(Received for publication, August 30, 1995)
From the
Reduced peptide bond pseudopeptide analogues have been examined
for their ability to bind murine class I molecules of the major
histocompatibility complex (MHC). Eight pseudopeptide analogues of an
antigenic peptide derived from Plasmodium berghei (H-Ser-Tyr-Ile-Pro-Ser-Ala-Glu-Lys-Ile
-OH)
were obtained by systematically replacing one peptide bond at a time by
a reduced peptide bond
(CH
-NH). The resulting
analogues were then tested for their binding to a recombinant single
chain SC-K
class I molecule. The comparative results show
that five analogues can efficiently mimic the parent peptide while the
introduction of the reduced bond between P3-P4, P7-P8, and
P8-P9 is deleterious for SC-K
binding. The fact that
more stable pseudopeptides containing reduced peptide bonds can bind
major histocompatibility complex molecules is of great interest for the
design of peptidomimetics with potential therapeutical properties. Such
peptide analogues may prove useful for the development of peptide-based
cytotoxic T lymphocyte vaccines.
MHC ()class I molecules bind short peptides derived
from the cytosol, which are then presented at the cell surface to
CD8-expressing T lymphocytes(1) . MHC molecules are highly
polymorphic. Each MHC allele selects a unique subset of peptides (2) , but while a single MHC allele can bind up to 10
different peptides and perhaps even more(3) , few
peptides have been reported to bind to multiple MHC alleles. This
exquisite selectivity associated with a loose specificity is achieved
through conserved interactions with the peptide backbone and termini on
one hand (4) and multiple combinations of dominant negative
interactions involving polymorphic residues of the MHC on the other
hand(5) . Extensive hydrogen bonding between conserved residues
of the MHC and common features of the peptide were revealed by
crystallographic analysis of MHC molecules complexed with a single
peptide. On the other hand, polymorphic residues with a bulky side
chain, which are rather frequent in the peptide binding site, restrict
the size of the repertoire of bound peptides for example by steric
hindrance. In this model, conserved interactions would guarantee high
affinity binding with every peptide of correct length while polymorphic
negative interactions would minimize peptide repertoire overlapping
between the different MHC alleles. This view stems from
crystallographic studies of MHC molecules bound to single
peptides(4, 6, 7, 8, 9, 10) and has
only been partially confirmed by mutagenesis of the MHC molecule and by
amino acid substitution in the peptide. These studies did not directly
examine the peptide backbone for which a role has been postulated from
the MHC crystal structure.
In order to directly test the
contribution of atoms from the peptide backbone in the binding
affinity, we decided to introduce sequential chemical changes at each
peptide bond (CO-NH) of the peptide. Starting from a
9-residue-long antigenic peptide derived from Plasmodium berghei and known to bind to SC-K(11) , we
systematically replaced one peptide bond at a time by a reduced peptide
bond
(CH
-NH). The eight resulting analogues
were then tested in vitro for their binding to a recombinant,
single-chain SC-K
molecule. We found that out of the eight
peptide bonds, five can be reduced with only a modest effect on the
binding affinity. Our results specify the contribution of each peptide
bond and the importance of the peptide backbone as postulated by
crystallographers(4, 6, 8, 9, 10) .
All the coupling procedures
following the reduced peptide bond formation were performed using a
2-fold excess of N-Boc--amino acid and activation
reagents and were monitored with the ninhydrin test. At the end of the
synthesis and after the last deprotection step, the peptide resin was
washed twice with ether and dried under vacuum in a desiccator. The
peptides were cleaved from the resin by treatment with anhydrous
hydrogen fluoride (HF) containing 10% (v/v) anisol and 1% (v/v)
1,2-ethanedithiol. After removal of HF in vacuo, the peptides
were extracted from the resin and lyophilized. The crude peptides were
purified by reverse phase HPLC using a Perkin-Elmer preparative HPLC
system on an Aquapore ODS 20-µm column (100
10 mm) by
elution with a linear gradient of aqueous 0.06% trifluoroacetic acid
(A) and 80% acetonitrile, 20% A (B) at a flow rate of 6 ml/min with UV
detection at 220 nm.
Analytical HPLC was run on a Beckman instrument
(Gagny, France) with a Nucleosil C18 5-µm column (3.9 150
mm) using a linear gradient of 0.1% trifluoroacetic acid and
acetonitrile containing 0.08% trifluoroacetic acid at a flow rate of
1.2 ml/min. Mass spectra were obtained by fast atom bombardment-mass
spectrometry in the positive mode on a VG analytical ZAB-2S.E. double
focusing instrument and recorded on a VG 11-250 data system (VG
Analytical, Manchester, UK). Proton spectroscopy was acquired at 300 K
on a Bruker ASP200 spectrometer (Bruker Spectrospin, Wissembourg,
France).
Table 1shows the sequences of the antigenic peptide
252-260 of P. berghei circumsporozoite (PbCS) and of
eight pseudopeptides (1 to
8), which were used in this
study. The reduced peptide bond
(CH
-NH) was
formed by reductive amination of N-Boc-
-amino aldehydes
in acidic dimethylformamide as described previously(13) . All
the
-amino aldehydes and intermediates were characterized by
H NMR spectroscopy and showed expected features. Stepwise
elongation of peptide chains using t-butyloxycarbonyl
chemistry led to the fully protected peptide resin. HF cleavage and
HPLC purification on a C18 column yielded the final products. All the
compounds were identified by fast atom bombardment spectroscopy, and
their purity was assessed by analytical HPLC. HPLC retention times and
fast atom bombardment-mass spectroscopy values of the peptides are
reported in Table 1.
The eight different analogues were then
tested for their binding to purified soluble, single-chain SC-K produced by transfected Chinese hamster ovary cells. This
recombinant protein contains the three external domains of the mouse
MHC class I molecule H-2K
connected through a
15-amino-acid-long spacer to the N terminus of the
-microglobulin(20) . The resulting fusion
protein is soluble in the absence of detergent(21) . It
contains
-microglobulin of mouse origin exclusively,
unlike heterodimeric MHC class I molecules purified from tissue
cultures, which include bovine
-microglobulin that may
alter the heavy chain conformation(22) . It selects a peptide
repertoire indistinguishable from that of cell surface-associated
SC-K
(19) .
Parent peptide or pseudopeptide
binding to SC-K was determined using a competition assay
with the
I-labeled peptide S9I of known equilibrium
affinity value for SC-K
(K
= 30
10
M(19) ). This assay was
previously used to identify antigenic epitopes derived from mouse
mammary tumor virus sequences(18) . Fig. 1shows the
competitive binding activity of PbCS peptide and of six analogues,
1-
6. With increasing concentrations of the
1,
2,
4,
5, and
6 competitors, the binding of
labeled peptide was reduced. For the parent peptide (PbCS) and each
analogue, the concentration leading to 50% inhibition binding of S9I
(IC50) was determined and used to calculate the relative affinity
following the Cheng and Prussoff (23) relationship, K
= IC
/(1 +
[S9I]/K
), where [S9I] is the
concentration of the labeled peptide and K
is
its equilibrium affinity value for SC-K
(30 nM). Table 2shows the effect of introducing reduced peptide bonds in
the parent peptide on its binding to SC-K
. While every PbCS
analogue consistently displayed a reduced binding to SC-K
,
broad differences were observed. Out of eight pseudopeptides tested,
five (
1,
2,
4,
5, and
6) demonstrated
strong binding (K
between 150 and 280 nM as compared with 50 nM measured with PbCS peptide). In
contrast,
7 and
8 showed weak activity and
3 did not
compete at all for SC-K
. Thus, introduction of a reduced
amide bond between Ile
and Pro
(P3-P4), Glu
and Lys
(P7-P8),
and Lys
and Ile
(P8-P9) was
deleterious for binding to SC-K
while modifications between
P1-P2, P2-P3, P4-P5, P5-P6, and P6-P7 had
only a minor effect.
Figure 1:
Binding of analogues to K.
Peptide binding was measured as described under ``Materials and
Methods'' with different concentrations of test peptides. Peptide
analogues were compared with unlabeled positive control peptide PbCS (open circle). The means and standard deviations of three
independent experiments are presented.
,
1;
,
2;
,
3;
,
4;
,
5;
,
6.
The tendency for a reduced affinity of
pseudopeptides in binding SC-K may be related to the loss
of a potential intermolecular hydrogen bonding at the point of
replacement of the carbonyl moiety by a methylene group. This
observation is consistent with crystallographic studies showing the
importance of extensive hydrogen bonding between the MHC residues and
carbonyl oxygen of the peptide backbone(6) . The structure of
H-2K
with a peptide from vesicular stomatitis virus reveals
that, in addition to hydrogen bonds involving the N and C termini of
the peptide, nine direct hydrogen bonds are formed between main chain
atoms of the peptide and side chain atoms of cleft
residues(6, 7) . Similar hydrogen bonding patterns
were found with H-2K
complexed with a peptide from
Sendaï virus(6) , with H-2D
complexed to a peptide derived from of the nucleoprotein (NP) of
influenza virus (10) and HLA-Aw68 complexed with another
influenza NP peptide. For example, in these four different MHC-peptide
complexes, the indole nitrogen atom of the highly conserved Trp
was found in hydrogen bonding distance of the C=O peptide
bond linking residues P8 and P9. This carbonyl moiety is reduced in
8 and accordingly a reduced affinity of
8 for SC-K
is observed. In the structure of H-2D
complexed with
the influenza virus peptide NP 366-374, the carbonyl oxygen atoms
of both Met
and Glu
hydrogen bond directly to
the indole nitrogen atom of the Trp
residue. Moreover, the
side chains of residues Trp
, Tyr
, and
Trp
form a ridge across the cleft of
H-2D
(10) . This peculiar structural feature is
shared by the H-2K
molecule(24) , and one should
expect a similar hydrogen bonding pattern for Trp
in the
SC-K
PbCS complex and hence a decrease in affinity
binding of
6 and
7 to SC-K
. On the other hand,
the hydroxyl group of the conserved Tyr
was proposed to
be hydrogen bonded to the carbonyl of the first peptide
bond(6, 7, 8, 10) . This conserved
hydrogen bond may not be as critical as suggested by these previous
studies, since affinity of
1 is only reduced by a factor of 6 as
compared with the parent peptide PbCS.
It should be kept in mind
that, in addition to preventing a particular carbonyl from forming a
hydrogen bond, the presence of a secondary amine has conformational
effects on reduced peptide bond pseudopeptide analogues. The neutral
amine form introduces an increased flexibility due to the higher
rotation around the C-N bond. However, it is known that the
reduced amide bond is protonated at physiological pH, and it has been
shown (25) using pseudodipeptide units that the resulting
ammonium link (CH2-N
H2) could stabilize a
folded structure by acting as a strong proton donor. Therefore, the
total lack of reactivity of the
3 peptide toward the SC-K
molecule may be related to a major conformational change of the
analogue, which is no longer able to mimic the extended bioactive
conformation of the parent PbCS peptide. Alternatively, the MHC may not
be able to adapt to the
3 peptide.
Under certain conditions, synthetic peptides are highly immunogenic in animals and might constitute chemically defined, safe, and cheap vaccines. Recently, tumor- and virus-specific cytolytic T lymphocytes have been generated using appropriate peptide immunization protocols. However, the rapid clearance of the peptide in the blood circulation may represent a severe limitation to human applications since high doses of peptide would be required. Pseudopeptides with altered peptide bond are less susceptible to natural proteases. Thus it would be tempting to use such pseudopeptides to induce cytolytic effector cells in vivo. But it was not obvious that such analogues could bind to MHC class I molecules, particularly given the postulated crucial role of the backbone in binding to the MHC molecules. The reduced amide bond has been used for the design of various analogues of bioactive peptides, but only two recent investigations have dealt with the immunological impact of this modification in the context of peptide-antibody (15) and peptide-class II HLA molecule interaction(26) . We report here that certain reduced peptide bond pseudopeptide analogues are bound almost as well as the parent peptide by MHC class I molecule and thus that reduced bond pseudopeptide analogues can mimic the parent peptide. Our results confirm the major role of the peptide backbone in the binding of peptide to MHC molecule but reveal a differential contribution for each carbonyl. By selectively introducing reduced peptide bonds in pseudopeptides, it might be possible to produce peptidomimetics with full biological activity but reduced sensitivity to proteases. It should be pointed out that the effects of such backbone modifications on the MHC-ligand recognition by the T cell receptor still need to be addressed. This study presents the first unequivocal example for the potent applicability of reduced peptide bond pseudopeptides in the context of peptide-MHC class I recognition.