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,*
*
First Department of Surgery and
Department of Parasitology and Immunology, Okayama University Medical School, Okayama, Japan;
Department of Medical Zoology and Immunology, Nagasaki University School of Medicine, Nagasaki, Japan;
§
Department of Microbiology, Okayama University Dental School, Okayama, Japan; and
¶
Center for Immunotherapy of Cancer and Infectious Diseases, University of Connecticut School of Medicine, Farmington, CT 06030
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1 and its precursors are associated with the chaperones
hsp90 and hsp70 in the cytosol and gp96 in the lumen of the endoplasmic
reticulum. Hsp70 was associated with only final sized octamer, while
hsp90 was found to associate with the octamer and two distinct
precursor peptides. The gp96 was associated with the octamer and one of
the two precursors. Thus, each of the hsps bound a distinct set of
peptides. Our results have demonstrated for the first time that the
hsps associate not only with final sized tumor Ag peptide but also with
its precursors. The implication of this evidence is also discussed in
terms of the roles of hsps in MHC class I Ag
processing/presentation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the tumor immunology, the original thesis that the immune response
was elicited by tumor Ag peptides associated with hsps but not by hsps
themselves remained uncertain because the real entity of the peptides
associated with hsps that cause the tumor regression was not known;
that is, whether it is possible to purify exact CTL epitopes or their
precursors from hsps that could sensitize targets for lysis by
tumor-specific CTLs. To answer this question as well as to show the
generality of the observations of Nieland et al. (11) and Breloer et
al., we examined here a tumor system that demonstrates association of
an Ld-restricted peptide (IPGLPLSL, or pRL1a) and its
longer precursors with the three hsps, hsp70, hsp90, and gp96, of the
BALBRL1. The pRL1a epitope is derived from the 5' untranslated
region of the akt gene (13, 14) expressed in this leukemia
and acts as a tumor rejection Ag for this leukemia (15).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RL1 and Meth A cells were expanded in ascitic form in BALB/c
mice. The mice were obtained from The Jackson Laboratory (Bar Harbor,
ME) and were bred in pathogen-free condition at the animal facility of
Okayama University Medical School (Okayama, Japan). RL1-specific CTL
clone Y-15 was maintained by weekly stimulation with irradiated RL1
cells and feeder cells as described previously (16).
Purification of gp96, hsp90, hsp70, and Ld molecules
The gp96 and hsp90 were purified simultaneously as described
previously (17). Briefly, a 10-ml cell pellet (
1010
cells) of ascitic RL1 cells of BALB/c mice was homogenized in 40 ml
of a hypotonic buffer (30 mM NaHCO3, and 0.5 mM PMSF (pH
7.1)), and its 100,000 x g supernatant was applied to
a Con A-Sepharose (Pharmacia Biotech, Uppsala, Sweden) column followed
by elution with 10% 
-methyl-mannopyranoside. For isolation of gp96,
Con A-bound material was resolved on a Mono Q FPLC (Pharmacia) system
equilibrated with 5 mM phosphate buffer (pH 7.0) and eluted by a 0–1 M
NaCl gradient. For purification of hsp90, the Con A-unbound material
was dialyzed against 20 mM of sodium phosphate (pH 7.4), 1 mM EDTA, and
250 mM NaCl and then resolved on the Mono Q system equilibrated with 20
mM sodium phosphate (pH 7.4), 1 mM EDTA, and 200 mM NaCl. The proteins
were eluted by a 200–600 mM NaCl gradient. Hsp70 was purified as
described previously (18, 19) with some modifications. For purification
of hsp70, a 100,000 x g supernatant of the homogenate
was dialyzed against buffer D (20 mM Tris acetate (pH 7.5), 20 mM NaCl,
0.1 mM EDTA, 15 mM 2-ME, and 3 mM MgCl2) with a PD10 column
(Pharmacia Biotech) and loaded onto an ADP-agarose (Sigma, St. Louis,
MO) column. The column was extensively washed with 5 gel vol of buffer
D plus 500 mM NaCl and with 2 vol of buffer D. Bound material was
eluted with 3 mM ADP in buffer D and dialyzed with a PD10 column to
FPLC buffer, then resolved on a Mono Q FPLC system equilibrated with 20
mM Tris acetate (pH 7.5), 20 mM NaCl, 0.1 mM EDTA, and 15 mM 2-ME. The
proteins were eluted by a 20–500 mM NaCl gradient. Otherwise, the
eluate was dialyzed against buffer E (20 mM sodium phosphate and 20 mM
NaCl (pH 7.0)), resolved on a DEAE-Sepharose column (Sigma), and eluted
by buffer E plus 130 mM NaCl. After this, fractions (1 ml) eluted by
the Mono Q system or on a DEAE-Sepharose column were probed by SDS-PAGE
followed by silver staining (Silver Stain Plus, Bio-Rad, Hercules, CA),
and the fractions containing single bands of each hsps were used in the
experiments. By these procedure approximately 1.5–2 mg of gp96, 2–2.5
mg of hsp70, and 4–6 mg of hsp90 were recovered from 1 x
1010 RL1 cells, respectively. These proteins were
resolved on SDS-PAGE, blotted to nitrocellulose, and probed with
anti-grp94 mAb (SPA-850, StressGen, Victoria, British Columbia),
anti-hsp90 mAb (3G3, Affinity BioReagents, Golden, CO), and
anti-hsp70 mAb (SPA-820, clone N27F3–4, StressGen), and alkaline
phosphatase-labeled second Ab (Bio-Rad) was used for visualization.
Ld molecules were purified from Nonidet P-40-treated RL1
cell lysate with an immunoaffinity column as described previously (13).
Dissociation of the endogenous peptides from gp96, hsp90, hsp70, and Ld
Approximately 2 mg of the purified hsp70 preparations in a 1-ml volume were exposed to 10 mM ATP and 3 mM MgCl2 at 37°C for 1 h. Purified gp96 and hsp90 preparations (2 mg each) and immunoaffinity-purified Ld molecules were exposed to 0.2% trifluoroacetic acid (TFA) at 4°C for 1 h to strip peptides followed by centrifugation through an assembly with a molecular cut-off membrane (m.w. 10,000; Centricon 10, Amicon, Beverly, MA). The low m.w. fractions were pooled, concentrated by evaporation with a Speed-Vac (Savant, Farmingdale, NY), and dissolved in 0.1% TFA. These materials were applied to a C18 reverse phase HPLC column (Vydac, Hesperia, CA) pre-equilibrated with 0.1% TFA and water, and the bound materials were eluted at a flow rate of 0.5 ml/min. Solvent A was 0.1% TFA and water, and solvent B was 79.9% acetonitrile, 0.1% TFA, and 20% water. The gradient for chromatography was 20–60% B over 60 min, and fractions at 1-min intervals were collected. To avoid the cross-contamination of peptides between synthetic peptides and naturally processed peptides, we purchased two new C18 column; one column was used only for the separation of naturally processed peptides stripped from hsps, and the another was used for separation of synthetic peptides.
Cytotoxicity assay
The fractions collected by reverse phase HPLC were completely
evaporated by the Speed-Vac and dissolved in 150 µl of 0.1% TFA.
P-815 target cells were labeled by incubating 2 x 106
cells with 2 MBq of Na251CrO4 in
0.3 ml of RPMI (with 10% FCS) for 1 h at 37°C under 5%
CO2 in air. The cells were washed and cultured in 100 µl
of medium at 5 x 103 cells/well in a 96-well
round-bottom plate with sensitization by the peptides containing each
10 µl of each HPLC fractions for 1 h. Then the RL1-specific
CTL clone Y-15 cells were added and cultured for 4 h. Finally the
supernatants (100 µl) were removed, and their radioactivities were
measured. The percent specific 51Cr release was calculated
by the following equation: [(a -
b)/(c - b)] x 100, where
a is the radioactivity in the supernatant of target cells
mixed with effector cells, b is the spontaneous release, and
c is the maximum release after lysis of target cells with
1% Nonidet P-40.
Peptide synthesis
Peptides were synthesized by standard solid phase methods using F-moc chemistry in a peptide synthesizer (model 430A, Applied Biosystems, Foster City, CA). The peptides were purified by reverse phase HPLC on a preparative C8 column (10 x 100 mm, 20-mm particle size; Applied Biosystems) in 0.1% TFA with an acetonitrile gradient and freeze-dried for stock.
Mass spectrometry analysis
The masses of the peptides were determined on-line by a tandem quadrupole mass spectrometer (TSQ 700, Finnigan MAT, San Jose, CA) equipped with an electrospray ion source. The peptides were identified by their molecular mass as the m/z peaks of single charged ion.
Competition assay by pRL1a and pRL1b of complex formation between hsp70 and lactalbumin
Peptide pRL1a and pRL1b binding to hsp70 was evaluated by their
competition of complex formation between hsp70 and the unfolded form of
lactalbumin as described previously (20). Four micrograms of hsp70
(final concentration, 2.8 mM) and 11 µg of lactalbumin (final
concentration, 40 mM) suspended in PBS, pH 7.2 were incubated at 37°C
for 30 min with or without gradient doses of pRL1a and PRL1b as
indicated in Fig. 3
, then resolved by 6% native PAGE and visualized by
staining with Coomassie Brilliant Blue R-250. The bands of the stained
hsp70-lactalbumin complex were quantitated by densitometry.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 and fractionation of the peptides by reverse phase
HPLC
The CTL epitope of the RL1 mouse leukemia has been identified
previously as the octamer epitope (pRL1a), derived from a mutated
akt gene product (13). A putative precursor of pRL1a, an
amino terminal-extended 10 mer peptide (pRL1b), was also identified in
this previous study. Association of pRL1a, pRL1b, and other possible
precursors with the chaperones hsp70, hsp90, and gp96 was investigated.
Fig. 1A shows apparently
homogeneous preparations of gp96 (lane a), hsp90
(lane b), and hsp70 (lane c)
purified from RL1 cells and examined by SDS-PAGE and silver
staining. In our preparation, another member of the hsp70 family, grp78
was not detected. Immunoblotting of preparations by anti-grp94,
anti-hsp90, and anti-hsp70 mAbs was used to confirm their
identity and the lack of cross-contamination of the preparations (Fig. 1B). Peptides associated with three hsps were isolated as
described in Materials and Methods and were separated in a
C18 reverse phase column. The chromatograms of peptides
dissociated from gp96, hsp90, and hsp70 are shown in Fig. 2, a–c. A distinct profile
was observed for the peptides eluted from each hsps preparation. The
synthetic 8 mer peptide pRL1a and its putative 10 mer precursor pRL1b
were also analyzed, and the chromatograms were shown to elute in
fractions 39 and 46, respectively (Fig. 2, d and
e).
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All peptide fractions from each of the hsps, Ld
molecules, and synthetic peptides pRL1a and pRL1b were used to pulse
P815 cells and were tested for recognition by CTL clone Y-15, which
recognizes the 8 mer peptide, pRL1a, in association with Ld
molecules. We gave careful attention to avoid the carried over peptides
with each other; thus, a blank run before running the biological
samples was performed in each experiment and confirmed there were no
carried over peptides in each preparation. Among RL1-derived,
hsp70-associated peptides eluted by treatment with ATP (Fig. 3
A-a) or TFA (Fig. 3A-b), significant sensitization activity was observed in
fraction 39, which corresponds to the precisely eluted position of
Ld-associated or the synthetic 8 mer pRL1a (Fig. 3, A-e and A-f). A molecule with a molecular mass
(m/z) of 809, which is precisely the mass of pRL1a, was
identified in fraction 39 of the hsp70-associated peptides (Fig. 3, B-I and B-II). As shown in Fig. 3A-c,
hsp90 was found associated with three different peptides eluted in
fractions 32, 39, and 46, which were able to sensitize P815 cells for
lysis by the CTL clone Y-15. The position of fraction 39 has been shown
to be identical with that of pRL1a, while fraction 46 contains the
synthetic putative precursor 10 mer pRL1b (Fig. 3A-g). A
molecule with an m/z value of 1008, precisely equal to that
of pRL1b, was identified in fraction 46 of the hsp90-associated
peptides from RL1 (Fig. 3, B-III and B-IV).
Thus, hsp90 was associated with the final sized CTL epitope as well as
with two other putative precursor peptides, one of which is pRL1b and
the other of which was unknown. The size of the precursor peptide
eluted in fraction 32 was deduced to be larger than a 10 mer on the
basis of the following observations. Peptides shorter than 8 mer pRL1a
had no sensitizing activity (data not shown), while the 8 mer pRL1a and
10 mer pRL1b eluted in fractions 39 and 46, respectively. The 9 mer
intermediate between pRL1a and pRL1b was observed to elute in fraction
36 or 37 (data not shown). Thus, the sensitizing activity in fraction
32 was >10 mer. The chaperone gp96 was found associated with both
pRL1a and pRL1b eluted in fractions 39 and 46, respectively (Fig. 3A-d). No sensitizing activity was detected in fraction 32
or any other fraction among the peptides eluted from gp96. No positive
fractions were identified in any peptide fractions eluted
from hsp70, hsp90, and gp96 isolated from altered
akt-negative BALB/c fibrosarcoma Meth A (Fig. 3A-h).
Requirement of serum proteases for hsp90- and gp96-associated precursor peptides to sensitize P815 cells for lysis by the CTL clone
The pRL1a, but not the pRL1b, was detected in TFA-treated
immunoaffinity-purified Ld preparations (Fig. 3A-e) (13), indicating that this putative precursor peptide
pRL1b does not bind Ld. It was presumed that the 10-mer
pRL1b and the longer precursor in hsp90-eluted fraction 32 are able to
sensitize P815 cells for lysis by the CTL clone Y-15 because of an
exopeptidase activity in the serum-containing medium in which the assay
was performed. In fact, the synthetic peptide pRL1b was shown to lose
its sensitizing activity of P815 cells for lysis by CTLs in the
serum-free plain RPMI medium (18). This premise, especially in the case
of natural peptides associated with the hsps, was tested directly by
carrying out the assay in medium with or without serum. It was observed
that (Table I) synthetic peptide pRL1b
and fractions 32 and 46 from the hsp90 preparation or fraction 46 from
the gp96 preparation could not sensitize P815 cells for recognition by
CTL clone Y-15 in the AIM-V serum-free medium. However, the
sensitization activities of synthetic pRL1a peptide and peptides in
fraction 39 derived from hsp70, hsp90, and gp96 were not impaired in
this medium, indicating that the putative precursor peptides are real
precursors and must be trimmed at the N-terminus to bind Ld
molecules in the medium containing FCS. We cannot determine whether
C-terminal trimming of the peptide in fraction 32 from the hsp90
preparation is also necessary.
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The 8-mer pRL1a was added to a suspension of Meth A cells, which
do not express this Ag, and hsp70 preparations were derived from the
lysate of such Meth A cells. The concentration of peptide added to Meth
A (320 pM final concentration) was severalfold higher than an abundant
estimate of the natural peptide in RL1. If the association of pRL1a
peptide with hsp70 can occur after cell lysis, hsp70 preparations
derived from the Meth A cell lysate, to which large quantities of pRL1a
have been added, should be found associated with pRL1a. Peptides eluted
from such an hsp70 preparation were used to pulse P815 cells, which
were then tested for recognition of pRL1a by CTL clone Y-15. No
recognition was observed (Table II). In
contrast, peptides eluted from hsp70 preparations derived from the
RL1 could sensitize P815 cells for lysis by CTL clone Y-15
successfully (Table II). Similar studies with hsp90 and gp96 are
unnecessary, as association of these hsps with peptides in vitro
requires harsh conditions, such as high temperatures or the use of
denaturants (17), which were not used in their purification here (see
Materials and Methods).
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, A-a and A-b), studies in
vitro indicate that hsp70 can bind equally effectively with both pRL1a
and pRL1b; this was demonstrated by competition studies in which the
abilities of the two peptides to inhibit formation of the
hsp70-lactalbumin complex was tested quantitatively as described in
Materials and Methods (20). It was observed that generation
of the hsp70-lactalbumin complex, as judged by native gel
electrophoresis, was inhibited equally in a dose-dependent manner by
pRL1a and pRL1b (Fig. 4). Thus, the
observed association of pRL1a, but not pRL1b, with hsp70 is significant
and occurs in vivo; if the observation were the result of artifactual
association of peptides with hsp70 after cell lysis, pRL1a and pRL1b
would both be found in association with hsp70 molecules. This is not
the case. Thus, pRL1b is not available to hsp70 in vivo.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), and pRL1b, although capable of binding hsp70 in vitro, was
not found in association with it (Fig. 4). In other studies, it has
been demonstrated that addition of a broad array of labeled peptides of
diverse sizes and sequences to cell suspensions before lysis does not
lead to their association with gp96 (A. Menoret and P. K.
Srivastava, manuscript in preparation). Further, as observed here, the
patterns of association of peptides were distinct and specific for each
hsps; while hsp70 was found associated solely with pRL1a, gp96 is
observed associated with both pRL1a and pRL1b, and hsp90 associated
with pRL1a, pRL1b, and the larger precursors. In three complete sets of
experiments repeated, no deviation from this pattern was detected.
Another issue that needs to be addressed has to do with the possibility
that the exact octamer or the putative precursor peptides found
associated with the three hsps could have dissociated from the MHC I
molecules and associated with the hsps. This possibility is ruled out
by a number of considerations. Firstly, the 10 mer pRL1b peptide has
been shown unable to bind the Ld molecule (Fig. 3A-e) (13), and thus, it cannot bind hsp90 or gp96 after
dissociation from Ld molecules. Secondly, the finding that
each hsps has a distinct spectrum of peptides associated with it argues
forcefully against the possibility of random association regardless of
source. Thirdly, the hsps preparations were derived from aqueous (i.e.,
nondetergent-containing) lysates, where they were purified away from
the MHC molecules at the very first step of cell lysis. In addition,
the results of other studies, which show that the exact MHC I-binding
peptides may be isolated from gp96 preparations derived from cells that
do not express the cognate MHC I molecules (11), provide structural
evidence that the hsps-associated peptides are not derived from the MHC
I-associated peptides. Finally, the observations by Arnold et al. (10)
and Suto and Srivastava (8) that hsps preparations can cross-prime
against minor histocompatibility Ags and VSV-derived epitopes across
the H-2 provide functional evidence that association of peptides with
hsps does not derive from or depend upon their association with MHC I
molecules.
The association of hsps with the exact-sized MHC I ligand and its
precursors might be implicated as the roles of the hsps in MHC I Ag
processing/presentation. Peptides associated with the MHC I molecules
are eight or nine amino acids long (21), and peptides with different
motifs bind specific MHC I alleles (22). The peptides or their
precursors originate in the cytosol (23, 24, 25) and are transported into
the ER, where the association with MHC I molecules occurs (26). The
events between the generation of peptides in the cytosol and their
final association with MHC I molecules are not fully understood. The
peptides are clearly not present in the cytosol in a freely diffusible
manner; it has been proposed that molecular chaperones of the hsps
family, such as hsp90 and hsp70 in the cytosol and gp96 in the lumen of
the ER, constitute a molecular relay line that chaperones the peptides
from their generation in the cytosol to their binding to MHC I in the
ER (27). Very recently, Shimbara et al. have shown that pRL1b precursor
peptide, but not pRL1a, was produced from synthetic peptide by
proteasome in the presence of the IFN-
-inducible activator PA28
(28). To date, pRL1b is the true precursor peptide and needs to be
trimmed to become Ld ligand-pRL1a by aminopeptidases. In
this context, our data may suggest that the precursor peptide pRL1b
(and presumably larger precursors, such as those in fraction 32 of
hsp90-associated peptides) are received by hsp90 first, followed by
N-terminal trimming of some of the precursor molecules through as yet
uncharacterized mechanisms in the cytosol. This idea is consistent with
recent reports that demonstrate physical association of hsp90 molecules
with the proteasomes (29, 30). However, not all the precursor molecules
delivered to hsp90 from the proteasome are completely processed, as the
ER luminal hsps gp96 is still observed associated with the pRL1b
precursor. In addition, a proportion of precursor peptides may bypass
hsp90 altogether and thus go on to associate with gp96. Interestingly,
hsp70 molecules are not observed associated with precursor peptides. In
light of the reported proteolytic activity of hsp70 (31), this
observation might suggest that any precursors that come to associate
with hsp70 are rapidly cleaved to the final product. If the transport
of pRL1a, pRL1b, and other precursor peptides into the ER is TAP
dependent, pRL1b would be expected to be transported at a higher
efficiency than pRL1a because of the inhibitory presence of proline at
position 2 (32) in the octamer but not in the 10-mer pRL1b.
Nevertheless, it is conceivable that the constraints on the ability of
TAP molecules to transport peptides do not reflect the situation in
vivo, as they have been deduced by monitoring the transport of
exogenously added free peptides to TAP-containing vesicles or
permeabilized cells. Thus, while free peptides with proline at position
2 may not be transported by TAP molecules, such peptides chaperoned by
hsps may be transported by the same TAP molecules more efficiently. The
recent observations of Lammert et al. (19, 33), Spee and Neefjes (34),
and Marusina et al. (35) are in accord with our present observation and
previous speculation that a relay line of hsps chaperones peptides from
the point of their generation in the cytosol to their association with
MHC I-ß2m molecules in the ER. These authors have
demonstrated that peptides associate with a number of peptide-binding
proteins in the cytosol (gp100) and in the lumen of the ER (gp96,
protein disulfide isomerase, gp120, and gp170). Our studies suggest
that hsps are in a position to play an important role in the transport
of antigenic peptides to MHC I molecules.
Regardless of the precise mechanisms by which the precursor peptides are transported into the ER, our results are the first evidence showing that hsps localized in distinct intracellular compartments are associated with different sets of precursors of MHC I-binding tumor Ag peptide.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Heiichiro Udono, Department of Medical Zoology and Immunology, Nagasaki University School of Medicine, 1-12-4 Sakamoto, Nagasaki 852-8523 Japan. E-mail address:
3 Abbreviations used in this paper: hsp(s), heat shock protein(s); gp, glycoprotein; FPLC, fast protein liquid chromatography; TFA, trifluoroacetic acid; ER, endoplasmic reticulum; grp, glucose-regulated protein; VSV, vesicular stomatitis virus.
Received for publication September 8, 1998. Accepted for publication October 14, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 leukemia recognized by cytotoxic T lymphocytes and its relation to the Akt oncogene. J. Exp. Med. 180:1599.1 leukemia recognized by cytotoxic T lymphocytes: derivation from the normally untranslated 5' region of the c-akt proto-oncogene activated by long terminal repeat. Cancer Res. 55:4780.1. Int. Immunol. 9:1195.1 rejection antigen peptide pRL1a by proteolysis of natural precursor pRL1b. Jpn. J. Cancer Res. 87:1165.[Medline]
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) RL
), hsp90 (
), and gp96 (
) isolated from Meth A
(h) did not show any sensitization of P815 cells.
B, The synthetic peptide pRL1a (I) and pRL1b (III) or
the peptides (II) dissociated from hsp70 in fraction 39 observed in
Fig. 2
) in I and
II indicate a peptide of 809 Da, and the
arrowheads in III and IV indicate a
peptide of 1008 Da.
