Lupus anti-ribosomal P autoantibody proteomes express convergent biclonal signatures

Abstract

SummaryLupus-specific anti-ribosomal P (anti-Rib-P) autoantibodies have been implicated in the pathogenesis of neurological complications in systemic lupus erythematosus (SLE). The aim of the present study was to determine variable (V)-region signatures of secreted autoantibody proteomes specific for the Rib-P heterocomplex and investigate the molecular basis of the reported cross-reactivity with Sm autoantigen. Anti-Rib-P immunoglobulins (IgGs) were purified from six anti-Rib-P-positive sera by elution from enzyme-linked immunosorbent assay (ELISA) plates coated with either native Rib-P proteins or an 11-amino acid peptide (11-C peptide) representing the conserved COOH-terminal P epitope. Rib-P-and 11-C peptide-specific IgGs were analysed for heavy (H) and light (L) chain clonality and V-region expression using an electrophoretic and de-novo and database-driven mass spectrometric sequencing workflow. Purified anti-Rib-P and anti-SmD IgGs were tested for cross-reactivity on ELISA and their proteome data sets analysed for shared clonotypes. Anti-Rib-P autoantibody proteomes were IgG1 kappa-restricted and comprised two public clonotypes defined by unique H/L chain pairings. The major clonotypic population was specific for the common COOH-terminal epitope, while the second shared the same pairing signature as a recently reported anti-SmD clonotype, accounting for two-way immunoassay cross-reactivity between these lupus autoantibodies. Sequence convergence of anti-Rib-P proteomes suggests common molecular pathways of autoantibody production and identifies stereotyped clonal populations that are thought to play a pathogenic role in neuropsychiatric lupus. Shared clonotypic structures for anti-Rib-P and anti-Sm responses suggest a common B cell clonal origin for subsets of these lupus-specific autoantibodies.

Extended boiling of peanut progressively reduces IgE allergenicity while retaining T cell reactivity

“…Total lane reactivity was quantified by drawing a transect through each lane, subtracting background and integrating the total reactivity in each lane as previously described [32]. 2D electrophoresis of 100 lg of raw or 2-h boiled peanut extracts using pI 3-11 nonlinear IPG strips (Bio-Rad) and 4-20% Criterion Stain Free polyacrylamide gels (Bio-Rad) was performed as previously described [33].…”

Molecular Profiling and Clonal Tracking of Secreted Rheumatoid Factors in Primary Sjögren's Syndrome

“…In this study, we performed the first proteomic profiling of secreted (serum) RF responses in primary SS using a simple purification method for RF heavy chains followed by de novo Ig sequencing and database matching. As reported for other systemic autoantibodies in lupus and primary SS, such as anti‐Ro/La, anti‐Sm, and anti–ribosomal P, serum IgM‐RF repertoires express shared sets of Ig V H subfamilies and reveal moderate levels of somatic hypermutation . While an early study of RF hybridomas reported a diverse usage of germline‐encoded Ig V H genes in primary SS, single‐cell studies provide a limited view of the actual secreted RF repertoire in comparison with direct high‐resolution mass spectrometric sequencing .…”

Precipitating anti-dsDNA peptide repertoires in lupus

“…In-gel tryptic and chymotryptic digests were performed on the 50 kD H-and 25 kD L-chain bands and peptides analysed using a TripleTOF 5600+ mass spectrometer (AB Sciex, Framingham, MA, USA) coupled to an Ekspert nano LC 415 high-performance liquid chromatography (HPLC) (Eksigent; AB Sciex), as detailed previously [17,18,[21][22][23][24]. Parameters for database searches, data refinement and IgV gene family assignments have been described previously [17,18,[21][22][23][24]31]; in brief, a maximum of two missed cleavages, precursor tolerance of < 15 parts per million, product ion tolerance of 0·02 Da, precursor charge state of +2 to +4, fixed modification carbamidomethylation, variable modifications oxidation and deamidation, a maximum of three modifications allowed and non-specific cleavage at one end. 1).…”

“…We have developed proteomic methodology within our facility to analyse immunoglobulin (Ig)V peptide repertoires of anti-Ro/La autoantibodies in primary Sjögren's syndrome (SS) and anti-Sm and anti-ribosomal P responses in SLE, based on high-resolution mass spectrometric sequencing of specific autoantibodies purified from stored serum samples [17][18][19][20][21][22][23]. In brief, anti-Ro/La proteomes comprised oligoclonal expression patterns of shared, heavily mutated heavy (H)-and light (L)-chains with anti-Ro60 IgV molecular peptide fingerprints of anti-Ro/La determinant spreading [22][23][24].…”

“…In brief, anti-Ro/La proteomes comprised oligoclonal expression patterns of shared, heavily mutated heavy (H)-and light (L)-chains with anti-Ro60 IgV molecular peptide fingerprints of anti-Ro/La determinant spreading [22][23][24]. Conversely, lupus-specific anti-SmD and anti-ribosomal P proteomes were biclonally restricted with shared and individual amino acid replacement mutations [17,18]. This evolving technology is potentially transformative because it can identify molecular fingerprints and clonal turnover of pathogenic autoantibodies that are invisible to immunoassays used in current clinical practice [20].…”