The PPGMRPP repetitive epitope of the Sm autoantigen: Antigenic specificity induced by conformational changes. Application of the Sequential Oligopeptide Carriers (SOCs)

Abstract

SUMMARYThe PPGMRPP sequence, found in several copies in the Sm and U1RNP autoantigens, is the main target of antiSm and anti-U1RNP antibodies in systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD) patient's sera. It is also recognized, to a lower extent, by anti-Ro/SSA and anti-LaJSSB specificities. The PPGMRPP-NH2 peptide amide and the PPGMRPP peptide, which is bound to a pentameric sequential oligopeptide carrier (SOC5), were examined by 1H-NMR spectroscopy and ELISA assays, using sera from patients with autoimmune rheumatic diseases. Among the three main conformers found for the free PPGMRPP, the extended one was also identified for PPGMRPP-NH 2 and (PPGMRPP)5-SOC 5. This can be attributed to the absence of ionic interactions between the Arg-guanidinium and the carboxylate group in the amide and SOC5-bound forms of the peptide. Immunoassays using sera from various specificities showed an enhanced anti-Sm and anti-U1RNP recognition of PPGMRPP-NH 2 and (PPGMRPP)5-SOC 5, and lowering of the anti-Ro/SSA and anti-La/SSB reactivity. The presence of multiple conformers of free PPGMRPP may explain the unexpected cross-reactivity to the anti-Ro/La positive sera, while the prevalence of the extended conformation in PPGMRPP-NH 2 and (PPGMRPP)5-SOC5 is mainly responsible for the enhanced recognition from the anti-Sm and anti-U1RNP autoantibodies. It is concluded that the antigenic specificity of PPGMRPP-NH 2 and (PPGMRPP)5-SOC 5 is mainly induced by conformational changes resulting from the conversion of the C-terminal carboxylate group to the amide form.

No Evidence of Epitope Spreading After Immunization with the Major Sm Epitope P-P-G-M-R-P-P Anchored to Sequential Oligopeptide Carriers (SOCs)

“…These prerequisites were fulfilled in the present study: Aminoacid analysis of the purified (PPGMRPP) 5 -SOC 5 gave aminoacid molarities as expected; the 1 H-NMR spectra confirmed further the purity of our material [25] as well as that the PPGM-RPP epitope exists on SOC in a unique conformation. Therefore it was not surprising that immunization of animals with the immunogen (PPGMRPP) 5 -SOC 5 elicited an immune response specific for the PPGM-RPP epitope, rather than spreading to other epitopes of the spliceosome.…”

“…In fact, we have observed by NMR experiments three conformers of the PPGMRPP epitope in solution, which are structurally [15] different from each other and show various cross reactivities. However, only one of these conformers predominates when the PPGMRPP peptide is anchored to SOCs [25].…”

Artificial Carriers: A Strategy for Constructing Antigenic/Immunogenic Conjugates

“…It was concluded that the occurrence of these multiple conformers may explain why the PPGMRPP epitope is recognized by autoantibody species other (antiRo/La) than anti-Sm and anti-U1RNP [123]. However, the prevalence of one conformer of PPGMRPP after anchorage to SOC 5 -I, as demonstrated by 1 H-NMR analysis, resulted in a complete loss of the unrelated reactivity (anti-Ro/La) and an almost quantitative recognition by anti-Sm autoantibodies [103,124]. Fig.…”

“…Taking advantage of the plethora of amino-protective groups and by applying an orthogonal protection strategy, it was feasible to shape SOC n -conjugates Ac-Aib-Lys-Aib-Gly-Aib-Lys-Aib-Gly-Aib-Lys-Aib-Gly Peptide Peptide Peptide with identical or alternate ABAB motifs, where A and B represent different epitopes, as for example two types of B cell epitopes, B and T cell epitopes or CD4+ and CD8+ T cell epitopes. Two N-protective groups, Boc-and Fmoc-, are required for the attachment of identical copies of epitopes to the Lys-N ε H 2 groups of the carrier on the Pam resin [96,103], whereas an orthogonal system with three N-protective groups as Boc-, Fmoc-, and Alloc-(Pam resin) or Fmoc-, Alloc-and Mtt-(Wang resin) is needed for grafting two different epitopes to the carrier (Fig. 7) [104,105].…”

Fullerene–biomolecule conjugates and their biomedicinal applications

“…It also appeared, in other studies, that conformation greatly guided the recognition process, as demonstrated by the fact that the amide form converted from the C-terminal carboxylate group of the parent peptide reduced anti-Ro/La recognition. 47,48 Bianco et al described a fulleropeptide 27 ( Figure 6C) which derived from the nucleosomal histone (H3) protein attached to a L-fulleropyrrolidino-glutamic acid residue. 49 Molecular modeling research suggested that this peptide analog conserved the same binding capacity to major histocompatibility complex molecules as the parent peptide; however, further studies are required to verify whether it stimulates or inhibits T-cell responses.…”