Mitsubishi Kasei Institute of Life Sciences, Tokyo, Japan;8Department of Material-Process Engineering and Applied Chemistry for Environment, Akita University Faculty of Engineering and R
Trang 1Characterization of the 105-kDa molecular chaperone
Identification, biochemical properties, and localization
Mika Matsumori1,2, Hideaki Itoh1, Itaru Toyoshima3, Atsushi Komatsuda4, Ken-ichi Sawada4, Jun Fukuda5, Toshinobu Tanaka5, Atsuya Okubo6, Hiroyuki Kinouchi6, Kazuo Mizoi6, Tokiko Hama7, Akira Suzuki1, Fumio Hamada8, Michiro Otaka3, Yutaka Shoji2and Goro Takada2
1
Department of Biochemistry,2Department of Pediatrics,3First Department of Internal Medicine,4Third Department of Internal Medicine,5Department of Gynecology, and6Department of Neurosurgery, Akita University School of Medicine, Akita City, Japan;7President’s Frontier Laboratory Mitsubishi Kasei Institute of Life Sciences, Tokyo, Japan;8Department of
Material-Process Engineering and Applied Chemistry for Environment, Akita University Faculty of Engineering and
Resource Science, Akita City, Japan
We have characterized the biochemical properties of the
testis and brain-specific 105-kDa protein which is
cross-reacted with an anti-bovine HSP90 antibody The protein
was induced in germ cells by heat stress, resulting in a protein
which is one of the heat shock proteins [Kumagai, J.,
Fuk-uda, J., Kodama, H., Murata, M., Kawamura, K., Itoh, H
& Tanaka, T (2000) Eur J Biochem 267, 3073–3078] In the
present study, we characterized the biochemical properties of
the protein The 105-kDa protein inhibited the aggregation
of citrate synthase as a molecular chaperone in vitro ATP/
MgCl2has a slight influence of the suppression of the citrate
synthase aggregation by the 105-kDa protein The protein
possessed chaperone activity The protein was able to bind to
ATP–Sepharose like the other molecular chaperone HSP70
A partial amino-acid sequence (24 amino-acid residues) of
the protein was determined and coincided with those of the
mouse testis- and brain-specific APG-1 and osmotic stress
protein 94 (OSP94) The 105-kDa protein was detected only
in the medulla of the rat kidney sections similar to OSP94
upon immunoblotting The purified 105-kDa protein was cross-reacted with an antibody against APG-1 These results suggested that APG-1 and OSP94 are both identical to the 105-kDa protein There were highly homologous regions between the 105-kDa protein/APG-1/OSP94 and HSP90 The region of HSP90 was also an immunoreactive site An anti-bovine HSP90 antibody may cross-react with the 105-kDa protein similar to HSP90 in the rat testis and brain
We have investigated the localization and developmental induction of the protein in the rat brain In the immuno-histochemical analysis, the protein was mainly detected in the cytoplasm of the nerve and glial cells of the rat brain Although the 105-kDa protein was localized in all rat brain segments, the expression pattern was fast in the cerebral cortex and hippocampus and slow in the cerebellum Keywords: molecular chaperone; 105-kDa protein; APG-1; OSP94
All living cells display a rapid induction of some proteins
known as molecular chaperones (heat-shock proteins or
stress proteins) when the cells are exposed to environmental
stresses such as lethal heat shock or variety of toxic reagents
[1] Among the molecular chaperones, HSP90 is a
cytoplas-mic protein in unstressed mammalian cells and has been
found in transient association with steroid hormone
recep-tors and regulates their activation mechanism [2] It has
been reported that HSP90 plays the role of capacitor for morphological evolution [3] and facilitates the synthesis and correct folding of other intracellular proteins [4] HSP90 contains two independent chaperone sites both in the N-terminal and C-terminal of the protein [5,6] Each chaperone activity of the protein will be inhibited by different antineoplastic agents of geldanamycin and cisplatin, respectively [7]
We reported before that a rat 105-kDa testis protein was cross-reacted with an antibody against bovine HSP90 [8] The 105-kDa protein was also detected in the brain, but not
in the liver, lung, spleen, kidney, ovarium, or uterus, in contrast to the wide distribution of HSP90 There was a high similarity between HSP90 and the 105-kDa protein on peptide mapping with trypsin digestion Except for the molecular mass, the physicochemical properties of the 105-kDa protein were similar to those of HSP90, and the protein seems to be a cognate protein of HSP90
On immunoblotting analysis, the 105-kDa protein appeared at approximately the age of 5 weeks and coincided with the appearance of spermatozoa during the development
of the rat testis The 105-kDa protein was more abundant in the spermatozoa but not in a somatic cell line derived from a
Correspondence to H Itoh, Department of Biochemistry,
Akita University School of Medicine, 1-1-1 Hondo, Akita City,
010-8543 Japan Tel.: + 81 18 884 6078, Fax: + 81 18 884 6078,
E-mail: hideaki@med.akita-u.ac.jp
Abbreviations: HSP110, 105, 90, HSP70 and HSP60, 110-, 105-,
90-, 70-, and 60-kDa heat shock proteins; GRP78, 78-kDa glucose
regulated protein; OSP94, osmotic stress protein 94; HSF-1, heat
shock transcription factor 1; CS, citrate synthase; AzC, L
-azetidine-2-carboxylic acid; PC12, rat phenochromocytoma cell; BCIP,
5-bromo-4-chloro-3-indolyphosphate p-toluidine salt; NBT, nitroblue
tetrazolium chloride.
(Received 29 July 2002, revised 13 September 2002,
accepted 18 September 2002)
Trang 2Leydig cell tumor in rat testis [9] These results indicate that
the 105-kDa protein is one of the sperm-specific proteins
Recently, we have reported that signals of the 105-kDa
protein were selectively detected immunohistochemically in
the germ cells and might translocate into the nuclei from the
cytoplasm in response to heat shock [10] Moreover, the
105-kDa protein formed a complex with p53 at 32.5C,
which is the scrotal temperature, but not at 37C, which is
the suprascrotal temperature; the 105-kDa protein is
sug-gested to contribute to the regulation of p53 function in
testicular germ cells [10]
It has been shown that testis-specific APG-1 (accession
no D49482) and osmotic stress protein 94 (OSP94)
(accession no U23291) cDNA were cloned independently
[11,12] Both APG-1 and OSP94 are members of the
HSP110/SSE subfamily The cDNA of both APG-1 and
OSP94 encodes 838 acid residues, and those
amino-acid sequences were the same To determine the interaction
between the 105-kDa protein and APG-1 or OSP94, we
investigated the amino-acid sequence and some biochemical
properties of the 105-kDa protein In the present study, we
discuss the biochemical properties of the 105-kDa protein in
the rat brain and the interaction between the 105-kDa
protein and APG-1 or OSP94
In our earlier studies, the 105-kDa protein was induced in
germ cells by heat stress, and the protein formed a complex
with p53 in a temperature-dependent manner [10] The
protein appeared at 5 weeks (postbirth), during the
devel-opment of the rat testis and coincided with the appearance
of spermatozoa [9] The 105-kDa protein may be involved in
the regeneration of p53 function in testicular germ cells In
contrast, the localization of the protein in the brain has not
yet been known We have also investigated the distribution
of the 105-kDa protein in the rat brain and the appearance
of 105-kDa protein in several sections of the post natal
developmental rat brain
M A T E R I A L S A N D M E T H O D S
ATP–Sepharose was prepared as described previously [13]
DE-52 was obtained from Whatman and Lysyl
endopep-tidase (EC 3.4.21.50) was from Wako Pure Chemical
Institute
Purification of the 105-kDa protein
The 105-kDa testis and brain protein was purified from rat
testis as described previously [8] The protocols for animal
experimentation described in this paper were previously
approved by the Animal Research Committee, Akita
University School of Medicine; the Guidelines for Animal
Experimentation of the University were completely adhered
to in all subsequent animal experiments
Antibody production
An antibody against to the rat 105-kDa protein was
produced by intramuscular injection into a rabbit of 1 mg of
the protein emulsified in complete Freund’s adjuvant
Booster shots were given three times in the same manner
as the original injection at 2-week intervals The rabbit was
bled 10 days after the last injection The antiserum raised
against rat 105-kDa protein (2 mL) was dialyzed against
10 mM Tris/HCl (pH 7.4) The serum was applied to a DEAE–cellulose column (1· 4 cm) pre-equilibrated in
10 mM Tris/HCl (pH 7.4) The pass-through fractions were collected and examined as IgG on SDS/PAGE gel electrophoresis An anti-(rat 105-kDa protein) IgG was used
in this study Antibodies against porcine HSP60, bovine HSP70, and bovine HSP90 were used as described previ-ously [8,13,14]
Measurement ofin vitro chaperone activity The influence of the 105-kDa protein on the thermal aggregation of mitochondrial citrate synthase (CS;
EC 4.1.37; Boehringer–Mannheim) at 43C was measured
as previously described [7] Light scattering CS (0.075 lM)
in 50 mMHepes buffer (pH 7.4) in the presence or absence
of bovine serum albumin (15 lM) and the 105-kDa protein (0.075 lM) was monitored for 90 min by the optical density
at 500 nm in a Pharmacia Ultospec 3000 UV/Vis spectro-photometer equipped with a temperature control unit using semi microcuvettes (1 mL) with a path length of 10 mm ATP–Sepharose column chromatography
Rat testes were homogenized in 9 vols of 50 mMTris/HCl (pH 7.4) containing 0.25M sucrose and centrifuged at
9000 g for 10 min at 4C The supernatant was collected, followed by centrifugation at 105 000 g for 60 min at
4C The supernatant obtained from ultracentrifugation was used as the rat testis cytosol in the present study ATP–Sepharose was equilibrated in buffer A (10 mM Tris/HCl, pH 7.4, 5 mM CaCl2, 5 mM MgCl2) Rat testis cytosols containing 5 mM CaCl2 and 5 mM MgCl2 were applied to the column and washed with buffer A containing 0.15M NaCl After washing the column, binding proteins were eluted with 5 mM ATP in buffer
A Eluted proteins were detected by SDS/PAGE and immunoblotting
Amino-acid sequence of 105-kDa protein The amino-acid sequence of 105-kDa protein was deter-mined using a protein sequencer as described previously [14,15] Briefly, purified 105-kDa protein from the testis was electrophoresed by SDS/PAGE, and the protein band was excised and digested using lysyl endopeptidase The reverse phase column (Wakosil 5C18, Wakopak) that was connected to an HPLC apparatus purified the digested peptides The peptides were applied onto the column and eluted with a linear gradient of 0–60% acetonitrile (v/v) in 0.1% trifluoroacetic acid (v/v) at a flow rate of 0.5 mLÆmin)1, and 0.5-mL fractions were collected Amino-acid sequencing of the purified peptides was performed with a 491 Procise protein sequence system (Perkin-Elmer)
Gel electrophoresis SDS/PAGE was carried out by the procedure of Laemmli [16] Gels were stained with 0.1% Coomassie Brilliant Blue (v/v) with 25% isopropyl alcohol (v/v) and 10% acetic acid (v/v) and destained with 10% isopropyl alcohol (v/v) and 10% acetic acid (v/v)
Trang 3Samples were electrophoresed on SDS/polyacrylamide gels,
transferred to a poly(vinylidene difluoride) membrane
(Bio-Rad) electrophoretically, and processed as described
by Towbin et al [17] The membrane was incubated with
anti-HSP90 antibody (diluted 1 : 1000 in 7% skim milk) or
anti-rat 105-kDa protein IgG (diluted 1 : 2000 in 7% skim
milk) The membrane was also incubated with an antibody
against APG-1 or an antibody against HSF-1 (Santa Cruz
Biotechnology, diluted 1 : 500 in 7% skim milk) The
membranes were treated with alkaline phosphatase
anti-(rabbit IgG) Ig (Bio-Rad) (diluted 1 : 1000 in 7% skim
milk) Antigen-antibody complexes were visualized by
reacting alkaline phosphatase using
5-bromo-4-chloro-3-indolyphosphate p-toluidine salt (BCIP) and nitroblue
tetrazolium chloride (NBT)
Water-restriction of rat
Control Wister rats (Male, 6 weeks) were provided ad
libitum access to water, and dehydrated rats were restricted
from drinking water for 3 and 5 days All rats were
sacri-ficed, the kidneys were removed, and the renal cortex,
medulla, and papilla were dissected Tissues were,
respect-ively, homogenized in 3 vols of 10 mM Tris/HCl (pH 7.4)
containing 0.15M NaCl and centrifuged at 20 000 g for
10 min at 4C The supernatants were used for SDS/PAGE
and immunoblotting as the soluble fraction The
precipi-tates were washed with the same buffer, collected by
centrifugation at 20 000 g for 10 min at 4C, and used for
SDS/PAGE and immunoblotting as the insoluble fraction
Homology search, hydropathy profile, and secondary
structure prediction
DNASIS(version 2.1, Hitachi Software Engineering Co, Ltd)
was used for the amino-acid sequence homology search,
hydropathy profiles, and secondary structure prediction of
the 105-kDa protein, HSP90, APG-1, and OSP94 For the
hydropathy profiles of APG-1 or OSP94, we used the
hydrotable of Hopp and Woods The secondary structure
prediction of the proteins has been performed by Chou and
Fasman prediction methods
Detection of the 105-kDa protein in rat brain during
the development
Female and male Wistar rats were born from the same
parents, 3.5 days old, 1-, 2-, 3-, 4-, 5-, and 6-weeks-old,
and dissected to obtain several brain sections The sections
were the olfactory lobe, cerebral cortex, hippocampus,
mid brain, cerebellum, and medulla oblongata Each
tissue section was homogenized in 3 vols of 10 mM Tris/
HCl (pH 7.4) containing 0.15M NaCl and centrifuged at
40 000 g for 10 min at 4C The supernatants were used
for SDS/PAGE and immunoblotting as the soluble
fraction The precipitates were washed with the same
buffer, collected by centrifugation at 40 000 g for 10 min
at 4C, and used for SDS/PAGE and immunoblotting as
the insoluble fraction Control Wister rat (Female,
6 weeks) were sacrificed and the brain was removed
The olfactory lobe, cortex of the cerebrum, hippocampus,
mid brain, cerebellum, and medulla oblongata were dissected Tissues were, respectively, homogenized as described above The supernatant and precipitates were used for SDS/PAGE or immunoblotting
Immunohistochemistry Nonfixed and quickly frozen rat brains were sectioned in a cryostat Sections (15 lm thick) were fixed 3.7% formalde-hyde in NaCl/Pifor 10 min at room temperature After the sections were washed with NaCl/Pi(5 min, 3 times), they were incubated with anti-rat 105-kDa protein IgG (diluted
1 : 200 in 0.2% BSA/0.01% Saponin NaCl/Pi) overnight at
4C and then washed again with NaCl/Pi (15 min, threefold) Bound antibodies were visualized with horse-radish peroxidase-labeled anti-(rabbit IgG) Ig conjugated with amino-acid polymer (Histofine simple stain MAX-PO, Nichirei) according to the manufacturers instructions Cell culture
PC12 and PC12h cells (provided from T Hama, Mitsubishi Kasei Institute of Life Sciences) were grown in RPMI-1640 supplemented with 10% fetal calf serum in a culture flask at
37C in a humidified atmosphere containing 5% CO2 Upon approaching confluence (c 1· 106 cells/culture flask), the medium was removed, and the cells were stressed
by the addition of 5 mM L-azetidine 2-carboxylic acid (AzC)
in fresh medium containing 10% fetal bovine serum for 6 h The cells were stressed by increasing the temperature to
43C for 30 min followed by growth at 37 C for 6 h The cells were harvested and washed twice with 10 mM Tris/ HCl, pH 7.4) containing 0.125M NaCl After the centrif-ugation at 2000 g for 10 min, the cells were collected The pellet was then sonicated in SDS sample buffer and centrifuged at 18 000 g for 10 min The supernatant was used for SDS/PAGE and immunoblotting
R E S U L T S
Specificity of antibody against rat 105-kDa protein Two types of sample were prepared as the soluble and insoluble fractions from the cortex of the cerebrum Samples were separated on SDS/PAGE following to immunoblotting using anti-bovine HSP90 antibody [8] or anti-rat 105-kDa protein IgG (Fig 1) An antibody against bovine HSP90 was recognized as rat HSP90 (Fig 1B) HSP90 was detected mainly in the soluble fraction of the rat brain The antibody recognized another protein with a molecular mass of
105 kDa Anti-bovine HSP90 reacts mainly with HSP90 and also reacts faintly with 105-kDa protein On the contrary, anti-rat 105-kDa protein IgG was cross-reacted with only 105-kDa protein (Fig 1C) No other protein bands were detected both in the soluble and insoluble fractions of the rat brain The antibody is highly specific for the antigen Thus, an anti-rat 105-kDa protein IgG can strongly recognize the protein compared to an anti-bovine HSP90 antibody Chaperone activity of the 105-kDa protein
To analyze the functional properties of the 105-kDa protein,
we studied its action in protein folding in vivo As shown
Trang 4Fig 2, spontaneous aggregation of CS has been obserbed at
43C Although there was no effect on the reaction in the
presence of 200-fold molar excess bovine serum albumin, an
equimolar amount of the 105-kDa protein suppressed the
aggregation of CS Next, we investigated the influence of
ATP on the assay system ATP/MgCl2has a slight influence
on the suppression of CS aggregation by the 105-kDa
protein The 105-kDa protein apparently interacts
transiently with the highly structured early unfolding inter-mediates
ATP–Sepharose column chromatography Among the mammalian molecular chaperones, HSP70, GRP78, and HSP60 are able to bind to an ATP–Sepharose column [13,14] Although HSP90 is not able to bind to ATP–Sepharose, the protein has two independent ATP-binding sites in both the N- and C-terminals [18] As mentioned above, almost all molecular chaperones can interact with ATP On the contrary, there are no reports for ATP-binding of 105-kDa protein To investigate interaction between the 105-kDa protein and ATP, we analyzed the ATP-binding proteins of rat using ATP–Sepharose column chromatography Rat testis cytosols were applied onto the column; eluted proteins were detected by SDS/PAGE and immunoblotting as described in Materials and methods As shown in Fig 3A, we detected some protein bands with molecular masses of 70-, 78-, and 105-kDa on the gel On immunoblotting using anti-HSP90 antibody, the 105-kDa protein was detected only in the one eluted fraction to some extent (Fig 3B) HSP90 was also detected in the same fraction with a very faint protein band On the other hand, the 105-kDa protein was detected in all eluted fractions on immunoblotting using an anti-rat 105-kDa protein IgG (Fig 3C) Because of a high titer of an anti-(rat 105-kDa protein) Ig, IgG was recognized in all eluants in spite of the low concentration on immunoblotting Due to the low
Fig 2 Chaperone activity of the 105-kDa protein Thermal
aggrega-tion of CS (0.075 l M ) in the absence of additional components (open
circle), in the presence of a 200-fold molar ratio of albumin (closed
circle), an equimolar ratio of the 105-kDa protein (open triangle), an
equimolar ratio of the 105-kDa protein and 5 m M ATP/MgCl 2 (closed
triangle) ATP/MgCl 2 was added (down arrow) after 30 min of
ther-mal aggregation of CS and the 105-kDa protein (open square).
Fig 3 ATP–Sepharose column chromatography of testis cytosols The eluted fractions from an ATP–Sepharose column were electrophoresed
on SDS-polyacrylamide gels (9% gel), which were stained with Coo-massie Brilliant Blue (A), by immunoblotting with an antibody against bovine HSP90 (B), or by immunoblotting with an antibody against the 105-kDa protein IgG (C).
Fig 1 Specificity of an antibody against the 105-kDa protein Soluble
and insoluble fraction of rat brain were electrophoresed on
SDS-polyacrylamide gels (10% gel), which were stained with Coomassie
Brilliant Blue (A), by immunoblotting with an antibody against bovine
HSP90 (B), or by immunoblotting with an antibody against the
105-kDa protein IgG (C).
Trang 5concentration, the protein band was faintly recognized only
in one fraction on the Coomassie Brilliant Blue stained gel
An anti-HSP90 antibody could barely recognize the protein
in only one eluted fraction These results suggested that the
105-kDa protein is an ATP-binding protein the same as the
other molecular chaperones like a HSP70 and GRP78
Amino-acid sequence of the 105-kDa protein
To investigate the biochemical properties of the 105-kDa
protein, the amino-acid sequence of the protein was
determined using a peptide sequencer The peptides of the
105-kDa protein digested with lysyl endopeptidase were
purified using the reverse phase column connected to an
HPLC As shown in Fig 4, the two peptides (#40 and #70)
were sequenced Nine amino-acid residues were obtained
from peptide #40, and 15 amino-acid residues were obtained
from peptide #70 The total of 24 amino-acid sequences
from the two peptides of the protein had complete similarity
to APG-1, a testis-specific protein [11], and OSP94, a renal
medulla-specific protein [12] No homology has been shown
between the 105-kDa protein and HSP105 [19] The
amino-acid sequence of the 105-kDa protein showed that the
protein is identical to APG-1 and OSP94
Renal localization of the 105-kDa protein during water
restriction
It has been shown that OSP94 mRNA is induced in the
renal inner medulla of a water-restricted mouse [12] We
could detect the 105-kDa protein only in the brain and testis
on immunoblotting Although a partial amino-acid
sequence of the 105-kDa protein coincides with that of
OSP94, we could not detect the 105-kDa protein in the rat whole kidney until now We investigated the detailed localization of the protein using water-restricted rat kidney segments, cortex, medulla, and papilla, on immunoblotting using an anti-rat 105-kDa protein IgG As shown in Fig 5, the protein was detected only in the soluble fraction of the renal medulla We could not detect the 105-kDa protein in the insoluble fraction of renal medulla None of the protein was detected in the soluble and insoluble fractions of renal cortex and renal papilla The 105-kDa protein in the kidney
is specifically located in the medulla, and the localization of the 105-kDa protein is the same as that ofOSP94 These results suggested that the 105-kDa protein is identical
to OSP94 in partial amino-acid sequence and renal localization
Identification of APG-1/OSP94 and the 105-kDa protein
To confirm the identity of APG-1/OSP94 and the 105-kDa protein, we studied the cross-reactivity of an antibody against APG-1 with the 105-kDa protein As shown in Fig 6, the purified 105-kDa protein was recognized by an antibody against the 105-kDa protein on immunoblotting The 105-kDa protein was recognized by an antibody against APG-1 antibody, the same as an anti-105 kDa antibody Based on these results, APG-1 and OSP94 are identical to the 105-kDa protein Biochemical properties of the 105-kDa protein, APG-1, and OSP94 are shown in Table 1
Sequence homology between the 105-kDa protein (APG-1/OSP94) and HSP90
We analyzed the sequence homology between the 105-kDa protein (APG-1/OSP94) and mouse HSP90a (accession number P07901) and HSP90b (accession number P11499) There was a low homology (34.9 % match) between those
Fig 5 Interaction between the 105-kDa protein and OSP94 Control Wister rats or water-restricted rats (3 and 5 days) kidneys were dissected into renal cortex, medulla, and papilla The soluble and insoluble fractions of these sections were electrophoresed on SDS-polyacrylamide gels (7% gel), which were stained with Coomassie Brilliant Blue (A), by immunoblotting with an antibody against the 105-kDa protein IgG (B) C, P, and M in panel A indicate renal cortex, renal papilla, and renal medulla, respectively In panel A, 0, 3, and 5 indicate water-restriction time (days).
Fig 4 RT-HPLC fractionation of lysyl endopeptidase digests and the
amino-acid sequence of the 105-kDa protein A Lysyl endopeptidase
digests of the 105-kDa testis protein were separated by reverse phase
chromatography on a C 18 column with a linear gradient of 0–60%
acetonitrile in 0.1% TFA at a flow rate of 0.5 mL per minute The
purified peptides indicated in the panel (#40 and #70) were sequenced
by a peptide sequencer B The two purified peptides of lysyl
endo-peptidase digests (#40 and #70) were sequenced and compared with
APG-1 and OSP94 Identical residues are denoted by a dash (–).
Parentheses indicate the position of APG-1 and OSP94.
Trang 6two proteins Interestingly, we could detect a highly
homologous region between APG-1/OSP94 (680–702) and
mouse HSP90a or HSP90b (Fig 7A) For the hydropathy
profiles of APG-1/OSP94, the region showed hydrophilicity
(Fig 7B) Moreover, the domain may consist of a b-sheet
and a b-turn on the secondary protein structure-prediction
(Fig 7C) Based on the analysis, the domain (670–700 from
the N-terminal) may exist on the surface of APG-1/OSP94
On the contrary, we reported before that an antibody
against bovine HSP90 recognizes mainly the N-terminal
immunoreactive site of HSP90 (2–282 in the N-terminal
region of human HSP90) [20] The immunoreactive site is
almost the same as the highly homologous site (264–284
from the N-terminal of HSP90) vs those of APG-1/OSP94
(680–702 from the N-terminal) Based on these reasons, an
anti-bovine HSP90 antibody may cross-react with the 105-kDa protein (APG-1/OSP94) the same as HSP90 in rat testis and brain
Expression time of 105-kDa protein in rat brain
In order to clarify the localization of the 105-kDa protein in the brain, the rat brain was dissected into six sections and
Table 1 Properties of the 105-kDa protein, APG-1, and OSP94 ND, not determined.
Localization in organ Testis, brain Testis, brain Renal medulla renal medulla
Amino-acid sequence Partially same as APG-1and OSP94 Same as OSP94 Same as APG-1
Fig 6 Cross-reactivity of an antibody against APG-1 to the 105-kDa
protein Soluble fraction of rat brain and purified the 105-kDa protein
were electrophoresed on SDS-polyacrylamide gels (10% gel), which
were stained with Coomassie Brilliant Blue (A), by immunoblotting
with an antibody against the 105-kDa protein IgG (B) or
immuno-blotting with an antibody against APG-1 (C) Lane 1, purified
105-kDa protein; lane 2, soluble fraction of rat brain; lane 3, molecular
standard proteins.
Fig 7 Secondary structure of the APG-1/OSP94 (the 105-kDa pro-tein) (A) Sequence homology between APG-1/OSP94 and mouse HSP94a (accession no P07901) or HSP90b (accession no P11499) The same amino acid and homologous amino acid are shown in red square and in yellow square, respectively (B) Hydropathy profiles of APG-1/OSP94 In the panel, plus values indicate hydrophilicity of the amino-acid residues (C) Secondary structure prediction of APG-1/ OSP94 Helix indicates blue loop, sheet structure indicates red zigzag, and turn structure indicates green line Numbers in the panel indicate amino-acid residues of APG-1/OSP94.
Trang 7the protein was detected on immunoblotting using an anti-(rat 105-kDa protein) IgG The 105-kDa protein was detected in the soluble fraction As shown in Fig 1B, the 105-kDa protein was detected in all brain sections The protein was detected in large amounts in the cerebellum and medulla oblongata compared to the other sections On the contrary, the 105-kDa protein was detected as a faint protein band in the insoluble fractions (Fig 8C) We investigated the expression time of the protein in the postnatal Wistar rat brain (0.5-, 1-, 2-, 3-, 4-, 5-, and 6-weeks-old) on immunoblotting The 105-kDa protein could be detected at 3.5 s-day-old-in all sections of the rat brain However, the induction pattern was different in each case In the cerebral cortex and hippocampus, the expres-sion of the protein was strongly induced at 2-weeks-old (Fig 8E) In the olfactory lobe, mid brain and medulla oblongata, the expressions of the 105-kDa protein were induced at 3 weeks old In the cerebellum, the expression of the 105-kDa protein was induced at 4 weeks old (Fig 8E) Thus, the expression time of the 105-kDa protein has been shown with slight differences in rat brain sections
Immunohistochemistry of the 105-kDa protein
in rat brain
In order to investigate the localization of the 105-kDa protein in the rat brain, we performed immunohistochem-ical studies using an anti-(rat 105-kDa protein) IgG The localization of the 105-kDa protein in the rat brain is presented in Fig 9 The protein is localized predominantly
in the cytoplasm of nerve cells in the cerebral cortex, hippocampus, and cerebellum Some neurons showed
Fig 8 Localization and expression time of the 105-kDa protein in rat
brain Rat brain (female 6 weeks) was dissected into six sections and
soluble proteins were electrophoresed on SDS-polyacrylamide gels
(10% gel), which were stained with Coomassie Brilliant Blue (A), by
immunoblotting with an antibody against the 105-kDa protein IgG
(B) Insoluble proteins were electrophoresed and processed
immuno-blotting with an antibody against the 105-kDa protein IgG (C) Each
section of the postnatal rat brain (3.5-day-old, 1-, 2-, 3-, 4-, 5-, and
6-week-old) were homogenized and the supernatants were
electro-phoresed on SDS-polyacrylamide gels (10% gel), which were stained
with Coomassie Brilliant Blue (D, rat cerebral cortex), by
immuno-blotting with an antibody against the 105-kDa protein IgG (E).
Fig 9 Immunohistochemistry of the 105-kDa protein in rat brain The sections of rat brain were stained with anti-rat 105-kDa protein IgG Panels A and B, cerebral cortex; C and D, hippocampus; E and F, cerebellum Bar indicates 50 lm.
Trang 8nuclear staining Cell bodies and proximal dendrites were
intensely stained, whereas distal dendrites and axons were
obscure Glia cells were also reacted Purkinje cells were the
most intensely stained in the cerebral cortex
Detection of the 105-kDa protein in PC12
and PC12h cells
The 105-kDa protein is localized in the rat brain, especially
in nerve cells We investigated the induction pattern of the
protein in PC12 and PC12h (sub clone of PC12 cell) cells
under different stress conditions As shown in Fig 10,
HSP70 and HSP60 both were strongly induced under AzC
treatment or heat treatment in both cells We investigated
the induction patterns of the 105-kDa protein under the
same conditions Although the protein was slightly induced
under AzC treatment, the induction pattern of the protein
under heat treatment was quite different from those of
HSP70 and HSP60 The protein was slightly reduced under
heat treatment in PC12 cells Surprisingly, the protein was strongly reduced in PC12h cells The same pattern was obtained from HSP90 Under these conditions, HSF-1 was activated in both cells (Fig 10C)
D I S C U S S I O N
We reported before that a 105-kDa testis and brain protein was cross-reacted with an antibody against bovine HSP90 [8] On immunoblotting, the 105-kDa protein could not be detected in the liver, lung, spleen, kidney, ovarium and uterus, in contrast to the wide distribution of HSP90 [8] The physicochemical properties of the 105-kDa protein were similar to those of HSP90, and the protein seems to be a cognate protein of HSP90 [8] We produced a specific antibody against rat 105-kDa protein and the antibody was cross-reacted only with the protein in the rat brain Recently, we have shown that the 105-kDa protein is induced by heat stress and is able to bind to p53 in a temperature-sensitive manner in the rat testis [10] In the present study, we have characterized the biochemical properties of the 105-kDa protein Mitochondrial CS was chosen as a model substrate in the chaperone activity CS aggregates and is inactived rapidly upon incubation at
43C [21] The 105-kDa protein inhibited CS aggregation The protein binds transiently to unfolding intermediates of the thermal unfolding of CS Upon release from the 105-kDa protein, the intermediates are able to refold rapidly
to the native state Thus, the 105-kDa protein stabilizes the native CS Chaperone activity of the 105-kDa protein was the same as that of HSP90 [7]
Many molecular chaperones including HSP70 show ATPase activity or bind to ATP–Sepharose [13] For ATP binding of the 105-kDa protein, ATP–Sepharose column chromatography has been performed On immunoblotting, the protein was detected in all eluted fractions from the column These results suggest that the protein is an ATP-binding protein the same as the other molecular chaperones and may have an ATP-binding sequence It has been shown that the ATP-binding consensus sequence is divided into two short elements termed type A, the putative triphosphate binding sequence, and type B, on adenine-binding sequence [22,23] Type A is A/GXXXXGKT/SXXXXXXI/V On the contrary, type B is H/RKK(5)7)hXhhD/E, where h stands for a hydrophobic residue We searched for ATP-binding proteins with a molecular mass of about 100 kDa in the data base Two interesting proteins, APG-1 and OSP94, have been found
An HSP110-related gene, APG-1, has been isolated from the mouse testis cDNA library [11] APG-1 was abundantly expressed in the testis, and a lower level of expression was seen in the brain on Northern blot analysis [11] On the contrary, OSP94 cDNA, a member of the HSP110/SSE family, has been cloned from another group [12] Renal inner medullary OSP94 mRNA expression was increased in water restricted mice Both APG-1 and OSP94 cDNA encodes an 838-amino-acid residues protein, and the sequence is the same in each case Both genes show the putative ATP-binding sequence in the amino terminal Therefore, these two genes, APG-1 and OSP94, are the same The in vitro translated OSP94 product migrated as the 105–110-kDa protein on SDS/PAGE [12] The specific localization of APG-1/OSP94 in mouse organs and their
Fig 10 Induction pattern of the 105-kDa protein in PC12 and PC12h
cells PC12 and PC12h cells were stressed with 5 m M AzC for 6 h or
stressed by the increasing of temperature at 43 C for 30 min as
des-cribed under Materials and methods Cells were homogenized with
sample buffer and the samples were electrophoresed on
SDS-polyacrylamide gels (7% gel), which were stained with Coomassie
Brilliant Blue (A), by immunoblotting with an antibodies against
HSP60, HSP70, HSP90, and rat 105-kDa protein (B) or
immuno-blotting with an antibody against HSF-1 (C).
Trang 9apparent molecular masses are the same as the 105-kDa
protein
To analyze the partial amino-acid sequence of the
105-kDa protein, lysyl endopeptidase digested fragments
were separated on HPLC, and the partial amino-acid
sequence was determined The 24 amino-acid residues
obtained from two different peptides were the same as the
deduced amino-acid sequence of APG-1 and OSP94 An
important question regarding localization of the 105-kDa
protein in the kidney has remained unanswered Although
the protein could not be detected in the whole kidney sample
on immunoblotting, the protein could be clearly detected
only in the medulla of normal and 3- or 5-day
water-restricted rat kidney It has been shown that OSP94 mRNA
expression was increased in the renal medulla during water
restriction In the present study, we could not detect the
increasing expression of the protein in the water-restricted
rat kidneys This may reflect the difference in detection
systems between Northern blotting and immunoblotting
An anti-APG-1 cross-reacted with the 105-kDa protein
on immunoblotting The former detects mRNA and the
latter detect the protein These results allow us to conclude
that APG-1 and OSP94 are identical to the 105-kDa
protein
The most important question has remained unanswered
Why does an antibody against bovine HSP90 cross-react
with the 105-kDa protein (APG-1/OSP94)? As shown in
Fig 7, there were highly homologous regions between the
immunoreactive sites of HSP90 (2–282 in the N-terminal
region of human HSP90) and the 105-kDa protein (APG-1/
OSP94) An anti-HSP90 antibody may recognize the
105-kDa protein the same as HSP90 In 1997, APG-1 and
OSP94 cDNA has been isolated independently [11,12]
However, the biochemical properties of the two proteins
have not yet been fully understood until today In 1990, we
purified and reported some biochemical properties of the
105-kDa protein as a novel HSP90-related protein [8] Now,
we have shown the identity of APG-1 and OSP94 relative to
the 105-kDa protein and that the 105-kDa protein plays a
role as a molecular chaperone
We have reported the physiological functions of the
protein in the rat testis [9,10] The protein could bind to
p53 in a temperature-dependent manner in the
cyto-plasm of the germ cells The 105-kDa protein may
contribute to the stabilization of p53 and prevent the
potential induction of apoptosis by p53 On the
contrary, localization and expression time of the protein
in the brain are not yet known Immunoblotting using
an anti-105 kDa protein IgG revealed that the 105-kDa
protein was constitutively expressed in all sections of the
rat brain In the present study, we showed that the
105-kDa protein was localized in the cytoplasm of the nerve
cells and/or glia of hippocampus, cerebral cortex, and
cerebellum of the rat brain The 105-kDa protein may
play an important role in brain throughout postnatal
period
PC12h cells had been established as one of the sub clones
of PC12 cells [24,25] The cell was demonstrated to have
nerve growth factor (NGF)-responsive tyrosine hydroxylase
(TH) activity In the present investigation, HSP70 and
HSP60 were strongly induced by AzC, an amino-acid analog
(proline-synthesis inhibitor), or heat treatment of PC12h
cells HSF-1 was activated under these stress-conditions
Surprisingly, both 105-kDa protein and HSP90 both were reduced under the heat stress conditions in spite of the remarkable induction of HSP70 and HSP60 under the same conditions Although APG-1, OSP94, and the 105-kDa protein have homology to HSP110 and HSP70 in their primary amino-acid sequences, the induction pattern under the heat stress conditions in PC12h cells were quite different from those of HSP70 and HSP60 The reason why the 105-kDa protein and HSP90 were reduced under the stressed conditions of PC12h cells are not yet known at present The 105-kDa protein and HSP90 induced under the heat treatment might be quickly digested by protease in PC12h cells We investigated the induction pattern of HSP90 and the 105-kDa protein under the same conditions
in the presence of some kinds of protease inhibitors However, the induction patterns of the 105-kDa protein and HSP90 were the same as those in the absence of protease inhibitors (data not shown) Thus, the different induction patterns of the 105-kDa protein and HSP90 from those of the other molecular chaperones in PC12h cells might be dependent on the difference in transcriptional mechanisms It has been shown that the regulation of heat induction of the APG-1 transcript cannot be explained by the HSF1 activation alone and that some other mechanisms are responsible for the differential induction of HSP70 and APG-1 [11] The transcriptional mechanisms of the 105-kDa protein may be different from those of the other molecular chaperones including HSP70 in the PC12h cells By inves-tigating the biochemical properties of the 105-kDa protein in PC12h cells, we may be able to understand the physiological functions of the protein in nerve cells We have already started research to understand the biochemical properties of the protein using over-expression of the protein or HSP90 in PC12h cells
A C K N O W L E D G E M E N T S
This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas (C) (Advanced Brain Science Project:
12210033 to H I.) and by a Grant-in-Aid for Scientific Research on Priority Areas (Molecular Chaperone: 11153201 to H I.) and C2 (12670105 to H I., 14571011-00 to A K., and 14570442 to M O.) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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