 M sodium barbital (pH 8.6), and run for 30 min at 100 V. After electrophoresis, the sulfated polysaccharides were stained with 0.1% toluidine blue in 1% acetic acid and washed for about 4 h in 1% acetic acid. The numbers on the top of the gel indicates: standard dextran sulfates with molecular masses of 8 and 500 kDa (1and 10); sulfated polysaccharides extracted from either the sea urchin eggs without their egg jellies (2), the purified egg jelly (3) or the ER vesicles (4); fractions F1 (5) and F2 (6) obtained from the Mono Q chromatography (see Fig. 3); fractions F1-a (7) and F1-b (8) obtained from chromatography on Superose 12 (see Fig. 5) and the sialoglycoconjugate purified from the sea urchin egg jelly (9) (see Alves et al., 1997).
ide. A further purification of the fraction containing this new sulfated polysaccharide (F1, from Fig. 3) was achieved by gel filtration chromatography on Superose 12. Fig. 5 shows the elution pattern and revealed that fraction F1 is contaminated with small amount of the sulfated fucan from jelly coat (Fig. 5, fraction F1a) as indicated by the polyacrylamide electrophoresis (Fig. 4, lane 7) and an agarose gel electrophoresis (data not shown), while an other fraction (Fig. 5, fraction F1-b) contains
Fig. 5. Chromatography of the sulfated polysaccharides from the vesicles of A. lixula on Superose 12. The sulfated polysaccharide from the sea urchin vesicles (∼ 18 mg), partially purified on the Mono Q column (fraction F1 from Fig. 3) (●) and (∼ 1 mg) of the sulfated polysaccharides extracted from the egg jelly (○) were chromatographed on Superose 12. Fractions were eluted as described under Materials and methods, checked for their metacromasia and pooled as indicated by the horizontal bars in the panel. These fractions were dialyzed against distilled water and lyophilized.
A.M. Landeira-Fernandez et al. / Biochimica et Biophysica Acta 1760 (2006) 1529–1535
mainly the new sulfated polysaccharide with a molecular mass of approximately 8 kDa (Fig. 5, lane 8). Chemical analysis of the purified sulfated polysaccharide reveals a heterogeneous content of sugars, with galactose, glucose hexosamine and small amounts of manose. In order to determinate its chemical structure, the purified sulfated polysaccharide was analyzed using high field nuclear magnetic resonance. However, due to its high heterogeneity, the NMR analysis revealed a very complex spectrum, avoiding structural determination (data not shown). The attempt to characterize the purified sulfated polysaccharide using mass spectrometry was also unsuccessful, possibly due to the high molecular size and complexity of this polysaccharide (data not shown). Further structural characterization of this sulfated polysaccharide was not possible due to scarcity of material. The possibility of the studied sample to be a glycoconjugate is refuted due to the extraction procedure, exhaustive digestion with papain, and the absence of significant amounts of amino acids in the 1H-NMR spectrum. Therefore, we believe this is a polysaccharide. Overall, these results showed that vesicles from ER from the sea urchin unfertilized eggs contain a sulfated polysaccharide that presents a clear difference from the high-molecular-weight sulfated polysaccharide previous described in the sea urchin egg jelly. While egg jelly sulfated polysaccharides present simple and repetitive sequences, composed of fucose units, the sulfated polysaccharide associated to the endoplasmic reticulum vesicles (ERV) display a highly heterogeneous sugar composition but a low and homogeneous molecular size. Interestingly, the unique sulfated polysaccharides associated with the Ca2+-ATPases found in sea urchins and sea cucumbers [24] have a complex sugar composition but present a sharp electrophoretic migration on polyacrylamide gel, denoting a more homogeneous molecular size. In the case of the sea urchin we observed a single electrophoretic band while the sea cucumber showed diverse sharps bands. These results suggest that, although highly complex, those sulfated polysaccharide
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may have a regular structure, which indicates a complex physiological function. 3.5. Effect of sulfated polyssacharide on Ca2+ -transport by rabbit muscle vesicles We tested the effect of the sea urchin sulfated polysaccharide (fraction F1-b) on the Ca2+-transport catalyzed by the rabbit SERCA. Fig. 6A shows that increasing concentrations of the purified sulfated polysaccharide from the ERV (fraction F1-b) was able to inhibit the rabbit Ca2+-transport. This inhibition was totally antagonized by 100 mM KCl, as observed in Fig. 6B. In the absence of the sulfated polysaccharide, K+ has no effect on rabbit Ca2+-transport. However, in the presence of 100 μg/ml of ERV sulfated polysaccharide, the activity was totally inhibited and the rabbit SERCA became dependent on K+ to reach maximal activity (Fig. 6B). Increasing concentrations of the purified sulfated fucan extracted from the jelly coat from A. lixula was also able to inhibit the rabbit Ca 2+ -transport, but it required higher concentrations than the ERV polysaccharide (Fig. 6A and Table 1). We expressed the inhibitory effect of the polysaccharides on weight rather than on molar basis because the repeating structure of these compounds is capable of multiple interactions. An expression of our results on molar basis could reflect a “false” difference between the high and low-molecular weight polysaccharides isolated from the sea urchin egg. 3.6. Specificity of the inhibitory effect of the sulfated polysaccharide on ATPase The in vitro experiments showed that the inhibitory effect of the sulfated polysaccharides on different P-types ATPases can be related to the sulfated content of the polysaccharide [19–21]. For the P-types ATPases, the inhibitory effects of low and highmolecular-weight dextran sulfates and of heparin were about the
Fig. 6. Effect of sulfated polysaccharides extracted from sea urchin unfertilized eggs (A) and of K+ (B) on Ca2+-uptake catalyzed by rabbit muscle vesicles. The reaction medium was composed of 50 mM Mops–Tris (pH 7.0), 1 mM MgCl2, 1 mM ATP, 10 μM CaCl2 and 10 mM Pi. On Panel A symbols represent increasing concentrations of (○) crude egg jelly, (▴) unfractionated and (●) superose 12-purified (fraction F1-b) sulfated polysaccharides. On panel B symbols represent incubations in the absence (○) or in the presence of either 100 mM KCl (Δ), 100 μg/ml of sulfated polysaccharide (fraction F1-b) (●) or the mixture of 100 μg/ml the purified sulfated polysaccharide plus 100 mM KCl (■).
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Table 1 Concentration required for half-maximal (K0.5) inhibition promoted by sulfated polysaccharide on Ca2+-uptake catalyzed by rabbit muscle vesicles Sulfated Polysaccharide
K0.5 (μg/ml)
Heparin
4.0
Chondroitin 6-sulfate
1.0
Sulfated polysaccharide from sea cucumber vesicles Egg Jelly Crude sulfated polysaccharide from the sea urchin unfertilized eggs Purified sulfated polysaccharide from the sea urchin unfertilized eggs
0.1 40.0 40.0 4.0
References de Meis and Suzano (1994) de Meis and Suzano (1994) Landeira-Fernandez et al. (2000a) This work This work This work
same, but it required significantly higher concentrations of dermatan sulfate to achieve the same inhibitory effect. However, this does not apply to the sulfated polysaccharides extracted from sea urchin. The sulfated fucan derived from the egg jelly seems to have higher sulfate content than the ERV sulfated polysaccharide, since it is eluted from the anion exchange column with higher NaCl concentration than the ERV polysaccharide (Fig. 3). However, the concentration necessary to achieve the half-maximum inhibition is approximately 10 times higher for the egg jelly sulfated polysaccharide than for the ERV polysaccharide (Fig. 6A and Table 1). Furthermore, the sulfated fucan from the egg jelly has a significant higher molecular size than the polysaccharide from the ERV. The inhibitory effect of the ERV sulfated polysaccharide from sea urchin on the mammal SERCA activity is about the same as heparin, low-molecular-weight dextran sulfate and fucosylated chondroitin sulfate from the sea cucumber (Table 1). The sulfated polysaccharide with more potent inhibitory effect is the one extracted from the smooth muscle of sea cucumber [24]. In a previous work we showed that the inhibitory effect of the sulfated polysaccharide is independent of its molecular size [24]. Together these data indicate that the inhibitory effect of the ERV sulfated polysaccharide from the sea urchin unfertilized eggs on Ca2+-uptake is not a single consequence of its sulfated content or molecular size but depends on the particular structure of the molecule. 3.7. “Physiological importance of SERCA regulation by K+ and sulfated polysaccharides” Early studies about the cytoplasmic K+ concentration in sea urchin eggs have shown that basal cytoplasmic [K+] is in the range of 200–250 mM [37] and only a modest increases in [K+]i have been reported to occur during fertilization [38]. Thus, apparently at physiological conditions the SERCA pump from sea urchin eggs would be always working at maximal activity. However, different authors have shown that a substantial decrease in cytoplasmic [K+] can occur during cell apoptosis [39,40]. Barbiero et al. [41] used the K+-sensitive fluorescent dye PBFI to estimate that the [K+]i in apoptotic mouse L fibro-
blast cells falls below 50 mM. Also, measurements performed on lymphoma cells during dexamethasone-induced apoptosis also confirmed early K+ loss [42], which may account to 50–75% of the initial [K+]i [43,44]. These results raise the possibility that during programmed cell death the activity of the sea urchin SERCA is inhibited leading to an increase in the cytoplasmic [Ca2+]. Apoptosis was also demonstrable in sea urchin oocytes, eggs and early embryos [45,46], and is a key event during oocyte maturation in mammals [47]. Thus, maybe the K+ regulation on sea urchin SERCA activity can be involved in the programmed cell death pathway. Nevertheless, more studies in sea urchin eggs must be done and the regulation of SERCA by K+ and sulfated polysaccharides still remains an open question. In conclusion, echinoderms are the only group, so far studied, that have sulfated polysaccharides in the membrane of the ER or SR, conferring natural SERCA K+ dependence. Further studies with other organisms are necessary to elucidate the evolutionary and physiological significance of this observation. Nevertheless, our results emphasize a particular mechanism for regulation of the Ca2+ transport found in marine organisms based on the expression of a sulfated polysaccharide together with the Ca2+ ATPase. Acknowledgments We are grateful to Valdecir Suzano and Antonio Carlos Miranda for the technical assistance. We are also grateful to Antonio Galina for snorkeling and helpful to get the gametes from sea urchins. This work was supported with grants from Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). Rafael S. Aquino is a PhD fellow from CNPq. References [1] M.J. Berridge, A tale of two messengers, Nature 365 (1993) 388–389. [2] A. Galione, A. McDougall, W.B. Busa, N. Willmott, J. Gillot, M. Whitaker, Redundant mechanisms of calcium induced calcium release underlying calcium waves during fertilization of sea-urchin eggs, Science 261 (1993) 348–352. [3] A. Galione, S. Patel, G.C. Churchill, NAADP-induced calcium release in sea urchin eggs, Biol. Cell 92 (2000) 197–204. [4] T.P. Dousa, E.N. Chini, K.W. Beers, Adenine nucleotide diphosphate: emerging second messengers acting via intracellular Ca2+ release, Am. J. Phys. 271 (1996) C1007–C1024. [5] J.M. Cancela, G.C. Churchill, A. Galione, Coordination of agonist-induced Ca2+-signalling patterns by NAADP in pancreatic acinar cells, Nature 398 (1999) 74–76. [6] A.N.K. Yusufi, J. Cheng, M.A. Thompson, E.N. Chini, I.P. Grande, NAADP elicits specific microsomal Ca2+ release from mammalian cells, Biochem. J. 353 (2001) 531–536. [7] E.N. Chini, Interactions between intracellular Ca2+ stores: Ca2+ released from the NAADP pool potentiates cADPRinduced Ca2+ release, Brazil. J. Med. Biol. Res. 35 (2002) 543–547. [8] V.D. Vacquier, The isolation of intact cortical granules from sea urchin eggs: Calcium ions trigger granule discharge, Dev. Biol. 43 (1975) 62–74. [9] R.S. Zucker, R.A. Steinhardt, Prevention of the cortical reaction in fertilized sea urchin eggs by injection of calcium-chelating ligands, Biochim. Biophys. Acta 541 (1978) 459–466.
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