Immediate early response of the marine sponge Suberites domuncula to heat stress: reduction of trehalose and glutathione concentrations and glutathione S-transferase activity

Journal

ELSEVIER

of Experimental Marine Biology 210 (1997) 129-141

JOURNAL OF EXPERIMENTAL MARINE BIOLOGY AND ECOLOGY

and Ecology,

Immediate early response of the marine sponge Suberites domuncula to heat stress: reduction of trehalose and glutathione concentrations and glutathione S-transferase activity Nilza Bachinski”‘b, Claudia Koziol”, Renato Batel”, Zeljka Labura’, C. Schriider”, Werner E.G. Miiller”‘”

Heinz

‘Institut fiir Physiologische Chemie, Universitiit, Duesbergweg 6, D-55099 Muinz, Germany ‘Departamento de Bioquimica, Institute de Quimica, Centro de Tecnologia, Universidade Federal do Rio de Janeiro, 21941-900 Rio de Janeiro, Brazil ‘Center for Marine Research, ‘Rudjer Boskovic’ Institute, 52210 Rovinj, Croatiu Received

13 May 1996; revised 24 July 1996; accepted

7 August

1996

Abstract The marine sponge Suberites domuncula was used to identify early markers for thermal stress. Cubes from sponges have been kept, for 30 min at 31°C (10°C higher than the ambient temperature). After this treatment the sponge cubes were kept again at 2 1°C. To demonstrate that the animals reacted to the elevated temperature, the expression of heat shock protein (HSP) was determined. Using an antibody raised against HSP70, it was found by Western blotting that the animals specifically express a 45 kDa polypeptide after heat treatment. It was shown that even after 10 min of heat treatment the steady-state concentration of trehalose drops by 40% from a base level of 13 nmol/mg protein. The activity of the trehalose-degrading enzyme, trehalase, remained unchanged. Additional early biomarkers for thermal stress include the enzyme activity of glutathione S-transferase (GST) and the concentration of glutathione (GSH). After 5-min the activity of GST decreased by 40%. Similarly, the concentration of GSH dropped by 50% after 15 to 20 min exposure. The orginal levels of the biomarkers, trehalose, GSH and GST, were reached again after a recovery period of about 180 min. By contrast, the steady-state concentration of polyphosphates did not change during heat treatment. These data show that in S. domunculu the concentrations of trehalose, GSH, and the activity of GST, are biomarkers for immediate early response towards heat stress. Copyright 0 1997 Elsevier Science B.V. All rights reserved. Keywords:

Sponge: Suberites

*Corresponding

domunculu;

Heat shock; Trehalose;

Glutathione;

author.

0022-0981/97/$17.00 Copyright PII SOO22-098 I (96)02705-O

0 1997 Elsevier Science B.V. All rights reserved

S-transferase

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N. Bachinski et al. I J. Exp. Mar. Biol. Ecol. 210 (1997) 129-141

1. Introduction Sponges (Porifera) are one of the major phyla found in the marine hard-substrate benthos, both with respect to the number of species and biomass (Sara and Vacelet, 1973). Adult specimens are sessile filter-feeders able to ingest particles of sizes between 5-50 p,m through the cells of the mesohyl and the pinacoderm; microparticles (0.3-l pm) are taken up by the cells of the choanocyte chambers (Simpson, 1984). A specimen of 1 kg of a small siliceous sponge Geodia cydonium, filters every day = 24 000 1 of seawater (Vogel, 1977). Therefore, sponges have to be provided with effective protection systems against environmental stress. Sponges are increasingly used as model organisms to study biomarkers both at the biochemical (induction of mixed function oxidases) and molecular levels (expression of genes coding for stress proteins) (Miiller, 1994; Mtiller and Miiller, 1996) e.g. with respect to heat shock response (expression of the gene coding for the heat shock protein of M, 70 000 (HSP70) as well as its protein product, HSP70) (Mtiller et al., 1995; Koziol et al., 1995), response towards detergents (Zahn et al., 1977) or heavy metals (Bate1 et al., 1993). Recently, it was shown, that sponge cells are suitable systems to detect a hitherto unrecognized group of pollutants, the inactivators of the multixenobiotic resistance pump (Kurelec et al., 1995; Miiller et al., 1996). It is the aim of the present investigation to identify immediate early markers of thermal stress in the sponge S.&rites domuncula. The study focuses on four potential defense systems; trehalose, glutathione S-transferase (GST), glutathione (GSH) and polyphosphates. The disaccharide trehalose is known to accumulate in a variety of plants and animals (Elbein, 1974). Coutinho et al. (1988) demonstrated that a mutant strain of Succharomyces cerevisiae which was unable to synthesize this disaccharide, failed to survive storage in a frozen and dried state. By contrast, cells of S. cerevisiae that accumulate trehalose during a temperature shift are able to survive otherwise lethal temperature stress (Hottinger et al., 1987; Eleutherio et al., 1993). Accumulation of trehalose is not only protective against heat stress, but also against high osmotic pressure to cells of Escherichia coli (Dinnbier et al., 1988). Trehalose breakdown yielding glucose is catalyzed by trehalase (summarized in Ribeiro et al., 1994). GSH plays a major role in several cell processes, such as drug detoxification, peroxide metabolism and amino acid transport (Meister and Anderson, 1983) and is involved in the maintenance of a wide number of physiological functions in vertebrate and invertebrate cells (Vina et al., 1986; Felton, 1995; Alcutt and Pinto, 1994). Little information on GSH concentration and distribution in marine animal species has been published (Alcutt and Pinto, 1994; Braddon et al., 1985). With respect to pollutants, an adaptive alteration of the GSH system in response to mercury and other metals in fish tissues (Romero et al., 1990) as well as in response to lead in the hard clam, Mercenaria mercenaria, has been reported (Alcutt and Pinto, 1994). GSH is used as a substrate for GST to detoxify xenobiotics via conjugation (Habig et al., 1974). GST has been proposed as a biomarker of pollution exposure in the mussel Mytilus edulis (Sheehan et al., 1995). GSTs are a family of enzymes which are involved in conjugation of xenobiotic substances as well as peroxide degradation (Mannervik et al., 1985; Meyer et

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129-141

131

al., 1991). So far, it is unknown which form(s) is predominant in S. domuncula since the substrate used in the present study (CDNB) is not specific for a particular class of GST. A further detoxification enzyme system in marine invertebrates consists of the mixed function oxidases which have been described for the mussel Mytilus edulis (Livingstone et al., 1988). Polyphosphates [poly(P)] have been reported in almost all classes of organisms studied, including bacteria, fungi, plants, insects and mammals (Kulaev, 1979; Kulaev and Vagabov, 1983; Wood and Clark, 1988). The biological function of poly(P) remains to be determined. They have been suggested to serve (i) as a source of energy, (ii) as an easily available phosphate reserve, (iii) as chelators for divalent cations, (iv) as counterions for basic amino acids in vacuoles or (v) as donors of phosphate for certain sugar kinases. More recently, it has been proposed that (vi) hydrolysis of poly(P) may provide a pH-stat mechanism to counterbalance alkaline stress (summarized in Leitao et al., 1995). In this paper we show that decreases in (i) trehalose level, (ii) GST activity as well as (iii) amount of GSH are immediate early response markers in S. domunculu for heat stress, while poly(P) concentration and trehalase activity remain unchanged in this model system during temperature shift and the subsequent recovery period.

2. Materials

and methods

2.1. Materials The monoclonal antibody (McAb) against bovine brain HSP70 (anti-HSP70 Ab; raised in mice) (H5147), HSWO from bovine brain (H1523), secondary antibodies (anti-mouse raised in goat), Na-phosphate glass (polyphosphate of an average chain length of 35 residues; S4504), 1-chloro-2,4_dinitrobenzene (CDNB) (C6396), glutathione S-transferase (G6511; from equine liver) were from Sigma (Deisenhofen, Germany) and PVDF Immobilon from Millipore (Saint-Quentin, France). 2.2. Animals Live specimens of the marine sponge Suberites domuncula (Demospongiae) were collected near Rovinj (Croatia). For adaptation the animals were kept in aquaria at 21°C for two weeks. For comparative reasons some experiments were performed with the mussel Dreissena polymorpha, the clam Corbicula jluminea and the golden ide (fish) Leuciscus idus. 2.3. Heat shock treatment The sponges were cut into cubes with 0.5-cm edges and incubated in filtered, oxygenated seawater at 21°C. In controls, the tissue samples were continously kept at 21°C. For heat shock treatment, cubes were incubated at 31°C in seawater for 0 to 30 min. Then samples were kept at 21°C for up to 180 min to identify immediate early

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response of the animals or for 12 h to identify HSP70 induction. At the end of the experiment the samples were immediately frozen in liquid nitrogen until further use. In comparison, the mussel D. polymorpha, the clam C. @minea and the golden ide (fish) L. idus were heat shocked in the same manner: 30 min at 10°C above the ambient temperature (3 1“C) and for 12 h at 2 1“C. 2.4. Sample preparation Frozen material was treated with 3 volumes of ice-cold NaC1/Pi (137 mM NaCl, 2.7 mM KCl, 8 mM Na,HPO,, 1.5 mM KH,PO,; pH 7.4), supplemented with 1 mM phenylmethylsulfonyl fluoride and 2 mM EDTA. After thawing, the sponge samples were homogenized (on ice) and centrifuged (12 000 X g; 60 min; 0°C). The supematant fraction (protein content 3 to 5 mg/ml) was collected and kept at - 20°C until use (two to three wk). 2.5. Enzyme assays 2.5.1. Trehalase The reaction was carried out in a final volume of 0.2 ml in 50 mM Tris-maleate buffer (pH 6.0) containing 500 nmol of trehalose and 50 ~1 of sponge extract. The reaction mixture was incubated at 30°C for 60 min and terminated by heating at 100°C for 3 min. Subsequently, 0.3 ml of the Tris-maleate buffer was added and glucose was determined applying the glucose-oxidase-peroxidase method (Zimmermann and Eaton, 1974). One unit of trehalase activity is defined as the amount of enzyme that catalyzes the conversion of 1 pmol of glucose in 1 min under the assay conditions used. 2.5.2. Glutathione S-transferase GST activity was determined with CDNB as substrate according to Habig et al. (1974) at 30°C. Briefly, the assay mixture contained in a 0.1 M K-phosphate buffer (pH 7.0) 1 mM GSH, 50 p,M CDNB and 50 pl of sponge extract in a total volume of 0.2 ml. The assays were linear for at least 10 min. The difference in the extinction between the assays with sponge extracts and the controls (without enzyme) was determined. The molecular extinction coefficient for CDNB (AE: 9.6 mM’ X cm-‘) was used to calculate the amount of substrate converted. The determinations were performed at a wavelength of 340 nm. The enzyme assays were performed under conditions which are linear with respect to protein content and time. 2.6. Western blotting Gel electrophoresis of the protein extracts was performed in 10% polyacrylamide gels containing 0.1% NaDodSO, (PAGE) according to Laemmli (1970). For calibration, the size markers ovalbumin (M, 45 000), bovine serum albumin (M, 66 000) and phosphorylase b (M, 97 000) were used. Protein samples were subjected to gel electrophoresis in

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133

the presence of 2-mercaptoethanol. Semi-dry electro-transfer was performed according to Kyhse-Andersen (1984) onto PVDF-Immobilon membranes. The membranes were blocked and incubated with McAb anti-HSP70 Ab (diluted 1: 1000) for 1.5 h at room temperature (Bachmann et al., 1986). After blocking the membranes with 5% bovine serum albumin, the immune complexes were visualized by incubation with anti-mouse IgG (alkaline phosphatase conjugated) followed by staining with bromochloroindolyl phosphate-nitro blue tetrazolium. 2.7. Extraction

and determination

of polyphosphates

contents

Poly(P) was extracted from the tissues as described (Leitao et al., 1995; Clark et al., short-chain 1986). Following this procedure ‘step 1 extracts’ contain acid-soluble poly(P), ‘step 2 extracts’ represent the easily extractable, ‘soluble’ portion of long-chain poly(P) and ‘step 3 extracts’ the remaining long-chain poly(P) (Clark et al., 1986). Protein was removed from the extracts obtained in steps 2 and 3 by one extraction with phenol-chloroform (1: 1, v/v), followed by three successive extractions with chloroform. The content of long-chain poly(P) of the ‘step 2-’ and ‘step 3 extracts’ was estimated by measuring the change in the absorption spectrum of toluidine blue at 530 nm and 630 nm due to the metachromatic effect produced by polyphosphate, as described (Leitao et al., 1995), with a Beckman DU-64 spectrophotometer. A calibration curve was used that was obtained by determination of the ratio of the absorbance values at 530 nm and those at 630 nm for various amounts of the poly(P) with poly(P),, as standard. 2.8. Analytical

methods

Trehalose was extracted and quantified from 300 mg of sponge tissue with 0.5 M trichloroacetic acid at 0°C as described (Brin, 1966). GSH was measured with CDNB as substrate and glutathione-S-transferase from equine liver as enzyme (Brigelius et al., 1983). Protein was determined as described by Bradford (1976) with bovine serum albumin as standard. 2.9. Statistics The results were analysed

by paired Student’s

r-test (Sachs,

1984).

3. Results 3.1. Expression

of the HSP70 in S. domuncula

after thermal stress

To induce expression of HSP70, sponge cubes were heat treated for 30 min at 10°C above the ambient temperature, followed by a 12 h ‘recovery’ phase at ambient temperature. The expression of HSP70 protein in S. domunculu was monitored by Western blotting. For these experiments an anti-HSP70 Ab which has previously been

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found to immunologically cross-react with the HSP70 protein from Geodiu cydonium was used (Koziol et al., 1995). Control samples or preparations from S. domuncuZa after thermal stress were isolated, electrophoretically separated (Fig. 1 A) and subjected to Western blotting (Fig. 1 B). In samples obtained from heat-treated sponge, a strong expression of HSP (Fig. 1 B, lane a (sample from animals maintained at ambient temperature) and lane b [after thermal stress)) was obtained; however, unexpectedly the mass of the polypeptide that was recognized was not 70 kDa but 45 kDa. If the animals were heat treated for only 15 min, instead of 30 min, the extent of HSP expression was lower (lane c). To rule out the possibility that this anti-HSP70 Ab-reaction was nonspecific, two series of experiments were performed. Firstly, the anti-HSP70 Ab was adsorbed with purified HSP70 from bovine brain prior to Western blotting. After this pretreatment no immunoreaction occurred. Secondly, in a comparative approach this McAb was used in studies with distantly related species; the mussel D. polymorpha, the clam C. jluminea and the golden ide (fish) L. idus. As summarized in Fig. 1 B, the fish extract (lane d versus e (control)) showed expression of both HSP70 as well as HSP72 after heat treatment; this double band is characteristic for inducible HSPs (Sanders, 1990). The clam also reacted with expression of HSP but, as with S. domunculu, of a mass of 45

6

A a

b

abcdefgh kDa -97 -66 -

+

-+++-+-+

-45

hs

Fig. 1. Expression of HSP in the marine sponge S. domuncula by Western blot analysis after thermal stress. The animals remained either the entire incubation period at the ambient temperature of 21°C [hs: - ] or were heat stressed [hs: + ] as described under Section 2. A: The proteins (5 pg of protein per lane) from nonstressed- (lane a) or stressed sponges (lane b) were size-separated by PAGE using a 10% polyacrylamide gel and stained with Coomassie brilliant blue. B: Identically, the proteins from the sponge S. domunculu (lane a, b; heat treated as described above for 30 min) and lane c, heat treated only for 15 min), the fish L. idus (lane d and e), the mussel D. polymorpha (lane f) and the clam C. jluminea (lane g and h) were transferred to Immobilon sheets, and incubated with McAb anti-HSP70 Ab. The immunocomplexes were visualized by labelled secondary antibodies.

N. Bachinski et al. I .I. Exp. Mar. Biol. Ecol. 210 (1997) 129-141

kDa (lanes g (control) and h (heat-treated)). The mussel did not synthesize under the incubation and detection conditions used (lane f). 3.2. Alteration

of trehalose

level and trehalase

135

any HSP

activity during heat shock

Sponge cubes were treated for 0 to 30 min at 10°C above ambient temperature and were subsequently maintained again at the ambient temperature. Trehalase activity was determined. Neither during heat stress nor during the following recovery were significant changes measured; the activity in the controls, extracts from nonstressed tissue, was 1.8kO.5 mU/mg protein (Fig. 2). In parallel, trehalose concentration in the samples was determined. The results revealed that during the initial period of thermal stress the level of trehalose dropped significantly (P
oJ, 0

10

I 20

I 30

/h-&O 120

180

Incubationtime (min) Fig. 2. Effect of thermal stress on trehalose concentration (0) and trehalase activity (x) in cubes from S. domunculu. The cubes were treated for the first 30 min at 31°C and subsequently maintained again at 21°C. the ambient temperature. After the indicated incubation period, analyses were performed. The values are means (%SD) from ten parallel experiments.

136

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0

et al. / J. Exp. Mar. Biol. Ecol. 210 (1997) 129%14I

IO

20

30

120

180

Incubationtime (min) Fig. 3. Effect of heat stress on the activity of glutathione S-transferase. Cubes from S. domuncula remained either at ambient temperature of 21°C (0) or were heat treated for the initial 30 min period and subsequently maintained again at 21°C (0). At the time indicated, the enzyme activity was determined. The mean values (+SD) from 10 parallel experiments are given.

In parallel the effect of heat treatment on the concentration of GSH was determined (Fig. 4). Already 15 min after heat stress the amount of GSH dropped from 21.822.2 nmol/mg to 12.42 1.7 nmol/mg (P < O.OOl>, a level which remained stable even after prolonged heat treatment. This finding suggests that any lower value of GSH would be incompatible with life of the animals. In the recovery phase, control values are reached again.

251

0

10

20

30

120

180

Incubationtime (min) Fig. 4. Change of GSH concentration in S. domuncula tissue samples after treatment at elevated temperature and subsequent incubation at recovery temperature for up to 3 h. Mean values (tSD) are given; n = 10.

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3 :

01, 0

I,

I

I

1

10

20

30

rr,120

180

Incubationtime (min) Fig. 5. Effect of heat treatment on poly(P) concentration in cubes from S. domuncula. At different times, samples were taken and analysed for poly(P) content. MeanskSD from 10 experiments are given. Both the easily extractable, ‘soluble’ portion of long-chain poly(P) (0) and the remaining long-chain poly(P) fractions (x) were analysed.

3.4. Poly(P)

content in tissue samples from S. domuncula

in response to heat stress

The two fractions of poly(P), the easily extractable, ‘soluble’ portion of long-chain poly(P) and the remaining long-chain poly(P) fractions were analysed in tissue samples from controls and from heat treated animals. In the nontreated controls, the concentration of ‘soluble’ portion poly(P) was determined to be 428538 ng/mg, and in the remaining long-chain poly(P) fraction, 313?28 ng/mg protein. During heat stress and the subsequent recovery phase these levels remained unchanged (Fig. 5).

4. Discussion Sponges (Porifera) represent the oldest multicellular animal phylum, which diverged 800 million years ago from a common ancestor for all Metazoa (Miiller, 1995). Therefore, it can be expected that these animals are provided with protection system(s) against environmental stress which are likewise phylogenetically ancient. One of the most ubiquitously existing protection systems, in pro- and eukaryotes, are the stress proteins also termed heat shock proteins (HSPs). From the marine sponge Geodia cydonium two of these have been cloned, HSP70 (Koziol et al., 1995) and ubiquitin (Pfeifer et al., 1993); both of which are inducible genes which respond to environmental stress. The stress response in these systems requires a period of more than 1 h (Sanders, 1990). In the present study, it was investigated whether sponges have a protection system that acts immediately after heat stress. At first it had to be established that the sponge species selected, S. domuncula, reacts to thermal stress with the expression of HSPs. Applying a

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N. Bachinski et al. I J. Exp. Mar. Biol. Ecol. 210 (1997) 129-141

routine temperature stress schedule it was observed that anti-HSP70 Ab reacted specifically with a polypeptide of M, 45 000. This finding is interesting with respect to future cloning investigations. Until now the HSPs have been subdivided into the following classes, HSP90, HSP70, HSP60 and HSP20-30 (Sanders, 1990). Since the same or a similar protein of 45 kDa is present also in the clam C. @minea, but not in the fish L. idus, we assume that we have identified a novel member of proteins belonging to this family of heat stress proteins. One prime candidate for immediate response is the trehalose-trehalase system (summarized in Panek, 1995). In plants, yeast and invertebrates trehalose is known to be an effective protection system during anhydrobiotic period (Panek, 1995). Now it is shown that in S. domunculu the content of trehalose dropped immediately after thermal stress. The steady-state intracellular concentration of trehalose in this sponge is 13.3 nmol/mg protein and hence approximately 50-fold lower than that in S. cerevisiue under induced conditions (Ribeiro et al., 1994). Unlike in S. cerevisiae (Ribeiro et al., 1994), trehalase activity remains unchanged in 5. domunczdu under the stress conditions used. It remains to be studied, in future, if this enzyme is induced at a later stage during stress response. The results observed with the enzyme GST immediately after thermal stress are interesting and surprising. GST is known to be an ubiquitous detoxification enzyme in metazoa and plants (surveyed in Felton, 1995). Considering the fact that GST comprises a family of enzymes (Mannervik et al., 1985; Meyer et al., 1991), it remains to be studied which form(s) of GST is (are) present in S. domunculu and affected during heat treatment. Besides its protective function against toxic xenobiotics, GST is, due to its peroxidase activity, also involved in removal of peroxides generated during oxidative stress (Felton, 1995). In S. domunculu GST has an activity of 25 nmol/min X mg; this is the same range as found in higher animals, e.g. the crayfish Procambarus clurkii (Almar et al., 1988). The activity of this enzyme is known to change in response to physiological and pathophysiological conditions, as demonstrated in P. clurkii (Romero et al., 1990; Almar et al., 1988). The finding presented here, shows that the activity of GST drops by 40%, 5 min after heat treatment (from 25 nmol/min X mg to 15 nmol/min X mg). Parallel with this drop in GST activity, the concentration of GSH decreases after heat stress (from 22 nmol/mg to 12 nmol/mg during a 20 min incubation period) indicating that GST displays a lower activity after heat treatment. This conclusion must be drawn from the fact that in the in vitro assay for GST, used here, saturating conditions are used; hence, any decrease in activity reflects decrease in enzymic activity. In a recent study, it has been reported that sponges contain both poly(P) and their metabolizing enzymes (Lorenz et al., 1995). In algae the poly(P) content varies in response to osmotic stress (Leitao et al., 1995). Furthermore, it was proposed that poly(P) might be involved in signal transduction reactions in sponges (Imsiecke et al., to be published). In the species Tethya lyncurium, steady-state concentrations for ‘soluble’ long-chain poly(P) as well as for the ‘insoluble’ long chain fraction were determined which are on the same level as those found in S. domuncula with 430 ng/mg and 310 nglmg, respectively. These concentrations remained unchanged in S. domuncula during temperature stress.

N. Bachinski et al. I J. Exp. Mar. Biol. Ecol. 210 (1997) 129-141

The experiments

reported

here demonstrate

cuZa to heat, the sponge reacts with a decrease

in the activity of GST and the concentration expression of HSP(s) (Koziol et al., 1995).

139

that during early response of S. domunin the steady-state level of trehalose and of GSH; all these changes precede the

Acknowledgments N.B. is a recipient of a fellowship from the Brazilian Ministry of Education, CAPES. We thank Prof. Dr. F.J. Romero (Universitat de Valencia), Valbncia (Spain) for his advise in the performance of the experiments. Supported by grants from the Bundesministerium fur Bildung und Forschung (STRESSTOX-Verbundprojekt) and from the Commission of the European Communities, 93/Avi 001 and MED-CAMPUS Project C034.

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proteins

during the assembly

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