Serum SmD autoantibody proteomes are clonally restricted and share variable-region peptides

Abstract

Recent advances in mass spectrometry-based proteomic methods have allowed variable (V)-region peptide signatures to be derived from human autoantibodies present in complex serum mixtures. Here, we analysed the clonality and V-region composition of immunoglobulin (Ig) proteomes specific for the immunodominant SmD protein subunit of the lupus-specific Sm autoantigen. Precipitating SmD-specific IgGs were eluted from native SmD-coated ELISA plates preincubated with sera from six patients with systemic lupus erythematosus (SLE) positive for anti-Sm/RNP. Heavy (H)-and light (L)-chain clonality and V-region sequences were analysed by 2-dimensional gel electrophoresis and combined de novo database mass spectrometric sequencing. SmD autoantibody proteomes from all six patients with SLE expressed IgG1 kappa restricted clonotypes specified by IGHV3-7 and IGHV1-69 H-chains and IGKV3-20 and IGKV2-28 L-chains, with shared and individual V-region amino acid replacement mutations. Clonotypic sharing and restricted V-region diversity of systemic autoimmunity can now be extended from the Ro/La cluster to Sm autoantigen and implies a common pathway of anti-Sm autoantibody production in unrelated patients with SLE.3

Lupus anti-ribosomal P autoantibody proteomes express convergent biclonal signatures

“…The IGHV3·7‐JH6/IGKV3·20‐JK2 clonotype, which binds an epitope outside the COOH‐terminus yet to be mapped, is notable for its immunological cross‐reactivity and shared germline H/L chain pairing signature with a recently identified anti‐SmD clonotype . These findings provide a novel molecular explanation for cross‐reactivity between two lupus‐specific autoantibodies based on shared clonotypic structures, suggesting a common clonal origin for at least a subset of these autoantibodies.…”

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].…”