The fingerprint of antimitochondrial antibodies and the etiology of primary biliary cholangitis

Abstract

The identification of environmental factors that lead to loss of tolerance has been coined the Holy Grail of autoimmunity. Our work has focused on the reactivity of antimitochondrial autoantibodies (AMA) to chemical xenobiotics and has hypothesized that a modified peptide within PDC-E2, the major mitochondrial autoantigen, will have been immunologically recognized at the time of loss of tolerance. Herein we successfully applied intein technology to construct a PDC-E2 protein fragment containing amino acid residues 177–314 of PDC-E2 by joining a recombinant peptide spanning residues 177 to 252 (PDC-228) with a 62 residue synthetic peptide from 253 to 314 (PP), which encompasses PDC-E2 ILD. We named this intein-constructed fragment PPL. Importantly, PPL, as well as lipoic acid conjugated PPL (LA-PPL) and xenobiotic 2-octynoic acid conjugated PPL (2OA-PPL), are recognized by AMA. Of great importance, AMA has specificity for the 2OA modified PDC-E2 ILD peptide backbone distinct from antibodies that react with native lipoylated PDC-E2 peptide. Interestingly, this unique AMA subfraction is of the IgM isotype and more dominant in early stage PBC, suggesting that exposure to 2OA-PPL-like compounds occurs early in the generation of AMA. To understand the structural basis of this differential recognition we analyzed PPL, LA-PPL and 2OA-PPL using electron paramagnetic resonance spectroscopy, with confirmations by ELISA, immunoblotting and affinity antibody analysis. We demonstrate that the conformation of PDC-E2 ILD is altered when conjugated with 2OA, compared to conjugation with lipoic acid. In conclusion a molecular understanding of the conformation of xenobiotic modified PDC-E2 is critical for understanding xenobiotic modification and loss of tolerance in PBC with widespread implications for a role of environmental chemicals in the induction of autoimmunity.

The interplay of type I and type II interferons in murine autoimmune cholangitis as a basis for sex‐biased autoimmunity

“…IgM and IgG anti‐PDC‐E2 (pyruvate dehydrogenase complex component E2) assays were performed by enzyme‐linked immunosorbent assay (ELISA) with data presented as optical density (O.D.) ± SEM, as described, with known positive and negative controls and standardized recombinant PDC‐E2 . Antibodies to SP100 were evaluated by ELISA using INOVA kits (INOVA Diagnostics, San Diego, CA) and again including known controls …”

“…6 SEM, as described, with known positive and negative controls and standardized recombinant PDC-E2. (5,13,14) Antibodies to SP100 were evaluated by ELISA using INOVA kits (INOVA Diagnostics, San Diego, CA) and again including known controls. (15,16) TOTAL BILE ACID ANALYSIS Total bile acid (TBA) was analyzed using freshly collected serum and a Total Bile Acid Enzymatic Cycling Assay Kit (Diazyme, Poway, CA), as described.…”

Pathogen infections and primary biliary cholangitis

“…Furthermore, another xenobiotic, 2-nonyamide, provided an optimal chemical structure of the xenobiotics modified epitope, which demonstrated enhanced recognition by AMA-positive PBC sera [88]. These findings illustrate that xenobiotic modification of PDC-E2 by chemical exposure in everyday life can generate immunogenic neoantigens that result in the loss of tolerance to human PDC-E2 in subjects who are genetically susceptible to PBC [89]. This hypothesis is supported by animal studies in which animals immunized with 2OA-BSA developed AMA and autoimmune cholangitis 90,91.…”

Rheumatic Manifestations in Autoimmune Liver Disease

“…PBC is a chronic cholestatic disease characterized by high-titer serum anti-mitochondrial antibodies (AMA) in nearly 100% of patients when sensitive techniques are used. 64 It results in autoimmunemediated destruction of the small and medium-sized intrahepatic bile ducts 17,65 . PBC prevalence varies substantially according to geography; the highest rates appear in the northern US with a point prevalence of 402 per million in Minnesota 66 .…”