(Received for publication, March 1, 1995; and in revised form, June 19, 1995)
From the
Retro-inverso peptides which contain NH-CO bonds instead of CO-NH peptide bonds are much more resistant to proteolysis than L-peptides. Moreover, they have been shown recently to be able to mimic natural L-peptides with respect to poly- and monoclonal antibodies (Guichard, G., Benkirane, N., Zeder-Lutz, G., Van Regenmortel, M. H. V., Briand, J. P., and Muller, S. (1994b) Proc. Natl. Acad. Sci. U. S. A. 91, 9765-9769). We have further tested the capacity of retro-inverso peptidomimetics to serve as possible targets for antibodies produced by lupus mice and by patients with rheumatic autoimmune diseases. Several retro-inverso peptides corresponding to sequences known to be recognized by autoantibodies were synthesized, namely peptides 28-45 and 130-135 of H3, 277-291 of the Ro/SSA 52-kDa protein, and 304-324 of the Ro/SSA 60-kDa protein, and tested with autoimmune sera by enzyme-linked immunosorbent assay. We have found that retro-inverso peptides are recognized as well as or even better than natural peptides by antibodies from autoimmune patients and lupus mice. This new approach may lead to important progress in the future development of immunodiagnostic assays, particularly in the case of diseases characterized by inflammatory reactions in the course of which the level of degradative enzymes is increased.
Over the last decade, solid-phase immunoassays such as
enzyme-linked immunosorbent assay (ELISA) ()and solid-phase
radioimmunoassays have become increasingly popular, and these assays
are now widely used for measuring the antigenic activity of synthetic
peptides for both diagnostic and experimental purposes. In particular,
a number of immunoassays based on the use of synthetic peptides for the
detection and quantification of autoantibodies in human autoimmune
diseases have been developed (Elkon, 1992; Muller, 1994). It is
important to realize that during the test, peptides used either free in
solution or applied directly to plastic microtiter plates can be
altered by proteases which are present in the patients' sera. The
release of various proteolytic enzymes is indeed one of the
characteristics of acute inflammatory reactions leading to tissue
damage and further release of proteinases (Kaplan and Silverberg,
1988). In autoimmune diseases, such as rheumatoid arthritis or systemic
lupus erythematosus (SLE), inflammatory tissue damage may be caused by
circulating immune complexes (Levinson, 1994). Proteolytic degradation
of peptides may to a certain extent be circumverted by their
conjugation to a carrier protein. However, this step can represent a
technical hurdle in some laboratories or, depending on the sequence of
the peptide, a coupling strategy can be difficult to adopt when
residues outside the epitope are not available. As an alternative, it
was tempting to try to convert antigenic peptides into peptidomimetics
resistant to proteolytic degradation while retaining a high antigenic
activity.
Recently, we have described the first immunological application of modified peptides containing D-amino acid residues (Benkirane et al., 1993), reverse peptide bonds (Guichard et al., 1994b; Benkirane et al., 1995; Muller et al., 1995), and reduced peptide bonds (Guichard et al., 1994a). In particular, we have established that the retro-inverso analogue of the model peptide of sequence IRGERA corresponding to the COOH-terminal residues 130-135 of histone H3 could mimic the natural L-peptide with respect to poly- and monoclonal antibodies (Guichard et al., 1994b; Benkirane et al., 1995). The affinity of a monoclonal antibody induced against the L-hexapeptide was 100-fold higher when measured toward the retro-inverso peptide than toward the L-peptide. Antibodies to retro-inverso analogues of peptide IRGERA and of a 19-residue-long peptide of foot and mouth disease virus VP1 cross-reacted equally well with homologous retro-inverso analogues and with the respective L-peptides (Benkirane et al., 1995; Muller et al., 1995). Furthermore, and of importance in the present context, we have also shown that the IRGERA retro-inverso peptide containing NH-CO bonds instead of CO-NH peptide bonds was much more resistant to proteolysis than the L-peptide. Its half-life in the presence of trypsin was at least 7 times longer (Guichard et al., 1994b). In this study, we report the synthesis and evaluation with murine and human autoimmune sera of several retro-inverso peptidomimetics. We show that retro-inverso peptides are recognized as well as and in many cases better than natural peptides by autoimmune sera. This new approach may lead to important progress in the future development of immunodiagnostic assays.
Assembly of the protected peptide chains was carried out on a 25-µmol scale according to a classical Fmoc methodology. Peptide resins were cleaved with reagent K (King et al., 1990) for 2 h, and each peptide was collected in a tube filled with cold t-butyl methyl ether. After centrifugation, pellets were washed twice with cold ether. After the last centrifugation, each peptide was dissolved in an aqueous solution for lyophilization. The crude peptides were finally purified as described above.
All peptides used in this study were controlled by HPLC and fast atom bombardment (FAB)-MS analysis.
An alternative to limit the end group problem consists of preparing blocked peptides and blocked retro-inverso analogues. This solution has been adopted for peptides 304-324 of Ro60 and 28-45 of H3. The use of this strategy implies, however, that peptides lacking free termini are still recognized by antibody probes.
Another problem in the retro-inverso approach is that secondary chiral centers in threonine and isoleucine side chains should retain their correct chirality. We investigated the question with peptide 304-324 of Ro60 which was synthesized in its retro-inverso form with either D-Ile residues or D-allo-Ile residues (Table 1).
The purity of 13 analogue peptides used in this study was greater than 80% as checked by analytical HPLC. (FAB)-MS analysis gave the expected results for all compounds (data not shown).
Figure 1: Reactivity in ELISA of SLE and SS serum samples with the parent and RIb analogue 277-291 of Ro52. Patients' sera were diluted 1:1000. Only IgG activity was measured. The median absorbance values are indicated. The dotted line represents the upper limit of normal population corresponding to the average absorbance value of 20 normal human sera + 2 S.D. (0.30 A unit).
Figure 2:
Reactivity in ELISA of two SLE sera (A and B) with the parent L-peptide 277-291
of Ro52 (--
) and the two diastereomers RIa
(
--
) and RIb (
--
).
Patients' sera were diluted 1:1000 and allowed to react with
various concentrations of peptide.
Competition
experiments were performed with 11 patients' sera showing in
direct ELISA absorbance values 0.40 with the 277-291 RIb
peptide. When the 277-291 L-peptide was used as
inhibitor and the RIb peptide as antigen for coating plastic plate, the L-peptide was found to possess inhibitory activity in all
cases. Depending on the sera tested, up to 75.8% inhibition was found.
The homologous peptide tested in parallel in the same conditions
inhibited up to 79.1% of the binding of antibodies to the RIb peptide.
We thus demonstrated that the 277-291 RIb peptide not only mimics the L-peptide but is generally better recognized than the parent peptide by patients' antibodies. This allowed us to detect antibodies in 50% of patients' sera tested while only 17% of sera reacted with the 277-291 L-peptide (Fig. 1).
Figure 3: Schematic structure of the L-peptide 304-324 of Ro60: Reaction in ELISA of sera from 20 patients with SLE and SS with analogues of the peptide 304-324 of Ro60. Patients' sera were diluted 1:1000. Only IgG activity was measured. The median absorbance values are indicated. The dotted line represents the upper limit of normal population corresponding to the average absorbance value of 20 NHS + 2 S.D. (0.30 A unit).
We then investigated whether Ro60
304-324 peptide analogues with acetylated NH terminus
and carboxaminated COOH terminus were recognized by autoantibodies. In
comparison to the natural L-peptide, the blocked L-peptide was found to be more strongly recognized by
antibodies from autoimmune patients (Fig. 3). Fourteen sera
(70%) reacted with the blocked L-peptide (mean A 0.86, S.D. 0.87). The mean absorbance and the number of positive
sera were still enhanced in using the retro-inverso analogue with
blocked termini (15 positive (75%) sera out of 20; mean A 1.15, S.D. 0.97). Depending on the sera, positive reaction with
the blocked retro-inverso analogue was still detected at a 1:4000
dilution of the serum. The binding of autoantibodies to the blocked
retro-inverso analogue could be inhibited by both the homologous
blocked retro-inverso analogue and by the heterologous blocked L-peptide (data not shown).
It has been shown recently that
peptide 304-324 of Ro60 protein which contains a zinc-finger
motif effectively binds radioactive zinc (Muller et al.,
1994). We thus attempted to check whether the analogue peptides, and
most particularly retro-inverso peptides, were able to bind Zn. As shown in Fig. 4, the blocked L-peptide as well as the three retro-inverso analogues were
readily labeled with
Zn. This result implies that
retro-inverso analogues, as the natural L-peptide, can easily
form a finger structure stabilized by a central tetrahedrally
coordinated atom of zinc using the two cysteine and two histidine
residues of the sequence as ligands (residues 305, 309, 320, and 323; Fig. 3).
Figure 4:
Dot immunoassay of the L-peptide
304-324 of Ro60 and peptide analogues incubated with Zn. A control peptide which does not contain the zinc
binding motif (peptide 56-77 of U1-RNP polypeptide A) was used as
control (lane 1). Increasing amounts of the 6 peptides were
spotted on nitrocellulose sheets (0.22 µm) and incubated as
described previously (Mazen et al., 1989; Muller et
al., 1994). The nitrocellulose filters were autoradiographed for 5
h (A) and 14 h (B) at -70 °C. Lanes
2,2`, L-peptide; lanes 3,3`, RI peptide; lanes 4,4`, D-allo-Ile RI peptide; lane
5, blocked L-peptide; lanes 6,6`, blocked RI
peptide. Results are from two independent experiments (A and B).
Figure 5:
Reaction in ELISA of monoclonal antibody
LG2-1 generated from an autoimmune lupus mouse with several analogues
of peptide 28-45 of histone H3. A, each analogue (2
µM) in carbonate buffer, pH 9.6, was allowed to incubate
in wells of microtiter plates and tested with various concentrations of
antibody. B, inhibition experiments of LG2-1 monoclonal
antibody binding. LG2-1 (0.3 µg/ml) was preincubated with various
concentrations of inhibitor peptide and allowed to react with
antigen-coated plates (H3 protein, 100 ng/ml). Whole histone H3 was
isolated and purified as described (Van der Westhuyzen and Von Holt,
1971). Peptide analogues: L-peptide
(--
), carboxaminated L-peptide
(
--
), blocked L-peptide
(
--
), blocked retro-inverso peptide
(
--
).
Further investigation of the peptide analogues of the region 28-45 of H3 was then undertaken with autoimmune sera. In previous studies, peptide 30-45 of H3 was not found to possess antigenic activity (Muller and Van Regenmortel, 1993). We screened in ELISA 69 sera from patients with various systemic rheumatic diseases, including 22 sera from lupus patients. Among these sera, 12 sera (17.4%) were positive with the 28-45 natural L-peptide (8 out of 22 SLE sera), 20 sera (29%) were positive with the 28-45 blocked L-peptide (12 out of 22 SLE sera), and 21 sera (30.4%) reacted with the blocked retro-inverso analogue (12 out of 22 SLE sera).
This study is the first to report the use of retro-inverso peptides in replacement of natural linear L-peptides for the detection of antibodies in the serum of autoimmune patients. In all cases examined so far (antigenic regions 28-45 and 130-135 of H3, 277-291 of Ro52, and 304-324 of Ro60), we found that retro-inverso peptidomimetics displayed the same or superior antigenic activity as compared with the natural L-enantiomer peptide. This allowed us to both detect much more positive sera reacting with each peptide and, in general, significantly enhance the absorbance level of individual sera toward peptide probes without changing the cut-off line for positivity determined with normal sera. It is noteworthy that the antibodies reacting with retro-inverso peptides do not constitute a particular antibody subset present in patients' sera since the binding of patients' antibodies to retro-inverso analogue was inhibited equally well by retro-inverso and L-peptides. This new development of retro-inverso peptidomimetics in diagnostics seems therefore extremely promising.
We should lay stress, however, on the fact that in order to develop better immunodiagnostic procedures based on the use of antigenic retro-inverso structures, particular attention has to be paid to the reversal of end groups which may alter the antigenic reactivity of retro-inverso peptidomimetics. For example, many retro-inverso analogues of biologically active peptides described in the literature have been found to be totally inactive. A number of approaches have been designed to circumvent this problem (Goodman and Chorev, 1979; Chorev and Goodman, 1993). For example, a gem-diaminoalkyl residue can be introduced at the amino terminus of the retro-inverso peptide and a 2-substituted malonic acid can be introduced at the carboxyl terminus. However, monoacyl gem-diaminoalkyls are hydrolyzed, and one should expect the half-life of peptides incorporating such residues to be 10-50 h at 25 °C (Loudon et al., 1981). Consequently, in the case of retro-inverso peptides 130-135 of H3 and 277-291 of Ro52, we chose to use a carboxaminated termination instead of free amino group of the parent peptides. A C-2-substituted malonic acid was incorporated into these peptides as a racemate to mimic the COOH terminus of the L-peptides. In the case of the retro-inverso analogue of Ro60 304-324 peptide, we found that the retro-inverso analogue with unblocked reversed termini was better recognized than the respective L-peptide (Fig. 3) suggesting that for this particular peptide, reversal of the end groups did not affect its antigenicity.
During the course of this work, we found that compared to parent
peptides with free NH and COOH termini, blocked peptides
304-324 of Ro60 and 28-45 of H3 were better recognized by
antibodies from autoimmune sera. This finding may be related to the
fact that autoantibodies are most likely induced by proteins complexed
with other proteins or nucleic acids (DNA and RNA). When internal
sequences in the primary structure of the protein are specifically
recognized by autoantibodies, it may be that peptides with blocked
amino and carboxyl groups better mimic the inner fragment in the
protein (Gras-Masse et al., 1986). Regarding the presence of
reversed termini in blocked retro-inverso analogues, we found again
that reversal of termini had no effect on their antigenicity. However,
this lack of effect should be checked for any particular peptides newly
investigated.
The presence of amino acid residues such as threonine and isoleucine which contain two chiral centers can also represent a problem with respect to the correct chirality of the retro-inverso compound. The Ro60 304-324 peptide contains two isoleucine residues in positions 319 and 324; two retro-inverso analogues were thus synthesized with either D-Ile or D-allo-Ile-residues, and we found that the D-allo-Ile retro-inverso analogue was slightly better recognized by patients' antibodies than the retro-inverso analogue containing D-Ile residues. Since protected D-allo-threonine is not commercially available, the problem cannot be completely overcome yet. It has to be pointed out however that both peptide 277-291 of Ro52 and peptide 28-45 of H3 contain two threonine residues and, apparently, this does not seem to affect their antigenic activity.
Finally, reversing the peptide sense with D-proline residue generates different local conformational constraints on the peptide (Shemyakin et al., 1969) which may influence antibody binding. Since the Ro60 304-324 retro-inverso peptide is strongly recognized by autoantibodies and can efficiently bind zinc, it means that the proline residue present in the zinc finger motif (Fig. 3) only serves to maintain the two histidine residues at an appropriate distance and does not compromise either binding of metal ions or interaction with antibodies.
LG2-1 monoclonal antibody did not cross-react with the retro-inverso analogue of the blocked 28-45 peptide of H3. Several possibilities can explain this lack of recognition. In particular, this peptide contains 3 proline and 2 threonine residues. In retro-inverso peptides, as discussed above, these two types of amino acid residues may affect both the peptide backbone and side chain conformation.
Since the field of applications of retro-inverso peptides as diagnostic tools is in its early stages, more examples of antigenically active peptides are needed to gain a better understanding of the scope and limitations of the approach. However, the results presented in this study show that retro-inverso peptidomimetics could be very useful for enhancing the activity of peptides used as antigenic probes.