Journal

ELSEVIER

of Experimental Marine Biology 217 (1997) 225-235

and Ecology,

JOURNAL OF EXPERIMENTAL MARINE BIOLOGY AND ECOLOGY

Stress-70 protein induction in Mytilus edulis: Tissue-specific responses to elevated temperature reflect relative vulnerability and physiological function
J. Paul Chapple*,
Plymouth Marine

Gary R. Smerdon,
Prospect

Anthony J.S. Hawkins

PLI 3DH. UK

Lahoruto~~,

Place, West Hoe, Plymouth

Received

23 July 1996; received

in revised form 29 January

1997; accepted

19 February

1997

Abstract
The exposure of organisms to environmental stressors affects the expression levels of certain stress proteins that play an important role in protein homeostasis and stress tolerance. We have used an antibody to monitor this response to elevated temperature in the gill, mantle and muscle tissues of the mussel Mytilus edulis. The antibody detected four isoforms of the highly conserved stress-70 family of proteins, which appear homologous with the hsp70, hsc70, hsp72, and grp78 proteins that are detected by the same antibody in humans. Compared with mantle and adductor muscle tissues, gill tissue showed much the greatest increase in levels of both the 70 and 72 kDa proteins. Levels of the 70 and 72 kDa proteins increased for the first 48 hours of heat stress in the gill, and subsequently decreased between 48 and 72 hours. A 78 kDa protein was present in the gill and mantle tissues, but was absent from the adductor muscle. Alternatively, the 70 kDa protein was more abundant in unstressed adductor muscle than in unstressed gill or mantle tissues. Results are discussed in terms of the proposed cellular locations and function of these proteins, the processes contributing to thermally-induced death, and their implications for our understanding of how temperature affects the physiology, ecology and distribution limits of marine organisms. 0 1997 Elsevier Science B.V.
Keywords:

Heat shock proteins; Myrilus

edulis;

Physiological

ecology; Temperature

stress

1. Introduction
Intertidal organisms live in a rapidly fluctuating environment level, organisms where thermal stress can

be a determinant

of survival.

At the molecular

respond

to the protein
ECIV 9EL.

*Corresponding author. Present address: The Institute of Ophthalmology, Bath Street, London U.K. Tel.: 0171 608 6808; fax: 0171 608 6828; e-mail: j.chapple@ucl.ac.uk 0022-0981/97/$17.00 P/f SOO22-098 0 1997 Elsevier Science B.V. All rights reserved

1(97)00057-9

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damage that results from thermal stress by synthesising a number of highly conserved stress proteins. These proteins play an irn~~~t role in protein homeostasis and have been shown to promote thermotolerance (reviewed in Parse11 and Lindquist, 1994). The role of stress proteins in conferring tolerance to organisms in extreme environments is directly relevant to the understanding of their ecology, physiology and distribution. For example, it has been suggested that the presence of heat stress proteins in all tissues of the teleost Fundulus heteroclitus at non stressful temperatures preadapt it to rapid environmental temperature change (Koban et al., 1991). It has also been shown that differential expression of low molecular mass stress proteins in intertidal and subtidal populations of the sea anemone, Ane~~~j~ viridis, correlates with the~otoler~ce (Sharp et al., 1994). The stress-70 family of proteins is the largest and most highly conserved group of stress proteins (Sanders, 1993). Members of the stress-70 family carry out a range of molecular chaperone functions, that include aiding assembly, proper folding, and the intracellular transport of proteins, thereby helping to protect organisms from thermal or other s~ess-induced damage (Lindquist and Craig, 1988; Morimoto et al., 1990; Gething and Sambrook, 1992; Gupta and Golding, 1993). The stress-70 family of proteins is not generic and varies considerably between organisms. The 70 kDa heat-shock protein (hsp70) is an important and highly conserved member of the stress-70 family. Hsp70 is thought to function in conjunction with the 72 kDa cognate protein hsc70, which is active in keeping polypeptides in an unfolded state for translocation across the membranes of the mitochondria and endoplasmic reticulum (Chirico et al., 1988; Deshaies et al., 1988; Beckmann et al., 1990). During environmental stress, hsp70 is synthesised rapidly and augments the action of hsc70, countering disassembly and unfolding of damaged proteins. Hsp72 is also a 72 kDa protein, but one which can be induced by heat stress. The glucose regulated 78 kDa protein (grp78) is identical to the immunoglobulin heavy-chain binding protein (BiP) (Munro and Pelham, 1986; Hendershot et al., 1988), and shares 60% amino acid identity with hsp70 (Munro and Pelham, 1986). Grp78 is localised in the endoplasmic reticulum of stressed and unstressed cells, where it forms stable complexes with misfolded, modified or unassembled proteins, preventing them from leaving the endoplasmic reticulum (Little et al., 1994). Mussels have been the subject of several stress protein studies (Steinert and Pickwell, 1988; Sanders et al., 1991) and are one of the few organisms for which elevated levels of stress proteins have been established at natural environmental temperatures (Hofmann and Somero, 1995). We have previously described the simultaneous immunological detection of four stress-70 isoforms (70, 72, 72 and 78 kDa) in the mussel Mytilus edulis, using a commercially available monoclonal antibody (Affinity BioReagents, IgG clone 5A5) (Smerdon et al., 199.5) that recognises a region of stress-70 proteins thought to be involved in ATP binding (Mushy et al., unpubl. data; Affinity BioReagents data sheet). In this study, we describe tissue-specific expression of 70, 72, and 78 kDa proteins in the gill, mantle, and adductor muscle tissues of M. edulis exposed to various regimes of heat stress. The relative amounts of these proteins are discussed in the context of their use as indicators of comparative protein damage, thereby establishing differential sensitivity between tissues to thermal stress and helping to explain associated consequences that are both of physiological and ecological relevance.

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2. Materials

and methods

h4ytilu.s edulis individuals were collected from Whitsand Bay, Cornwall, U.K., in May 1993. Thirty-five individuals of 50-55 mm shell length were selected and maintained for 8 days in a system of recirculating sea water at 15C. After this period, thirty of the animals were transferred to sea water at a temperature of 28C; the other five were maintained at 15C. Five animals were removed following treatment at 28C after 2, 6, 12, 24, 48 and 72 hours, and immediately dissected. Mussels from the 15C treatment were dissected after 72 hours. The gill, mantle and adductor tissues were excised separately from each individual and immediately homogenised (Ultraturrax) in 6 ml of homogenisation buffer (20 mM HEPES pH 7.3, 0.5 M NaCl and 12.5 mM KCl). The homogenisation buffer contained the proteolitic inhibitors leupeptin, phenylmethylsulfonyl fluoride and tosyl-L-lysine chloromethyl ketone), each of which were added to the buffer to a final concentration of 1 mM immediately before use. Homogenates were transferred to microfuge tubes and SDS added to a final concentration of 2%. After mixing, the samples were boiled for 6 min prior to centrifugation (RCF = 16 000) for 4 min. Pellets were discarded and supematant fractions stored at - 70C. Aliquot of supernatants were diluted 5 X in homogenisation buffer, thus reducing the SDS concentration to 0.4%, and the concentrations of protein were determined using the Bio-Rad DC assay. Samples containing equivalent total protein (20 pg) were boiled for 10 min in SDS-PAGE sample buffer (Laemmli, 1970) and centrifuged (RCF = 16 000) for 4 min. Proteins were separated by one dimensional SDS-PAGE on 10% resolving gels, with 5% stacking gels. Electroblotting of the proteins onto supported nitrocellulose (Sartorius) was performed for 6 h at 100 mA as described by Towbin et al. (1979). After blotting, immunological detection of stress-70 proteins was performed using IgG clone 5A5 (Affinity BioReagents), an alkaline phosphatase-conjugated secondary antibody and p-nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate visualisation system, as described by Smerdon et al. (1995). Densitometric analysis of developed westerns blots was performed using an Enhanced Laser Densitometer with GelScan XL Version 2.0 software (Pharmacia LKB Biotechnology, Bromma, Sweden). Where comparisons were necessary between samples on different membranes, the same controls were included on each, and the relative intensities of the control bands were calculated such that data could be normalised for any variations resulting from processing. Differences between the mean intensities of protein bands were tested using the Students r-test.

3. Results 3. I. Variation in levels of stress- 70 proteins between gill, mantle and adductor tissue

Results of western analysis of gill, mantle and adductor tissue from each of four M. edulis individuals that had been maintained at 15C are shown in Fig. 1. Results of densitometric analysis of the developed membrane are shown in Fig. 2. For the 70 kDa

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1 2 3 4 5 Std L-II Hsp70 Adduetor

IO

I1

12 13 Gill

Mantle

Fig. 1. Western blot analysis of stress-70 proteins in adductor (lanes 2-5) mantle (lanes 6-9) and gill (tanes 10-13) tissues from four M. edulis maintained at 15C. Lane 1 contains 0.5 pg of hsp70 protein standard (Sigma Chemical Company). The positions of fluorescently stained molecular weight markers (Integrated Separation Systems) are indicated in kDa.

protein, both gill and mantle tissue showed similar intensities that were low when compared to the values obtained in adductor tissue, which showed an approximately L&fold greater level of the 70 kDa protein. The 72 kDa protein was present in the gill tissue, mantle and adductor tissues. The protein band of 78 kDa was present in gill and mantle tissue, but was undetectable in adductor muscle tissue. The level of this 78 kDa protein was approximately Z-fold greater in gill than mantle. A band of approximately 50 kDa was detected in adductor, but was not present in gill or mantle tissues. Immunoblotting in the absence of the 5A.5 primary antibody established that this 50 kDa band was not an artifact resulting from nonspecific binding of the secondary antibody. The levels of stress-70 proteins in pooled samples (5 individuals) of adductor, mantle and gill tissues from animals exposed to different periods of heat stress are shown in Fig. 3. The 70 kDa protein was not at detectabfe levels in the gill tissue of animals immediately after transfer to sea water at 28C or in animals maintained at 15C under experimental conditions for 72 h, but was present in the gill tissue of animals held at 28C for 24 or 72 h. This protein was, however, present in all mantle and adductor tissue samples taken. The 72 kDa protein was present in all of the samples, including those taken from unstressed animals. The 78 kDa protein was evident in gill and mantle, but absent from adductor tissue. The band of 50 kDa was again detected in adductor tissue samples.

70 kDa .i
0.8 0.6

72 kDa

78 kDa

d
3

g 0.4 0 .$ 0.2 SO ~ AM Tiisue G AMG Tisue

_Q-L

AMG

Fig. 2. Mean intensities (relative optical density) of the 70, 72 and 78 kDa bands in the adductor (A), mantle (M) and gill (G) tissues determined by densitometric analysis of the western blot presented in Fig. 1. Error bars represent one standard error of the mean.

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229

1
-70

IO

11

12

13

Std I Adductor

I/I MalttIe Gill

Fig. 3. Western blot analysis of stress-70 levels in adductor (lanes 2-S), mantle (lanes 6-9) and gill (lanes IO- 13) tissues pooled from 5 M. edulis, sampled after 0 h (lanes 4, 8 and 12), 24 h (lanes 3, 7 and I I ) and 72 h (lanes 2, 6 and lo), respectively, of temperature elevation from 15C to 28C and 72 h under experimental conditions at 15C lanes (5, 9 and 13). Lane I contains 0.5 kg of hsp70 protein standard. The positions of molecular weight markers are indicated in kDa.

3.2. Effect of exposure gill tissue

time at elevated temperature

on levels of stress-70

proteins

in

Western analyses of gill tissue from M. edulis exposed over a time course to an elevated temperature of 28C (four individuals per exposure period) are shown in Fig. 4(a-c). Densitometric analyses of the 70 kDa, 72 kDa and 78 kDa bands are summarised in Fig. 5. For the 70 kDa protein, there was an increase in average protein levels between 2 and 6 hours after the temperature shift, although this increase was only clear in two of the four sampled mussels (lanes 9 and 12 of Fig. 4(a)). A positive correlation (r2 = 0.944, p = 0.001) between average relative optical density and time confirms that this increase in 70 kDa protein continued in sampled mussels for up to 48 h after the temperature shift, after which the level appeared to decline. The level of 72 kDa protein shows a similar trend, although the initial protein increase appeared to occur between 6 and 12 h after the temperature shift, as confirmed by a comparison between the grouped mean relative optical densities from those observed over O-6 h and those observed over 12-48 h after the temperature shift (t-value = - 4.297, df = 11, p = 0.001). The levels of 78 kDa protein remained unchanged upon transfer to 28C.

3.3. Comparison tissues following

of the increase in levels of stress- 70 protein 72 hours at elevated temperature

in gill and mantle

Western analyses of the mantle tissues from five M. edulis maintained at 28C for 72 h and five similar individuals maintained at the control temperature of 15C are shown in Fig. 6. Results of the densitometric analyses of the ensuing blot are shown in Fig. 7. The intensity of the 70 kDa band was seen to increase significantly in the heat-treated mussels (t-value = 3.646, df = 4, p = 0.022). After 72 h of exposure to 28C there was no significant difference in intensities of either the 72 kDa protein (t-value = 0.892, df = 4, p = 0.423), or the 78 kDa protein (t-value = 0.302, df = 4, p = 0.777). Comparisons of the relative amounts of stress-70 proteins in gill and mantle tissue after 72 h at the elevated temperature, taken from Fig. 4(c) and Fig. 6, are shown in Table 1.

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_ . ,

10

11

12 13

1
C0a

I
2h

I
6h

Std
Hsp70

I I

3 COII

4 I

7 12h

9 I

10 11 12 24h

13 Std w70

7 48b

10

11

12

13

con

I
72h

Std
Hsp70

Fig. 4. Western blot analysis of gill tissue of M. edulis individuals exposed to a temperature of 28C for increasing durations of time (4 individuals per exposure period). (a) Animals exposed for 0 h (lanes l-4), 2 h (lanes 5-8), 6 h (lanes 9-12), respectively. (b) Animals exposed for 0 h (lanes i-4), 12 h (lanes 5-Q 24 h (lanes 9-12), respectively. (c) Animals exposed for 0 h (lanes l-4), 48 h (lanes 5-8), 72 h (lanes 9-12). respectively. Lane 13 of (a), (b) and (c) contains 0.5 p,g of hsp70 protein standard. The positions of molecular weight markers are indicated in kDa.

4. Discussion We have shown that abundances of the stress-70 protein isoforms detected by a monoclonal antibody differed between mussel tissues, as well as with the duration of exposure to elevated temperature. In heat stressed MytiEus edulis gill tissue, 2D-SDS PAGE western analysis showed clone 5A5 cross-reacted with four stress-70 isoforms (Smerdon et al., 1995). The two-dimensional gel pattern for these isoforms in M. edulis was similar to that detected in human (HeLa) cells, where it was shown that the four proteins were hsp70, hsc70, hsp72 and grp78. (Murphy et al., unpubl. data; Affinity BioReagents data sheet). Therefore, it is likely that the four stress-70 isoforms detected in M. ed~lis have similar functions to those that have been established in HeLa cells (Smerdon et al., 1995).

J.P. Chupple

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231

,g
B a ?I 8 .; a g

70kDa
0.4 0.3 0.2 0.1 0
0 2 6 12 24 4812 0 2

72kDa

78kDa

6 12244872

2 6 12244872

Duration

of heat stress

Duration

of heat stress

(hours~

@oJln)

Duration of heat stress olours)

Fig. 5. Densitometric data for the mean intensities of the 70, 72 and 78 kDa bands in gill tissue of M. r&t/is as detected by western blot analysis at 0, 2, 6, 12, 24, 48 and 72 h respectively, after temperature elevation from 15C to 28C (see Fig. 4(a), (b) and (c)). Error bars represent one standard error of the mean.

Fig. 6. Western blot analysis of stress-70 protein levels in mantle tissues sampled from 5 individual M. edulis at 0 h (lanes 2-6), 72 h (lanes 7-11). respectively, after transfer from 15C to 28C. Lane 1 contains 0.5 pg of hsp70 protein standard. The position of molecular weight markers are indicated in kDa.

Gill and mantle tissue had relatively similar stress-70 protein compliments, with the 70, 72 and 78 kDa proteins being expressed. The adductor tissue had a signiticantly different stress 70 protein compliment, with undetectable levels of the 78 kDa protein and markedly higher level of the 70 kDa protein in animals which had not been exposed 70 kDa 72 kDa 78 kDa

__&A-&
0
Duration of heat stress (hours)

_L!q!! jLL_~
i

[!t

72

Duration of heat stress (hours)

Duration of heat stress fho&

Fig. 7. Densitometric data for the mean intensities of the 70, 72 and 78 kDa bands in mantle tissue of M. dulis as detected by western blot analysis (see Fig. 6) at 0 and 72 hours, respectively, after transfer from 15C to 28C. Error bars represent one standard error of the mean.

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Table 1 Comparison of the increase in levels of stress-70 elevated temperature (28C) Tissue and band Mean Relative Oh Gill 70 Mantle Gill 72 Mantle Gill 78 Mantle kDa 70 kDa kDa 72 kDa kDa 78 kDa 0.003 *0.003 0.007~0.007 0.133?0.024 0.143+0.015 0.103t0.045 0.061%0.027

in gill and mantle tissue caused by maintenance

for 72 h at

Optical Density 12 h 0.170t0.011 0.064~0.220 0.216rtO.041 0.197r0.067 0.108+0.02.5 0.~8~0.0~9

Factor of Change

+57 +9 + 1.6 + 1.4 + 1.04 - 1.2

to elevated temperature. A protein of approximately 50 kDa was detected in adductor tissue, however, it seems unlikely that this 50 kDa band was caused by proteolytic degradation of the 78 kDa protein, as adductor tissue is the least proteolytic of the tissues examined. It is to be expected that differing protein composition of tissues may influence the extent of protein damage and therefore the relative requirements for regulatory stress-70 proteins between tissues. Certainly, tissue-specificity of stress-70 proteins has previously been observed in both vertebrates and invertebrates. For example, a 74 kDa stress-70 protein was induced by heat stress in the gill and heart tissues of the eu~the~al teleost, ~~~~~1~s ~etero~~if~s, but was not present in the liver, skeletal tissue or brain (Koban et al., 1991). In the fathead minnow (Pimephales promelm) neuronal tissue synthesised the fewest stress proteins and had the lowest capacity for their synthesis at the upper thermal limit of the organism, when compared with muscle and gill tissues (Dyer et al., 1991). Dyer et al. (1991) suggest that this result supports the suggestion that neural tissue may be rate limiting in the ability of poikilotherms to survive thermal stress, due to its limited capacity to synthesis stress proteins. It has also previously been suggested that differences in the accumulation of stress proteins might be useful in identifying tissues which are particularly vulnerable to damage by a specific stressor and identifying the extent of the damage (Sanders et al., 1994). In this study, compared with mantle and adductor muscle tissues, gill tissue showed the largest increase in stress-70 proteins between unstressed and stressed mussels (Table 1). Histopathological studies have shown that high temperature causes degeneration of the gill filaments in M. edulis, which may result in whole animal death (Gonzalez and Yevich, 1976), and that changing temperatures may induce elevated heat resistance in the gill epithelium (Huppert and Laudien, 1980). Therefore, the amount of protein damage and the subsequent requirement for stress proteins is likely to be greater in gill than other tissues in the mussel, and our findings are consistent with this. However, it remains uncertain whether stress protein responses signify an adverse affect by stressors or a successful compensatory response which protects from damage (Vedel and Depledge, 1995). The absence of the 78 kDa protein in adductor muscle is not wholly unexpected. If the

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78 kDa protein observed in this study is a molluscan form of grp78, it is unlikely to be present in the adductor muscle. This is because grp78 is expressed and localised in the endoplasmic reticulum (Little et al., 1994), which is absent from muscle tissue where it is replaced by the sarcoplasmic reticulum. The absence of the 78 kDa protein in adductor muscle tissue therefore adds to the argument that the stress proteins observed here in M. edulis are homologous with those observed previously in humans. Studying the time-course of induction in gill tissue alone, elevation in stress-70 was first detected in some mussels 6 h after transfer from 15C to 28C with a consistent significant increase in average 70 kDa protein abundance thereafter (Fig. 5). Sanders et al. (1992) described an approximate IO-fold increase in hsp70 6 h after transfer from 17C to 27C, using a polyclonal antibody. In the present study, the level of hsp70 had increased by more than lOO-fold 48 h after the start of heat stress. However, between 48 and 72 h, the level of hsp70 fell by approximately half. Levels of the 72 kDa protein showed a similar pattern of change over time as for the 70 kDa protein, although the first increase in levels of 72 kDa protein was not detected until 12 h after the initial temperature elevation, and the overall induction was less notable. The reduction in levels of 70 and 72 kDa protein between 48 and 72 h may have been an early indication that the mussels were unable to acclimate to 28C. Mussels certainly die if maintained at 28C for more than about 12 days (Hawkins et al., 1987), and it is likely that decreased synthesis of hsp70 and all other proteins immediately precedes death. In summary, this study has demonstrated differences in stress-70 compliment, as well as tissue-specific inducibility, despite those tissues being subject to the same temperature increase. The greatest induction of stress-70 proteins was shown to occur in gill tissue, where protein damage is histopathologically detectable. The absence of a 78 kDa protein from adductor muscle is consistent with the normal localisation of grp78 in endoplasmic reticulum. The fact that the stress response differs among tissues supports the hypothesis that the thermal limits of an organism are governed by certain tissues more than others (Dyer et al., 1991). We suggest that decreasing levels of 70 and 72 kDa proteins, after an initial increase over the first 48 h of heat stress, reflected metabolic stress that was associated with the mussels inability to acclimate at 28C. Stress-70 proteins function in an ATP-dependent manner (reviewed in McKay et al., 1994). This dependency, together with the energy requirements of stress protein synthesis, represents costs associated with the metabolic response to thermal stress (Coleman et al., 1995). Results reported here indicate that these energy costs will vary according to both the relative vulnerability and the physiological function of different tissues, with likely consequences for the ecology and distribution of different marine organisms.

Acknowledgements
This work forms part of research projects SRP.5 (Stress Effects and Health in the Oceans) and SRPl (Productivity and Physical Structure in Pelagic Ecosystems) of the Plymouth Marine Laboratory, a component of the Centre for Coastal Marine Science within the U.K. Natural Environmental Research Council.

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