Download Constitutive heat shock protein 70 (HSC70) expression in rainbow

Comparative Biochemistry and Physiology Part C 132 (2002) 223–233
Constitutive heat shock protein 70 (HSC70) expression in rainbow
trout hepatocytes: effect of heat shock and heavy metal exposure
Adrienne N. Boone, Mathilakath M. Vijayan*
Department of Biology, University of Waterloo, Waterloo, Ont., Canada N2L 3G1
Received 28 October 2001; received in revised form 11 April 2002; accepted 12 April 2002
Abstract
The 70-kDa family of heat shock proteins plays an important role as molecular chaperones in unstressed and stressed
cells. The constitutive member of the 70 family (hsc70) is crucial for the chaperoning function of unstressed cells,
whereas the inducible form (hsp70) is important for allowing cells to cope with acute stressor insult, especially those
affecting the protein machinery. In fish, the role of hsc70 in the cellular stress response process is less clear primarily
because of the lack of a fish-specific antibody for hsc70 detection. In this study, we purified hsc70 to homogeneity from
trout liver using a three-step purification protocol with differential centrifugation, ATP-agarose affinity chromatography
and electroelution. Polyclonal antibodies to trout hsc70 generated in rabbits cross-reacted strongly with both purified
trout hsc70 protein and also purified recombinant bovine hsc70. Two-dimensional electrophoresis followed by Western
blotting confirmed that the isoelectric point of rainbow trout hsc70 was more acidic than hsp70. Using this antibody, we
detected hsc70 content in the liver, heart, gill and skeletal muscle of unstressed rainbow trout. Primary cultures of trout
hepatocytes subjected to a heat shock (q15 8C for 1 h) or exposed to either CuSO4 (200 mM for 24 h), CdCl2 (10
mM for 24 h) or NaAsO2 (50 mM for 1 h) resulted in higher hsp70 accumulation over a 24-h period. However, hsc70
content showed no change with either heat shock or heavy metal exposure suggesting that hsc70 is not modulated by
sublethal acute stressors in trout hepatocytes. Taken together, we have for the first time generated polyclonal antibodies
specific to rainbow trout hsc70 and this antibody will allow for the characterization of the role of hsc70 in the cellular
stress response process in fish. 䊚 2002 Elsevier Science Inc. All rights reserved.
Keywords: Heat shock protein 70; Liver; Hsc70 antibody; Trout; Fish; Oncorhynchus mykiss; Stress; Copper; Cadmium; Arsenite
1. Introduction
Heat shock proteins (hsps) are a family of
highly conserved proteins playing an important
role in the functioning of unstressed and stressed
cells (Parsell and Lindquist, 1993). The hsp70
family, the most widely studied of the hsps, is
constitutively expressed (hsc70) in unstressed cells
and is also induced in response to stressors
(hsp70), especially those affecting the protein
*Corresponding author. Tel.: q1-519-888-4567, ext. 2305;
fax: q1-519-746-0614.
E-mail address:
[email protected] (M.M. Vijayan).
machinery. The hspyhsc70 proteins act as molecular chaperones and are crucial for protein functioning,
including
folding,
intracellular
localization, regulation, secretion, and protein degradation (Feder and Hofmann, 1999; Fink, 1999).
Different genes encode for hsp70 and hsc70 and
the coding sequences are continuous for hsp70,
whereas for hsc70 the sequences are interrupted
with several introns. The human hsc70 (Dworniczak and Mirault, 1987) and hsp70 amino acid
sequences (Hunt and Morimoto, 1985) are 81%
homologous with differences between the two
sequences mainly attributed to stretches of one to
at most five consecutive amino acid substitutions.
1532-0456/02/$ - see front matter 䊚 2002 Elsevier Science Inc. All rights reserved.
PII: S 1 5 3 2 - 0 4 5 6 Ž 0 2 . 0 0 0 6 6 - 2
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In fish, hsp70s have been sequenced in different
species including zebrafish (hsc70: Graser et al.,
1996; Santacruz et al., 1997), tilapia (hsp70: Molina et al., 2000), medaka (hsp70 and hsc70: Arai
et al., 1995), and rainbow trout (hsp70 partial
sequence: Kothary et al., 1984; Zafarullah et al.,
1992). The trout hsc71 and hsp70 amino acid
sequences are 80% identical, whereas the human
and trout hsc70 sequences are 94% identical (Zafarullah et al., 1992). In fish, there appear to be
different protein isoforms for hsp70, however, no
variation in protein isoforms was evident for hsc70
(White et al., 1994; Norris et al., 1995; Place and
Hofmann, 2001).
Like mammals, fish hsp70 expression is induced
by stressors including heat shock and chemical
shock, however, the stressor-induced hsc70 expression is not very clear. Hsc70 induction has been
observed in response to cadmium in rat brain
tumor cells (Hung et al., 1998), electrical shock
in mouse brain (Kaneko et al., 1993), transient
global ischemia in gerbil brain (Kawagoe et al.,
1993), hypoxia in a human kidney cell line (Turman et al., 1997), and by heat shock in rat kidney
cells (Sakakibara et al., 1992) or human melanoma
cell lines (Dressel et al., 1998). Alternatively,
decreased hsc70 expression has been observed
following treatment of IB3-1 cells with sodium 4phenylbutyrate (Rubenstein and Zeitlin 2000), or
mice with D-galactosamine and lipopolysaccharide
(Morikawa et al., 1998). Zebrafish hsc70 mRNA
expression was induced in embryos by heat shock
(Santacruz et al., 1997) and during caudal fin
regeneration (Tawk et al., 2000). Slightly enhanced
hsc70 mRNA and protein expression was also
observed after heat shock of two medaka cell lines
(Arai et al., 1995), whereas heat shock did not
affect hsc70 expression in topminnow hepatocytes
(White et al., 1994; Norris et al., 1995). In other
fish studies, no change in hsc70 mRNA expression
was observed following heat shock of RTG cells
(Zafarullah et al., 1992), exposure of CHSE cells
to cadmium or zinc (Zafarullah et al., 1992), or
exposure of rainbow trout red blood cells to azide,
hypoxia or zinc (Currie et al., 1999). While most
studies examined hsc70 mRNA changes, very few
studies have characterized hsc70 protein expression with stressors in fish. Changes in mRNA
expression do not necessarily correspond to changes in protein levels, and since proteins are functionally important, it is critical to examine hsc70
protein expression in order to understand the chap-
eroning role of these proteins in the stress tolerance
process in fish. The characterization of hsc70 in
fish has been limited primarily due to the lack of
hsc70-specific antibody because all the antibodies
currently available recognize both the inducible
and the constitutive form of the protein. While
mammalian hsp70 and hsc70 appear as two distinct
bands on SDS–PAGE gels (Hung et al., 1998),
these bands overlap with our fish samples even
with 6–10% gels and 8–16% gradient gels. It is
therefore, very difficult to study hsc70 expression
patterns with an antibody that recognizes both
hsp70 and hsc70, especially under conditions that
induce hsp70, such as following heat shock or
metal exposure.
Our objective, therefore, was to purify hsc70
from rainbow trout and develop antibodies in order
to characterize the hsc70 protein expression in
rainbow trout. To this end, our two major options
were to either purify rainbow trout hsc70 or to
synthesize peptides to a portion of the hsc70
sequence for antibody production. Peptide
sequences have been used successfully for hsp70
antibodies and total hsp70 antibodies (hsp70q
hsc70), but not for hsc70 antibody generation
(Dunlap and Matsumura 1997; Green et al., 1995).
Indeed, human hsp70 (Hunt and Morimoto 1985)
and hsc70 (Dworniczak and Mirault 1987) are
;81% homologous and the sequence differences
between the two are not great enough to design a
peptide sequence specific for hsc70. Consequently,
we chose to purify trout liver hsc70 and used the
entire protein as the antigen for antibody production. This trout-specific antibody was used as a
probe to examine the expression pattern of hsc70
in response to stressors that are known to induce
hsp70 in rainbow trout hepatocytes.
2. Materials and methods
2.1. Materials
ATP-Agarose beads, L15 media, sodium arsenite, protease inhibitor cocktail (P2714), 2-phenoxyethanol, and Freunds Adjuvant were obtained
from Sigma (St. Louis). Most general laboratory
chemicals and the BCIP and NBT were from
Fisher Scientific (Ontario). The 6-well Primaria
plates were from Falcon (Becton Dickinson Labware, NJ). The empty Econo column and flow
adaptor used for the ATP-agarose column were
from Bio-Rad. All electrophoresis reagents and
A.N. Boone, M.M. Vijayan / Comparative Biochemistry and Physiology Part C 132 (2002) 223–233
supplies, including the molecular weight markers
and the secondary antibody were from BioRad.
The protein A column was from Amersham Pharmacia Biotech. The total trout hsp70 antibody was
from Dr E. Peter M. Candido (Biochemistry
Department, UBC). Bicinchoninic acid (BCA)
solution was from Pierce Chemical Co. (IL).
Purified recombinant bovine hsc70 protein (SPP751) was from StressGen Biotechnologies Corp.
(Victoria, BC).
2.2. Animal
Rainbow Trout (Oncorhynchus mykiss) were
obtained from Rainbow Springs Trout farm (Thamesford, Ont.) and were maintained in tanks with
running well water (12"1 8C) under a 12-h lighty
dark photoperiod. Trout were acclimated for at
least 2 weeks prior to experimentation and were
fed once daily to satiety (3 pt sinking food; Martin
Mills Inc., Elmira, Ont).
2.3. Constitutive heat shock protein 70 (hsc70)
purification
2.3.1. Arsenite treatment
Fish (;300 g) were anaesthetized with an
overdose of 2-phenoxyethanol (1:1000) and the
liver quickly removed and placed in L15 medium
on ice. The fresh livers were minced (20 ml L15
mediumyliver), transferred to a 50-ml centrifuge
tube, and were slowly rocked in the presence of
50 mM sodium arsenite (NaAsO2) for 4 h at
13 8C. At the end of the 4 h, the arsenitecontaining media was replaced with fresh media
and the tissue was allowed to recover overnight
(typically 16–20 h) at 13 8C with gentle rocking.
Following recovery, the minced liver was pelleted
by centrifugation (300=g) for 5 min at 4 8C, the
media removed, and the liver protein either purified immediately or stored frozen at y70 8C.
2.3.2. Sample preparation
Arsenite-induced liver pieces were homogenized
(4 mlyg tissue) with a PowerGen tissue homogenizer (Fisher Scientific, Ont.) in ice-cold buffer A
(pH 7.5) containing Tris (20 mM), NaCl (20
mM), EDTA (0. 1 mM), MgCl2 (3 mM) and 2mercaptoethanol (15 mM) (Srivastava, 1997) and
supplemented with protease inhibitor cocktail (4(2-aminoethyl)-benzenesulfonyl fluoride, trans(4-guanidino)epoxysuccinyl-L-leucylamido
225
butane, bestatin, leupeptin, aprotinin and sodium
EDTA; Sigma). Homogenates were centrifuged
(10 000=g for 10 min followed by 100 000=g
for 1 h) at 4 8C and the high-speed supernatant
filtered (0.22-m syringe filter) prior to
chromatography.
2.3.3. ATP-agarose affinity chromatography
Lyophilized adenosine 59-triphosphate-agarose
(ATP linked via carbon-8 to cross-linked 4%
agarose beads) was hydrated in buffer A and a
column (5.5 ml; 1=7 cm) was prepared and
equilibrated. The column was fitted with a flow
adaptor and attached to a Bio-Rad DuoFlow FPLC
system equipped with a BioLogic QuadTec UV–
Vis detector (3 mm pathlength) and conductivity
detector. The filtered high-speed supernatant was
loaded onto the ATP-agarose column at 0.5 mly
min. The column was washed (1 mlymin) with 5
column volumes (CV) of buffer A, 10 CV buffer
A supplemented with 0.5 M NaCl, and then 5 CV
buffer A. The column was incubated (20 min)
with buffer A supplemented with 4 mM ATP and
the released proteins were eluted (0.5 mlymin).
The first 2.5 CV of eluted protein were pooled
and concentrated by dialysis against dry polyethylene glycol 8000 (overnight at 4 8C). The conductivity and proteinyATP absorbance (280 nm)
were monitored throughout the purification.
2.3.4. Electroelution
ATP-agarose-purified proteins were electrophoresed on 8% mini-gels at 200 mA for 90 min. The
gels were stained with Bio-Safe Coomassie stain
and the single hsc70 band from many lanes were
pooled and electroeluted with a BioRad model 422
Electro-Eluter according to the manufacturer’s
instructions. The hsc70 was electroeluted from the
gel (7 mA, 16 h, room temperature) into ammonium bicarbonate (50 mM) buffer with SDS (0.
1% wyv). The electroeluted hsc70 was dried on
low heat in a Savant Speedvac and then stored
frozen at y70 8C. Approximately 15–20 mg of
purified electroeluted hsc70 was recovered per
gram of liver. The specificity of the purified
protein was confirmed with SDS–PAGE followed
by immunodetection using a trout-specific total
hsp70 antibody (see below).
2.4. Antibody production
Two female New Zealand white rabbits (Oryctolagus cuniculus) (4.9 and 3.9 kg) (Charles River
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Canada, Ont.) were maintained in accordance with
Canadian Council of Animal Care guidelines. Lyophilized hsc70 (100 mg) was resuspended in 600
ml PBS (20 mM sodium phosphate pH 7.4, 150
mM NaCl), mixed with Complete Freunds Adjuvant (600 ml) and injected into the two rabbits,
each receiving a total of 50 mg hsc70 over four
initial injection spots. Three and 6 weeks after
injections, blood was withdrawn and the rabbits
were given booster shots (50 mg hsc70yrabbit).
Protein for booster shots was prepared in the same
manner as for the initial injections except Incomplete Freunds Adjuvant was used. Rabbits were
exsanguinated either 9 or 12 weeks after the initial
hsc70 injections. Blood collected before each
injection and during exsanguination was clotted (1
h at 37 8C and overnight at 4 8C) and the serum
recovered by centrifugation (10 000=g, 10 min,
4 8C). Purified hsc70 protein blots were probed
with the immunized serum (and goat anti-rabbit
secondary antibody) to determine the specificity
of hsc70 antibodies. The serum was frozen (y
70 8C) for later purification.
2.5. Antibody purification with protein A
The serum was purified with a HiTrap protein
A column (5 ml) attached to a Bio-Rad DuoFlow
FPLC system according to the manufacturer’s
(Amersham Pharmacia Biotech.) instructions.
Purified hsc70 protein blots were probed with
column fractions to determine the fractions containing hsc70 antibodies. Absorbance was monitored at 280 nm and the entire eluted protein peak
contained hsc70 antibody as determined by Western blotting (data not shown). Fractions containing
hsc70 antibodies were pooled, aliquoted and frozen
(y70 8C).
2.6. SDS–PAGE and Western blotting
The tissue protein concentrations were determined by the BCA method with bovine serum
albumin (BSA) as the standard. The samples were
analyzed on 8% polyacrylamide gels using the
discontinuous buffer system of Laemmli (1970).
The gels were either stained with Bio-Safe Coomassie stain or were transferred (20 V for 30 min)
onto nitrocellulose membranes with a SemiDry
Transfer Unit (Bio-Rad) using transfer buffer consisting of 25 mM Tris (pH 8.3), 192 mM glycine,
and 20% (vyv) methanol. Membranes were
blocked (60 min) with 5% skim milk in TBS-t
(20 mM Tris pH 7.5, 300 mM NaCl, 0.1% (vyv)
Tween 20) with 0.02% sodium azide. Primary and
secondary antibodies were diluted in the blocking
solution to the appropriate concentrations as indicated below. For total hsp70, a polyclonal rainbow
trout gonadal RTG-2 antibody was used at 1:3000
dilution. This antibody recognized both the hsp70
and hsc70 in rainbow trout tissues (Forsyth et al.,
1997; Vijayan et al., 1997, 1998). Our hsc70
antibody, obtained in the present study, was used
at 1:3000 dilution. The secondary antibody was
alkaline phosphatase-conjugated goat anti-rabbit
(1:3000) antibody. The blots were incubated with
primary antibody (60 min) at room temperature,
washed (3=5 min) with TBS-t, incubated with
secondary antibody (60 min), washed with TBS-t
(2=5 min), and finally washed with TBS (1=10
min). The bands were visualized with NBT
(0.033% wyv) and BCIP (0.017% wyv) and the
molecular weight was visualized using prestained
low range molecular weight markers (phosphorylase B 112 kDa, bovine serum albumin 81 kDa,
ovalbumin 49.9 kDa, carbonic anhydrase 36.2 kDa,
soybean trypsin inhibitor 29.9 kDa, lysozyme 21.3
kDa). The protein bands were scanned and the
band intensities quantified using the AlphaEase
software (AlphaEase Innovatech, CA).
2.7. 2D-electrophoresis
Rainbow trout hepatocytes were heat shocked
(q15 8C for 1 h) and then allowed to recover
(13 8C for 23 h) as described below. The cells
were harvested and stored frozen (y70 8C).
Hepatocytes (1.5 million cells) were sonicated in
25 mM Tris (pH 7.5) with 0.4% SDS and 10 mM
MgCl2. Samples were heated (90 8C for 5 min),
treated with DNase (room temperature for 10 min),
and the proteins were precipitated with acetone
(on ice for 10 min) and collected by centrifugation
(18 000=g, 3 min). Proteins (20 mgylane) were
solubilized in IEF sample buffer (9.25 M urea,
1.5% pH 5–8 Bio-Lytes, 2.5% 2-mercaptoethanol,
1% triton X-100, 0.00125% bromophenol blue)
and separated in the first dimension by isoelectric
focussing in 5% vertical denaturing mini-slab gels
with 250 ml BioLyte 5–8y10 ml of gel solution.
IEF gels were electrophoresed at 150 V for 30
min and then 200 V for 5 h with 10 mM H3PO4
and 20 mM NaOH as the lower and upper electrophoresis buffers, respectively. The measured pH
A.N. Boone, M.M. Vijayan / Comparative Biochemistry and Physiology Part C 132 (2002) 223–233
227
range of the IEF gel was 7.4–6.3. Proteins were
separated in the second dimension by 8% SDS–
PAGE. Gels were transferred for Western blotting
as mentioned above.
tioned above. The proteins (30 mgylane) were
separated on 8% SDS–PAGE gels and probed with
trout-specific hsc70 (present study) and total hsp70
antibodies as outlined above.
2.8. Constitutive heat shock protein 70 expression
in rainbow trout tissues
3. Results and discussion
Trout (;300 g) were anaesthetized with an
overdose of 2-phenoxyethanol and pieces of liver,
gill, heart and skeletal muscle were quickly frozen
on dry ice for detection of hsc70 expression.
Tissues were sonicated in 50 mM Tris (pH 7.5)
with protease inhibitor cocktail (see above), centrifuged (15 000=g, 2 min), and the protein
concentration determined in the supernatant as
mentioned above. The proteins (60 mgylane) were
separated on 8% SDS–PAGE gels and probed with
hsc70 antibodies as outlined above.
2.9. Hepatocyte preparation
Rainbow trout hepatocytes were isolated using
collagenase perfusion according to established protocols (Sathiyaa et al., 2001) and the cells were
plated in 6-well Primaria plates at a density of
1.5=106 cellsywell (0.75=106 cellsyml) in L15
media. The cells were allowed to recover overnight
(13 8C) prior to experimentation.
2.10. The effect of heat shock and metal exposure
on hsc70 expression
Hepatocytes were heat shocked (q15 8C for 1
h) and then allowed to recover at 13 8C for up to
47 h as indicated. Samples were taken before heat
shock (0 h), during heat shock (0.25, 0.5 h),
immediately following heat shock (1 h) and during
recovery (2, 4, 8, 24, 48 h). For comparison,
parallel control samples in the absence of heat
shock were also taken at 0, 1, 2, 4, 8, 24, 48 h.
For metal treatment, hepatocytes were incubated
with either CuSO4 (200 mM; 13 8C; 24 h),
CdCl2 (10 mM; 13 8C; 24 h) or NaAsO2 (50 mM;
13 8C; 1 h then 23 h with fresh media) and
preliminary studies established these dosages to be
sublethal to cells. The cells were pelleted by
centrifugation (13 000=g, 20 s) and stored at y
70 8C for later analysis. Hepatocytes were thawed
and sonicated in 100 ml 50 mM Tris (pH 7.5)
with protease inhibitor cocktail (see above) and
the protein concentrations determined as men-
We have purified to homogeneity the constitutive heat shock protein 70 (hsc70) in rainbow trout
and developed antibodies specific for trout hsc70.
This is the first known hsc70-specific antibody in
fish and allowed the characterization of hsc70
protein expression following acute heat shock and
metal exposure in rainbow trout hepatocytes.
3.1. Purification of trout hsc70
A trout-specific total hsp70 antibody that recognizes both the inducible (hsp70) and the constitutive (hsc70) hsp (see Vijayan et al., 1997;
Forsyth et al., 1997) was used to chart the progress
of our protein purification and also to characterize
the specificity of our antibody. The purification of
hsc70 was carried out following established protocols using ATP-agarose affinity chromatography
(Srivastava, 1997; Welch and Feramisco, 1985).
Although our intention was to simultaneously purify both hsp70 and hsc70 from arsenite-treated trout
liver pieces, preliminary studies confirmed that
there was no increased synthesis of total hsp70
with arsenite treatment compared with the control
liver pieces (data not shown). Consequently, the
arsenite-treated liver homogenate was used for the
purification of hsc70 protein. The majority of
proteins in the high-speed supernatant obtained
from arsenite-treated trout livers did not bind to
the ATP-agarose affinity column and were
removed by washing (Fig. 1a). There were three
major bands that eluted from the ATP-agarose
column (Fig. 1a) and immunodetection using
trout-specific total hsp70 antibody identified the
middle band as the 70-kDa heat shock protein
(Fig. 1b). This protein band, from multiple lanes,
was electroeluted and the protein appeared as a
single band on a Coomassie-stained gel (Fig. 1a)
and cross-reacted strongly with the trout total
hsp70 antibody (Fig. 1b).
3.2. Generation of polyclonal and bodies for hsc70
The rabbits were immunized with the purified
(electroeluted) protein and the serum from the
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A.N. Boone, M.M. Vijayan / Comparative Biochemistry and Physiology Part C 132 (2002) 223–233
Fig. 1. Hsc70 purification and antibody production. Hsc70 was purified from rainbow trout livers via differential centrifugation, ATPagarose chromatography and electroelution as detailed in Section 2. Progression of hsc70 purification was documented by (a) Coomassiestained SDS–PAGE and by (b) Western blotting with a trout-specific antibody that recognizes total hsp70 (hsp70qhsc70). The observed
results are for the high speed supernatant (Sup, 40 mg) that was loaded onto the ATP-agarose column, the fraction that flowed through
the column (FT, 40 mg), the concentrated eluted fraction (E, 2.25 mg), and the fraction further purified by electroelution from SDS–
PAGE gels (EE, 0.7 mg for (a) and 0.28 mg for (b)). The purified electroeluted protein was used for polyclonal antibody production
and the resultant rabbit serum was purified by protein A chromatography. (c) A representative Western blot showing that the purified
antibody cross-reacts with the ATP-eluted fraction (lane 1, 2.25 mg) and purified recombinant bovine hsc70 (lanes 2 and 3, 2 mg).
Parallel detection under the same conditions as with the purified antibody revealed no detectable labeling of purified recombinant bovine
hsc70 with the trout-specific total hsp70 antibody (lanes 4 and 5, 2 mg). (d) Hsc70 and hsp70 were separated by 2D electrophoresis
from heat-shocked (q 158 HS 1 h followed by 23 h recovery at ambient) trout hepatocytes and the proteins were visualized by Western
blotting with either the purified hsc70 antibody, the trout-specific total hsp70 antibody or a combination of both these antibodies mixed
together. The dark arrow and hollow arrow indicates the major and minor hsc70 spots, respectively; the thin arrow indicates hsp70
spot.
A.N. Boone, M.M. Vijayan / Comparative Biochemistry and Physiology Part C 132 (2002) 223–233
Fig. 2. Hsc70 expression in rainbow trout tissue. Trout heart,
gill, skeletal muscle and liver were excised and immediately
frozen on dry ice. Samples (60 mg) were separated by 8%
SDS–PAGE, transferred to a nitrocellulose membrane and
probed with trout-specific hsc70 antibody. Tissues were taken
from two fish.
immunized rabbits was partially purified by protein
A affinity chromatography. The antibody crossreacted strongly with the ATP-eluted fractions
confirming that the antibodies were specific to the
purified protein (Fig. 1c). We further confirmed
the specificity of our hsc70 antibody by probing
purified recombinant bovine hsc70. Our antibody
showed clearly very high cross-reactivity with
bovine hsc70, whereas the total hsp70 antibody
showed no cross-reactivity (Fig. 1c) providing
further evidence that our antibody is highly specific to hsc70 with much stronger cross-reactivity
for hsc70 than the total hsp70 antibody. Despite
the substantial purification achieved with the protein A column, some minor vertical streaks were
still observed when using the antibody for Western
blotting (Figs. 1c, 2, 3d). The non-specific streaks
did not disappear with either ammonium sulfate
precipitation or drying followed by reconstitution
of the antibody, although further dilution of the
antibody did slow the appearance of streaks. Interestingly, once the antibody solution was used to
probe a number of blots, the streaks and nonspecific binding disappeared. The antibody was
very stable at 1:3000 dilution in 5% skim milk
(with sodium azide) and the diluted antibody could
be used to probe numerous blots for many months.
Our hsc70 antibody also recognized one and sometimes two faint bands of approximately 55–65
kDa in the ATP-agarose-purified fraction (Figs.
1c, 3d) suggesting that these bands were ATPbinding proteins. The electroeluted purified hsc70
used for antibody production was a single band
and contained no detectable 55–60-kDa proteins
(Fig. 1a). A likely explanation is that the lower
bands may contain epitopes similar to those of
hsc70 or perhaps the antibody was detecting a
breakdown product of hsc70.
229
The specificity of the hsc70 antibody was further
assessed by 2D-gel electrophoresis and Western
blotting. While mammalian hsp70 and hsc70
appear as two distinct bands on SDS–PAGE gels
(Hung et al., 1998), these bands overlap with our
fish samples even with 6–10% gels and with 8–
16% gradient gels. However, we were able to
separate hsc70 and hsp70 by 2D-gel electrophoresis of heat-shocked hepatocytes and Western blotting with the hsc70 antibody showing a prominent
spot (dark arrow) with a pI more acidic than hsp70
(thin arrow) (Fig. 1d). This agrees with other
studies that showed hsc70 to be more acidic than
hsp70 in fish (Norris et al., 1995). Also, our
antibody also labeled a minor spot with a pI that
was more basic than the prominent hsc70 spot
(hollow arrow; Fig. 1d). The total hsp70 antibody
showed a large streak (Fig. 1d) that may be due
to the fact that the antibody recognized both hsc70
and hsp70 and also because of the presence of
multiple hsp70 isoforms with slightly different pI
values (Norris et al., 1995). However, alignment
of the two blots (probed with hsc70 and total
hsp70 antibody) showed some overlap of spots
and it is not clear if the minor more basic spot
labelled with the hsc70 antibody represents an
additional hsc70 isoform or is labeling hsp70. The
observed lack of hsc70 induction with heat shock,
copper, cadmium, and arsenite (Figs. 3 and 4)
strongly suggests that the hsc70 antibody is recognizing at least two separate hsc70 isoforms in
rainbow trout.
3.3. Tissue distribution of hsc70
The constitutive member of the hsp70 family of
proteins, hsc70, plays an important role as molecular chaperone in unstressed cells (see Fink, 1999).
Recently, hsc70 was purified from the white muscle of Goby and was shown to exhibit chaperoning
function at temperature ranges far above their
physiological tolerance (Place and Hofmann,
2001) suggesting a role for this protein in the heat
shock response in fish tissue. Using our antibody
we were able to detect hsc70 in different tissues,
including liver, heart, gill and white muscle of
rainbow trout (Fig. 2). The lower hsc70 content
in the skeletal muscle may be due to the lower
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Fig. 3. The effect of heat shock on hsc70 protein expression. (A,B) Hepatocytes were heat shocked for 1 h (q15 8C) and sampled
following recovery at 3 (lane 2) or 23 h (lane 3) at ambient temperature (13 8C). Control hepatocytes (lane 1) were sampled just
prior to stressor exposure. Samples (30 mg) were probed with either trout-specific purified hsc70 antibody (a) or trout-specific total
hsp70 antibody (b). The experiment was repeated four times with similar results. The temporal changes in hsc70 protein expression
was further studied with three separate fish over a longer period of time and samples were probed with trout-specific hsc70 antibody.
(c) A representative Western blot of control cells in the absence of heat shock and sampled at 0, 1, 2, 4, 8, 24, 48 h. (d) A representative
Western blot of heat-shocked hepatocytes (1 h at q15 8C) and sampled either during the heat shock (0.25, 0.5 h), immediately after
heat shock (1 h) or during recovery from heat shock (2, 4, 8, 24, 48 h). (e) Hsc70 expression prior to heat shock (0) and at 48 h
either in the absence (48yHS) or presence of heat shock (48qHS); values represent mean"S.E.M. (ns3); there was no statistically
significant difference (P-0.05, paired t-est).
protein turnover in this tissue compared to liver,
gill and heart. Studies are underway to characterize
the temporal pattern of hsc70 expression in various
trout tissues in response to different stressors,
including heat and chemical shock.
3.4. The effect of heat shock on hsc70 protein
expression in trout hepatocytes
Heat shock (HS; q15 8C) exposure induced
total hsp70 protein accumulation by approximately
A.N. Boone, M.M. Vijayan / Comparative Biochemistry and Physiology Part C 132 (2002) 223–233
231
Fig. 4. The effect of metals on hsc70 protein expression. Rainbow trout hepatocytes were incubated either in the absence (Control;
Con) or presence of copper (Cu; 200 mM CuSO4), cadmium (Cd; 10 mM CdCl2) or arsenite (As; 50 mM NaAsO2) as outlined in
Section 2. Samples (30 mg) were separated by 8% SDS–PAGE, transferred to nitrocellulose membrane, and probed with purified troutspecific hsc70 antibody (a,c) or trout-specific total hsp70 antibody (b,d). All blots are representative of three separate fish. (e) Hsc70
protein expression with metals were quantified and shown as mean"S.E.M. (ns3 fish); there was no statistically significant difference
(P-0.05; paired t-est).
fivefold over a 24-h period in trout hepatocytes in
primary culture (Fig. 3b) and this result concurred
with a recent study showing a similar increase in
hsp70 response with HS in trout hepatocytes
(Boone et al., 2002). When a parallel blot to that
shown in Fig. 3b was probed with our purified
trout-specific hsc70 antibody, no increase in hsc70
protein expression was observed (Fig. 3a). Also,
there were no temporal changes in hsc70 protein
expression either in the absence of heat shock
(Fig. 3c) or during heat shock and the recovery
period (Fig. 3d,e). These results concur with other
studies showing a similar lack of change in hsc70
expression (35S-labelling and 2D-electrophoresis)
with heat shock in fish hepatocytes (White et al.,
1994; Norris et al., 1995).
3.5. The effect of metal exposure on hsc70 protein
expression
Heavy metal exposure results in higher hsp70
expression in fish tissues, including hepatocytes
(Iwama et al., 1998; Boone et al., 2002). Our
results clearly show elevated hsp70 accumulation
(three to sixfold) following exposure of trout
hepatocytes to either CuSO4, CdCl2 or NaAsO2
(Fig. 4b,d). However, we show for the first time
that hsc70 protein expression is not altered by
232
A.N. Boone, M.M. Vijayan / Comparative Biochemistry and Physiology Part C 132 (2002) 223–233
metal exposure in fish cells (Fig. 4a,c,e). This lack
of higher hsc70 expression with metals in trout
hepatocytes (Fig. 4) may not be due to altered
protein turnover because previous studies also
failed to observe any changes in hsc70 mRNA
accumulation upon exposure of CHSE cells to
cadmium or zinc (Zafarullah et al., 1992), or
exposure of rainbow trout red blood cells to azide,
hypoxia or zinc (Currie et al., 1999). Taken together, these results suggest that hsc70 expression is
not modulated by sublethal acute stressors in fish.
However, although the hsc70 content does not
change with stressors, intracellular partitioning of
hsc70 content may be playing a key role in
allowing cells to cope with stress. Indeed nuclear
translocation of hsc70 was shown to be important
in allowing HeLa cells to cope with heat shock
(Chu et al., 2001). The availability of trout-specific
hsc70 antibody will, therefore, allow us to characterize the stressor-induced intracellular trafficking of hsc70 in fish cells and its implication in the
cellular stress response process.
Acknowledgments
This study was supported by the Natural Sciences and Engineering Research Council
(NSERC), Canada, operating grant to M.M. Vijayan. The authors would like to thank Dr P. Candido
for the generous gift of the trout-specific total
hsp70 antibody and Mr M. Ryan for assistance
with the generation of hsc70 antibodies.
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