©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Retro-Inverso Peptidomimetics as New Immunological Probes
VALIDATION AND APPLICATION TO THE DETECTION OF ANTIBODIES IN RHEUMATIC DISEASES (*)

(Received for publication, March 1, 1995; and in revised form, June 19, 1995)

Jean-Paul Briand Gilles Guichard Hélène Dumortier Sylviane Muller (§)

From the Institut de Biologie Moléculaire et Cellulaire, UPR 9021 CNRS, 67084 Strasbourg Cedex, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

Over the last decade, solid-phase immunoassays such as enzyme-linked immunosorbent assay (ELISA) (^1)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.


MATERIALS AND METHODS

Peptides

All peptide analogues were synthesized by the solid-phase methodology on a multichannel peptide synthesizer (Neimark and Briand, 1993) using a fully automatic mode for the L-peptides and a semiautomatic one for the retro-inverso peptides. t-Butoxycarbonyl- and Fmoc-protected L- and D-amino acid derivatives were from Neosystem (Strasbourg, France). The schematic representation of peptide analogues used in this study is shown in Table 1.



Peptide 130-135 of Histone H3

The synthesis of the L- and retro-inverso peptides has been described previously (Guichard et al., 1994b).

Peptide 277-291 of 52-kDa SSA/Ro (Ro52) Protein

The L- and retro-inverso peptides were assembled in t-butoxycarbonyl chemistry on a t-butoxycarbonyl Leu Pam resin and on a p-methylbenzhydrylamine resin, respectively. Assembly of the protected peptide chain was carried out on a 100-µmol scale using the in situ neutralization protocol described previously (Neimark and Briand, 1993). For the retro-inverso peptide, a close mimicry of the parent peptide COOH terminus was achieved by using a malonate derivative. The (R,S)-2-isobutyl malonic acid monobenzyl ester obtained by alcoholysis of 2,2-dimethyl-5-isobutyl-1,3-dioxane-4,6-dione (Chorev et al., 1983) was incorporated into the peptide chain as a racemate, thereby generating a pair of diastereomers. 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 then purified on an Aquapore C18 ODS column (100 10 mm) using a preparative HPLC apparatus (Perkin Elmer, Saint-Quentin en Yvelines, France). In the case of retro-inverso analogues, the two diastereomers could be separated and purified by HPLC. They were identified according to their retention time. The more rapidly eluted retro-inverso isomer was referred to as RIa peptide and the less rapidly eluted one as RIb peptide (Table 1). The rate of isomerization of the separated diastereomers was followed at 4 °C and 37 °C. Both isomerized when incubated during 12 h at 37 °C (conditions commonly used for coating ELISA plates), the RIa and RIb components leading to about a 50:50 (RIa/RIb) and a 70:30 (RIb/RIa) equilibrium mixture of diastereomers, respectively. In contrast, at 4 °C, the two isomers appeared very stable as we observed no change in the respective HPLC profiles. Coating of plastic plates with Ro52 277-291 peptide analogues was thus performed at 4 °C (see below).

Peptide 304-324 of 60-kDa SSA/Ro (Ro60) Protein

Two series of peptides were synthesized. In the first series, the NH(2) and COOH termini of the parent peptide and the reversed termini of the retro-inverso peptide were not protected. They were assembled in Fmoc chemistry on a p-alkoxybenzyl alcohol resin. In the D-allo-Ile retro-inverso analogue, D-allo-Ile was introduced instead of D-Ile. In the second series of analogues, the NH(2)- and COOH termini of the parent and retro-inverso peptides were acetylated and carboxaminated, respectively. These blocked peptides were assembled in Fmoc chemistry on a Fmoc-2,4-dimethoxy-4`-(carboxymethyloxy)-benzhydrylamine resin.

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.

Peptide 28-45 of Histone H3

Four peptide analogues were synthesized, namely the parent L-peptide, the L-peptide in which the free terminal -COOH group was replaced by a carboxamide group -CONH(2), and the L- and the retro-inverso peptide with blocked NH(2)- and COOH termini (Table 1). The four peptides were prepared in Fmoc chemistry as described above.

All peptides used in this study were controlled by HPLC and fast atom bombardment (FAB)-MS analysis.

Binding of Zinc to Ro60 304-324 Peptide Analogues

The capacity of Ro60 304-324 parent peptide and retro-inverso analogues to bind zinc ions was tested by using Zn and peptides immobilized on nitrocellulose as described previously (Mazen et al., 1989; Muller et al., 1994).

Coupling of Peptide 130-135 of H3

The 130-135 peptide of H3 and the retro-inverso analogue were conjugated to bovine serum albumin by means of N-succinimidyl 3-(2-pyridyldithio)propionate. The yield of coupling was obtained spectrophotometrically by release of 2-thiopyridone from N-succinimidyl 3-(2-pyridyldithio)propionate derivatized bovine serum albumin on interaction with cysteine-containing peptides (Muller, 1988).

Mouse and Human Autoimmune Sera; Monoclonal Antibody LG2-1

Serum samples from lupus (NZB/W) F1 female mice were obtained from S. Batsford (Freiburg, Germany). Human sera were from patients with SLE and Sjögren's syndrome (SS). They were obtained from D. A. Isenberg (London), O. Meyer (Paris), and G. Fournié (Toulouse, France). Sera from normal volunteers were obtained from J. L. Pasquali (Strasbourg). Most of these sera have been used in previous studies (Ricchiuti et al., 1994; Ricchiuti and Muller 1994). The monoclonal antibody LG2-1 was a kind gift from M. Monestier (Philadelphia, PA). LG2-1 reacts with histone H3 and peptide 30-45 of H3 (Monestier et al., 1993).

Enzyme-linked Immunosorbent Assay (ELISA)

Sera from lupus mice were tested by ELISA as described previously (Benkirane et al., 1993). Only the IgG antibody response was measured. The method for testing IgG antibodies reacting with Ro52 and Ro60 peptides was as described by Barakat et al.(1992) and Ricchiuti et al.(1994) except that in the case of Ro52 277-291 peptide, the coating of ELISA plates was performed at 4 °C instead of 37 °C (see above). The cut-off value of each test including that of tests based on retro-inverso peptide analogues was determined by using 20 sera from healthy individuals. The antigenic activity of peptides was also measured by ELISA using fluid-phase inhibition. The procedure was as described previously (Ricchiuti et al., 1994).


RESULTS

Synthetic Peptides and Retro-Inverso Analogues

With respect to the native peptide sequence, retro-inverso modification of linear polypeptides involves the synthetic assembly in a reverse order of amino acids with alpha-carbon stereochemistry opposite to that of the corresponding L-amino acids. The net result is that the position of carboxyl and amino groups in each peptide bond are exchanged while the topology of the side chains is similar to or identical with that of the natural peptide (Shemyakin et al., 1969). The reversal of the end groups creates a major problem in structural and charge complementarity. However, a number of approaches have been designed to treat this problem (Goodman and Chorev, 1979). For example, the retro-inverso analogues of peptides 130-135 of H3 and 277-291 of Ro52 were synthesized as end group modified retro-inverso isomers. We introduced a C-2-substituted malonic acid residue to closely mimic the COOH terminus of the parent peptides and a carboxamide group (NH(2)-CO-) instead of the free terminal amino group. The two diastereomers of the 277-291 Ro52 retro-inverso peptide (RIa and RIb analogues, Table 1) could be separated by HPLC.

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).

Recognition of the Retro-Inverso Analogue of H3 COOH-terminal Hexapeptide by Sera from Lupus Mice

The COOH-terminal hexapeptide of histone H3 constitutes a major epitope of the protein (Muller et al., 1982). This segment was used as a model peptide in our previous studies of modified peptides, in particular to show that retro-inverso analogues can be significantly more resistant to proteolytic enzymes than L-peptides (Guichard et al., 1994b). First, we have screened a series of serum samples from (NZB/W) F1 female mice with the parent H3 peptide 130-135 (^2)and selected a number of positive sera. These sera were then tested in ELISA for their ability to react with the 130-135 retro-inverso peptide. As shown in Table 2, sera from lupus mice reacted equally well with the L- and the retro-inverso peptides.



Recognition of the Retro-Inverso Analogue of Ro52 277-291 Peptide by Sera from Autoimmune Patients

Thirty sera from patients with SLE and SS were first tested for their ability to react with the parent 277-291 peptide of Ro52 which was shown previously to contain an epitope recognized by IgG antibodies from autoimmune patients (Ricchiuti et al., 1994). Using a cut-off value corresponding to 0.30 A unit (Ricchiuti et al., 1994), it was found that of these 30 sera, 5 contained IgG antibodies reacting with L-peptide (Fig. 1; coating of microtiter plates at 4 °C instead of 37 °C). All these sera (positive and negative with the L-peptide) were tested in parallel with the RIa and RIb analogues. All sera positive with the L-peptide were found positive with the retro-inverso peptides. The absorbance levels were significantly higher with the RIb analogue (Fig. 2). Most interestingly, among the 25 patients' sera negative with the L-peptide, 10 sera reacted with the RIb peptide (Fig. 1). The mean absorbance corresponding to the arithmetic mean of all absorbance values, including values under the cut-off line for positivity, was 0.21 (S.D. 0.25) and 0.51 (S.D. 0.62) with the L- and the RIb peptide, respectively.


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 (bullet--bullet) 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 geq0.40 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).

Recognition of the Retro-Inverso Peptide Analogue of Ro60 304-324 Peptide by Sera from Autoimmune Patients

The region 304-324 of Ro60 is known to contain a major epitope of the protein (Ricchiuti and Muller, 1994). Twenty sera from unselected patients with SLE and SS were first tested with the unblocked L- and retro-inverso analogues. Six sera (30%) reacted with the L-peptide (mean A 0.21, S.D. 0.26) while 15 sera (75%) reacted with the retro-inverso peptide (mean A 0.81, S.D. 0.54). Patients' sera reacted slightly better with the D-allo-Ile retro-inverso analogue: as shown in Fig. 3, 16 sera (80%) reacted with the latter analogue (mean A 0.81, S.D. 0.50).


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(2) 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).



Recognition of the Retro-Inverso Analogue of H3 28-45 Peptide

Peptide 28-45 of H3 and its retro-inverso analogue were first tested with a monoclonal antibody called LG2-1 obtained from a female MRL/Mp-lpr/lpr lupus mouse. LG2-1 (IgG2a, kappa) was found previously to react with histone H3 and more precisely with the domain 30-45 of this protein (Monestier et al., 1993). In a direct ELISA test in which the test peptides were immobilized to the solid-phase of microtiter plates, LG2-1 reacted strongly with the 28-45 parent L-peptide (Fig. 5A). In contrast, it bound the blocked L-peptide much more weakly. By using an analogue of the 28-45 peptide in which the COOH-terminal was carboxaminated, we determined that the drop of reactivity of LG2-1 with the blocked peptide was mainly due to the lack of charge at the NH(2) terminus since the peptide with carboxamide COOH-terminal group was strongly recognized by LG2-1 (Fig. 5A). In direct ELISA, the retro-inverso analogue was not recognized. A putative explanation for this lack of reactivity could be that this analogue was not efficiently attached to the solid-phase or that it adopted a particular conformation on plastic which was not recognized by LG2-1. We thus tested the different analogues free in solution in a competitive ELISA test. The capacity of peptide analogues to inhibit the binding of LG2-1 to H3 whole protein is shown in Fig. 5B. Almost complete inhibition could be achieved with the L- and the two blocked peptides (28-45 CONH(2) and Ac 28-45 CONH(2)). However, and in agreement with results obtained in direct ELISA test, no inhibition could be obtained with the blocked retro-inverso analogue.


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 (bullet--bullet), carboxaminated L-peptide (--), blocked L-peptide (up triangle, filled--up triangle, filled), 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).


DISCUSSION

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(2) 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.


FOOTNOTES

*
This work was supported by CNRS (Groupe de Coordination Chimie-Biologie, Project 28D4). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Tel.: 33-88-41-70-27; Fax: 33-88-61-06-80.

(^1)
The abbreviations used are: ELISA, enzyme-linked immunosorbent assay; RI, retro-inverso; SS, Sjögren's syndrome; SLE, systemic lupus erythematosus; Fmoc, N-(9-fluorenyl)methoxycarbonyl; HPLC, high performance liquid chromatography.

(^2)
C. Mézière, S. Batsford, and S. Muller, manuscript in preparation.


ACKNOWLEDGEMENTS

We are grateful to Drs. S. Batsford, G. Fournié, D. Isenberg, O. Meyer, and J. L. Pasquali for providing normal and autoimmune sera, and to Dr. M. Monestier for his gift of LG2-1 monoclonal antibody. We wish to thank Dr. M. Friede for critically reviewing the manuscript and Dr. M. H. V. Van Regenmortel for his support.


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