Al Kindi, Mahmood, 2016 Molecular Signatures of Lupus-Specific Autoantibody Proteomes, Flinders University, School of Medicine
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The structures of epitopes bound by autoantibodies against RNA-protein complexes have been well-defined over several decades, but little is known of the clonality, immunoglobulin (Ig) variable (V) gene usage and mutational status of the autoantibodies themselves at the level of the secreted (serum) proteome. The lupus-specific autoantibodies anti-Smith (anti-Sm) and anti-ribosomal P (anti-Rib-P) have been implicated in the pathogenesis of neurologic and renal complications in systemic lupus erythematosus (SLE), but these clinically important autoantibodies have yet to be characterized at a molecular level. The aim of this thesis is to determine the clonality and V-region usage of lupus-specific autoantibodies (anti-Sm and anti-Rib-P) with the hypothesis that secreted lupus autoantibody proteomes derive from restricted sets of public B cell clonotypes. In this thesis, a novel proteomic workflow is presented based on affinity purification of specific Igs from serum, high-resolution two-dimensional gel electrophoresis (2-DE), and de novo and database-driven sequencing of V-region proteins by mass spectrometry (MS). Anti-Sm and anti-Rib-P Igs were purified from six anti-Sm- and six anti-Rib-P-positive sera, respectively by elution from ELISA plates coated with either native SmD or native Rib-P proteins. Furthermore, an 11- amino acid peptide (11-C peptide) representing the conserved COOH-terminal P epitope was used to specifically purify anti-Rib-P Igs directed to the COOH-epitope. SmD, Rib-P- and 11-C peptide-specific Igs were analyzed 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 Igs were tested for cross-reactivity on ELISA and their proteome data sets analyzed for shared clonotypes. Anti-SmD and anti-Rib-P autoantibody proteomes were IgG1 kappa (K) restricted and comprised each two public clonotypes defined by unique H/L chain pairings. Analysis of purified anti-SmD Igs revealed a major clonotypic population specified by IGHV1.69/IGKV2.28. Whilst, the major clonotypic population in Rib-P response was specified by IGHV1.3-JH4/IGK1.39-JK4 for the common COOH-terminal epitope. The second clonotype in both SmD and Rib-P was shared and sequenced as IGHV3.7-JH6/IGKV3.20-JK2, accounting for two-way immunoassay cross-reactivity between these lupus autoantibodies. Sequence convergence of anti-SmD and 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 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. The discovery of shared sets of specific V-region peptides can be exploited for diagnostic biomarkers in targeted MS platforms and for tracking and removal of pathogenic clones.
Keywords: systemic lupus erythematosus,
Subject: Medicine thesis
Thesis type: Doctor of Philosophy
School: School of Medicine
Supervisor: Tom Gordon