From the Department of Applied Biosciences, Swiss
Federal Institute of Technology, Wintherthurerstrasse 190, CH-8057
Zürich, Switzerland, § Centro de Biologia Molecular
"Severo Ochoa", Universidad Autonoma de Madrid, Facultad de
Ciencias, Cantoblanco, E-28049 Madrid, Spain, and
Laboratoire de
Pharmacochimie de la Communication Cellulaire, Unité Mixte de
Recherches CNRS 7081, 74 route du Rhin, B.P.24, F-67401
Illkirch, France
Received for publication, March 29, 2001, and in revised form, May 7, 2001
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ABSTRACT |
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An HLA-B27-restricted self-octapeptide
known to react with an alloreactive T-cell receptor has been modified
by systematic substitution of a Major histocompatibility complex
(MHC)1 class I molecules are
proteins that present a large repertoire of peptides on the cell surface where they can be recognized by cytotoxic T-lymphocytes (CTLs)
(1). Under physiological conditions, only non-self-peptides, e.g. of viral origin, can activate CTL and
thereby trigger an immune response. In contrast to the normal function
of the immune system, CTLs can also be activated by self-antigens,
which represent a pathological state resulting in severe autoimmune
diseases such as rheumatoid arthritis, multiple sclerosis, or
autoimmune uveitis (2). The high association of several alleles with
autoimmune diseases, e.g. HLA-B*2705 with
spondyloarthropathy (3, 4), is not fully understood yet, but one of the
most favored models postulates the allele-specific selection and
presentation of autoantigenic peptides (5). For several of these
diseases, self-peptides triggering CTL are described (6, 7), whereas in
some cases like rheumatoid arthritis the relevant self-peptides are
still unknown. MHC class I molecules are also involved in the acute rejection of allogenic transplants (8, 9). Alloreactive T-cells are
stimulated by allogenic MHC molecules and often recognize epitopes
containing both the presenting molecule and specific peptides (9).
Several antigens have been described to be involved in alloreactivity
including MHC- and non-MHC-derived peptides. Current therapy for both
autoimmune diseases and alloreactivity includes suppression of the
entire immune system, e.g. by corticosteroids. However, the utilization of such immunosuppressive drugs is
characterized by severe side effects. Consequently, much effort has
been undertaken to establish alternative therapies that are based on
the selective, antigen-specific modulation of the immune response.
Several strategies have been developed including utilization of
antibodies, soluble MHC complexes, and peptide vaccines (10-12). Among
these strategies, peptide vaccines are of special interest as they have
been reported to induce tolerance in mice and humans (6, 7) and, in
contrast to protein-based therapeutics, are accessible by solid-phase
synthesis. However, the therapeutic application of peptides is hindered
by the rapid clearance from the serum because of the degradation by
proteases (13). Therefore, it would be highly desirable to obtain
peptidomimetics, which are enzymatically stable and bind with high
affinity to MHC molecules. Furthermore, these compounds have to be
recognized by the same pool of CTL as the parent peptide and induce the
desired immunomodulatory effect. Many attempts have been undertaken to
obtain peptidomimetics that fulfill these requirements, but only few
successful results have been reported (14). The utilization of
Here, we report the systematic variation of the HLA-B*2705-restricted
self-peptide by single replacement of all amino acids using the
corresponding Peptide Synthesis--
Peptides were obtained by automated
solid-phase peptide synthesis on an automated multiple peptide
synthesizer (Syro Multi-Syn-Tech, Bochum, Germany) using standard Fmoc
(N-(9-fluorenyl)methoxycarbonyl) protecting strategy.
Protected Protein Expression and Purification--
HLA-B*2705 heavy chain
(HC) and human Thermal Denaturation--
The thermal denaturation assay was
performed as described earlier (16). Briefly, the
HLA-B*2705-peptide complex was refolded by dialysis of 10 ml of
reconstitution buffer (20 mM Tris, 150 mM NaCl,
2 mM EDTA, 3 mM Binding Experiments--
The HLA-B*2705-peptide complex was
refolded by dilution of HC (1 µM), Peptide Sensitization Cytotoxicity Assay--
TAP
(transporter in antigen
processing)-deficient B*2705-T2 transfectant cells were
incubated for 18-20 h at 26 °C in RPMI 1640 medium supplemented
with 10% fetal calf serum (both from Life Technologies, Inc.)
in the absence of peptide. Cells were then labeled for 90 min at
37 °C with 50 µCi of 51Cr, washed four times,
resuspended in the same medium with 1% fetal calf serum, seeded in
96-well plates, blocked previously with bovine serum albumin at 1 mg/ml
in sterile phosphate-buffered saline, and incubated for 30 min at room
temperature with variable amounts of synthetic peptides. Effector CTL
27S69 cells (20) were then incubated with peptide-sensitized targets
for 5 h at 37 °C at an effector:target ratio of 1:1 in the
continuous presence of peptide, and the supernatants were subjected to
Peptide Degradation--
Peptide susceptibility to enzymatic
cleavage was assessed using a previously reported protocol (23). Human
sera were obtained by centrifugation at 2000 × g/g of
blood samples collected from two healthy donors. The sera were stored
at Modified Peptides Bind to Recombinant HLA-B*2705 with
High Affinity--
The affinities of the modified peptides for
recombinant HLA-B*2705 were evaluated by determination of
IC50 values using an fluorescence-polarization-based
competition assay. All peptides were able to compete for binding to
HLA-B*2705 with the fluorescent peptide, which is reported to be a good
binder.2 The
specificity of ligand binding was confirmed by several control experiments (data not shown) and is reflected by the different dose
dependences of competition observed for the modified ligands. A typical
curve resulting from titration with the Altered HLA-B*2705-Peptide Complexes Are Recognized by A Specific
Alloreactive T-cell Clone with Decreased Efficiency--
The influence
of
The effect of
These results indicate that substitutions of Two Altered Peptides Show Enhanced Resistance to Proteolytic
Cleavage--
The influence of the peptide modification on stability
against proteolysis was evaluated by monitoring the successive
degradation of the peptides by incubation with human blood serum. The
degradation kinetics were followed by HPLC analysis using the
corresponding peak area for peptide quantification. The unmodified
reference peptide was rapidly degraded, less than 10% of the initial
peak area being detected after 18 min. For the modified peptides, the time course of degradation showed to be highly dependent on the position of the substitution. No stabilization was observed for all
modifications occurring between P3 and P8 positions of the parent
peptide (Fig. 4). However, a significant
resistance to proteolytic cleavage is observed for the peptides
modified at the first two N-terminal residues (Fig. 4). For the In this study we investigated the influence of systematic
modification of a HLA-B*2705-restricted octapeptide by single
substitution of all amino acids with their corresponding
Interestingly, the substitution of the C-terminal residue induced the
opposite effect resulting in an increased stability for RRFFPY In addition to the thermal stability, the affinity of a ligand to its
host MHC molecule represents a parameter of high importance for the
development of non-natural class I MHC ligands. Thus, we evaluated the
IC50 values of the modified peptides using a new
fluorescence-polarization-based competition assay. The IC50 value of the parent peptide (3.2 µM) is comparable with
previously reported affinities for medium binders (27). All modified
peptides were able to compete for binding with a fluorescent peptide.
Structure properties observed for the binding affinity were very
similar to that reported previously for the thermal stability of the
resulting MHC-peptide complexes. Alteration of the first two N-terminal residues increases IC50 values by a factor of 15-20 (see
Fig. 2 and Table I) whereas modification of the peptide sequence from P3 to the C terminus did not alter the binding properties of the altered peptides to the target MHC protein (Fig. 2B).
Interestingly, our data are in contradiction with a recent report (28)
indicating that P1, P2, PC-1, and PC residues (PC: C-terminal
residue) of an HLA-A2 binding tumoral peptide are not permissive for
Molecular modeling of the RRFFPYYV epitope in complex with B*2705 (20)
suggests that Arg2, Phe3,
Tyr6, and Val8 are anchor residues, whereas
Arg1, Phe4, Pro5, and
Tyr7 are exposed at the surface of the complex. Ala
scanning further indicated that Arg1 is not significantly
involved in recognition by CTL 27S69, but Phe4,
Pro5, and Tyr7 are critical for allorecognition
(29). There was no correlation between the effect of The rapid clearance from blood serum, e.g. by
enzymatic cleavage, is one of the major limitations for the
pharmaceutical application of peptides. As peptides composed solely of
Our data reveal a strong influence of single -amino acid for the natural
-amino acid residue, over the whole length of the parent epitope.
All modified peptides were shown to bind to recombinant
HLA-B*2705 and induce stable major histocompatibility
complex-peptide complexes, but with some variation depending on the
position of the
-amino acid on the peptide sequence. Alteration of
the natural peptide sequence at the two N-terminal positions (positions
1 and 2) decreases binding affinity and thermodynamic stability of the
refolded complex, but all other positions (from position 3 to the
C-terminal residue) were insensitive to the
-amino acid
substitution. All modified peptides were recognized by an alloreactive
T-cell clone specific for the parent epitope with decreased efficiency,
to an extent dependent of the position that was modified. Furthermore,
the introduction of a single
-amino acid at the first two positions of the modified peptide was shown to be sufficient to protect them
against enzymatic cleavage. Thus,
-amino acids represent new
interesting templates for alteration of T-cell epitopes to design
either synthetic vaccines of T-cell receptor antagonists.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-amino acids represents a systematic approach suited for the
successive development of peptidomimetics (15). The side chains of
-amino acids are identical to the parent
-amino acids, which is
of particular importance regarding the great influence of side chains
on the complex stability (16). The modification of the backbone by
introduction of the methylene moiety results in complete resistance of
peptides composed solely of
-amino acids against proteolytic
cleavage (15). Successful use of
-homoalanine for the design of
non-natural MHC ligands has been reported previously by our group (17),
but the introduction of the
-amino acid was limited to the middle
part of the peptide, which is known to bulge out of the peptide binding
groove (18) and thus only weakly interacts with the host MHC molecule
(19).
-amino acid analogues. The presentation of the
self-peptide chosen for modification (RRFFVYYV; one-letter code) was
shown previously to be restricted by the HLA-B*2705 molecule and
specifically recognized by the 27S69
T-cell receptor of
an alloreactive T-cell clone (20). The influence of substitutions on
complex stability, ligand affinity, proteolitic degradation, and CTL
recognition was evaluated using circular dichroism, fluorescence polarization, HPLC, and a chrome-release cytotoxicity assay.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-amino acids were purchased from Fluka, Buchs,
Switzerland. For synthesis of fluorescein-labeled peptide
GRAFVTIK*K (*, fluorescein) a lysine with a Dde-protected side
chain was inserted at position 8. After synthesis and selective deprotection, Lys8 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 described previously (16).
2-microglobulin (
2m) were
cloned, expressed, and analyzed as described previously (16). Briefly,
the proteins were expressed in an Escherichia coli
BL21-Codonplus (DE3)-RIL strain (Stratagene) as
polyhistidine-tagged fusion proteins. Purification of the
proteins was performed by nickel affinity chromatography. The
N-terminal polyhistidine tag of
2m was cleaved by
thrombin after purification.
-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 by 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 are averaged from at
least two independent refolding experiments, and experimental error was
estimated to be lower than 1 °C.
2m (2 µM), fluorescent peptide GRAFVTIGK*K (* = fluorescein) (8 nM), and various amounts of competitor (typically 1 nM-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 by a modified size-exclusion HPLC assay (22).
The ratio between bound and unbound labeled ligand was determined by
fluorescence polarization. Polarization values were measured on a
Polarion fluorescence-polarization system (Tecan, Salzburg, Austria) using 200 µl of sample in a 96-well black quartz microtiter plate (Hellma, Müllheim, Germany). Number of flashes was set to
200, and total intensity was held at 65000 relative fluorescence units. IC50 values were obtained by fitting polarization
values versus total concentration of competitor to a
dose-response model. The IC50 values are averaged from
three independent experiments.
-counting. Percent-specific 51Cr release was calculated
as follows: (experimental lysis
spontaneous lysis/maximum
release
spontaneous lysis) × 100. Recognition of the
natural CTL 27S69 epitope was quantified as the peptide concentration
required to obtain half of the maximum lysis observed with this peptide
in the concentration range used. Recognition of the
-amino acid
analogs was measured as the peptide concentration required to obtain
the half-maximal lysis of the octamer epitope (LC50).
20 °C. Prior to use, the sera were incubated for 5 min at room
temperature followed by 5 min at 37 °C. 100 µl of serum was added
to 20 µl of peptide stock solution (1 mM) and incubated
at 37 °C. The reaction was stopped after 0, 1, 3, 6, 9, 12, and 18 min by addition of 12 µl of trifluoroacetic acid. Precipitated
serum proteins were pelleted by centrifugation. Degradation was
monitored by HPLC at a wavelength of 218 nm using 50 µl of the
supernatants per injection. Separation was performed on a LiChrospher
RP-18 column (Merck) at a flow rate of 0.6 ml/min. The following
solvent system was used: 0.08% trifluoroacetic acid in acetonitrile
and 0.1% trifluoroacetic acid in water with a gradient of 10 to 40%
0.08% trifluoroacetic acid in acetonitrile for 30 min. The
presence of the non-degraded peptide was confirmed by mass spectrometry
analysis of the corresponding HPLC peak (data not shown).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-Amino Acid Substitution Does Not Impair Formation of Stable
MHC-Peptide Complexes--
To characterize the influence of amino acid
substitution by corresponding
3-amino acids we
mutated separately all positions of the HLA-B*2705-restricted octapeptide RRFFPYYV (Table I).
The influence of
3-amino acids on the stability of
HLA-B*2705-peptide complexes was evaluated by thermal denaturation
experiments using CD spectroscopy to monitor complex unfolding. A
typical denaturation curve resulting from unfolding of the
B*2705-RRFFPYYV complex is shown in Fig. 1A. The midpoint of unfolding
or melting points (Tm) observed for the different complexes
are highly variable, which is reflected by differences between the
Tm values of up to 12 °C (Fig. 1B). Furthermore, the influence of the substitutions depends strongly upon
the position of the mutated amino acid. A decreased stability is
observed upon modification of the two N-terminal residues of the
peptide. For the
RRFFPYYV
R1 peptide, the Tm
value (39.7 °C) is 9 °C lower than that of the parent peptide.
Mutation of the neighboring position 2 (P2) results in only minor
destabilization; a Tm value of 46 °C was observed for
R
RFFPYYV (
R2), which is a thermal destabilization of 2.5 °C
when compared with the parent peptide (Tm = 48.5 °C).
All other modifications from P3 to the C terminus result in altered
peptides that stabilize the HLA-B27-peptide complex. An increase of
stability of about 2 °C is observed for mutation of the amino acids
Phe4 (50.2 °C), Pro5 (50.4 °C), and
Val8 (50.8 °C). The highest Tm values were
measured for the peptides RR
FFPYYV (51.5 °C) and RRFFPY
YV
(51.8 °C), which reflects a stabilization of about 3 °C. No
significant effect on complex stability is observed for substitution of
Tyr6 by its
3-amino acid analogue, the
observed Tm of 49.1 °C being comparable with the that of
the parent epitope.
Amino acid sequence of modified peptides
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Fig. 1.
Influence of peptide mutation on
thermal stability. Thermal denaturation was monitored by CD
spectroscopy (218 nm). A, typical denaturation profile of
the HLA-B*2705 complexed with RRFFPYYV. B, influence of the
position of the mutated amino acid on thermal complex stability. The
experimental error is 1 °C or less.
F3 peptide (Table I) is
shown in Fig. 2A. The final
fluorescence-polarization value of about 55 millipolarization
units corresponds to the value obtained for the free labeled
ligand and mirrors complete competition by the non-labeled molecule. As
seen in Fig. 2B and Table I, the effect of the
3-amino acid scan is highly dependent upon the position
of the substitution. A significant decrease in affinity is observed for mutations at the N-terminal part of the peptide. For the
R1 and
R2 peptides IC50 values of 45.1 and 65.0 µM have been determined, respectively, which
represents a 15-20-fold decreased affinity when compared with the
parent peptide. In contrast, the substitution of other positions in the
peptide results in IC50 values in the µM
range that are comparable with that of the parent peptide (3.2 µM).
View larger version (16K):
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Fig. 2.
Influence of peptide mutation on ligand
affinity. IC50 values were determined by a
fluorescence-polarization-based competition assay. A,
typical titration curve for the peptide RR( F)FPYYV (IC50 = 1.9 µM). The experimental error is 5 millipolarization
units or less. B, influence of the position of the mutated
amino acid on the IC50 values.
-amino acid substitutions at individual positions of the
alloreactive peptide epitope RRFFPYYV on recognition by CTL 27S69 was
analyzed by means of a peptide sensitization assay using the
TAP-deficient B*2705-T2 transfectant cell line. All of the
-amino
acid analogs were recognized by CTL 27S69 but less efficiently than the
natural epitope (Fig. 3). Significant differences were observed in the effect of substitutions at different positions (Table II). The lowest effect,
a decrease of about two orders of magnitude in LC50, was
obtained with
R1,
P5, and
V8. The most drastic decrease, about
four orders of magnitude, corresponded to
Y6 and
Y7, followed by
F4. Intermediate affects, LC50 about three orders of
magnitude lower than the unmodified epitope, were observed with
R2
and
F3.
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Fig. 3.
Specific cytotoxicity of CTL 27S69 against
B*2705-T2 cells sensitized with various amounts of the natural RRFFPYYV
peptide epitope (solid circles) or each of the
-amino acid analogs. The natural B*2705
ligand, RRIYDLIEL, was used as a negative control (cross
symbols). The effector:target ratio was 1:1. Data are means of
three to seven independent experiments.
Specific lysis of B*2705-T2 cells sensitized with the alloreactive
peptide or modified analogs
-amino acid substitutions on CTL recognition did not
correlate with stability (Tm) or affinity
(IC50) of the corresponding B*2705-peptide complexes (Table
I). For instance
Y6, which was among the analogs recognized with
lowest efficiency, had similar Tm and IC50
values as the natural epitope. Conversely
R1, which showed the
smallest effect on CTL recognition, had significantly lower stability
and affinity than the natural epitope for B*2705. Furthermore,
significantly decreased recognition of
F3 was observed despite
increased stability of the B*2705-
F3 complex.
-amino acids at
individual positions of an allospecific peptide epitope decrease, but
do not abrogate, CTL recognition. The effect is strongly dependent on
the peptide position but is not a direct consequence of decreased affinity of the peptide analog for B*2705 or lower stability of the
B*2705-peptide complex.
R2
peptide, about 74% of the initial peak area was recovered after 18 min. The substitution of the first amino acid showed to have the
strongest influence on the degradation. For
R1, no significant
decrease of the peptide area was observed after 18 min.
View larger version (17K):
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Fig. 4.
Enzymatic degradation of the peptides.
Peptide degradation is monitored by HPLC at a wavelength of 218 nm.
A, typical chromatogram overlay for degradation of the
reference peptide (RRFFPYYV) after 0, 3, 6, 9, and 12 min of incubation
with human blood serum. The unmodified peptide elutes after 21.3 min.
B, time course of enzymatic cleavage for the series of
altered peptides (Table I).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
3-amino acid analogues. As the thermal stability of MHC
class I-peptide complexes is reported to be a good descriptor of the
in vivo immunogenicity of the antigenic complex (24), all
altered B27-peptide complexes were subjected to thermal denaturation
experiments. The Tm values obtained showed to be highly
dependent on the position of the mutation within the peptide. Upon
modification of the central positions 4-6 a minor stabilization of
about 1-2 °C is observed. These results are in agreement with the
expected minor contributions of these residues to the total free energy
of binding as the middle part of HLA-B*2705-bound peptides bulges out
of the peptide binding groove (18) and does not strongly interact with
the host MHC molecule. This structural feature explains why replacing
this peptide region by organic spacers (polyesters (25),
-amino acids (17), polyethylene (19, 25)) does not influence either ligand
binding or thermodynamic stability of the resulting MHC-ligand complexes. The importance of the correct hydrogen bonding at the N
terminus of the peptide (18) is reflected by the observed destabilization upon mutation of the first (
9 °C) and the second (
2.5 °C) N-terminal residue. The
-amino acid scan shifts
important hydrogen bond donors/acceptors (first two peptide bonds,
arginine side chain) in the C-terminal end direction. This effect is
more deleterious for the
R1 peptide for which only the terminal
ammonium and first side chain could be located in the binding groove,
in an orientation similar to that of the reference peptide. All other atoms are translated toward the C-terminal residue in a region (pocket A, pocket B) of HLA-B*2705 very sensitive to small
topological changes of the bound ligand. For the
R2 peptide, the
destabilization noticed upon
-amino acid substitution is less
pronounced as the important arginine side chain at P2, as well as the
N-terminal P1 residue, share a bound orientation that should be
analogous to that of the parent epitope. The increased stability
observed upon substitution at P3 probably results from an improved
accommodation of the aromatic side chains in the D pocket of the
HLA-B*2705, a subsite known to favor bulky aromatic substituents (19,
25).
YV (+ 3.3 °C) and RRFFPYY
V (+ 2.3 °C). Some variation around the
binding mode of the C-terminal residue has already been showed by x-ray
diffraction of class I MHC molecules (18). The last peptide amino acid
can even be partly located outside the binding groove (26). Thus,
alteration of the starting peptide at the C-terminal residues can be
well accommodated and even enhance interactions with the binding
groove. As the C-terminal residue controls for a large part to the
overall thermodynamic stability of the MHC-peptide complex (16),
-amino acid substitution represents an interesting alternative for
designing altered ligands.
-amino acid substitution. The P1 modification was found to be
less detrimental for binding than the P2 change, by opposition to
measured thermal stabilities of the antigenic complexes. However, this
contradiction can be explained by the differing set of protein-ligand
interactions contributing to affinity and complex stability (27). In
our binding assay, competitors compete with the reference peptide for
promoting the refolding of the HC-
2m-ligand
heterotrimer. Thus, MHC-peptide interactions constitute only one aspect
of the multistep refolding procedure in which both entropic and
enthalpic contributions of the ligand play an important role. By
contrast, thermal unfolding of already formed MHC-peptide complexes
only takes into account the ligand release from the peptide binding groove. Our data illustrate a stronger contribution of residue P2 to
complex refolding but a lower influence on complex stabilization compared with that of the N-terminal residue.
-amino acid
substitutions on CTL recognition and the involvement of the
corresponding residue in anchoring to B*2705 or in TCR interactions.
Thus, of the residues for which
-amino acid substitutions had the
lowest effect on CTL recognition, Arg1 is not critical for
alloreactivity (29) but makes a significant contribution to affinity
and stability of the B*2705-peptide complex (Table I), Pro5
is exposed and critical for CTL recognition, and Val8 is an
anchor residue (20, 29). The highest effect on CTL recognition was
obtained upon substitution of two exposed residues (Phe4,
Tyr7) or a hidden one (Tyr6). The lack of
correlation between the effect of
-amino acid substitutions on CTL
recognition and the nature of the peptide positions, or their
contribution to affinity or stability of the B*2705-peptide complex,
suggests that the conformational changes induced in the peptide
following introduction of
-amino acids have independent effects on
peptide binding and T-cell recognition.
3-amino acids are completely stable against proteases
(15), we investigated whether single
3-amino acids have
an influence on the degradation upon incubation of the peptides with
human blood serum. For the majority of the peptides a decrease similar
to that of the parent peptide was observed. Typically, less than 20%
of the initial amount was detected after 18 min of incubation. However,
the
R2 peptide was significantly more resistant to degradation than
the parent epitope. The strongest effect was induced for mutation of
the N-terminal residue, which resulted in complete stability during the
time of observation. This is particularly significant, because the
R1 substitution had the lowest effect on CTL allorecognition among
those tested in this study. These data confirm the relevance of
aminopeptidases for the cleavage of short peptides in blood serum (23).
Moreover, our results show that single substitution by
3-amino acids analogues can already induce a significant
protective effect against enzymatic degradation.
3-amino
acids on parameters of high relevance for the immunogenic properties of
the ligand. Furthermore, we could show that the introduction of
3-amino acids may be used as a general method to
increase the resistance of short peptides against enzymatic
degradation. Thus, the results of this study strongly suggest the
utilization of
3-amino acids for the design of altered
MHC ligands for therapeutic application.
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ACKNOWLEDGEMENTS |
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We thank Dr. Richard Söll for help with peptide synthesis, Reto Bader for measuring mass spectra, and AlexanderHeckel for collecting blood samples.
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FOOTNOTES |
---|
* This work is supported in part by the Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Project 31-57307.99) and by Grants SAF99-0055 and PM99-0098 from the Spanish Ministry of Science and Technology (to J. A. L. C.).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.
¶ Present address: Pharmaceuticals Division, F. Hoffmann-La Roche Ltd., CH-4070 Basel, Switzerland.
** To whom correspondence should be addressed. Tel.: 33-3-90-24-42-35; Fax: 33-3-90-24-43-10; E-mail: didier.rognan@pharma.u-strasbg.fr.
Published, JBC Papers in Press, May 7, 2001, DOI 10.1074/jbc.M102772200
2 Unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
MHC, major
histocompatibility complex;
CTL(s), cytotoxic T-lymphocyte(s);
HPLC, high pressure liquid chromatography;
HC, heavy chain;
2m, human
2-microglobulin;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
Tm, melting point;
Pn, position n;
LC50, half-maximal lysis of the octamer epitope;
Dde, 1-(4-4-dimethyl-2,6-dioxocyclohex-1- ylidene)ethyl.
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REFERENCES |
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