Electrophile-modified lipoic derivatives of PDC-E2 elicits anti-mitochondrial antibody reactivity

Abstract

Our laboratory has hypothesized that xenobiotic modification of the native lipoyl moiety of the major mitochondrial autoantigen, the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2), may lead to loss of self-tolerance in primary biliary cirrhosis (PBC). This thesis is based on the finding of readily detectable levels of immunoreactivity of PBC sera against extensive panels of protein microarrays containing mimics of the inner lipoyl domain of PDC-E2 and subsequent quantitative structure-activity relationships (QSARs). Importantly, we have demonstrated that murine immunization with one such mimic, 2-octynoic acid coupled to bovine serum albumin (BSA), induces anti-mitochondrial antibodies (AMAs) and cholangitis. Based upon these data, we have focused on covalent modifications of the lipoic acid disulfide ring and subsequent analysis of such xenobiotics coupled to a 15mer of PDC-E2 for immunoreactivity against a broad panel of sera from patients with PBC and controls. Our results demonstrate that AMA-positive PBC sera demonstrate marked reactivity against 6,8-bis(acetylthio)octanoic acid, implying that chemical modification of the lipoyl ring, i.e. disruption of the S-S disulfide, renders lipoic acid to its reduced form that will promote xenobiotic modification. This observation is particularly significant in light of the function of the lipoyl moiety in electron transport of which the catalytic disulfide constantly opens and closes and, thus, raises the intriguing thesis that common electrophilic agents, i.e. acetaminophen or non-steroidal anti-inflammatory drugs (NSAIDs), may lead to xenobiotic modification in genetically susceptible individuals that results in the generation of AMAs and ultimately clinical PBC.

Figure 1

Preparation of xenobiotic-coupled derivatives of the 15-amino-acid-PDC-E2 mimic.

Figure 2

Comparison of IgM (A) and IgG (B) reactivity against xenobiotics-modified PDC-E2 peptide between AMA-positive patients with PBC and healthy controls. Asterisks indicate significant differences (*, p < 0.05; **, p < 0.01; ***, p < 0.001; two-tailed unpaired t-test with Welch’s correction).

Figure 2

Comparison of IgM (A) and IgG (B) reactivity against xenobiotics-modified PDC-E2 peptide between AMA-positive patients with PBC and healthy controls. Asterisks indicate significant differences (*, p < 0.05; **, p < 0.01; ***, p < 0.001; two-tailed unpaired t-test with Welch’s correction).

Figure 3

Schematic representation showing the physiological function of lipoylated PDC-E2 during ATP synthesis.

Figure 4

Hypothetical mechanism of electrophile-modified lipoylated PDC-E2 leading to AMA production. Covalent modification at the thiol group of lipoylated PDC-E2 by electrophilic agent (E⁺) inhibits acyl transfer to CoA, disrupts ATP synthesis, and leads to cell death. Subsequently, the immune exposure to modified PDC-E2 in genetically susceptible individuals results in the generation of AMAs by molecular mimicry and epitope spreading. Examples of E⁺ are listed in the left lower panel.