Infidelity in the exogenous lipoylation pathway : a potential mechanism for the generation of neo-antigens

Primary biliary cirrhosis (PSC) is a chronic liver disease with an autoimmune aetiology, which leads to the destruction of biliary epithelial cells causing fibrosis, cirrhosis and eventually liver failure. Major autoimmune responses seen in PSC patients are directed against lipoylated members of the 2-oxo-acid dehydrogenase family of multi-enzyme complexes with lipoic acid (LA) itself forming a direct part of key autoepitopes. It has been hypothesised that small changes in a self-antigen, such as the aberrant binding ofaxenobiotic, could render a self-antigen immunogenic and potentially lead to autoimmunity. Xenobiotics synthetically bound to a Iipoyl domain peptide in the place of LA have previously been found to react very strongly with PSC patient antibodies. When used as a sensitising agent in animal models one of these compounds has been. shown to induce PSC like immune responses, however the ability of these substances to be produced in vivo has not been demonstrated. The exogenous Iipoylation pathway consists of two enzymes; Iipoate activating enzyme (LAE), a medium-chain acyl-CoA synthetase (MACS1) highly expressed in liver tissue, which converts LA to Iipoyl-AMP and Iipoyl transferase (LT) which catalyses the transfer of Iipoyl-AMP to Iipoyl-domains. Importantly, some evidence suggests that these enzymes are capable of substrate promiscuity. This work details the development of a novel, recombinant exogenous Iipoylation system. Recombinant LT and LAE were shown capable of Iipoylating the relevant domains in the same way as the in vivo system. The fidelity of the system was tested . . using a number of potential xenobiotics, several of which were found to be incorporated into Iipoyl domains in the place of LA. These additions were validated by mass spectrometry. Further investigations with one such xenobiotic, 6-bromohexanoic acid, showed that it could not only compete with LA, but also identified conditions in which it was preferentially incorporated. Xenobiotic incorporation was demonstrated in both the inner Iipoyl domain of the E2 subunit of pyruvate dehydrogenase complex, the major autoantigen of PSC, and in all Iipoyl domain containing subunits of pyruvate dehydrogenase and oxo-glutarate dehydrogenase complexes. The data presented demonstrate infidelity in the exogenous Iipoylation system, and reveals conditions in which certain xenobiotics may be incorporated into Iipoyl domains in preference to LA. This is an important proof of concept observation as it validates a potential mechanism for the development of altered-self auto-immunogenicity.EThOS - Electronic Theses Online ServiceGBUnited Kingdo