Precipitating anti‐dsDNA peptide repertoires in lupus

• Published in: Clinical and experimental immunology Vol. 194; no. 3; pp. 273 - 282
• Main Authors: Wang, J. J., Colella, A. D., Beroukas, D., Chataway, T. K., Gordon, T. P.
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.12.2018; John Wiley and Sons Inc
• Subjects: Adult; Aged, 80 and over; Amino Acid Sequence; Amino Acid Substitution - genetics; Amino acids; Antibodies, Antinuclear - blood; Antibodies, Antinuclear - genetics; Antibodies, Antinuclear - immunology; anti‐dsDNA; Arginine; Autoantibodies; Complementarity Determining Regions - genetics; Complementarity-determining region 3; DNA - immunology; Female; Germinal centers; Humans; IgG; Immunoglobulin G - genetics; Immunoglobulin G - immunology; Immunoglobulin kappa-Chains - genetics; Immunoglobulin kappa-Chains - immunology; Immunoglobulin Variable Region - genetics; Immunoglobulin Variable Region - immunology; Immunoglobulins; Lupus; Lupus Erythematosus, Systemic - immunology; Male; mass spectrometric sequencing; Mass Spectrometry; Middle Aged; Original; Peptides; Precipitins; Proteomes; Systemic lupus erythematosus; Variable region; Young Adult; IgG; systemic lupus erythematosus; proteomes; mass spectrometric sequencing; anti-dsDNA
• ISSN: 0009-9104; 1365-2249; 1365-2249
• DOI: 10.1111/cei.13197

Summary Anti‐double‐stranded (ds)DNA autoantibodies are prototypical serological markers of systemic lupus erythematosus (SLE), but little is known about their immunoglobulin variable (IgV) region composition at the level of the secreted (serum) proteome. Here, we use a novel proteomic workflow based on de novo mass spectrometric sequencing of anti‐dsDNA precipitins to analyse IgV subfamily expression and mutational signatures of high‐affinity, precipitating anti‐dsDNA responses. Serum anti‐dsDNA proteomes were oligoclonal with shared (public) expression of immunoglobulin (Ig)G heavy chain variable region (IGHV) and kappa chain variable region (IGKV) subfamilies. IgV peptide maps from eight subjects showed extensive public and random (private) amino acid replacement mutations with prominent arginine substitutions across heavy (H)‐ and light (L)‐chains. Shared sets of L‐chain complementarity determining region 3 (CDR3) peptides specified by arginine substitutions were sequenced from the dominantly expressed IGKV3‐20 subfamily, with changes in expression levels of a clonal L‐chain CDR3 peptide by quantitative multiple reaction monitoring (MRM) paralleling the rise and fall of anti‐dsDNA levels by Farr radioimmunoassays (RIA). The heavily mutated IgV peptide signatures of precipitating anti‐dsDNA autoantibody proteomes reflect the strong selective forces that shape humoral anti‐dsDNA responses in germinal centres. Direct sequencing of agarose gel precipitins using microlitre volumes of stored sera streamlines the antibody sequencing workflow and is generalizable to other precipitating serum antibodies. This study reports the first mass spectrometric sequencing of high‐affinity, precipitating anti‐dsDNA antibodies purified from dsDNA‐anti‐dsDNA precipitins. Proteomic analyses reveal shared expression of oligoclonal heavy and light chains and personalised somatic mutational signatures. We then identify clonotypic light‐chain complimentary determining region (CDR)3 peptides for application in high resolution quantitative proteomics, showing that changes in CDR3 clonotypic expression parallel anti‐dsDNA levels by conventional precipitating Farr radioimmunoassays.