From the Department of Applied BioSciences, Swiss
Federal Institute of Technology, Winterthurerstrasse 190, CH-8057
Zürich, Switzerland and the § Laboratoire de
Pharmacochimie de la Communication Cellulaire, UMR CNRS-ULP 7081, 74 route du Rhin, F-67401 Illkirch, France
Received for publication, February 9, 2001
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ABSTRACT |
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The B pocket of the class I major
histocompatibility complex-encoded protein HLA-B*2705 has recently been
suggested to be responsible for the misfolding of this HLA
haplotype and thus to induce susceptibility to autoimmune inflammatory
diseases. Four mutants of the B*2705 heavy chain were refolded in the
presence of three control peptides. The monitoring of the thermal
unfolding of the B*2705-peptide complexes by circular dichroism
spectroscopy showed that all heterotrimeric mutants were markedly less
stable than the corresponding complexes with the wild-type heavy chain. Among the four heavy chain mutations, the C67S change was investigated for unfolding and peptide binding properties because this position may
mediate disulfide pair bridging and alter T-cell recognition of
HLA-B*2705. Wild-type heterotrimers completely unfold in a single
transition at mild acidic pH whereas increase of the pH to mild basic
conditions induce only a partial biphasic unfolding. Cys-67 seems to
play a crucial role in controlling the thermodynamic stability of the
B*2705-peptide complexes as the C67S mutant unfolds faster and with a
single transition, independent of pH. Fluorescence polarization
and size exclusion chromatography of unfolding intermediates suggest
that the peculiar unfolding of the B*2705 wild-type heavy chain cannot
be explained by modified peptide binding properties but more likely by
the formation of high molecular weight species.
There is a striking association between susceptibility to
spondyloarthropathies (e.g. ankylosing spondylitis or AS)
and expression of the class I
MHC1 HLA-B27 allele (1, 2).
Despite intensive research over the last decade, the pathogenic
mechanism of the association is still unknown (3, 4). One of the most
favored models for disease association postulates the binding to
HLA-B27 of autoantigenic peptides, thus mimicking foreign antigens (5).
This hypothesis is also supported by a recent finding for a specific
T-cell reactivity to a self-peptide for AS patients (6). However, it
cannot account for the differential susceptibility to several HLA-B27
alleles even though they select very similar peptide repertoires (7). Recent studies on transgenic mice seem to contradict the arthritogenic hypothesis. Mice lacking The B pocket of the HLA-B27 protein is considered to be the domain with
the most influence on peptide selection. HLA-B27 shows a high
specificity for Arg (position 2 of the peptide) in the B pocket of the
binding site (11). In addition to the role played by the B pocket in
peptide selection, this subsite is also believed to play a predominant
role in protein misfolding. Mear et al. (12) recently
showed that the replacement of the HLA-B27 B pocket with one from
HLA-A2 could totally impair the misfolding phenotype of the protein.
They proposed that misfolding and its consequences are a basis for
susceptibility to spondyloarthropathies rather than allele-specific
peptide presentation. The misfolding behavior of the protein could
cause endoplasmic reticulum stress responses (13, 14) and therefore
influence intracellular signaling pathways. HLA-B27 presents specific
features because of a free Cys-67 at the top of the B pocket. This free
Cys-67 has been shown to be highly chemically reactive (15), probably
because of polarization from a neighboring Lys-70 (16). It has also
been hypothesized that oxidative modification of this residue may play
a role in B27-related disease (17). Recently Allen et al.
(18) showed that a B*2705 heavy chain (HC) homodimer can form and be
presented at the cell surface, which is dependent on Cys-67. The
recognition of the HLA-B*2705 However, it is still unknown which amino acids of the B pocket controls
the peculiar misfolding properties of HLA-B*2705. Therefore, we
engineered a series of four pocket B mutants of the B*2705 HC and
investigated peptide binding properties as well as thermal unfolding of
the corresponding complexes with the three peptides. Circular
dichroism-monitored thermal unfolding, fluorescence polarization
spectroscopy, and size exclusion chromatography suggest that the
thermodynamic stability of the B*2705 heavy chain is significantly
decreased upon mutation of any of the pocket B residues. Cys-67 seems
to play a major role in promoting unfolding to high molecular weight
species while not affecting peptide binding properties.
Cloning of the HLA-A*0201 Heavy Chain--
The heavy chain was
cloned into pET-24b plasmid (Novagen) using the same protocol as
described previously (20) for HLA-B*2705. Briefly, the gene was
amplified by PCR starting from pQE-30 (Qiagen) containing the A*0201
heavy chain.2 The same
primers that were used for B*2705 were used to introduce the two
restriction sites NdeI and XhoI flanking the
gene. After restriction, the fragment was ligated into the expression
plasmid, which was then transformed into Escherichia coli
DH5 B Pocket Mutants of the HLA-B*2705 Heavy Chain--
All the
mutations of HLA-B*2705 heavy chain were introduced by the QuickChange
site-directed mutagenesis kit (Stratagene) because it required only the
change of a maximum of three neighboring base pairs. For this purpose,
we amplified the pET-24b plasmid (Novagen) containing the B*2705
wild-type heavy chain with PCR. The corresponding primer pairs (forward
and reverse) used for the four reactions are listed in Table
I. The PCR products were then digested
with DpnI and transformed into E. coli XL2-Blue strain (Stratagene) for selection. The sequence was confirmed by DNA
sequencing (ABI 310 PRISM, Perkin Elmer).
V67C Mutant of HLA-A*0201--
This mutation was introduced by
the same method using the pET-24b-A0201 plasmid as template. The
primers used are listed in Table I. The mutation was confirmed by DNA sequencing.
Protein Expression and Purification--
The expression of the
HLA-B*2705 or HLA-A*0201 heavy chains and of the relative mutants were
performed in E. coli BL21-Codonplus(DE3)-RIL strain
(Stratagene). The proteins were purified on a
Ni2+-nitrilotriacetate-agarose column (Qiagen) as
previously described (20). Peptide Synthesis--
Peptides were obtained by automated
solid-phase peptide synthesis on an automated multiple peptide
synthesizer (Syro Multi-Syn-Tech, Bochum, Germany) using the standard
Fmoc (N-(9-fluorenyl)-methoxycarbonyl) protecting strategy.
For synthesis of the fluorescein-labeled peptide GRAFVTIK*K (* is the
fluorescein label), a lysine with a Dde-protected side-chain was
inserted at position 8. After synthesis and selective deprotection,
Lys-8 was coupled to fluorescein isothiocyanate. Complete deprotection
and cleavage from the resin was achieved by trifluoroacetic acid.
Peptides were analyzed and purified by mass spectrometry and HPLC as
previously described (20).
Thermal Denaturation--
The thermal denaturation CD
experiments were performed as previously described (20). Briefly, the
HLA-B*2705/peptide complex was refolded upon dialysis of 10 ml of
reconstitution buffer (20 mM Tris, 150 mM NaCl,
2 mM EDTA, 3 mM Competition Experiments--
The HLA-B*2705/peptide complex was
refolded by dilution of HC (1 µM), Size Exclusion Chromatography--
Size exclusion chromatography
was performed using a Superdex 200 HR 10/30 FPLC column (Amersham
Pharmacia Biotech) pre-equilibrated in TBS containing 20 mM
Tris, 150 mM NaCl, pH 8.0. Experiments were performed at
25 °C and concentrations were held at 1 µM. 100 µl
of sample was loaded onto the column and eluted at 0.7 ml/min in TBS.
Elution profiles of the proteins were monitored by UV-absorbance at 280 and 220 nm. Molecular weight was calculated by comparison of the
elution retention times of MHC samples to those of marker proteins
(data not shown).
Peptide Dissociation from the MHC/Peptide Complex--
Details
of these experiments will be described
elsewhere.3 Briefly,
the complexes were refolded following the dialysis protocol previously
described (20) of 1 ml of protein-containing buffer against 100 ml
buffer. We used the fluorescent-labeled peptide GRAFVTIK*K as ligand.
FPLC chromatography was performed for purification, without a prior
concentration step. After addition of a large excess of unlabeled
competitor, the time course of peptide dissociation from the complex
was monitored at 26 °C. All measurements were duplicates of
independent experiments.
Mutation of Any Pocket B Amino Acid Decreases the Thermal Stability
of HLA-B*2705/Peptide Complexes--
To determine the stabilizing
properties of B pocket amino acids, we first mutated separately the
following four residues of the HLA-B*2705 HC: Thr-24 to Val, Glu-45 to
Met, Tyr-99 to Phe, and Cys-67 to Ser. T24V and Y99F changes were
chosen to be as conservative as possible. Met-45 is present in the
HLA-A*0201 B pocket, which when substituting the wild-type B*2705 B
pocket impairs misfolding of this allele (12). Furthermore, position 45 is known to control allele-specific peptide binding (23). Last, the
C67S mutation was chosen to be as conservative as possible while
removing the free Cys.
For refolding HC- Thermal Denaturation of HLA-B*2705 Is Dependent on the pH and
Residue at Position 67--
The profile of thermal denaturation curves
of HLA B*2705/peptide complexes was strongly dependent on pH value but
independent of the nature of the complexed peptide. The complex of
HLA-B*2705 with a reference peptide (I9; Ref. 20) was therefore
denaturated under mild alkaline, neutral, and mild acidic conditions
(Fig. 2). Changes of the second
transition are more easily monitored if the first transition is below
60 °C; therefore, the I9 peptide was chosen as ligand. Under mild
alkaline conditions (pH 8.3) a single transition was observed, which
led to partial unfolding, reflected by the loss of only a third of the
original CD signal (Fig. 2A). Under neutral conditions, an
additional step of unfolding was evident that resulted in an almost
complete unfolding. Interestingly, the first transition, which is
clearly peptide-dependent (data not shown), was unaffected
by a pH modification; neither midpoint of transition nor degree of
unfolding was changed. The novel intermediate state can be destabilized
by lowering the pH to mild acidic conditions (pH 6.4) where both
transitions seem to be merged (Fig. 2A). Only a single
unfolding step was apparent with an almost complete degree of unfolding
that was observed under neutral conditions.
The observation of the strong influence of pH on unfolding raised the
question of whether this effect is common to other HLA alleles or if it
is a specific property of the B*2705 subtype. Thus, we investigated the
unfolding of HLA-A*0201, an allele for which no misfolding has been
described (12). The thermal denaturation curves of HLA-A*0201 proteins
complexed with the natural epitope ILMEHIHKL (25) are presented in Fig.
2B. Under neutral conditions (pH 7.3) unfolding occurred in
a single step with the degree of unfolding (~65%) significantly
decreased compared with that observed for the reference
HLA-B*2705/peptide complex under acidic conditions (~85%). Moreover,
for HLA-A*0201, an increase of pH induced a two-step unfolding process.
But in contrast to HLA B*2705, the second step led to only minor
additional unfolding of the complex with a total degree of unfolding of
~33%. For HLA-B*2705, the intermediate state could be stabilized by
increasing the pH. At pH 9.4, the second transition was still observed
but was shifted toward a higher Tm value.
Among the amino acids differing between HLA-B*2705 and HLA-A*0201 HC,
Cys-67 was thought to have a major influence on the physiological
properties of HLA-B*2705. We hypothesized that the observed differences
in unfolding between HLA-B*2705 and HLA-A*0201 might be related to this
unpaired cysteine. Thus, we engineered two HLA mutants for which a
single point mutation had been introduced (C67S HLA-B*2705, V67C
HLA-A*0201). As anticipated, these mutations induced a dramatic change
in the unfolding process of the related HLA-peptide complexes.
HLA-B*2705 C67S complexed with the I9 peptide now showed a single
transition under neutral conditions (pH 7.3) resulting in about 65%
unfolding (Fig. 2C). As for the HLA-B*2705 WT complex, an
increase of pH value led to a two-step unfolding process, but similar
to the HLA-A*0201 WT complex, with only a minor contribution by the
second step toward the final degree of unfolding of ~40%. No
completed unfolding similar to that observed for the wild-type B*2705
HC (Fig. 2A) could be recorded for the C67S HC.
The unfolding of HLA-A*0201 V67C complexes under neutral conditions
seemed to be unaffected by the mutation (Fig. 2D). However Cys-67 had a strong influence on the second unfolding step under alkaline conditions. The final degree of unfolding was significantly increased (to ~50%) at pH 8.4 and 9.4 compared with the HLA-A*0201 WT complex (~30%), thus shifting toward the unfolding
characteristics of HLA-B*2705 WT complexes.
Cys-67 Promotes Unfolding of B*2705 toward High Molecular Weight
Species--
To gain further insight into the consequences of B*2705
unfolding, we characterized B*2705 WT and C67S folding intermediates. HLA-B*2705 WT and HLA-B*2705 C67S complexes were subjected to thermal
denaturation under mild alkaline conditions (pH 8.0) to obtain the
different unfolded states observed by CD spectroscopy and then
subjected to SEC and native gel electrophoresis. Under native
conditions, both the WT and the mutant HLA-I9 peptide complexes had
identical elution times, indicating that both heavy chains form
similarly folded complexes (data not shown). After heating to 73 °C,
a difference between WT and mutant can again be observed (Fig.
3). For the WT protein, a peak at 11.1 min (corresponding to a mass of >500 kDa) reflects formation of
high molecular mass aggregates. No peak corresponding to the
heterotrimer, peptide-free B*2705 or HC could be detected and only a
small
For the C67S complex, aggregation also takes place, but the aggregates
formed have a significant lower apparent molecular mass (~100 kDa).
The peak at 20.8 min corresponds to 39 kDa, representing either native
complex or peptide-free HC- The C67S Mutation Does Not Affect Peptide Binding Properties
of B*2705--
To determine whether destabilization of B*2705-peptide
complexes upon C67S mutation was correlated with peptide binding
affinity, we measured the affinity of a series of four related peptides (Table II) to either the WT or C67S
B*2705 mutant by a fluorescence polarization-based assay using a
fluorescein-labeled reference peptide (GRAFVTIK*K). Analysis of binding
data revealed no significant difference in binding between the WT and
mutant (data not shown). For this reason, the IC50 values
of the peptides can be compared directly between WT and mutant
complexes.
A typical dose-response curve is shown in Fig.
4. The IC50 values obtained
by fitting the experimental curve to a dose-response model are listed
in Table II. As expected, the higher affinity (in the submicromolar
range) was observed for the natural epitope (GRAFVTIGK) and its F3
analog. Replacement of Lys by Ile and Ser at P9 significantly decreased
affinity by 1 and up to 2 orders of magnitude, respectively (Table II).
Because of the low affinity of the S9 peptide, no complete competition
could be observed in the concentration range used for this assay.
Therefore, IC50 values for the S9 analog are given as an
approximation only. It can be noted that the changes in peptide binding
affinity are related to the melting temperatures of the corresponding
B*2705/peptide complexes.
Surprisingly, the thermal destabilization recorded upon C67S mutation
was not mirrored by a decrease in peptide binding affinities (Table
II). Very similar IC50 values were observed for all four peptides to either the WT or the C67S B*2705 mutant. It can be noted
that IC50 and Tm values are only
related when a series of protein-peptide complexes containing identical
HC are taken into account.
The C67S Mutation Increases Peptide Dissociation From the
HC- In the current study we investigated the influence of the amino
acids of the B pocket (Thr-24, Glu-45, Cys-67, Tyr-99) that are
relevant for peptide binding (11) on the thermodynamic stability of
several MHC-peptide complexes. Mutation of any of these amino acids
resulted in loss of stability of up to 22 °C, which reflects an
extreme influence of even a single amino acid mutation. There are no
evident relationships between the number of intermolecular hydrogen
bonds lost upon mutation and the observed destabilization. For example,
the most destabilizing change (T24V) is the consequence of a single
H-bond loss, according to the crystal structure of HLA-B*2705 (27). The
Y99F mutation resulting in the loss of one direct and two
water-mediated H-bonds is also very destabilizing and illustrates the
importance of a bound water molecule in the B pocket (27).
Surprisingly, modification of the polarity of the B pocket upon E45M
mutation did not alter refolding of the B*2705 HC in the presence of
Arg-2-containing peptides, although Arg-2 has been shown by x-ray
diffraction to make a salt bridge to Glu-45, and despite the lack of
evidence for an Arg-2 motif after pool sequencing, the peptide pool
naturally bound to this mutant (28). Among the three peptides
evaluated, the S9 complex was always the one that induced the least
stable complexes. It has to be noted that the WT complexes loaded with
S9 already have a low stability (20). It has been proposed that until a
certain threshold of stability (Tm of
~36 °C) has been reached, the mutation of such weakly stable
complexes will have little influence on their thermodynamic properties.
However, it should be stated that lower in vitro
thermodynamic stability might not always be related to lower in
vivo stability, because some potentially weakly stable complexes
are not refolded in vivo, or the corresponding peptide is
presented in very low amounts.
Interestingly, the C67S mutant destabilized the complex less
drastically than the other three mutants of the B pocket. Some recent
publications give great importance to Cys-67, because this residue is
implicated in the formation of heavy chain homodimers. For this reason,
we decided to focus our experiments on the C67S mutant. Our data show a
significant difference between thermal unfolding of HLA-B*2705 and
HLA-A*0201 complexes with regard to response to variation of pH.
Shifting the pH toward alkaline conditions induces a two-step thermal
unfolding. The first one is clearly peptide-dependent and
is probably the consequence of peptide release from the heterotrimer.
The second one, which appears at basic pH, is significantly increased
for MHC proteins bearing a free cysteine (HLA-B*2705 WT, HLA-A*0201
V67C mutant). A simple explanation for these different unfolding
processes can be clearly attributed to the amino acid at position 67 of
HC with Cys-67 responsible for an increased final degree of unfolding
after the second transition. Mutation of the amino acid at position 67 of the HC is reflected by almost complete interchange of the unfolding
profiles of HLA-B*2705 and HLA-A*0201/peptide complexes. It is already
known that Cys-67 is more acidic than expected, because it can be
alkylated by an aziridine-containing peptide ligand whereas free Cys in
solution cannot (15). Thus, any increase in the pH would favor the
existence of Cys-67 as a very nucleophilic thiolate anion promoting
oligomerization of the B*2705 HC.
Our hypothesis is supported by size exclusion chromatography of
unfolded intermediates of B*2705 and its C67S mutant, obtained at basic
pH. It clearly indicates that the mutation of Cys-67 results in the
loss of high molecular weight unfolded states that characterize the
unfolded B*2705 WT heavy chain. Surprisingly, the observed
destabilization of the heterotrimer upon Cys-67 mutation is not
reflected by a decreased affinity of peptide ligands to the mutant
(Table II). We have shown that IC50 values for three analogous peptides are not significantly affected by mutation of Cys-67
to Ser. These data are contradictory to previous results (29) for which
a correlation between the equilibrium dissociation constant
(KD)and thermal stability
(Tm) of the complex is described. They can
however easily be explained by the different experimental protocols
used in both studies. Whereas Morgan et al. (29) correlates
KD values obtained from equilibrium dialysis
experiments using a refolded peptide-free HC (29), our assay includes
the refolding of the heterotrimer in the presence of the peptide and
therefore does not lack the important contributions of protein-peptide
interactions for complex refolding. Furthermore, as peptide loading of
class I MHC complexes occurs in vivo during folding of the
proteins in the endoplasmic reticulum (30), our assay is more likely to
mirror physiological conditions and therefore is more suited to compare
peptide selection of complexes containing different heavy chains subtypes.
The different contributions of Cys-67 to thermal stability and peptide
binding suggest that Cys-67 is not directly involved in MHC-peptide
interactions participating in the refolding of the
HC- Furthermore, the results presented here confirm the important role of
Cys-67 for in vitro properties of HLA-B*2705 complexes. This
observation is an interesting example of the enormous influence of a
single amino acid on the unfolding process of a protein. This finding
is even more important in regard to the recently reported theory that
postulates that misfolded MHC-proteins are causative for the
association of HLA-B*2705 and autoimmune diseases (7, 30). Our data
show that the HLA-B*2705 complex unfolds in a subtype-specific manner
upon thermal denaturation. This observation suggests that not only
heavy chain dimers (18) but also abnormally unfolded complexes may be
the species responsible for autoimmune disease. Dimerization of class I
MHC-HC seems to be a general property of HC containing unpaired
Cys-residues (32) and therefore it cannot explain the peculiar
association of B*2705 expression with autoimmune diseases. Triggering
could either occur by altered TCR recognition or endoplasmic reticulum
stress due to an increased level of protein aggregation (4, 33). It
will be important to evaluate whether these specific properties of the
complex are relevant for activation of CTL resulting in autoreactivity.
However, the peculiar structural influence of Cys-67 on the
thermodynamic property of HLA-B*2705 does not explain why the
expression of a few B27 alleles is not associated with autoimmune
disorders (10) even though Cys-67 is conserved for all B27 alleles.
Rats transgenic for the C67S HLA-B*2705 mutant still develop arthritis, albeit with less severity than WT B*2705 rats (34). Furthermore, it
remains to be determined whether only Cys-67 has the described influence or if mutation of further B pocket amino acids specific for
HLA-B*2705 can induce similar effects.
Acknowledgments--
We thank Dr. T. Reitinger for synthesis of
the fluorescent-labeled peptide and Prof. J.A. López de
Castro for critical reading of the manuscript.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2m and thus a functional
antigen presentation pathway can develop a spontaneous inflammatory
arthritis (8). Moreover, this is also observed in the absence of MHC
class II genes (9), indicating that B27-derived peptides might not be responsible for the disease association. This evidence has led to an
increased number of alternative hypotheses that have been presented
over the last few years (4, 10).
1 helix by the ME1 mAb is
impaired by the formation of homodimers, consistent with a partial
unwinding of the helix that could be caused by a disulfide bonding. The
presentation of such a non-classical homodimer on the cell surface
could induce altered T-cell responses, while potentially mimicking a
MHC class II binding site, and lead to recognition by CD4 lymphocytes
and autoimmunity. Peptides longer than 9 amino acids characteristic of
MHC class II ligands have already been eluted from B27, which supports this theory (19).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
strain for selection.
Primers used for the mutagenesis of HLA-B*2705 or HLA-A*0201
2-microglobulin was expressed
and purified using the protocol described in the same report.
-mercaptoethanol, 0.3 mM 2,2'-dithiodiethanol, pH 8.0) containing HLA-B*2705 HC
(10 µM),
2m (20 µM), peptide (100 µM), and urea (6 M) against 1 liter of
reconstitution buffer. After purification and concentration, the
complex was used immediately for denaturation experiments. The
stability of the complex was examined by thermal denaturation, unfolding was monitored by CD spectroscopy at a wavelength of 218 nm.
The temperature was raised from 20 to 85 °C at a rate of 40 °C/h.
The concentration of the complex was held constant at 1 µM. The melting points were determined following a
standard protocol for thermal denaturation experiments (21). The
melting points were averaged from at least two independent refolding
experiments. Experimental error was estimated to be lower than
1 °C.
2m (2 µM), labeled peptide (8 nM), and various
amounts of competitor (typically 1 nM to 100 µM) into 1 ml of dilution buffer (20 mM Tris,
150 mM NaCl, 2 mM EDTA, 0.1 mM
CHAPS, 0.3 mM 2,2'-dithiodiethanol, pH 8.0). After 36 h of incubation at room temperature, complex formation was confirmed
according to a size exclusion HPLC assay (22). The ratio between bound
and unbound labeled ligand was determined using fluorescence
polarization. Polarization values were measured on a Polarion
fluorescence-polarization system (Tecan, Austria) using 200 µl of
sample in a 96-well black quartz microtiter plate (Hellma, Germany).
The number of flashes was set to 200, total intensity was held at
65,000 rfu. Concentrations at half-maximal inhibition (IC50
values) were obtained by fitting polarization values versus
total concentration of competitor to a dose-response model. The
IC50 values were averaged from three independent experiments.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2m-peptide heterotrimers, we used a
peptide from the B27-restricted T cell epitope from the HIV1 gp120
protein GRAFVTIGK (K9) (24) and two analogs (GRAFVTIGI or I9 and
GRAFVTIGS or S9). The mutant-peptide complexes were assembled by a
refolding assay starting from the recombinant proteins and were
analyzed for their thermal stability by means of CD spectroscopy. The
wild-type HLA-B*2705/peptide complexes represent a wide range of
thermal stabilities, with values varying from a high melting point of unfolding Tm (60.1 °C for K9), through medium
(53.3 °C for I9) and to low values (43.4 °C for S9) (20). Each
mutation appeared to be prejudicial to the thermal stability of the
mutant-peptide complex with a detrimental effect ranging from about
2.8 to
22.6 °C in comparison to the wild-type complexes (Fig.
1). The strongest effects were observed
for I9 and K9 complexes for which the destabilization values ranged
from
8.1 °C to a maximum of
22.6 °C. The T24V or E45M
mutations seemed to be extremely detrimental for the complexes loaded
with K9 (16.1 to 22.6 °C destabilization) and less so for the
complexes with I9 peptide (around
12 °C). Interestingly, the E45M
mutant could still be refolded in the presence of Arg-2-containing peptides. The Y99F and C67S mutations destabilized more the complexes with I9 (
18.1 and
12.4 °C, respectively) than with K9 (
11.7 and
8.1). The smallest destabilization was observed for the complexes with S9. For all mutant/S9 complexes, only a modest destabilization ranging from 3-6 °C could be observed.
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Fig. 1.
Influence of the mutation of B pocket amino
acids on the thermal stability of HLA-B*2705 in complex with three
peptides from the HIV1 gp120 parent sequence GRAFVTIGK (K9), GRAFVTIGI
(I9), GRAFVTIGS (S9). The thermal denaturation of the complexes is
monitored by CD spectroscopy at 218 nm, pH 8.2. Tm, Tm
(wild type)
Tm (mutant).
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Fig. 2.
Influence of pH and Cys-67 on the unfolding
of HLA-B*2705 and HLA-A*0201 heavy chains. HLA-B*2705
(A) and HLA-B*2705 C67S (C) are loaded
with I9 peptide, HLA-A*0201 (B) and HLA-A*0201 V67C
(D) are loaded with a known epitope (ILMEHIHKL). Percentage
of unfolding, monitored by CD-spectroscopy, is plotted against
temperature (°C).
2m peak was observed.
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Fig. 3.
Size exclusion chromatography of HC
intermediates obtained after thermal unfolding. Upon heating to
73 °C at pH 8.0, the complexes of the GRAFVTIGI peptide with
HLA-B*2705 WT (dotted line) and C67S (straight
line) form species of high molecular mass. Retention of WT
aggregates (peak A) indicates significantly higher molecular
mass (>500 kDa) than aggregated species formed by the C67S mutant
(peak B, ~100 kDa). Peak C corresponds to
native or peptide-free complex; peaks D and E,
respectively, correspond to 2m.
2m complex. Upon denaturation to 95 °C, precipitation occurred for the WT and mutant proteins, and
only minor peaks indicating the native or peptide-free complex and
2m were observed. No peak corresponding to the recently described B*2705 HC dimer (18) could be identified in our experiments. Analysis
of thermal unfolding by native-polyacrylamide gel electrophoresis confirmed these observations (data not shown).
Affinity (IC50 in µM) of four related peptides
for HLA-B*2705 WT or C67S mutant and the midpoint of unfolding
(Tm in °C) of the corresponding heterotrimers
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Fig. 4.
Determination of peptide binding affinity for
HLA-B*2705 IC50 values. Competition between a
fluorescent peptide (GRAFVTIGK*K; *, fluorescein) and a peptide
competitor for refolding the HC-peptide- 2m heterotrimer
is monitored by fluorescence polarization. A typical titration curve is
shown for the F3 peptide (GRFFVTIGK, Table II). The experimental error
of a single data point is 5% or less.
2m-Peptide Heterotrimer at 26 °C--
The data
from these experiments refer only to the dissociation of the labeled
peptide from the MHC complex, as confirmed by complementary FPLC
experiments (data not shown). Monitoring fluorescence polarization
(FP) versus time is particularly well suited for studying peptide dissociation from B*2705 because FP is directly dependent on changes in molecular weights, and this technique does not
require any washing or separation step (26). Thus, any release of the
labeled peptide from the heterotrimer can be recorded and immediately
translated into lower FP values. The complex between the fluorescent
peptide (GRAFVTIK*K) and B*2705 is remarkably stable at 26 °C as
shown by the FP time course of the corresponding complex at 26 °C
(Fig. 5). Upon C67S mutation, the same
labeled peptide dissociates faster. This result is in agreement with
the thermal denaturation experiments, again showing a higher stability
for the HLA-B*2705 WT complex.
View larger version (12K):
[in a new window]
Fig. 5.
Measurement of MHC-peptide complex stability
at 26 °C by fluorescence polarization. For comparison of
HLA-B*2705 WT and C67S, the labeled GRAFVTIK*K peptide was used. The
peptide dissociation kinetics was monitored in solution after addition
of a large excess of the same unlabeled peptide to prevent peptide
re-association. FP values (in mP units) decreases with release of the
fluorescent peptide from the heterotrimer.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2m-peptide heterotrimer. However, once the
MHC-peptide complex has been formed, Cys-67, as well as any amino acid
of the B pocket, plays an important role in maintaining a stable heterotrimer. Regarding these findings, our study strongly suggests that the nature of the selected peptide repertoire is not sufficient to
explain the different associations of HC subtypes with autoimmune diseases (31). Whereas the unfolding behavior of HLA-B*2705 is clearly
different from that of HLA-A*0201 because of the presence of a free
cysteine, it is still not clear why the expression of several alleles
has not been associated with high susceptibility to autoimmune
disorders, Cys-67 being conserved in all 20 HLA-B27 alleles reported to
date (10).
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FOOTNOTES |
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* This work was supported by the Schweizerischer Nationalfonds zur Förderung der wissenschaftlichen Forschung (Project 31-57307.99).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 333 9024 4235; Fax: 333 9024 4310; E-mail: didier.rognan@pharma.u-strasbg.fr.
These authors contributed equally to this work.
Published, JBC Papers in Press, February 26, 2001, DOI 10.1074/jbc.M101282200
2 D. Rognan and S. Krebs, unpublished results.
3 S. Dédier, T. Reinelt, S. Rion, G. Folkers, and D. Rognan, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are:
MHC, major
histocompatibility complex;
2m,
-2 microglobulin;
HLA, human leukocyte antigen;
PCR, polymerase chain reaction;
WT, wild
type;
HC, heavy chain;
FP, fluorescence polarization;
FPLC, fast
protein liquid chromatography;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
mAb, monoclonal antibody;
HPLC, high performance liquid chromatography;
SEC, size exclusion chromatography;
HIV, human immunodeficiency virus.
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REFERENCES |
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