No abstract

Three analogues of the model peptide of sequence IRGERA corresponding to the COOH-terminal residues 130-135 of histone H3 were synthesized, and their antigenicity, immunogenicity, and resistance to trypsin were compared to those of the natural L-peptide. The three analogues correspond to the D-enantiomer, containing only D-residues, and two retro-peptides containing NH-CO bonds instead of natural peptide bonds. The chirality of each residue was maintained in the retro-peptide and inverted in the retroinverso-peptide. Antibodies to the four peptide analogues were produced by injecting BALB/c mice with peptides covalently coupled to small unilamellar liposomes containing monophosphoryl lipid A. Each of the four peptide analogues induced IgG antibodies of various subclasses. The IgG3 antibodies reacted similarly with the four analogues, whereas antibodies of the IgGl, IgG2a, and IgG2b isotypes showed strong conformational preferences for certain peptides. The retro-inversopeptide IRGERA mimicked the structure and antigenic activity of the natural L-peptide but not of the D-and retro-peptides, whereas the retro-peptide IRGERA mimicked the D-peptide but not the L-and retro-inverso-peptides. The equilibrium affnity constants (Ka) of three monoclonal antibodies generated against the L-and D-peptides with respect to the four peptide analogues were measured in a biosensor system. Large differences in Ka values were observed when each monoclonal antibody was tested with respect to the four peptides. The use of retro-inverso-peptides to replace natural L-peptides is likely to find many applications in immunodiagnosis and as potential synthetic vaccines.The development of neuropeptides, peptide hormones, peptide antibiotics, or peptide-based synthetic vaccines is strongly impaired by the high susceptibility of peptides to proteolysis, which limits, inter alia, parental and oral administration. For many years intense work has been focused on the synthesis of peptide analogues in the search for mimics with enhanced activity and biological half-lives. Examples of modifications introduced in peptides are the replacement of L-amino acid residues by D-amino acids or by unnatural residues (e.g., sarcosine and 3-alanine) and the modification of peptide bonds (1-3). These changes provide pseudopeptides or peptidomimetics with a higher metabolic stability, since most natural proteases cannot cleave D-amino acid residues and nonpeptide bonds. An important problem encountered with such peptide analogues is the conservation of their biological activity. Recently, the D-form of human immunodeficiency virus type 1 protease has been synthesized (4). As could be expected, the enantiomeric protein displayed reciprocal chiral specificity as the enzyme was unable to cleave the normal L-substrate but did hydrolyze its D-enantiomer. In contrast, Wen and Laursen (5) showed that both the L-and D-form of an a-helical antifreeze polypeptide bound equally well to the same achiral ice substrate, whereas Wade et al. (6) found that the L-and D-e...

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.