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Detection of a major stress protein using a peptide antibody
Marine Environmental Research 28 (1989) 471~475
Detection of a Major Stress Protein Using a Peptide Antibody
Brian P. Bradley & Joni B. Ward Department of Biological Sciences, University of Maryland, Baltimore, Maryland 21228, USA
ABSTRACT The synthesis of a major stress protein ( hsp70 ) is induced by extremes of a wide variety of environmental variables, both physical and chemical. This protein of approximately 70 000 Da is a widespread 'heat shock'protein and is present in various isomorphs in man), species. Nevertheless it is highly conserved among organisms from bacteria to man. The production of hsp 70 in a natural population of amphipods ( G a m m a r u s sp.) varied with ambient temperature. Protein extracts were probed on immunoblots using a polyclonal antibody, which we were given, made against a highly conserved peptide q[" hsp 70. Relative protein production was estimated using reflectance scanning. These results and others using immunoblots, autoradiography and immunosorbence assays suggest that the stress protein response may be a useful quantitative and qualitative environmental indicator.
Living cells respond to environmental contamination and to stress in general by synthesizing what are known as stress proteins. Some of these proteins are present in unstressed cells, but are produced in larger quantities under stress. Other proteins appear as the stress increases, and yet others stop being synthesized. Recent work on stress proteins from an ecological perspective is reviewed by Huey & Bennett.1 The heat shock phenomenon itself, the most studied part of the stress protein response, has been reviewed by Lindquist & Craig. 2 The response occurs in all species studied and to virtually all agents is considered stressful. 3'4 We and others have even observed it in response to a parasite. 5 Among the most common heat shock proteins are the family with molecular weights around 70000Da (hsp70). Some of these are present 471 Marine Environ. Res. 0141-1136/90/$03'50 ~ 1990 Elsevier Science Publishers L.td, England. Printed in Great Britain
Brian P. Bradley, Joni B. Ward
472
under normal conditions, others are inducible. 6 Unlike proteins such as cytochromes and metallothioneins, induced by chemical contaminants, the functions of hsp70 and other stress proteins seem to be varied. 2 Whatever the mechanism, they appear to have a protective role in the cell during stress. We have used an anti-hsp70 peptide serum provided by Stephen Ullrich 7 to detect hsp70. The antiserum was prepared against a 23-amino acid sequence at the C-terminal end of the protein which is highly conserved among several vertebrates including man. The antiserum cross-reacts with hsp70 and related proteins in a variety of organisms including copepods and Artemia, 8 cladocerans, shellfish, finfish, Lepidoptera and soybeans (unpublished data). In testing organisms with this peptide antibody we have routinely used immunoblots. One such test is shown in Fig. 1, using samples of a natural population of amphipods (Gammarus sp.) collected over a 3 month period. The proteins were extracted by homogenizing in cold phosphate-buffered saline (PBS) (150mM NaC1, 10mM NaH2PO 4, pH 7.2) with lmM phenyl-
A
B
C
D
E
F
6
H
1 9 4 , 0 " / 5 "'=
I I 1,300
"="
58,550
.m
35,325
i.
25,700
.m
20,075 16,250
.m ~='
m
~=m~w=~
~-...'-'--
'
- -
~,,,..j,.m.=-
....
Fig. 1. Immunoblot of hsp70 from Gammarus sp. collected below Conowingo Dam on the Susquehanna River in Pennsylvania, USA over a 3 month period in 1988. Blots were probed with antibody against a conserved peptide of hsp70. All lanes were loaded with 35/~g protein. Lane A: positive control (Mya arenaria); lane B: 15 October (16~C); lane C: 19 November (9C); lane D: 3 December (6.5' C); lane E: 17 December (2°C); and lane F: 31 December (3 C). Samples in lanes G and H were the same as those in lanes E and F but were stored at - 20 C, not - 80'C. Flow rates of water at the site were similar on each collection date, around 5000 cubic feet per second.
Stress protein detection using peptide antihmtv
473
methylsulfonyl fluoride (PMSF, a protease inhibitor). Samples were centrifuged at 13 000 x g and the supernatants were analyzed using sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblots, using the hsp70-directed antibody. The samples were applied to the gels at 35/xg per lane. Protein concentrations were determined by the Lowry method. A 12.5% S D S - P A G E gel was used to separate the proteins on a BioRad minigel apparatus using Laemmli buffer. 9 The gels were transferred to nitrocellulose membranes using an electrophoretic transfer system (Pharmacia). Blots were blocked with PBS Tween (0.3%) and incubated for 1 h with hsp70 antibody (1:500 in PBS-Tween), rinsed with PBS-Tween and incubated with goat anti-rabbit alkaline phosphataseconjugated antibody (Sigma) (1:1000 in PBS-Tween) for l h. The nitrocellulose was then rinsed and incubated in pH 9-5 buffer (100mM Tris, 100mM NaC1, 5mN MgCI2) with nitro-blue tetrazolium-5-bromo-4-chloro3 indolyl phosphate (nBT BCIP, a substrate for alkaline phosphatase) for colour development. Figure 1 shows hsp70 detected in amphipods (Gammarus sp.) collected below Conowingo Dam on the Susquehanna River in Pennsylvania between October and December 1988. The relative amounts of protein represented by areas of peaks from reflectance density scans (with the ambient temperatures at collection) were: lane B:0 (16~C); lane C :0'138 (9C): lane D:0.143 (6'5 C); lane E:0"195 (2 'C); and lane F:0"205 (3r'C). There seems to be greater stress as temperature decreases. Later samples in January, February and March continue the trend, with greater production ofhsp70 in the former two months and less in March. We have also examined the effect of starvation on hsp70 production in Gammarus. We observed either breakdown products or strong cross-reactivity with proteins of molecular weight around 30000 in the starved organisms but not in normally fed organisms. A sample prepared from Mya arenaria collected in upper Chesapeake Bay expressed hsp70 very strongly and so was used as the positive control (Lane A). We do not know at present whether or not the antibody is reacting to a constitutive form of hsp70 in Mva. The importance of storing samples at 80:C rather than - 20°C is illustrated in Fig. 1 by the decrease in protein detected in lane G (0"022) and lane H (0"158) from samples stored at - 20~ C, compared to lanes E (0.195) and F (0"205) from the same samples stored at -
_
8 0 ~ C .
We have investigated three other methods for detecting stress proteins: autoradiography and two immunosorbence assays. Autoradiography detects all newly synthesized proteins by incorporation of isotope-labeled amino acid, 5"8"1° Immunosorbent assays depend on immobilization of either antigen or antibody and the detection of antigen antibody complexes.
474
Brian P. Bradley, Joni B. Ward
The enzyme-linked immunosorbent assay (ELISA) is based on the detection of antigen-antibody complexes by linking an enzyme to a secondary antibody and detecting the enzyme with appropriate substrate. Some results are given in Ref. 8. A variant of ELISA, using liposome rather than enzyme labels, the liposome immunosorbent assay (LISA), has been tested by Plant et al., 11 with great success. Whatever the detection method used, ELISA, LISA, dot blot, immunodiffusion or perhaps a natural biosensor, the induction of stress proteins in general, and of so ubiquitous a family as hsp70 in particular, seems to have potential as a sensitive and repeatable assay for environmental contamination, meeting all the usual criteria for sublethal bioassays) More recent (unpublished) results show both qualitative and quantitative responses in several contaminant gradients. The key to the success of an immunoassay of any kind is a good antibody. Even with the polyclonal antibody to the conserved peptide, perhaps binding to different shorter sequences, the response obtained has been rapid, quantitative and clear. The method may be improved with a set of monoclonal antibodies, each binding to a specific sequence in the antigen and prepared against a number of major stress proteins.
A C K N O W L E D G E M ENTS The research was funded by grants from E. I. DuPont and Co. and the Maryland Department of the Environment. We are grateful for technical advice from Dr Thomas Elthon of this department.
REFERENCES 1. Huey, R. B. & Bennett, A. F., In The Role of Heat Shock and Stress Response in Biology and Human Disease, eds R. Morimoto & A. Tissieres. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (in press). 2. Lindquist, S. & Craig, E. A., Ann. Rev. Genet., 22 (1988) 631 77. 3. Nover, L. (ed.), Heat Shock Response of Eukao'otic Cells. Springer-Verlag, Berlin, 1984. 4. Atkinson, B. G. & Walden, D. B. (eds), Changes in Gene Expression in Response to Environmental Stress. Academic Press, New York, 1985. 5. Hakimzadeh, R. & Bradley, B. P., J. Thermal Biol. (in press). 6. Welch, W. J. & Feramisco, J. R., Molec. Cellular Biol., fi (1985) 1229--37. 7. Ehrhart, J. C., Duthur, A., Ullrich, S., Appella, E. & May, P. Oncogene (1988) 3595-603, 8. Bradley, B. P., 13th Symposium Aquatic Toxicology. American Society Testing and Materials Publ. (in press).
Stress prote& detection using peptide antibody
475
9. Laemmli, U. K., Nature (London), 227 (1970) 680-5 10. Bradley, B. P., Hakimzadeh, R. & Vincent, J. S., Hydrobiologia, 176[177 (1988) 187-90. 11. Plant, Anne L., Brizgys, M. V., Locasio-Brown, L. & Durst, R. A., Anal. Biochem., 176 (1989) 420-6.
Detection of a Major Stress Protein Using a Peptide Antibody
Brian P. Bradley & Joni B. Ward Department of Biological Sciences, University of Maryland, Baltimore, Maryland 21228, USA
ABSTRACT The synthesis of a major stress protein ( hsp70 ) is induced by extremes of a wide variety of environmental variables, both physical and chemical. This protein of approximately 70 000 Da is a widespread 'heat shock'protein and is present in various isomorphs in man), species. Nevertheless it is highly conserved among organisms from bacteria to man. The production of hsp 70 in a natural population of amphipods ( G a m m a r u s sp.) varied with ambient temperature. Protein extracts were probed on immunoblots using a polyclonal antibody, which we were given, made against a highly conserved peptide q[" hsp 70. Relative protein production was estimated using reflectance scanning. These results and others using immunoblots, autoradiography and immunosorbence assays suggest that the stress protein response may be a useful quantitative and qualitative environmental indicator.
Living cells respond to environmental contamination and to stress in general by synthesizing what are known as stress proteins. Some of these proteins are present in unstressed cells, but are produced in larger quantities under stress. Other proteins appear as the stress increases, and yet others stop being synthesized. Recent work on stress proteins from an ecological perspective is reviewed by Huey & Bennett.1 The heat shock phenomenon itself, the most studied part of the stress protein response, has been reviewed by Lindquist & Craig. 2 The response occurs in all species studied and to virtually all agents is considered stressful. 3'4 We and others have even observed it in response to a parasite. 5 Among the most common heat shock proteins are the family with molecular weights around 70000Da (hsp70). Some of these are present 471 Marine Environ. Res. 0141-1136/90/$03'50 ~ 1990 Elsevier Science Publishers L.td, England. Printed in Great Britain
Brian P. Bradley, Joni B. Ward
472
under normal conditions, others are inducible. 6 Unlike proteins such as cytochromes and metallothioneins, induced by chemical contaminants, the functions of hsp70 and other stress proteins seem to be varied. 2 Whatever the mechanism, they appear to have a protective role in the cell during stress. We have used an anti-hsp70 peptide serum provided by Stephen Ullrich 7 to detect hsp70. The antiserum was prepared against a 23-amino acid sequence at the C-terminal end of the protein which is highly conserved among several vertebrates including man. The antiserum cross-reacts with hsp70 and related proteins in a variety of organisms including copepods and Artemia, 8 cladocerans, shellfish, finfish, Lepidoptera and soybeans (unpublished data). In testing organisms with this peptide antibody we have routinely used immunoblots. One such test is shown in Fig. 1, using samples of a natural population of amphipods (Gammarus sp.) collected over a 3 month period. The proteins were extracted by homogenizing in cold phosphate-buffered saline (PBS) (150mM NaC1, 10mM NaH2PO 4, pH 7.2) with lmM phenyl-
A
B
C
D
E
F
6
H
1 9 4 , 0 " / 5 "'=
I I 1,300
"="
58,550
.m
35,325
i.
25,700
.m
20,075 16,250
.m ~='
m
~=m~w=~
~-...'-'--
'
- -
~,,,..j,.m.=-
....
Fig. 1. Immunoblot of hsp70 from Gammarus sp. collected below Conowingo Dam on the Susquehanna River in Pennsylvania, USA over a 3 month period in 1988. Blots were probed with antibody against a conserved peptide of hsp70. All lanes were loaded with 35/~g protein. Lane A: positive control (Mya arenaria); lane B: 15 October (16~C); lane C: 19 November (9C); lane D: 3 December (6.5' C); lane E: 17 December (2°C); and lane F: 31 December (3 C). Samples in lanes G and H were the same as those in lanes E and F but were stored at - 20 C, not - 80'C. Flow rates of water at the site were similar on each collection date, around 5000 cubic feet per second.
Stress protein detection using peptide antihmtv
473
methylsulfonyl fluoride (PMSF, a protease inhibitor). Samples were centrifuged at 13 000 x g and the supernatants were analyzed using sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblots, using the hsp70-directed antibody. The samples were applied to the gels at 35/xg per lane. Protein concentrations were determined by the Lowry method. A 12.5% S D S - P A G E gel was used to separate the proteins on a BioRad minigel apparatus using Laemmli buffer. 9 The gels were transferred to nitrocellulose membranes using an electrophoretic transfer system (Pharmacia). Blots were blocked with PBS Tween (0.3%) and incubated for 1 h with hsp70 antibody (1:500 in PBS-Tween), rinsed with PBS-Tween and incubated with goat anti-rabbit alkaline phosphataseconjugated antibody (Sigma) (1:1000 in PBS-Tween) for l h. The nitrocellulose was then rinsed and incubated in pH 9-5 buffer (100mM Tris, 100mM NaC1, 5mN MgCI2) with nitro-blue tetrazolium-5-bromo-4-chloro3 indolyl phosphate (nBT BCIP, a substrate for alkaline phosphatase) for colour development. Figure 1 shows hsp70 detected in amphipods (Gammarus sp.) collected below Conowingo Dam on the Susquehanna River in Pennsylvania between October and December 1988. The relative amounts of protein represented by areas of peaks from reflectance density scans (with the ambient temperatures at collection) were: lane B:0 (16~C); lane C :0'138 (9C): lane D:0.143 (6'5 C); lane E:0"195 (2 'C); and lane F:0"205 (3r'C). There seems to be greater stress as temperature decreases. Later samples in January, February and March continue the trend, with greater production ofhsp70 in the former two months and less in March. We have also examined the effect of starvation on hsp70 production in Gammarus. We observed either breakdown products or strong cross-reactivity with proteins of molecular weight around 30000 in the starved organisms but not in normally fed organisms. A sample prepared from Mya arenaria collected in upper Chesapeake Bay expressed hsp70 very strongly and so was used as the positive control (Lane A). We do not know at present whether or not the antibody is reacting to a constitutive form of hsp70 in Mva. The importance of storing samples at 80:C rather than - 20°C is illustrated in Fig. 1 by the decrease in protein detected in lane G (0"022) and lane H (0"158) from samples stored at - 20~ C, compared to lanes E (0.195) and F (0"205) from the same samples stored at -
_
8 0 ~ C .
We have investigated three other methods for detecting stress proteins: autoradiography and two immunosorbence assays. Autoradiography detects all newly synthesized proteins by incorporation of isotope-labeled amino acid, 5"8"1° Immunosorbent assays depend on immobilization of either antigen or antibody and the detection of antigen antibody complexes.
474
Brian P. Bradley, Joni B. Ward
The enzyme-linked immunosorbent assay (ELISA) is based on the detection of antigen-antibody complexes by linking an enzyme to a secondary antibody and detecting the enzyme with appropriate substrate. Some results are given in Ref. 8. A variant of ELISA, using liposome rather than enzyme labels, the liposome immunosorbent assay (LISA), has been tested by Plant et al., 11 with great success. Whatever the detection method used, ELISA, LISA, dot blot, immunodiffusion or perhaps a natural biosensor, the induction of stress proteins in general, and of so ubiquitous a family as hsp70 in particular, seems to have potential as a sensitive and repeatable assay for environmental contamination, meeting all the usual criteria for sublethal bioassays) More recent (unpublished) results show both qualitative and quantitative responses in several contaminant gradients. The key to the success of an immunoassay of any kind is a good antibody. Even with the polyclonal antibody to the conserved peptide, perhaps binding to different shorter sequences, the response obtained has been rapid, quantitative and clear. The method may be improved with a set of monoclonal antibodies, each binding to a specific sequence in the antigen and prepared against a number of major stress proteins.
A C K N O W L E D G E M ENTS The research was funded by grants from E. I. DuPont and Co. and the Maryland Department of the Environment. We are grateful for technical advice from Dr Thomas Elthon of this department.
REFERENCES 1. Huey, R. B. & Bennett, A. F., In The Role of Heat Shock and Stress Response in Biology and Human Disease, eds R. Morimoto & A. Tissieres. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (in press). 2. Lindquist, S. & Craig, E. A., Ann. Rev. Genet., 22 (1988) 631 77. 3. Nover, L. (ed.), Heat Shock Response of Eukao'otic Cells. Springer-Verlag, Berlin, 1984. 4. Atkinson, B. G. & Walden, D. B. (eds), Changes in Gene Expression in Response to Environmental Stress. Academic Press, New York, 1985. 5. Hakimzadeh, R. & Bradley, B. P., J. Thermal Biol. (in press). 6. Welch, W. J. & Feramisco, J. R., Molec. Cellular Biol., fi (1985) 1229--37. 7. Ehrhart, J. C., Duthur, A., Ullrich, S., Appella, E. & May, P. Oncogene (1988) 3595-603, 8. Bradley, B. P., 13th Symposium Aquatic Toxicology. American Society Testing and Materials Publ. (in press).
Stress prote& detection using peptide antibody
475
9. Laemmli, U. K., Nature (London), 227 (1970) 680-5 10. Bradley, B. P., Hakimzadeh, R. & Vincent, J. S., Hydrobiologia, 176[177 (1988) 187-90. 11. Plant, Anne L., Brizgys, M. V., Locasio-Brown, L. & Durst, R. A., Anal. Biochem., 176 (1989) 420-6.