Despite the fact that biomineralization is a fundamental process that occurs during the development of both vertebrates and invertebrates, there is still a great deal of uncertainty regarding basic aspects of this process (Decker and Lennarz, 1988; Lowenstam and Weiner, 1989; Simkiss and Wilbur, 1989). The presence of an organic matrix appears to be a prerequisite step for the formation and growth of biominerals. This organic matrix is thought to play a key role in crystal nucleation and growth as well as in the control of crystal type, size and orientation (Weiner and Addadi, 1991; Falini et al. 1996; Belcher et al. 1996); however, its role in
the regulation of skeletal structure formation is still poorly understood.
Scleractinian corals are among the major calcium carbonate producers (Barnes and Chalker, 1990). As in all other biominerals, the skeleton contains an organic fraction first identified by Goreau (1956 cited in Goreau, 1959), who demonstrated the presence of mucopolysaccharides using histochemical methods. Coral skeleton also contains proteins rich in acidic amino acids which can represent almost half of the protein, ranging from 30 to 50 mol % (Wainwright, 1963; Young, 1971; Mitterer, 1978; Constantz and Weiner, 1988; Printed in Great Britain © The Company of Biologists Limited 1998
JEB1423
The kinetics of organic matrix biosynthesis and incorporation into scleractinian coral skeleton was studied using microcolonies of Stylophora pistillata. [14C]Aspartic acid was used to label the organic matrix since this acidic amino acid can represent up to 50 mol % of organic matrix proteins. External aspartate was rapidly incorporated into tissue protein without any detectable lag phase, suggesting either a small intracellular pool of aspartic acid or a pool with a fast turn-over rate. The incorporation of 14C-labelled macromolecules into the skeleton was linear over time, after an initial delay of 20 min. Rates of calcification, measured by the incorporation of 45Ca into the skeleton, and of organic matrix biosynthesis and incorporation into the skeleton were constant. Inhibition of calcification by the Ca2+ channel inhibitor verapamil reduced the incorporation of organic matrix proteins into the skeleton. Similarly, organic matrix incorporation into the skeleton, but not protein synthesis for incorporation into the tissue compartment, was dependent on the state of polymerization of both actin and tubulin, as shown by the sensitivity of this process to cytochalasin B and colchicin. These drugs may inhibit exocytosis of organic matrix proteins into the subcalicoblastic space. Finally, inhibition
of protein synthesis by emetin or cycloheximide and inhibition of N-glycosylation by tunicamycin reduced both the incorporation of macromolecules into the skeleton and the rate of calcification. This suggests that organic matrix biosynthesis and its migration towards the site of calcification may be a prerequisite step in the calcification process. On the basis of these results, we investigated the effects of tributyltin (TBT), a component of antifouling painting known to interfere with biomineralization processes. Our results have shown that this xenobiotic significantly inhibits protein synthesis and the subsequent incorporation of protein into coral skeleton. This effect was correlated with a reduction in the rate of calcification. Protein synthesis was shown to be the parameter most sensitive to TBT (IC50=0.2µmol l−1), followed by aspartic acid uptake by coral tissue (IC50=0.6µmol l−1), skeletogenesis (IC50=3µmol l−1) and Ca2+ uptake by coral tissue (IC50=20µmol l−1). These results suggest that the mode of action of TBT on calcification may be the inhibition of organic matrix biosynthesis.
Key words: anthozoa, coral, biomineralization, organic matrix synthesis, organotin, TBT, tributyltin, Stylophora pistillata.
Summary
Introduction
ORGANIC MATRIX SYNTHESIS IN THE SCLERACTINIAN CORAL STYLOPHORA
PISTILLATA: ROLE IN BIOMINERALIZATION AND POTENTIAL TARGET OF THE
ORGANOTIN TRIBUTYLTIN
DENIS ALLEMAND1,*, ÉRIC TAMBUTTÉ1,2, JEAN-PIERRE GIRARD3 ANDJEAN JAUBERT1
1Observatoire Océanologique Européen, Centre Scientifique de Monaco, Avenue Saint Martin, MC-98000 Monaco, Principality of Monaco, 2Commissariat à l’Énergie Atomique – LDG, BP 12, F-91680 Bruyêres-Le-Châtel Cedex, France and 3Laboratoire de Physiologie et Toxicologie Environnementales, Faculté des Sciences, Parc Valrose,
F-06108 Nice Cedex, France
*e-mail: allemand@unice.fr
Cuif and Gautret, 1995; Dauphin and Cuif, 1997). The presence of calcium-binding phospholipids (Isa and Okazaki, 1987) and glycoproteins (Constantz and Weiner, 1988) has also been reported.
Although the presence and composition of the organic matrix are documented in scleractinian corals, information regarding its synthesis and role in skeleton formation is still scarce. By incubating corals in Na214CO3-labelled sea water, Muscatine and Cernichiari (1969) and Young et al. (1971) detected 14C-labelled molecules in the skeletal matrix of the coral Pocillopora damicornis, suggesting that part of the organic matrix is derived from algal photosynthetic products. However, by using 14C-labelled food, Pearse (1971) showed that some of the 14C derived from the food was also incorporated into the organic matrix of the skeleton. Previously, however, no attempts to follow organic matrix synthesis by direct labelling of precursor amino acids have been made in scleractinian corals.
Biosynthesis of proteins and N-linked glycoproteins is a prerequisite for sea urchin spicule formation (Decker and Lennarz, 1988; Mintz et al. 1981). Selective inhibition of collagen synthesis by actinomycin D generates plutei without spicules (Gould and Benson, 1978) and inhibits in vitro spiculogenesis by cultured primary mesenchyme cells (Mintz
et al. 1981). Similar experiments performed on scleractinian
corals by Young (1973) were inconclusive.
The present study was undertaken to determine the kinetics of organic matrix synthesis and deposition in coral skeleton and to investigate the role of the organic matrix in the regulation of skeleton formation. For this purpose, we followed a compartmental approach similar to our recent study of Ca2+ pathways related to calcification of the scleractinian coral Stylophora pistillata (Tambutté et al. 1996). Since acidic amino acids are the most abundant amino acids in the organic matrix of scleractinian corals (Young, 1971; Mitterer, 1978; Constantz and Weiner, 1988; Cuif and Gautret, 1995; Dauphin and Cuif, 1997), we used 14 C-labelled aspartic acid as a tracer according to Kingsley and Watabe (1984) and Allemand and Bénazet-Tambutté (1996) in addition to specific inhibitors of cellular protein synthesis and exocytotic pathways.
As early as 1974, organostannic compounds such as tributyltin (TBT), which are still used in antifouling paints, were known to alter oyster calcification (Alzieu et al. 1980, 1982; Héral et al. 1981; Rodriguez and Lopez, 1983). Although these compounds interfere with cellular Ca2+ homeostasis (Reader et al. 1993; Girard et al. 1997), the mechanisms of their action on calcification remain unknown (Alzieu, 1991). A possible target of these compounds was thought to be the organic matrix since Krampitz et al. (1983) demonstrated that the gelatinous substance contained in the chambers of malformed oysters differed from the organic matrix of healthy individuals by having a lower proportion of aspartic acid. To investigate the mechanism of action of TBT on the calcification process, we therefore studied its effects on organic matrix synthesis and coral calcification.
Materials and methods
Biological material
Biological materials used for the present study are cloned microcolonies of the coral Stylophora pistillata (Esper) developed in the laboratory from fragments (6–10 mm long) of terminal portions of branches collected from a parent colony and placed on a nylon net (1 mm×1 mm mesh) as described by Tambutté et al. (1995, 1996). After approximately 1 month, the coral fragments became entirely covered with new tissues. Parent colonies were collected at a depth of 5 m in front of the Marine Science Station, Gulf of Aqaba, Jordan. They were packed into plastic bags and transported under humid conditions to the Oceanographic Museum of Monaco. Once in the laboratory, they were stored in an aquarium (300 l) supplied with Mediterranean sea water (exchange rate 2 % h−1) heated to 26±0.1 °C (mean ± S.D.) and illuminated with a constant irradiance of 175µmol m−2s−1 using metal halide lamps (Philips HQI-TS, 400 W) on a 12 h:12 h L:D photoperiod. Microcolonies were kept in the same conditions of light and temperature as the parent colonies.
Measurement of total 14C-labelled aspartic acid absorbed into the tissue and incorporated into the skeleton Incorporation of labelled aspartic acid was adapted from the protocol of Al-Moghrabi et al. (1993) for valine uptake and of Tambutté et al. (1995, 1996) for 45Ca uptake. Microcolonies were placed in plastic holders and incubated for periods varying from 30 min to 5 h in beakers containing 6 ml of sea water labelled with 30 kBq of [14C]aspartic acid (NEN). A sample of sea water (100µl) was removed from the incubation medium for the determination of the external radioactivity. Unless otherwise specified, the external concentration was adjusted to 0.5µmol l−1 with unlabelled aspartic acid. Water motion was provided by a small stirring bar. The stress of handling was limited by keeping exposures to air to less than 5 s. Three replicate incubations were carried out for each experiment under light and temperature conditions similar to those of the culture aquarium.
At the end of the incubation period, each holder and its microcolony was washed for 20 s by complete immersion in a beaker containing 600 ml of filtered sea water. Five successive rinsings with 5 ml of an ice-cold glycine-rich Ca2+ medium (50 mmol l−1CaCl
2, 950 mmol l−1glycine, pH adjusted to 8.2 at 2 °C) were then completed to block any further uptake of [14C]aspartic acid. The total duration of the rinsing procedure was less than 1 min. Preliminary experiments had shown that additional washing was unnecessary.
1.5 ml of 12 mol l−1HCl overnight. Radioactivity was counted in 4 ml of Luma-gel (Packard) using a liquid scintillation counter (Tricarb 1600 CA, Packard).
Measurement of aspartic acid incorporation into tissue proteins
To determine the amount of [14C]aspartic acid incorporated into tissue proteins, radioactive free aspartic acid must be eliminated. For this purpose, the NaOH-soluble tissue fraction was dialyzed against 600 ml of 0.5 mol l−1 NaOH using Spectra/Por dialyzing membrane (molecular mass cut-off 1 kDa). Preliminary experiments showed that a dialysis time of 24 h eliminated free [14C]aspartic acid. Free [14C]aspartic acid was calculated as the difference between total [14C]aspartic acid content and [14C]aspartic acid incorporated into protein.
Extraction of organic matrix
Skeletal organic matrix was isolated according to Allemand
et al. (1994). Briefly, the labelled skeleton was rinsed in
distilled water, oven-dried, weighed and powdered in an agate mortar. The skeleton then was demineralized by titration with acetic acid, reaching an end-point of pH 4.0 (Cuif et al. 1989). To remove salts, the solution, which contained both soluble and insoluble matrix, was ultrafiltered with a 1 kDa (Amicon YM2) cut-off membrane under a nitrogen pressure of 506.6 kPa until no calcium could be detected by flame photometry (Eppendorf). Both organic (ultrafiltrate) and mineral (rinsing solution) fractions were sampled for radioactivity.
Measurement of 45Ca incorporation into the skeleton Calcification rate was measured using the protocol of Tambutté et al. (1995, 1996). Briefly, the microcolonies were placed in plastic holders and incubated in 6 ml beakers containing 240 kBq of 45Ca (as CaCl
2, 1.38 MBq ml−1, New England Nuclear) dissolved in sea water filtered using 0.45µm Millipore membranes (FSW). At the end of the labelling period, the microcolonies were processed as described above with a 30 min efflux step as the only difference (Tambutté et
al. 1995, 1996).
Media and chemicals
Sea water (SW) was filtered through a 0.45µm Millipore membrane (FSW). Protein concentrations were measured according to the method of Lowry et al. (1951) using an autoanalyzer (Alliance Instruments) with bovine serum albumin as the standard.
Unless otherwise specified, all chemicals were obtained from Sigma and were of analytical grade. The Ca2+channel inhibitor verapamil was dissolved in dimethyl sulphoxide (DMSO) and used at a final concentration of 100µmol l−1. Cytochalasin B and colchicine, inhibitors of microfilament and microtubule polymerization, respectively, were dissolved in DMSO and used at a final concentration of 20µmol l−1 (cytochalasin) and 250µmol l−1 (colchicine). Emetin and cycloheximide, inhibitors of protein synthesis, were dissolved in ethanol and used at a final concentration of 100µmol l−1.
Tunicamycin, an inhibitor of the synthesis of N-linked glycoproteins, was dissolved in hot methanol and used at a final concentration of 0.5µg ml−1. TBT was diluted in ethanol and used at final concentrations of 1–10µmol l−1. The final solvent concentration never exceeded 1 % (v/v). Preliminary experiments showed that this concentration of solvent had no effect on Ca2+or aspartic acid uptake (results not shown). The inhibitors were added simultaneously with [14C]aspartic acid and incubated for 2 h.
Statistical analysis and curve fitting
For [14C]aspartic acid experiments, the results are expressed as disints min−1mg−1tissue protein (disints min−1mg−1protein) or as a fraction of the total radioactivity initially present in the external medium (expressed as ‰) related to the protein content of the sample. For [14C]aspartic acid concentration-dependence experiments, the total radioactivity initially present in the external medium was expressed with respect to the initial aspartic acid concentration. For 45Ca experiments, results are expressed as nmol Ca mg−1tissue protein.
Exponential (y=a+be−cx) and linear (y=a+bx) functions were fitted to the experimental data using the Igor data analysis package (Wave Metrics, Inc.). Results are presented as mean ± S.D. of at least three measurements. Student’s t-tests were used to evaluate differences between means. Differences with
P<0.05 were considered statistically significant.
Results
Time course of [14C]aspartic acid uptake and incorporation
into tissue and skeletal proteins
Fig. 1A shows the time course of [14C]aspartic acid uptake into the different compartments of coral microcolonies. While the rate of increase of total and free [14C]aspartic acid incorporation rapidly became saturated, that of [14C]aspartic acid incorporation into tissue proteins was linear up to at least 5 h. At this time, approximately 30 % of the initial amount of external [14C]aspartic acid had been absorbed by the microcolony. The distribution in the tissue of [14C]aspartic acid was approximately 80 % as free [14C]aspartic acid and 20 % incorporated into tissue protein. No lag period was detected before the beginning of [14C]aspartic acid incorporation into tissue proteins, suggesting that the intracellular pool of this amino acid is small. Incorporation of [14C]aspartic acid into coral skeleton is shown in Fig. 1B. The rate of incorporation was linear and started after a lag period of approximately 20 min. Expressed as a percentage of total [14C]aspartic acid absorbed by the coral microcolony, incorporation into the skeleton displayed a linear pattern and did not saturate up to 5 h (Fig. 1C), indicating that incorporation in this compartment is cumulative. In contrast, [14C]aspartic acid incorporation into tissue proteins saturated rapidly.
the incorporation of labelled organic macromolecules and not from the incorporation of inorganic carbon through metabolization of [14C]aspartic acid and recycling via respiratory 14CO
2.
Effects of inhibitors of Ca2+transport, protein synthesis and
N-glycosylation
To determine the role of organic matrix synthesis on the
control of the biomineralization process in corals, we tested the effects of various inhibitors known to interfere with either Ca2+ transport or protein synthesis (Fig. 2). None of the inhibitors tested altered cellular uptake of [14C]aspartic acid (results not shown). Previous work from our laboratory showed that, at 100 µmol l−1, the Ca2+channel inhibitor verapamil inhibited 90 % of 45Ca incorporation into coral skeleton (Tambutté et al. 1996). The same concentration of verapamil had a small but insignificant effect on [14C]aspartic acid incorporation into tissue protein and inhibited 14C-labelled protein incorporation into the skeleton by 50 %. Tambutté et al. (1996) showed that 45Ca incorporation into coral skeleton was sensitive to
inhibitors of cytoskeleton polymerization. A mixture of cytochalasin B and colchicine inhibited 45Ca incorporation into
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Uptake of [
14C]aspartic acid (‰ RAV mg −1 protein)
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䊊
(% mg
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Incorporation of [
14
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(% mg
−1 protein)
A Fig. 1. Time course of [14C]aspartic acid uptake and incorporation
into coral tissue (A,C) and skeletal organic matrix (B,C). (A) Total [14C]aspartic acid (䊉), free [14C]aspartic acid (*) and [14C]aspartic
acid incorporated into proteins (䊊) of coral tissue. (B) [14
C]Aspartic-acid-labelled protein incorporation into skeletal organic matrix. Results are expressed as the fraction of total radioactivity (RAV) initially present in the external medium (expressed as ‰) with respect to the protein content of the sample. (C) Time course of [14C]aspartic acid incorporation into skeletal organic matrix (left
axis, 䊏) and into tissue proteins (right axis, 䊊) expressed as a percentage of total [14C]aspartic acid absorbed by the microcolony.
Values are means ±S.D. (N=3); some errors bars have been omitted for clarity.
Control Vp CB+Col Em CH Tm
0 10 20 30 40 50 60 70 80 90 100
*
*
[image:4.609.55.286.67.634.2]Incorporation (% of control value)
Fig. 2. Effect of inhibitors on [14C]aspartic acid incorporation into
tissue proteins (filled columns) and skeletal proteins (hatched columns) and on 45Ca incorporation into skeleton (open columns).
Incorporation is expressed as a percentage of the value measured in the absence of inhibitor during a 2 h incubation period. The effects of a Ca2+channel inhibitor, verapamil (Vp, 100µmol l−1), a mixture of
inhibitors of the polymerization of actin and tubulin, cytochalasin B (CB, 20µmol l−1) and colchicine (Col, 250µmol l−1), the protein
synthesis inhibitors emetin (Em, 100µmol l−1) and cycloheximide
(CH, 100µmol l−1), and tunicamycin (Tm, 0.5µg ml−1), an inhibitor
of the synthesis of glycoproteins, are shown. [14C]Aspartic acid and 45Ca incubations lasted for 2 h. Values are means + S.D. (N=3).
Values marked with an asterisk are not significantly different from control values.
B
[image:4.609.331.552.242.385.2]coral skeleton by 80 %, while tissue protein synthesis was inhibited by 25 % and [14C]aspartic acid incorporation into the
skeleton by approximately 70 %. Two well-known protein synthesis inhibitors, emetin and cycloheximide, inhibited protein synthesis by approximately 60 % and 85 %, respectively, and [14C]aspartic acid incorporation into skeletal matrix by approximately 50 %. Simultaneously, they inhibited 45Ca incorporation into coral skeleton by approximately 50 %.
In the presence of tunicamycin, an inhibitor of the synthesis of N-linked glycoproteins (Schneider et al. 1978), protein synthesis was not significantly depressed, while the incorporation of 14C-labelled protein into the skeletal matrix was reduced by approximately 70 % and calcification by approximately 50 %.
Effects of tributyltin (TBT) on organic matrix synthesis and coral calcification
To determine the mode of action of TBT, we tested its effect on [14C]aspartic acid and 45Ca uptake, tissue protein synthesis, the incorporation of protein into the skeletal organic matrix and 45Ca deposition into the mineral skeletal fraction. Fig. 3 shows
the kinetics of action of 10µmol l−1TBT on these variables compared with controls. TBT reduced the synthesis of proteins and their subsequent incorporation into skeleton and the calcification process as measured by 45Ca incorporation into skeleton. A dose–response curve for the effects of TBT is shown in Fig. 4. The process most sensitive to TBT was protein synthesis (IC50=0.2µmol l−1), followed by [14C]aspartic acid uptake by coral tissue (IC
50=0.6µmol l−1), skeletogenesis (IC50=3µmol l−1) and 45Ca uptake by coral tissue (IC50=20µmol l−1). To compare the action of TBT with that of the protein synthesis inhibitor cycloheximide, we tested the effect of these inhibitors, either alone or in combination, on protein synthesis and incorporation into skeleton and calcification. TBT was used at 1µmol l−1, a concentration that does not cause complete inhibition. Table 1 shows that their
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Incorporation of [
45Ca]
(nmol Ca
2+
mg
−1 protein)
[image:5.609.86.255.73.585.2]Time (h)
Fig. 3. Effect of tributyltin (TBT) on [14C]aspartic acid incorporation
into tissue proteins (A) and skeletal proteins (B) and 45Ca
incorporation into coral skeleton (C). 䊉, control; 䊊, 10µmol l−1
TBT. TBT was added at time zero. The results are expressed as fraction of the total radioactivity (RAV) initially present in the external medium (expressed as ‰) with respect to the protein
content of the sample. Values are means ±S.D. (N=3). The effect of
TBT became significantly different from the control value at 30 min in all cases.
100
80
60
40
20
0
Uptake (% of control value)
10-11 10−10 10−9 10−8 10−7 10−6 10−5 10−4 10−3 [TBT] (mol l−1)
0
Fig. 4. Dose–response curve for the effect of TBT on [14C]aspartic
acid uptake by coral tissue (*) and incorporation into tissue proteins
(+) and on 45Ca absorption by coral tissue (䊏) and incorporation into
the skeleton (䊉). Arrows indicate the concentration causing 50 %
inhibition (IC50). Results are means expressed as a percentage of
[image:5.609.313.567.74.235.2]effect was in no case significantly additive, suggesting a common target.
Discussion
The synthesis of organic matrix necessitates a heterotrophic source of amino acid
While the presence of organic matrix in the skeleton of scleractinian corals is now widely documented (Goreau, 1956 cited in Goreau, 1959; Wainwright, 1963; Young, 1971; Young et al. 1971; Mitterer, 1978; Isa and Okazaki, 1987; Constantz and Weiner, 1988; Cuif and Gautret, 1995; Dauphin and Cuif, 1997), the dynamics of its synthesis and deposition remain virtually unknown. Several authors have shown that the photosynthetic products of zooxanthellae may be used as precursors for skeletal organic matrix biosynthesis (Muscatine and Cernichiari, 1969; Pearse and Muscatine, 1971; Young et
al. 1971; Barnes and Crossland, 1978); in addition, Pearse
(1971) showed that heterotrophic sources may be also used. Our results confirm that, in a symbiotic coral, a heterotrophic source of nutrient may be used as precursor for organic matrix biosynthesis, as is known in some non-symbiotic marine invertebrates. For example, Ellis and Winter (1967) and Pucci-Minafra et al. (1972) showed that sea urchin larvae of, respectively, Arbacia punctulata and Paracentrotus lividus incorporate proline as protein-bound hydroxyproline into spicules. Similarly, leucine or aspartic acid incorporation into octocoral skeleton has been demonstated by several authors (Leversee, 1980; Kingsley and Watabe, 1984; Allemand and Bénazet-Tambutté, 1996).
The presence of acidic amino acids is a characteristic of the organic matrix of mineralized tissues (Wheeler and Sikes, 1984; Mitterer, 1986; Weiner and Addadi, 1991). While the aspartic acid content of macromolecules is usually of the order of a few moles per cent (Lehninger, 1982), that of proteins present in biominerals may be as high as 50 mol % (Lowenstam
and Weiner, 1989). The time course of [14C]aspartic acid incorporation into tissue macromolecules, as shown in Fig. 1A, has no detectable lag period, suggesting the presence of either a small intracellular pool, mainly supplied by uptake from the external medium, and/or a pool with a fast turnover rate, supplied simultaneously by cellular biosynthesis and external uptake. In any case, this result is quite surprising because it suggests, at least in our experimental conditions, the need for a constant supply of external origin for this amino acid, which is known to be important in the control of the calcification process. Such a dependence may be a limiting factor in skeletogenesis unless, in the absence of an external source of aspartic acid, autotrophic aspartic acid could be used. However, by using Na214CO3in the absence of added external aspartic acid, Young et al. (1971) demonstrated that, even under these conditions, aspartic acid represented only 1.8 % of the total labelled amino acid, in spite of its high proportion in organic matrix proteins, and therefore did not originate from the photosynthetic products of zooxanthellae. This supports the major involvement of heterotrophic sources for aspartic acid incorporation into organic matrix macromolecules.
There was a lag period of approximately 20 min before the beginning of incorporation of labelled molecules into the skeleton. Such a lag period corresponds to the time required for protein synthesis and its transport to the site of skeletogenesis. In the gorgonian Leptogorgia virgulata, Kingsley and Watabe (1984) found that the entire process of matrix synthesis and transport lasts approximately 2 h. The shorter time measured in Stylophora pistillata can be related to its high calcification rate (Tambutté et al. 1996). This time is, however, comparable to the time required for the synthesis of cartilage proteins in mammals and their subsequent incorporation, i.e. 20–25 min (Paulsson et al. 1983). It is also similar to the time from the introduction of precursor labelled amino acids into the extracellular medium to the export of labelled protein when determined for typical secretory proteins. For example, the lag period for [3H]leucine labelling of serum albumin has been determined to be 19–21 min in isolated hepatocytes and 22–26 min in perfused rat liver (Feldhoff et al. 1977).
The synthesis of tissue protein controls the formation of calcified skeleton
[image:6.609.40.293.138.221.2]Taken together, the results of the present study suggest that organic matrix biosynthesis, rather than calcium deposition, may be the limiting factor controlling skeletogenesis, as suggested previously by Wainwright (1963) in hexacorallians and by Kingsley and Watabe (1984) in octocorallians. Calcification in scleractinian corals was dependent on Ca2+ entry into calicoblastic cells via a Ca2+channel sensitive to verapamil, a blocker that also affects the incorporation of macromolecules into the skeleton (Fig. 2). This suggests a link between Ca2+ deposition and organic matrix exocytosis and/or incorporation either to maintain a ratio between protein and Ca2+or to allow protein exocytosis to control the precipitation of calcium carbonate.
Table 1. Comparative effects of tributyltin (TBT, 1µmol l−1) and cycloheximide (100µmol l−1) on [14C]aspartic acid incorporation into tissue protein, incorporation of labelled
macromolecules into coral skeleton and calcification as measured by incorporation of 45Ca for 1 h
Incorporation of Protein macromolecules
Experimental synthesis into skeleton Calcification
condition (%) (%) (%)
TBT 77±4 55±7 25±9
Cycloheximide 84±7 49±8 45±5
TBT + cycloheximide 81±5 56±4 41±6
Results are given as a percentage of control values (without inhibitors).
Values are means ±S.D. (N=3).
Rates of incorporation of 14C-labelled macromolecules into the organic matrix and calcification are constant over the time range tested, demonstrating that the ratio Ca2+/aspartic acid is kept constant and that both organic and inorganic skeletal components are deposited simultaneously. However, they cannot be compared directly since it remains difficult to evaluate the specific radioactivity of aspartic acid in the cellular compartment. Nevertheless, by taking into account the low cellular content of aspartic acid (see above), it could be assumed that the specific radioactivity of [14C]aspartic acid in tissue is comparable to that in the external medium. This allows us to calculate a rate of 0.13 pmol aspartic acid h−1mg−1tissue protein incorporated in the skeleton, which is 4×105 times slower than the rate of calcium deposition (Tambutté et al. 1996). This gives a molar ratio of Ca2+/aspartic acid of approximately 3.8×106.
Further arguments were raised in favour of the hypothesis that the organic matrix controls of the rate of calcification by the use of specific inhibitors of protein synthesis and migration. Emetin and cycloheximide, inhibitors of protein synthesis, reduced both [14C]aspartic acid incorporation into tissue macromolecules and their subsequent incorporation into the skeleton. Simultaneously, these inhibitors reduced 45Ca deposition to a similar extent. It should be noted that this effect is not the consequence of a reduction in the rate of metabolism since these inhibitors did not affect, in the time and concentration range tested, O2production by photosynthesis or O2 consumption by respiration (P. Furla and D. Allemand, unpublished data). Therefore, this suggests that the inhibition of calcification by emetin and cycloheximide results specifically from an inhibition of the synthesis of the organic matrix. This dependence of skeletogenesis on protein synthesis observed in scleractinian corals has been demonstrated previously for spiculogenesis in the sea urchin Strongylocentrotus purpuratus (Gould and Benson, 1978; Decker and Lennarz, 1988; Dubois and Chen, 1989). Furthermore, tunicamycin, a specific inhibitor of N-glycosylation (Schneider et al. 1978), reduced the incorporation of macromolecules into coral skeleton without any detectable effect on protein synthesis. This suggests a requirement for post-translational modification of proteins (glycosylation) during skeleton formation, as has been demonstrated for spicule formation in the sea urchin
Strongylocentrotus purpuratus (Mintz et al. 1981; Decker and
Lennarz, 1988).
In Stylophora pistillata, the incorporation of organic matrix is sensitive to cytochalasin B and colchicine (70 % inhibition), suggesting a dependence on the state of polymerization of the cytoskeleton components actin and tubulin. The target of the inhibitors could be the exocytotic pathway guiding the migration of the organic matrix outwards from calicoblastic epithelial cells (Johnston, 1980). This may explain the inhibition of calcification by these drugs that Tambutté et al. (1996) previously attributed to an effect on the intracellular movement of Ca2+. In conclusion, the synthesis of proteins (glycoproteins) and their subsequent exocytotic movement control the synthesis of calcified coral structures.
Organotin inhibition of calcification occurs via inhibition of protein synthesis
The potential role of the organic matrix in the regulation of skeletogenesis prompted us to compare the action of TBT with that of inhibitors of protein synthesis. Organotin compounds are known to produce oyster shell abnormalities consisting of wafer-like chambering with the formation of an interlamellar jelly (Alzieu, 1991). The observation by Krampitz et al. (1983) that the gelatinous contents of the chambers differed from the normal organic matrix protein in having lower levels of aspartic acid, glycine and serine, and a higher level of threonine, led to the hypothesis that the target of TBT could be the organic matrix. Such involvement of the organic matrix in a perturbation of the calcification process has been demonstrated previously in epizootic diseases such as that described in the black lip pearl oyster
Pinctada margaritifera (Marin and Dauphin, 1991; Cuif and
Dauphin, 1996). The present results showed that TBT and inhibitors of protein synthesis inhibited protein synthesis, incorporation of protein into skeleton and calcification to a comparable extent.
Furthermore, the effect of TBT was not additive to the action of cycloheximide, suggesting that a common target is affected by these compounds. In addition, tissue protein synthesis shows an 80-fold higher sensitivity to TBT than Ca2+ absorption and incorporation. This result agrees with the results of Girard et al. (1997), who demonstrated that protein synthesis following egg fertilization in the sea urchin
Paracentrotus lividus is at least 100 times more sensitive to
TBT than is the Ca2+permeability of the egg membrane. Taken together, these arguments suggest that organotin inhibition of events leading to the building of coral structures results from its effects on protein synthesis.
Organic matrix biosynthesis is thus a prerequisite step in the calcification process. Heterotrophic sources of aspartic acid are rapidly incorporated into skeletal proteins. Owing to the importance of this process, the primary target of TBT in calcifying animals could be protein synthesis, whose inhibition leads to the inhibition of organic matrix formation and consequently reduced skeletogenesis.
This study was conducted as part of the O.O.E. 1995–2000 research program. It was supported by the Council of Europe (Open Partial Agreement on Major Natural and Technological Disasters) and belongs partly to the PNEM research program funded by IFREMER (95.1.450051). We thank the Marine Station of Aqaba (Jordan) for facilitating the initial collection of corals. We wish to thank A.-L. De Rosa and A. Durieux for performing preliminary experiments, and Professor M. Shick and Dr T. Tentori for their helpful comments on the manuscript. We also thank C. Emery for his technical assistance.
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![Fig. 4. Dose–response curve for the effect of TBT on [14the skeleton (inhibition (IC(+) and on C]asparticacid uptake by coral tissue (*) and incorporation into tissue proteins45Ca absorption by coral tissue (�) and incorporation into�)](https://thumb-us.123doks.com/thumbv2/123dok_us/1151404.636399/5.609.86.255.73.585/response-skeleton-inhibition-asparticacid-incorporation-proteins-absorption-incorporation.webp)
![Table 1. Comparative effects of tributyltin (TBT, 1 µmoll−1)and cycloheximide (100 µmol l−1) on [14C]aspartic acidincorporation into tissue protein, incorporation of labelledmacromolecules into coral skeleton and calcification asmeasured by incorporation of 45Ca for 1 h](https://thumb-us.123doks.com/thumbv2/123dok_us/1151404.636399/6.609.40.293.138.221/comparative-tributyltin-cycloheximide-acidincorporation-incorporation-labelledmacromolecules-calcication-incorporation.webp)
