Cross-reactivity of Antibodies to Retro-Inverso Peptidomimetics with the Parent Protein Histone H3 and Chromatin Core Particle

Abstract

A series of monoclonal antibodies has been generated against an hexapeptide of sequence IRGERA corresponding to the C-terminal residues 130-135 of histone H3 and three analogues of this model peptide. The analogues correspond to the D-enatiomer, containing only D-residues, and two retro-peptides containing NH-CO bonds instead of natural amide peptide bonds. The chirality of each residue was maintained in the retro-peptide and inverted in the retro-inverso-peptide. Monoclonal antibodies were generated from mice immunized with the analogues coupled to neutral small unilamellar liposomes containing monophosphoryl lipid A as adjuvant. The reactivity of antibodies with the four analogues and with the parent protein H3 was studied in enzyme-linked immunosorbent assay and in a biosensor system. The equilibrium affinity constant (Ka) toward the retro-inverso-peptide of two out of three antibodies of IgG1 isotype induced against the L-hexapeptide was 7-75-fold higher than toward the homologous L-peptide. The range of Ka values of four antibodies of IgG1 and IgG2a isotypes generated against the retro-inverso-peptide was 0.6-1.9 x 10(9) M-1 for both the retro-inverso- and L-peptides. Furthermore, antibodies to the L- and retro-inverso-peptides cross-reacted strongly (in some cases better than with the homologous peptide) with the parent histone H3 and with chromatin subunits containing H3. The results are thus promising in respect to the potential use of retro-inverso-analogues, which are particularly stable, in the design of much more potent synthetic vaccines or to generate antibody probes.

Antigenic mimicry of natural L-peptides with retro-inverso-peptidomimetics.

“…In contrast with these results, we have recently described the generation in mice of antibodies against an all-Dhexapeptide of sequence IRGERA corresponding to the COOH-terminal residues 130-135 of histone H3 (9). Antibodies of the IgG3 isotype induced against either the D-or L-peptide were able to bind both the homologous peptide and its mirror enantiomer.…”

“…Three analogues of the model peptide of sequence IRGERA corresponding to the COOH-terminal residues 130-135 of histone H3 were produced. Preparation and purification of L-and all-D IRGERA peptides have been described (9). The two new analogues, the retro-inverso-and retro-peptides, were synthesized as the L-and D-peptides by the solid-phase methodology in Boc chemistry on a p-methylbenzhydrylamine resin (Applied Biosystems) (11).…”

“…To allow the coating of peptides in a direct solid-phase ELISA test, IRGERA analogues were conjugated to bovine serum albumin (BSA) as described (9). For immunization of mice, peptides were covalently coupled to preformed small unilamellar vesicles containing monophosphoryl lipid A (9,14).…”

“…The (RS)-2-methylmalonic acid monobenzyl ester obtained by alcoholysis of 2,2,5,-trimethyl-1,3-dioxane-4,6-dione (12) was incorporated into the peptide chain as a racemate. HF cleavage, purification, HPLC, fast atom bombardment (FAB)-MS analysis, and circular dichroism measurements were performed as described (9,11,13).…”

The partial retro–inverso modification: A road traveled together

“…Furthermore, antibodies to the parent peptide and its RI isomer cross-reacted strongly with human histone H3 and with chromatin units containing histone H3. 40 Indeed, Briand and coworkers reported that end-group RI analogs of histone H3[130 -135] were strongly recognized by autoantibodies from lupus mice and from sera of patients with rheumatoid arthritis, diseases characterized by inflammatory reactions associated with increased levels of proteolytic enzymes. 41 These findings strongly suggest a potential role for RI peptides as antigens in immunodiagnostic assays.…”