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of Experimental Marine Biology 217 (1997) 225-235
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*,
Gary R. Smerdon,
Anthony J.S. Hawkins
PLI 3DH. UK
Place, West Hoe, Plymouth
23 July 1996; received
in revised form 29 January
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.
Heat shock proteins; Myrilus
Intertidal organisms live in a rapidly fluctuating environment level, organisms where thermal stress can
be a determinant
At the molecular
to the protein
*Corresponding author. Present address: The Institute of Ophthalmology, Bath Street, London U.K. Tel.: 0171 608 6808; fax: 0171 608 6828; e-mail: email@example.com 0022-0981/97/$17.00 P/f SOO22-098 0 1997 Elsevier Science B.V. All rights reserved
correlates with the~otoler~ce (Sharp et al. Morimoto et al. Gupta and Golding. I J. and adductor muscle tissues of M. Beckmann et al. The relative amounts of these proteins are discussed in the context of their use as indicators of comparative protein damage. thereby helping to protect organisms from thermal or other s~ess-induced damage (Lindquist and Craig. 1988. 1988. The stress-70 family of proteins is the largest and most highly conserved group of stress proteins (Sanders. Ecol. 1990). 1991). 1990. Deshaies et al. For example. Grp78 is localised in the endoplasmic reticulum of stressed and unstressed cells.. using a commercially available monoclonal antibody (Affinity BioReagents... The stress-70 family of proteins is not generic and varies considerably between organisms. 1993). Biol. and shares 60% amino acid identity with hsp70 (Munro and Pelham. Chupple et al. that include aiding assembly.. where it forms stable complexes with misfolded. Mar. 1994). 1988.. Hsp70 is thought to function in conjunction with the 72 kDa cognate protein hsc70. Mussels have been the subject of several stress protein studies (Steinert and Pickwell. proper folding. Hsp72 is also a 72 kDa protein. During environmental stress. 199. It has also been shown that differential expression of low molecular mass stress proteins in intertidal and subtidal populations of the sea anemone. 1994).5) that recognises a region of stress-70 proteins thought to be involved in ATP binding (Mushy et al. 1986. 1988.. mantle. preventing them from leaving the endoplasmic reticulum (Little et al. and the intracellular transport of proteins. 72 and 78 kDa) in the mussel Mytilus edulis. which is active in keeping polypeptides in an unfolded state for translocation across the membranes of the mitochondria and endoplasmic reticulum (Chirico et al. 1993). These proteins play an irn~~~t role in protein homeostasis and have been shown to promote thermotolerance (reviewed in Parse11 and Lindquist.. data. 72. Hendershot et al.P. The 70 kDa heat-shock protein (hsp70) is an important and highly conserved member of the stress-70 family.. modified or unassembled proteins. countering disassembly and unfolding of damaged proteins. thereby establishing differential sensitivity between tissues to thermal stress and helping to explain associated consequences that are both of physiological and ecological relevance. 217 (1997) 225-235 damage that results from thermal stress by synthesising a number of highly conserved stress proteins. but one which can be induced by heat stress. we describe tissue-specific expression of 70. Exp. IgG clone 5A5) (Smerdon et al.226 J. edulis exposed to various regimes of heat stress.. physiology and distribution. In this study. unpubl. We have previously described the simultaneous immunological detection of four stress-70 isoforms (70. 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. 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. Ane~~~j~ viridis. 1995). 1992.. The role of stress proteins in conferring tolerance to organisms in extreme environments is directly relevant to the understanding of their ecology. Members of the stress-70 family carry out a range of molecular chaperone functions. hsp70 is synthesised rapidly and augments the action of hsc70. 1994). Affinity BioReagents data sheet).. The glucose regulated 78 kDa protein (grp78) is identical to the immunoglobulin heavy-chain binding protein (BiP) (Munro and Pelham. 1986). and 78 kDa proteins in the gill. 1988). . Sanders et al. Gething and Sambrook. 72.
Pellets were discarded and supematant fractions stored at . and immediately dissected.4%.K. in May 1993. 6. Where comparisons were necessary between samples on different membranes. Proteins were separated by one dimensional SDS-PAGE on 10% resolving gels. (1995). 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. Electroblotting of the proteins onto supported nitrocellulose (Sartorius) was performed for 6 h at 100 mA as described by Towbin et al.. Results 3. After blotting. 24. an alkaline phosphatase-conjugated secondary antibody and p-nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate visualisation system. The homogenisation buffer contained the proteolitic inhibitors leupeptin. thus reducing the SDS concentration to 0. 3. Ed. Mussels from the 15°C treatment were dissected after 72 hours. Mar. U. 12. the same controls were included on each. Sweden). 1. I. and the relative intensities of the control bands were calculated such that data could be normalised for any variations resulting from processing. (1979). After this period. Results of densitometric analysis of the developed membrane are shown in Fig. For the 70 kDa .s edulis individuals were collected from Whitsand Bay.5 mM KCl). 48 and 72 hours. each of which were added to the buffer to a final concentration of 1 mM immediately before use. I J. Aliquot of supernatants were diluted 5 X in homogenisation buffer. the samples were boiled for 6 min prior to centrifugation (RCF = 16 000) for 4 min. as described by Smerdon et al. Chapple et al. mantle and adductor tissue Results of western analysis of gill. Thirty-five individuals of 50-55 mm shell length were selected and maintained for 8 days in a system of recirculating sea water at 15°C.70°C. Samples containing equivalent total protein (20 pg) were boiled for 10 min in SDS-PAGE sample buffer (Laemmli. thirty of the animals were transferred to sea water at a temperature of 28°C. 2. Variation in levels of stress. 0. immunological detection of stress-70 proteins was performed using IgG clone 5A5 (Affinity BioReagents). Exp. Homogenates were transferred to microfuge tubes and SDS added to a final concentration of 2%. Differences between the mean intensities of protein bands were tested using the Student’s r-test. edulis individuals that had been maintained at 15°C are shown in Fig. Materials and methods h4ytilu. 217 (1997) 225-23 221 2. Bromma. and the concentrations of protein were determined using the Bio-Rad DC assay. with 5% stacking gels. Bid.3. Five animals were removed following treatment at 28°C after 2.70 proteins between gill. the other five were maintained at 15°C. Cornwall. 1970) and centrifuged (RCF = 16 000) for 4 min.0 software (Pharmacia LKB Biotechnology. phenylmethylsulfonyl fluoride and tosyl-L-lysine chloromethyl ketone). After mixing. The gill.J.5 M NaCl and 12. Densitometric analysis of developed westerns blots was performed using an Enhanced Laser Densitometer with GelScan XL Version 2. mantle and adductor tissue from each of four M.P.
228 J. Chapple et al.5 primary antibody established that this 50 kDa band was not an artifact resulting from nonspecific binding of the secondary antibody. Exp. The positions of fluorescently stained molecular weight markers (Integrated Separation Systems) are indicated in kDa.4 0 . present in all mantle and adductor tissue samples taken. Lane 1 contains 0. mantle (M) and gill (G) tissues determined by densitometric analysis of the western blot presented in Fig. 72 and 78 kDa bands in the adductor (A).2 SO ~ AM Tiisue G AMG Tisue _Q-L AMG Fig. but was undetectable in adductor muscle tissue. This protein was. but absent from adductor tissue.i 0. 70 kDa . . I J. The 70 kDa protein was not at detectabfe levels in the gill tissue of animals immediately after transfer to sea water at 28°C or in animals maintained at 15°C under experimental conditions for 72 h. The level of this 78 kDa protein was approximately Z-fold greater in gill than mantle.8 0. The 78 kDa protein was evident in gill and mantle. both gill and mantle tissue showed similar intensities that were low when compared to the values obtained in adductor tissue. but was not present in gill or mantle tissues. Immunoblotting in the absence of the 5A.5 pg of hsp70 protein standard (Sigma Chemical Company). mantle and gill tissues from animals exposed to different periods of heat stress are shown in Fig. A band of approximately 50 kDa was detected in adductor. The 72 kDa protein was present in all of the samples. Error bars represent one standard error of the mean. however. protein. edulis maintained at 15°C. Mean intensities (relative optical density) of the 70. 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. 3. Ecol. 2. Biol. mantle and adductor tissues. 217 (1997) 225-235 1 2 3 4 5 Std L-II Hsp70 Adduetor 6 7 8 9 IO I1 12 13 Gill Mantle Fig. The protein band of 78 kDa was present in gill and mantle tissue. The 72 kDa protein was present in the gill tissue. including those taken from unstressed animals. which showed an approximately L&fold greater level of the 70 kDa protein.P.$ 0.6 72 kDa 78 kDa d 3 ‘g 0. 1. 1. The band of 50 kDa was again detected in adductor tissue samples. The levels of stress-70 proteins in pooled samples (5 individuals) of adductor. Mar. but was present in the gill tissue of animals held at 28°C for 24 or 72 h.
72 kDa and 78 kDa bands are summarised in Fig. there was an increase in average protein levels between 2 and 6 hours after the temperature shift. 3. taken from Fig. The intensity of the 70 kDa band was seen to increase significantly in the heat-treated mussels (t-value = 3. df = 4. Biol. p = 0. p = 0. For the 70 kDa protein. 4(a-c). although the initial protein increase appeared to occur between 6 and 12 h after the temperature shift. Exp. or the 78 kDa protein (t-value = 0. Mar.892.13) tissues pooled from 5 M. df = 4. The level of 72 kDa protein shows a similar trend. 7 and I I ) and 72 h (lanes 2. . 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 = . After 72 h of exposure to 28°C there was no significant difference in intensities of either the 72 kDa protein (t-value = 0. although this increase was only clear in two of the four sampled mussels (lanes 9 and 12 of Fig. are shown in Table 1. 4(c) and Fig. p = 0. mantle (lanes 6-9) and gill (lanes IO. Ecol.423). The positions of molecular weight markers are indicated in kDa.5 229 1 -70 2 3 4 5 6 7 8 9 IO 11 12 13 Std I Adductor I/I MalttIe Gill Fig. 3.5 kg of hsp70 protein standard. Chapple et al.022).302.P. of temperature elevation from 15°C to 28°C and 72 h under experimental conditions at 15°C lanes (5. 4(a)). 8 and 12).70 protein 72 hours at elevated temperature in gill and mantle Western analyses of the mantle tissues from five M. df = 11. The levels of 78 kDa protein remained unchanged upon transfer to 28°C.944.2. 6.297.J. edulis. Effect of exposure gill tissue time at elevated temperature on levels of stress-70 proteins in Western analyses of gill tissue from M. Comparison tissues following of the increase in levels of stress. 7. 3.001) 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. Western blot analysis of stress-70 levels in adductor (lanes 2-S). 6 and lo). 5. 217 (1997) 22_5-23. sampled after 0 h (lanes 4. I J.4. Comparisons of the relative amounts of stress-70 proteins in gill and mantle tissue after 72 h at the elevated temperature. df = 4. respectively. p = 0. edulis exposed over a time course to an elevated temperature of 28°C (four individuals per exposure period) are shown in Fig. edulis maintained at 28°C for 72 h and five similar individuals maintained at the control temperature of 15°C are shown in Fig. Densitometric analyses of the 70 kDa. 6. p = 0. 9 and 13). A positive correlation (r2 = 0.001). Lane I contains 0. 24 h (lanes 3.646. Results of the densitometric analyses of the ensuing blot are shown in Fig.3.777). after which the level appeared to decline.
4. (b) and (c) contains 0. Affinity BioReagents data sheet). 1 2 3 4 5 6 7 8 9 10 11 12 13 1 C0a I 2h I 6h Std Hsp70 I I 2 3 COII 4 I S 6 7 12h 8 9 I 10 11 12 24h 13 Std w70 1 2 3 4 5 6 7 48b 8 9 10 11 12 13 con I I 72h Std Hsp70 Fig. it is likely that the four stress-70 isoforms detected in M.P.. The two-dimensional gel pattern for these isoforms in M.5 p. Mar.g of hsp70 protein standard. edulis individuals exposed to a temperature of 28°C for increasing durations of time (4 individuals per exposure period). Lane 13 of (a). . 48 h (lanes 5-8).. as well as with the duration of exposure to elevated temperature. 217 (1997) 225-235 _’ . Therefore. edulis was similar to that detected in human (HeLa) cells. Western blot analysis of gill tissue of M. (Murphy et al. In heat stressed MytiEus edulis gill tissue. respectively. Exp.. 12 h (lanes 5-Q 24 h (lanes 9-12). (a) Animals exposed for 0 h (lanes l-4). 1995). 4. data. Chapple et al. 72 h (lanes 9-12). . respectively. (c) Animals exposed for 0 h (lanes l-4). 6 h (lanes 9-12). 2D-SDS PAGE western analysis showed clone 5A5 cross-reacted with four stress-70 isoforms (Smerdon et al. ed~lis have similar functions to those that have been established in HeLa cells (Smerdon et al. where it was shown that the four proteins were hsp70. 1995). I 1. (b) Animals exposed for 0 h (lanes i-4). hsp72 and grp78. 2 h (lanes 5-8). Discussion We have shown that abundances of the stress-70 protein isoforms detected by a monoclonal antibody differed between mussel tissues. B&l.230 J. hsc70. unpubl. Ecol. The positions of molecular weight markers are indicated in kDa. respectively.
4(a). / J. with the 70. Densitometric data for the mean intensities of the 70. 48 and 72 h respectively.P. 12. after temperature elevation from 15°C to 28°C (see Fig. Western blot analysis of stress-70 protein levels in mantle tissues sampled from 5 individual M. 72 and 78 kDa bands in mantle tissue of M.. 72 and 78 kDa proteins being expressed. Lane 1 contains 0. respectively. 5. a g 70kDa 0. Exp.1 0 0 2 6 12 24 4812 0 2 72kDa 78kDa 6 12244872 0 2 6 12244872 Duration of heat stress Duration of heat stress (hours~ @oJln) Duration of heat stress olours) Fig. Bid. 6) at 0 and 72 hours. 6.2 0. Ecol.g B a” ?I 8 . after transfer from 15°C to 28°C. respectively. after transfer from 15°C to 28°C. 72 h (lanes 7-11). The adductor tissue had a signiticantly different stress 70 protein compliment. 7.J. 24. Fig. 72 and 78 kDa bands in gill tissue of M. .4 0. 217 (1997) 225-235 231 . The position of molecular weight markers are indicated in kDa. dulis as detected by western blot analysis (see Fig. 6. 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 i __&A-& 0 Duration of heat stress (hours) _L!q!! jLL_~ ’ i I [!t 0 72 0 Duration of heat stress (hours) Duration of heat stress fho& Fig. edulis at 0 h (lanes 2-6).5 pg of hsp70 protein standard.3 0. Error bars represent one standard error of the mean. Mar. r&t/is as detected by western blot analysis at 0. Densitometric data for the mean intensities of the 70. Gill and mantle tissue had relatively similar stress-70 protein compliments. Chupple et cd. 2. (b) and (c)). Error bars represent one standard error of the mean.
due to its limited capacity to synthesis stress proteins. skeletal tissue or brain (Koban et al.061%0.103t0. Therefore.1. ~~~~~1~s ~etero~~if~s. For example. 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. Dyer et al. 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. 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.6 + 1.02. 217 (1997) 225-235 Table 1 Comparison of the increase in levels of stress-70 elevated temperature (28°C) Tissue and band Mean Relative Oh Gill 70 Mantle Gill 72 Mantle Gill 78 Mantle kDa 70 kDa kDa 72 kDa kDa 78 kDa 0.027 in gill and mantle tissue caused by maintenance for 72 h at Optical Density 12 h 0.220 0. Biof.~8~0.024 0. I 1..133?0. However. 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.. If the .015 0. 1994). but was not present in the liver. which may result in whole animal death (Gonzalez and Yevich.232 J. compared with mantle and adductor muscle tissues.04 . Mar. edulis.2 to elevated temperature.067 0.064~0. however.045 0. 1991). it seems unlikely that this 50 kDa band was caused by proteolytic degradation of the 78 kDa protein.003 *0. 1980).007 0. gill tissue showed the largest increase in stress-70 proteins between unstressed and stressed mussels (Table 1). 1991).0~9 Factor of Change +57 +9 + 1. Exp. 1995). 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.003 0. and that changing temperatures may induce elevated heat resistance in the gill epithelium (Huppert and Laudien. Certainly. A protein of approximately 50 kDa was detected in adductor tissue. tissue-specificity of stress-70 proteins has previously been observed in both vertebrates and invertebrates.216rtO.170t0. as adductor tissue is the least proteolytic of the tissues examined. Ecol. In this study.5 0. (1991) suggest that this result supports the suggestion that neural tissue may be rate limiting in the ability of poikilotherms to survive thermal stress.041 0.007~0. when compared with muscle and gill tissues (Dyer et al.108+0.011 0. ~~p~~e et ai.. The absence of the 78 kDa protein in adductor muscle is not wholly unexpected.4 + 1. and our findings are consistent with this. Histopathological studies have shown that high temperature causes degeneration of the gill filaments in M.143+0.P. a 74 kDa stress-70 protein was induced by heat stress in the gill and heart tissues of the eu~the~al teleost. 1976).197r0.
1987). Levels of the 72 kDa protein showed a similar pattern of change over time as for the 70 kDa protein. and the overall induction was less notable. We suggest that decreasing levels of 70 and 72 kDa proteins. represents costs associated with the metabolic response to thermal stress (Coleman et al. Exp. Acknowledgements This work forms part of research projects SRP. a component of the Centre for Coastal Marine Science within the U. with likely consequences for the ecology and distribution of different marine organisms. this study has demonstrated differences in stress-70 compliment.75 231 78 kDa protein observed in this study is a molluscan form of grp78. The absence of a 78 kDa protein from adductor muscle is consistent with the normal localisation of grp78 in endoplasmic reticulum. the level of hsp70 fell by approximately half. This is because grp78 is expressed and localised in the endoplasmic reticulum (Little et al. 1994). 1991). Studying the time-course of induction in gill tissue alone. 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... . the level of hsp70 had increased by more than lOO-fold 48 h after the start of heat stress. In summary. In the present study. Stress-70 proteins function in an ATP-dependent manner (reviewed in McKay et al. where “protein” damage is histopathologically detectable. edulis are homologous with those observed previously in humans. and it is likely that decreased synthesis of hsp70 and all other proteins immediately precedes death. as well as tissue-specific inducibility. Natural Environmental Research Council. I J. 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 28°C. elevation in stress-70 was first detected in some mussels 6 h after transfer from 15°C to 28°C with a consistent significant increase in average 70 kDa protein abundance thereafter (Fig. despite those tissues being subject to the same temperature increase.K. 1994). Ed. using a polyclonal antibody. although the first increase in levels of 72 kDa protein was not detected until 12 h after the initial temperature elevation. it is unlikely to be present in the adductor muscle. together with the energy requirements of stress protein synthesis.. Mar.5 (Stress Effects and Health in the Oceans) and SRPl (Productivity and Physical Structure in Pelagic Ecosystems) of the Plymouth Marine Laboratory. reflected metabolic stress that was associated with the mussels inability to acclimate at 28°C. However.. Results reported here indicate that these energy costs will vary according to both the relative vulnerability and the physiological function of different tissues. The absence of the 78 kDa protein in adductor muscle tissue therefore adds to the argument that the stress proteins observed here in M.J. 217 (1997) 22-G2. between 48 and 72 h. Mussels certainly die if maintained at 28°C for more than about 12 days (Hawkins et al. This dependency.. Sanders et al.P. 1995). after an initial increase over the first 48 h of heat stress. Chappie et al. (1992) described an approximate IO-fold increase in hsp70 6 h after transfer from 17°C to 27”C. The greatest induction of stress-70 proteins was shown to occur in gill tissue. Bid. which is absent from muscle tissue where it is replaced by the sarcoplasmic reticulum. 5).
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