Antimitochondrial Antibody Recognition and Structural Integrity of the Inner Lipoyl Domain of the E2 Subunit of Pyruvate Dehydrogenase Complex

Abstract

Antimitochondrial autoantibodies (AMA), the serological hallmark of primary biliary cirrhosis (PBC) are directed against the lipoyl domain of the E2 subunit of pyruvate dehydrogenase (PDC-E2). However, comprehensive analysis of the amino acid residues of PDC-E2 lipoyl beta sheet with AMA specificity is lacking. Herein, we postulated that specific residues within the lipoyl domain are critical to AMA recognition by maintaining conformational integrity. We systematically replaced each of 19 residue peptides of the inner lipoyl domain with alanine and analyzed these mutants for reactivities against 60 PBC and 103 control sera. Based on these data, we then constructed mutants with 2, 3, or 4 replacements and, in addition, probed the structure of the substituted domains using thiol-specific spin labeling and electron paramagnetic resonance (EPR) of a 5Ile-> Ala and 12Ile->Ala double mutant. Single alanine replacement at 5Ile, 12Ile and 15Glu significantly reduced AMA recognition. In addition, mutants with 2, 3, or 4 replacements at 5Ile, 12Ile and 15Glu reduced AMA reactivity even further. Indeed, EPR reveals a highly flexible structure within the 5Ile and 12Ile double-alanine mutant. Autoreactivity is largely focused on specific residues in the PDC-E2 lipoyl domain critical in maintaining the lipoyl loop conformation necessary for AMA recognition. Collectively, the AMA binding studies and EPR analysis demonstrate the necessity of the lipoyl beta sheet structural conformation in anti-PDC-E2 recognition.

Obeticholic acid for the treatment of primary biliary cholangitis in adult patients: clinical utility and patient selection

“…The major targets of the AMA have been identified and include the E2 subunits of the pyruvate dehydrogenase complex and related proteins 5,6. In fact, the target is restricted to lipoic acid modification of a specific lysine residue.…”

A contemporary perspective on the molecular characteristics of mitochondrial autoantigens and diagnosis in primary biliary cholangitis

“…Further, QSAR analysis on a focused panel of lipoic acid mimics in which the lipoyl S-S bond are modified provided evidence suggesting direct alteration of the lipoyl ring by xenobiotics – that is, disruption of the S-S linkage activates the lipoic acid and makes it receptive for xenobiotic modification and subsequent AMA recognition [52-57]. Furthermore, mutagenesis analysis of the PDC-E2 lipoyl domain revealed that specific amino acid residues are critical in maintaining the lipoyl loop conformation necessary for AMA recognition [58]. …”

“…Recombinant proteins are preferred over crude mitochondria from bovine heart, which can result in greater background (Figure 3). AMA ELISA against PDC-E2, BCOADC-E2, OGDC-E2, E3BP and PDC E1α have been demonstrated to be highly selective in the detection of AMA reactivity to each of these mitochondrial autoantigens [40,42,58,61,72,73]. Higher sensitivity can be achieved with the use of recombinant hybrid molecules, such as MIT3, which co-expresses the three immunodominant epitopes of PDC-E2, BCOADC-E2, and OGDC-E2 [73].…”

“…Although AMA is not associated with the severity of PBC, combination of AMA and discovery of other biomarkers in PBC [67,101-104] will increase the sensitivity and specificity in the diagnosis, disease prediction and prognosis of PBC. Finally, increasing knowledge on antigen specificity of AMA to lipoylated and non-lipoylated forms of the mitochondrial autoantigens, lipoic acid, xenobiotics and isotypes of the Ig molecule [52,58,101,105], may lead to discovery of AMA subpopulations which can be of indicative value for disease progression and prognosis.…”

The modulation of co-stimulatory molecules by circulating exosomes in primary biliary cirrhosis

“…9,10 The primary autoantigen of PBC has been identified as the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2). [11][12][13][14][15] Data from human studies and animal models demonstrate that the pathogenesis of PBC involves not only autoreactive T cells and other immune cells [16][17][18][19][20][21][22] but also biliary epithelial cells. 12,18,19,[23][24][25][26][27][28][29][30] Herein, we demonstrate that circulating exosomes from PBC could be taken up by antigenpresenting cells (APCs) and affect the expression of cell surface co-stimulatory molecules.…”