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Epitope Specificity Determines Pathogenicity and Detectability of Anti–Platelet‐Derived Growth Factor Receptor α Autoantibodies in Systemic Sclerosis
Author(s) -
Moroncini Gianluca,
Grieco Antonella,
Nacci Giulia,
Paolini Chiara,
Tonnini Cecilia,
Pozniak Katarzy.,
Cuccioloni Massimiliano,
Mozzicafreddo Matteo,
Svegliati Silvia,
Angeletti Mauro,
Kazlauskas Andrius,
Avvedimento Enrico V.,
Funaro Ada,
Gabrielli Armando
Publication year - 2015
Publication title -
arthritis and rheumatology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 4.106
H-Index - 314
eISSN - 2326-5205
pISSN - 2326-5191
DOI - 10.1002/art.39125
Subject(s) - epitope , autoantibody , recombinant dna , microbiology and biotechnology , monoclonal antibody , epitope mapping , antibody , biology , chemistry , immunology , biochemistry , gene
Objective To identify the epitopes recognized by autoantibodies targeting platelet‐derived growth factor receptor α (PDGFRα) in systemic sclerosis (SSc) and develop novel assays for detection of serum anti‐PDGFRα autoantibodies. Methods Epstein‐Barr virus–immortalized B cells from 1 patient with SSc (designated PAM) were screened for expression of IgG binding to PDGFRα and induction of reactive oxygen species in fibroblasts. The variable regions of anti‐PDGFRα IgG were cloned into an IgG expression vector to generate distinct recombinant human monoclonal autoantibodies (mAb), which were characterized by binding and functional assays. The epitopes of anti‐PDGFRα recombinant human mAb were defined by molecular docking, surface plasmon resonance binding assays, screening of a conformational peptide library spanning the PDGFRα extracellular domains, and expression analyses of alanine‐scanned PDGFRα mutants. Direct or competitive enzyme‐linked immunosorbent assays were established to detect all serum anti‐PDGFRα autoantibodies or, selectively, the agonistic ones. Results Three types of anti‐PDGFRα recombinant human mAb, with the same V H but distinct V L chains, were generated. Nonagonistic V H PAM–V κ 13B8 recognized 1 linear epitope, whereas agonistic V H PAM–V λ 16F4 and V H PAM–V κ 16F4 recognized 2 distinct conformational epitopes. Serum anti‐PDGFRα antibodies were detected in 66 of 70 patients with SSc, 63 of 130 healthy controls, 11 of 26 patients with primary Raynaud's phenomenon (RP), and 13 of 29 patients with systemic lupus erythematosus (SLE). Serum V H PAM–V κ 16F4‐like antibodies were found in 24 of 34 patients with SSc, but not in healthy controls, patients with primary RP, or patients with SLE. Peptides composing the V H PAM–V κ 16F4 epitope inhibited PDGFRα signaling triggered by serum IgG from SSc patients. Conclusion Agonistic anti‐PDGFRα autoantibodies are enriched in SSc sera and recognize specific conformational epitopes that can be used to discriminate agonistic from nonagonistic antibodies and block PDGFRα signaling in patients with SSc.