The role of N- or C-terminal biotinylation in autoantibody recognition of citrullin containing filaggrin epitope peptides in Rheumatoid arthritis

Here, we report on the synthesis, conformational analysis and autoantibody binding properties of new sets of Rheumatoid arthritis (RA) specific biotin-peptide conjugates derived from filaggrin epitope peptides. The biotin with or without a linker was attached to the Cit or Arg containing epitope core (311TXGRS315) or epitope region (306SHQESTXGXSXGRSGRSGS324) peptide (where X = Cit), through an amide bond at the N- or C-terminal of the epitopes. Antibody binding was detected by indirect enzyme-linked immunosorbent assay (ELISA) using sera from RA, Systemic lupus erythematosus (SLE) patients as well as healthy individuals and the secondary structure of conjugates was investigated by electronic circular dichroism (ECD). We found that autoantibodies from RA patients recognize specifically both filaggrin epitope region (306SHQESTXGXSXGRSGRSGS324) and short epitope core (311TXGRS315) peptides. Our data also indicate that the positioning of the biotin label within a peptide sequence can markedly influence the antibody binding, but the length of the linker incorporated has essentially no effect on the recognition. ECD experiments demonstrate that the Arg/Cit change does not influence the solution conformation of the peptide conjugates. However, the presence and position of the biotin moiety has a pronounced effect on the conformation of the 5-mer epitope core peptides, while it doesn’t alter the secondary structure of the 19-mer epitope region peptides

Identification of motives mediating alternative functions of the neomorphic moonlighting TPPP/p25

The disordered Tubulin Polymerization Promoting Protein (TPPP/p25), a prototype of neomorphic moonlighting proteins, displays physiological and pathological functions by interacting with distinct partners. Here the role of the disordered N- and C-termini straddling a middle flexible segment in the distinct functions of TPPP/p25 was established, and the binding motives responsible for its heteroassociations with tubulin and α-synuclein, its physiological and pathological interacting partner, respectively, were identified. We showed that the truncation of the disordered termini altered the folding state of the middle segment and has functional consequences concerning its physiological function. Double truncation diminished its binding to tubulin/microtubules, consequently the tubulin polymerization/microtubule bundling activities of TPPP/p25 were lost highlighting the role of the disordered termini in its physiological function. In contrast, interaction of TPPP/p25 with α-synuclein was not affected by the truncations and its α-synuclein aggregation promoting activity was preserved, showing that the α-synuclein binding motif is localized within the middle segment. The distinct tubulin and α-synuclein binding motives of TPPP/p25 were also demonstrated at the cellular level: the double truncated TPPP/p25 did not align along the microtubules in contrast to the full length form, while it induced α-synuclein aggregation. The localization of the binding motives on TPPP/p25 were established by specific ELISA experiments performed with designed and synthesized peptides: motives at the 178-187 and 147-156 segments are involved in the binding of tubulin and α-synuclein, respectively. The dissimilarity of these binding motives responsible for the neomorphic moonlighting feature of TPPP/p25 has significant innovative impact in anti-Parkinson drug research

Bead arrays for antibody and complement profiling reveal joint contribution of antibody isotypes to C3 deposition

The development of antigen arrays has provided researchers with great tools to identify reactivities against self or foreign antigens from body fluids. Yet, these approaches mostly do not address antibody isotypes and their effector functions even though these are key points for a more detailed understanding of disease processes. Here, we present a bead array-based assay for a multiplexed determination of antigen-specific antibody levels in parallel with their properties for complement activation. We measured the deposition of C3 fragments from serum samples to reflect the degree of complement activation via all three complement activation pathways. We utilized the assay on a bead array containing native and citrullinated peptide antigens to investigate the levels of IgG, IgM and IgA autoantibodies along with their complement activating properties in serum samples of 41 rheumatoid arthritis patients and 40 controls. Our analysis revealed significantly higher IgG reactivity against the citrullinated fibrinogen β and filaggrin peptides as well as an IgA reactivity that was exclusive for citrullinated fibrinogen β peptide and C3 deposition in rheumatoid arthritis patients. In addition, we characterized the humoral immune response against the viral EBNA-1 antigen to demonstrate the applicability of this assay beyond autoimmune conditions. We observed that particular buffer compositions were demanded for separate measurement of antibody reactivity and complement activation, as detection of antigen-antibody complexes appeared to be masked due to C3 deposition. We also found that rheumatoid factors of IgM isotype altered C3 deposition and introduced false-positive reactivities against EBNA-1 antigen. In conclusion, the presented bead-based assay setup can be utilized to profile antibody reactivities and immune-complex induced complement activation in a high-throughput manner and could facilitate the understanding and diagnosis of several diseases where complement activation plays role in the pathomechanism

Phosphorylation adjacent to the nuclear localization signal of human dUTPase abolishes nuclear import: Structural and mechanistic insights

Phosphorylation adjacent to nuclear localization signals (NLSs) is involved in the regulation of nucleocytoplasmic transport. The nuclear isoform of human dUTPase, an enzyme that is essential for genomic integrity, has been shown to be phosphorylated on a serine residue (Ser11) in the vicinity of its nuclear localization signal; however, the effect of this phosphorylation is not yet known. To investigate this issue, an integrated set of structural, molecular and cell biological methods were employed. It is shown that NLS-adjacent phosphorylation of dUTPase occurs during the M phase of the cell cycle. Comparison of the cellular distribution of wild-type dUTPase with those of hyperphosphorylation- and hypophosphorylation-mimicking mutants suggests that phosphorylation at Ser11 leads to the exclusion of dUTPase from the nucleus. Isothermal titration microcalorimetry and additional independent biophysical techniques show that the interaction between dUTPase and importin-alpha, the karyopherin molecule responsible for 'classical' NLS binding, is weakened significantly in the case of the S11E hyperphosphorylation-mimicking mutant. The structures of the importin-alpha-wild-type and the importin-alpha-hyperphosphorylation-mimicking dUTPase NLS complexes provide structural insights into the molecular details of this regulation. The data indicate that the posttranslational modification of dUTPase during the cell cycle may modulate the nuclear availability of this enzyme

Seropositivity and Antibody Profiling of Patients Are Dramatically Impacted by the Features of Peptides Used as Immunosorbents: A Lesson from Anti–Citrullinated Protein/Peptide Antibody

International audienceQuantification of Abs toward a single epitope is critical to understanding immunobiological processes. In autoimmunity, the prognostic value of the serological profiles of patients draws much attention, but the detection of Abs toward a single epitope is not well controlled. Particularly, the rheumatoid arthritis (RA)-specific anti-citrullinated protein/peptide Abs (ACPA) are specific to a two-atom change on arginyl residues and are considered a heterogeneous family of Abs. As a model, we studied ACPA to decipher how peptide features used as immunosorbent impact Ab detection. We synthesized 30 peptides encompassing immunodominant epitopes of citrullinated fibrin differing by their length and biotin location and tested them using ELISA with 120 sera from RA and non-RA rheumatic disease controls, generating over 3000 experimental measurements. We showed that minor molecular changes in peptide chemical structure had dramatic consequences. Even when peptides exhibited the same epitope, measured Ab titers were extremely variable, and patients' seropositivity was discordant in up to 50% of cases. The distance between epitope and biotin was the most critical parameter for efficient Ab detection irrespective of biotin position or peptide length. Finally, we identified a 15-mer peptide bearing a single citrullinated epitope detecting almost all ACPA-positive sera, thus revealing a high degree of homogeneity in RA autoimmune response. This integrative analysis deciphers the dramatic impact of the molecular design of peptide-based technologies for epitope-specific Ab quantification. It provides a model for assay development and highlights that the studies using such technologies can give a wrong perception of biological processes and therefore that medical use of data must be cautious

The fibrin-derived citrullinated peptide β60–74Cit(60,72,74) bears the major ACPA epitope recognised by the rheumatoid arthritis-specific anticitrullinatedfibrinogen autoantibodies and anti-CCP2 antibodies

Objectives To evaluate the proportions of rheumatoid arthritis (RA) sera containing anticitrullinated proteins autoantibodies (ACPA) reactive to α36–50Cit38,42 and/or β60–74Cit60,72,74, two peptides identified as bearing the immunodominant epitopes of their major target, citrullinated fibrin. To analyse the relationships of anti-α36–50Cit38,42 and anti-β60–74Cit60,72,74 autoantibodies with autoantibodies reactive to the complete citrullinated human fibrinogen molecule (AhFibA) and with anti-CCP2 antibodies. Methods 617 sera from 181 patients with established RA and 436 with non-RA rheumatic diseases were tested by ELISA for AhFibA, anti-CCP2, anti-α36–50Cit38,42, anti-β60–74Cit60,72,74 autoantibodies, and by nephelometry for rheumatoid factor (RF). Diagnostic indexes, correlations and concordances between tests were analysed. Crossreactivity of anti-α36–50Cit38,42 and anti-β60–74Cit60,72,74 autoantibodies was assessed in competition experiments. Results At a diagnostic specificity of 95%, the diagnostic sensitivity of AhFibA (83%) was significantly higher than that of all other tests. The diagnostic sensitivity of anti-β60–74Cit60,72,74 (71%) was significantly higher than that of anti-α36–50Cit38,42 autoantibodies (51%) but similar to that of anti-CCP2 (74%). Titres of RF, anti-α36–50Cit38,42 and anti-β60–74Cit60,72,74 autoantibodies were weakly correlated with each other, whereas titres of anti-β60–74Cit60,72,74 were strongly correlated with those of AhFibA (r=0.633) and anti-CCP2 (r=0.634). Anti-α36–50Cit38,42 and anti-β60–74Cit60,72,74 mainly corresponded to two non-crossreactive subfamilies of ACPA. More than 90% of AhFibA-positive or anti-CCP2-positive sera recognised the α36–50Cit38,42 and/or the β60–74Cit60,72,74 peptide. Conclusions Autoantibodies reactive to α36–50Cit38,42 and β60–74Cit60,72,74 form two distinct, non-overlapping subfamilies of ACPA that, together, cover practically all the ACPA reactivity to citrullinated fibrinogen and to CCP2 antigens. In established RA, anti-β60–74Cit60,72,74 autoantibodies show diagnostic indexes similar to those of anti-CCP2

Role of <i>N</i>- or <i>C</i>‑Terminal Biotinylation in Autoantibody Recognition of Citrullin Containing Filaggrin Epitope Peptides in Rheumatoid Arthritis

Here, we report on the synthesis, conformational analysis, and autoantibody binding properties of new sets of rheumatoid arthritis (RA) specific biotin–peptide conjugates derived from filaggrin epitope peptides. The biotin with or without a linker was attached to the Cit or Arg containing epitope core (³¹¹T<u>X</u>GRS³¹⁵) or epitope region (³⁰⁶SHQES­T<u>X</u>G<u>X</u>S<u>X­</u>GRSGR­SGS³²⁴) peptide (where X = Cit), through an amide bond at the <i>N</i>- or <i>C</i>-terminal of the epitopes. Antibody binding was detected by indirect enzyme-linked immunosorbent assay (ELISA) using sera from RA, Systemic lupus erythematosus (SLE) patients, as well as healthy individuals, and the secondary structure of conjugates was investigated by electronic circular dichroism (ECD). We found that autoantibodies from RA patients recognize specifically both filaggrin epitope region (³⁰⁶SHQEST­<u>X</u>G<u>X</u>S<u>X</u>­GRSGR­SGS³²⁴) and short epitope core (³¹¹T<u>X</u>GRS³¹⁵) peptides. Our data also indicate that the positioning of the biotin label within a peptide sequence can markedly influence the antibody binding, but the length of the linker incorporated has essentially no effect on the recognition. ECD experiments demonstrate that the Arg/Cit change does not influence the solution conformation of the peptide conjugates. However, the presence and position of the biotin moiety has a pronounced effect on the conformation of the 5-mer epitope core peptides, while it does not alter the secondary structure of the 19-mer epitope region peptides

Recognition of new citrulline-containing peptide epitopes by autoantibodies produced in vivo and in vitro by B cells of rheumatoid arthritis patients

Anti-citrullinated peptide/protein antibodies (ACPAs) are highly sensitive and specific markers of rheumatoid arthritis (RA). Identification of peptide epitopes that may detect different subgroups of RA patients might have diagnostic and prognostic significance. We have investigated citrulline- and arginine-containing peptide pairs derived from filaggrin, collagen or vimentin, and compared this citrulline-peptide panel with the serological assays conventionally used to detect ACPAs. Furthermore, we studied if the same citrulline-peptides identify antibody-secreting cells in in vitro cultures of RA B cells. Recognition of citrulline- and arginine-containing filaggrin, vimentin and collagen peptide epitopes were tested by Multipin ELISA system, by indirect ELISA and by a peptide-specific microarray. B cells were purified from blood by negative selection; antibody-producing cells were enumerated by ELISPOT assay. The panel composed of citrulline-peptide epitopes of filaggrin, collagen and vimentin was recognized by RA sera with a sensitivity and specificity comparable with the currently used tests. Moreover, the combined citrulline-peptide panel including the new short epitope peptide of filaggrin, fil311-315, also identified nearly one-third of RA cases that were negative for antibodies against cyclic citrullinated peptides, mutated citrullinated vimentin or for rheumatoid factor. The results with the peptide-specific microarray have shown that although most ACPAs recognizing the four citrulline peptides are IgG, some of them specifically recognizing citrulline-containing filaggrin peptides (fil311-315 and fil306-326) are IgM, and so may be produced either by newly formed activated B cells or by unswitched B memory cells. Furthermore, the citrulline-peptides of filaggrin and vimentin detect ACPA-producing cells, and so could also be applied to study the B cells of RA patients

Level of EBNA-1 specific antibodies and their complement activation.

<p>EBNA-1 coupled beads were incubated with sera of RA patients and controls. <b><i>A</i></b><i>)</i> Levels of bound human IgG, IgM and IgA antibodies along with the degree of complement activation are plotted for the two groups. Mann-Whitney non-parametric statistical test was used to calculate the statistical difference between the two groups. <b><i>B</i></b><i>)</i> Signal intensities for anti-IgM, anti-IgA and anti-C3 for the controls (upper panel) and RA patients (lower panel) are plotted in a 3D graph. The tables show the Spearman's Rho correlation coefficients between anti-C3, anti-IgG, anti-IgM and anti-IgA signal intensities calculated separately for the control and RA patient groups. Only statistically significant (p-value<0.05) correlation coefficients are shown and non-significant correlations are indicated by (n.s.).</p