Martin Blüthner

Characterization of the human autoimmune response to the major C-terminal epitope of the ribosomal P proteins

Autoantibodies to the ribosomal phospho (-P) proteins P0, P1, and P2, collectively referred to as Rib-P, are specifically found in 10–40% of patients with systemic lupus erythematosus (SLE). These antibodies are believed to be correlated with lupus nephritis, hepatitis, and central nervous system involvement. The major immunoreactive epitope of these ribosomal antigens has been localized to the carboxy terminus, which is a highly conserved domain of all three proteins and contains two phosphorylated serine residues. The phosphorylated amino acids of the P proteins are known not to be critical epitope determinants. Furthermore, epitope-mapping studies have shown that the major epitope is located within the last 11 C-terminal amino acids. Using peptide arrays we identified more precisely this shared epitope as the six C-terminal amino acids GFGLFD and elucidated the molecular recognition events of anti-Rib-P antibodies at the amino acid level. We identified Phe111 and Phe114 of Rib-P2 as the key residues for the interaction, with further contributions of Gly-112 and Asp-115. This amino acid stretch is also present in proteins of several pathogenic micro-organisms such as Trypanosoma cruzi, Brugia malayi, Pseudomonas aeruginosa, Candida albicans, several Leishmania species, and Bartonella henselae. Using newly developed ELISA systems with a C-terminal peptide (C22) and the recombinant proteins (P0, P1, and P2) as antigens we found a high specificity of anti-Rib-P antibodies for SLE and demonstrated positive correlations with anti-U1-C, anti-Sm-B/B′ and anti-D and anti-dsDNA antibodies. The sensitivity and specificity in the peptide (C22) based assay varied between 12.8%/100% and 23.4%/96.7% for SLE, depending on the assigned cutoff. In contrast to other studies, we found no significant correlation of anti-Rib-P reactivity with central nervous system manifestations or renal involvement in SLE patients. We conclude that the epitope motif GFGLFD in the C-termini of the ribosomal P proteins is the key determinant of anti-Rib-P antibodies, and that the C22 peptide and the recombinant proteins can be used equally well for the detection of anti-Rib-P antibodies. The role of the major Rib-P epitope in the development of anti-ribosomal P antibodies and in the pathogenesis of SLE remains a subject of further investigation.

Clinical evaluation of autoantibodies to a novel PM/Scl peptide antigen

Anti-PM/Scl antibodies represent a specific serological marker for a subset of patients with scleroderma (Scl) and polymyositis (PM), and especially with the PM/Scl overlap syndrome (PM/Scl). Anti-PM/Scl reactivity is found in 24% of PM/Scl patients and is found in 3–10% of Scl and PM patients. The PM/Scl autoantigen complex comprises 11–16 different polypeptides. Many of those proteins can serve as targets of the anti-PM/Scl B-cell response, but most frequently the PM/Scl-100 and PM/Scl-75 polypeptides are targeted. In the present study we investigated the clinical relevance of a major alpha helical PM/Scl-100 epitope (PM1-α) using a newly developed peptide-based immunoassay and compared the immunological properties of this peptide with native and recombinant PM/Scl antigens. In a technical comparison, we showed that an ELISA based on the PM1-α peptide is more sensitive than common techniques to detect anti-PM/Scl antibodies such as immunoblot, indirect immunofluorescence on HEp-2 cells and ELISA with recombinant PM/Scl polypeptides. We found no statistical evidence of a positive association between anti-PM1-α and other antibodies, with the exception of known PM/Scl components. In our cohort a negative correlation could be found with anti-Scl-70 (topoisomerase I), anti-Jo-1 (histidyl tRNA synthetase) and anti-centromere proteins. In a multicenter evaluation we demonstrated that the PM1-α peptide represents a sensitive and reliable substrate for the detection of a subclass of anti-PM/Scl antibodies. In total, 22/40 (55%) PM/Scl patients, 27/205 (13.2%) Scl patients and 3/40 (7.5%) PM patients, but only 5/288 (1.7%) unrelated controls, tested positive for the anti-PM1-α peptide antibodies. These data indicate that anti-PM1-α antibodies appear to be exclusively present in sera from PM/Scl patients, from Scl patients and, to a lesser extent, from PM patients. The anti-PM1-α ELISA thus offers a new serological marker to diagnose and discriminate different systemic autoimmune disorders.

Mapping of epitopes recognized by PMScl autoantibodies with gene-fragment phage display libraries

Sera from patients suffering from the polymyositis/scleroderma overlap syndrome (PM/Scl) recognize two antigenically non-related proteins with apparent molecular masses of 100 kDa and 75 kDa respectively. The two proteins are part of a particle termed PM/Scl localized in the granular component of the nucleolus. The predominant immunoreactivity of the PM/Scl sera was shown to be directed against the 100 kDa protein. The cDNA of the 100 kDa protein has been cloned recently and its immunogenic regions have been partially mapped using recombinant proteins. Thus far the localization of antigenic determinants on polypeptides has been done by expressing defined cDNA fragments in bacteria or by synthesizing overlapping short peptides and probing their immunoreactivity with antibodies. Here we present an alternative approach to localize autoimmune epitopes using sera containing polyclonal antibodies and gene-fragment phage display libraries. For epitope fine mapping of the PM/Scl-100 protein random fragments of the corresponding cDNA were cloned into the PIII protein of fUSE-5. These gene-fragment phage display libraries were incubated with affinity purified anti-PM/Scl-100 antibodies to enrich for epitope-displaying phages. All PM/Scl sera tested recognized 23 consecutive amino acids (229-251) encoded by four overlapping fUSE-5 clones, suggesting that a major epitope is contained within the 23 amino acids. In addition a minor epitope was localized in a region of 21 amino acids (775-795) encoded by two overlapping fUSE-5 clones since only three out of the seventeen sera reacted with this amino acid sequence. Additional fine mapping of the major epitope was done using synthetic oligopeptides. Thus, a stretch of 16 amino acids at position 229-244 could be identified as a major epitope on the deduced PM/Scl-100 amino acid sequence.

Identification of major linear epitopes on the sp100 nuclear PBC autoantigen by the gene-fragment phage-display technology.

Approximately 20-30% of sera from patients suffering from primary biliary cirrhosis contain autoantibodies against a nuclear protein termed sp100. By indirect cytoimmunofluorescence it was shown that the sp100 autoantigen is distributed in up to 20 dot-like structures per nucleus co-localizing with the so-called nuclear bodies. In western blots these sera react with a protein with an apparent molecular mass of 100kDa. By screening expression libraries with affinity-purified anti-sp100 antibodies we isolated a full-length sp100 cDNA whose sequence exactly matched the previously published sp100 sequence and encodes a protein of 481 amino acids with a deduced molecular mass of 53 kDa. In an attempt to determine immunoreactive regions on the sp100 antigen with the recently developed gene-fragment phage-display technology we were able to identify a stretch of sixteen amino acids (IKKEKPFSNSKVECQA) at position 296-311 as a major antigenic region (antigenic region 1) on the sp100-autoantigen. A second antigenic region (antigenic region 2) of twenty amino acids in length could be identified between amino acids 332-351 (EGSTDVDEPLEVFISAPRSE). By using immobilized synthetic peptides and various sp100-positive PBC patient sera the corresponding epitopes could be shown to be centered around epitope cores of six amino acids (SNSKVE, antigenic region 1) and nine amino acids (EPLEVFISA, antigenic region 2) respectively.

A population of autoantibodies against a centromere-associated protein A major epitope motif cross-reacts with related cryptic epitopes on other nuclear autoantigens and on the Epstein-Barr nuclear antigen 1.

Abstract. Autoimmune diseases arise from a hosts immune response against self-antigens. The triggering events ultimately resulting in such a break of tolerance are largely unknown. It is also not known why certain molecular structures become autoantigenic. The hypothesis has long been proposed that autoimmune diseases arise from molecular mimicry followed by an epitope spreading mechanism. Recently we have shown that the anti-centromere-associated protein A (CENP-A) immune response is directed against an autoantigenic motif, G/A-P-R/S-R-R, that occurs three times in the N-terminal amino acids of CENP-A. In the present study we used mutational analyses with immobilized oligopeptide arrays to identify the amino acids in this motif that are responsible for antibody binding. In particular, we found that surprisingly mimotopes of this motif are present in a vast number of autoantigens and in the Epstein-Barr nuclear antigen 1. With affinity-purified antibodies we show that the antibodies against this motif are polyclonal and cross-react with several autoantigens. However, in these autoantigens this motif often represents a cryptic epitope explaining the obvious conflict between our results and the known high specificity of autoantibodies. The presence of such an ubiquitous structure on autoantigens suggests a novel peptide-driven mechanism for the evolution of autoantibodies.

Immobilized Peptides to Study Protein-Protein Interactions — Potential and Pitfalls

In recent years, immobilized peptides synthesized on activated cellulose membranes (SPOT synthesis, Frank 1992) have become an important tool in the study of protein-protein interactions and numerous other aspects of molecular recognition (reviewed in Reineke et al. 2001). A broad range of applications has already been described and the rising interest in proteomics is bound to rely on the enormous potential of the (automated) method. When adapted to high-throughput screening, SPOTs, i.e. peptide arrays, will become an invaluable tool in pharmacogenomics and drug discovery.

Mutational Analysis and Structure Predictions

In Chapters 3 and 4, the technique of peptide synthesis on activated membrane supports was described in detail. In this chapter, a setup will be presented that allows the researcher to obtain limited structural data regarding the peptides under investigation. The data are derived from experimental procedures combined with theoretical considerations. These procedures and theoretical considerations are described for the epitope analysis of an autoantigen. However, since antibody-antigen systems represent special cases of protein-protein interactions, the same strategies may be applied when protein-protein interactions in general are investigated.

Affinity Purification and Competition Assays Using Solid-Phase Oligopeptides

The identification and characterization of linear binding sites of monoclonal antibodies on their corresponding antigens can usually be achieved by systematic epitope-mapping studies (see Chap. 5) and mutational analyses (see Chap. 9). When analyzing polyclonal sera containing a variety of antibody specificities directed against a certain antigen, it may be necessary to distinguish between different antibody populations of the B-cell immune response. When serum antibodies recognize adjacent or overlapping epitopes, discriminating between these antibody specificities may be required. Furthermore, it is often not clear whether antibody binding to related structures is mediated by cross-reactivity of the corresponding antibodies or by different antibody populations. All these questions might be answered by using affinity-purified antibodies from adjacent or immunologically related epitopes and (or) by performing competition assays (Valle et al. 1999; Mahler et al. 2000, 2001). Also, for small-scale applications immobilized peptides can be used as an alternative to on-column purification systems for proteins and antibodies. Thus, distinct antibody specificities induced by immunization with peptides or recombinant expression fragments can easily be purified from animal sera using the target peptide bound to cellulose membranes. Since the synthesis of a huge number of different soluble peptides is time-consuming and expensive, it is obvious that solid-phase oligopeptides represent a fast, efficient and practical method for the purification of different antibody specificities.

Epitope Mapping of Antibodies with Solid-Phase Oligopeptides

Epitope mapping represents a powerful tool not only for basic research but also for therapeutic applications. The knowledge of the exact binding site of antibodies on their antigens could ease the development of new vaccination strategies against pathogens. Furthermore, these insights could be used for the development of new therapeutic approaches to remove pathogenic autoantibodies from the serum of autoimmune patients.

Modification of Immobilized Peptides

Cells need to regulate protein activity in order to control processes such as cellular metabolism and growth. This can be achieved by directly modulating protein expression or protein activity. The latter is done by posttranslational protein modifications. In addition to modifications such as acetylation, protein phosphorylation is used in vivo especially to control the activity of cellular factors. Three amino acids (serine, tyrosine and threonine) can reversibly be phosphorylated within a given context. However, not all potential phosphorylation sites are actually phosphorylated in vivo and in vitro. Several distinct pathways that transmit extracellular signals to the nucleus or to the translational machinery use phosphorylation cascades to deliver the signal to the target molecule, thereby ensuring a fast, controlled response of the cell. The S6 kinases and the Raf/MAP pathway are well-established examples of these pathways. Cell-cycle checkpoints are controlled by kinases and regulatory phosphorylations.