Báo cáo Y học: Characterization of the 105-kDa molecular chaperone Identification, biochemical properties, and localization pptx

Characterization of the 105-kDa molecular chaperone
Identification, biochemical properties, and localization
Mika Matsumori
1,2
, Hideaki Itoh
1
, Itaru Toyoshima
3
, Atsushi Komatsuda
4
, Ken-ichi Sawada
4
, Jun Fukuda
5
,
Toshinobu Tanaka
5
, Atsuya Okubo
6
, Hiroyuki Kinouchi
6
, Kazuo Mizoi
6
, Tokiko Hama
7
, Akira Suzuki
1
,
Fumio Hamada
8
, Michiro Otaka

3
, Yutaka Shoji
2
and Goro Takada
2
1
Department of Biochemistry,
2
Department of Pediatrics,
3
First Department of Internal Medicine,
4
Third Department of Internal
Medicine,
5
Department of Gynecology, and
6
Department of Neurosurgery, Akita University School of Medicine, Akita City,
Japan;
7
President’s Frontier Laboratory. Mitsubishi Kasei Institute of Life Sciences, Tokyo, Japan;
8
Department 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/
MgCl
2
has 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
theproteinseemstobeacognateproteinofHSP90.
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:
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)
Eur. J. Biochem. 1–10 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03272.x
Leydig 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 32.5 °C,
which is the scrotal temperature, but not at 37 °C, 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 amino-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.
MATERIALS AND METHODS
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 ÔGuidelines for Animal
ExperimentationÕ 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 m
M
Tris/HCl (pH 7.4). The serum was applied to a
DEAE–cellulose column (1 · 4 cm) pre-equilibrated in
10 m
M
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 of

in 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 43 °C was measured
as previously described [7]. Light scattering CS (0.075 l
M
)
in 50 m
M
Hepes buffer (pH 7.4) in the presence or absence
of bovine serum albumin (15 l
M
) and the 105-kDa protein
(0.075 l
M
) 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 m
M
Tris/HCl
(pH 7.4) containing 0.25
M
sucrose and centrifuged at
9000 g for 10 min at 4 °C. The supernatant was collected,
followed by centrifugation at 105 000 g for 60 min at
4 °C. The supernatant obtained from ultracentrifugation

was used as the rat testis cytosol in the present study.
ATP–Sepharose was equilibrated in buffer A (10 m
M
Tris/HCl, pH 7.4, 5 m
M
CaCl
2
,5m
M
MgCl
2
). Rat testis
cytosols containing 5 m
M
CaCl
2
and 5 m
M
MgCl
2
were
applied to the column and washed with buffer A
containing 0.15
M
NaCl. After washing the column,
binding proteins were eluted with 5 m
M
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 5C
18
, 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).
2 M. Matsumori et al. (Eur. J. Biochem.) Ó FEBS 2002
Immnoblotting
Samples 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 m
M
Tris/HCl (pH 7.4)
containing 0.15
M
NaCl and centrifuged at 20 000 g for
10 min at 4 °C. 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 4 °C, 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 m
M
Tris/
HCl (pH 7.4) containing 0.15
M
NaCl and centrifuged at
40 000 g for 10 min at 4 °C. 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 4 °C, 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/P
i
for 10 min at room temperature. After the
sections were washed with NaCl/P
i
(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/P
i
)overnightat
4 °C and then washed again with NaCl/P
i
(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
37 °C in a humidified atmosphere containing 5% CO

2
.
Upon approaching confluence (c.1· 10
6
cells/culture
flask), the medium was removed, and the cells were stressed
by the addition of 5 m
ML
-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
43 °Cfor30minfollowedbygrowthat37°Cfor6h.The
cells were harvested and washed twice with 10 m
M
Tris/
HCl, pH 7.4) containing 0.125
M
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.
RESULTS
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
Ó FEBS 2002 Biochemical properties of the 105-kDa protein (Eur. J. Biochem.)3
Fig. 2, spontaneous aggregation of CS has been obserbed at
43 °C. 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/MgCl
2
has 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).
4 M. Matsumori et al. (Eur. J. Biochem.) Ó FEBS 2002
concentration, 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.
Ó FEBS 2002 Biochemical properties of the 105-kDa protein (Eur. J. Biochem.)5
two 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.
Parameters 105-kDa protein APG-1 OSP94
Apparent molecular mass 105 kDa ND 105–110 kDa
Chaperone activity + ND ND
ATP-binding + + (putative) + (putative)
Localization in organ Testis, brain Testis, brain Renal medulla renal medulla
Encoded amino acids ND 838 838
Amino-acid sequence Partially same as APG-1and OSP94 Same as OSP94 Same as APG-1
Localization in testis Spermatozoa ND ND
Binding protein p53 ND ND
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.
6 M. Matsumori et al. (Eur. J. Biochem.) Ó FEBS 2002
the 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.
Ó FEBS 2002 Biochemical properties of the 105-kDa protein (Eur. J. Biochem.)7
nuclear 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).
DISCUSSION
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
43 °C [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
thesameasthatofHSP90[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 °Cfor30minasdes-
cribed under Materials and methods. Cells were homogenized with
SDS-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).
8 M. Matsumori et al. (Eur. J. Biochem.) Ó FEBS 2002
apparent 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.
ACKNOWLEDGEMENTS
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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