The results obtained in this study reflect that the modified disulfide forms of lipoic acid reacted with the AMA-positive PBC sera at levels that were significantly higher than the native PDC-E2 itself. These xenobiotic-modified PDC-E2 did not react with sera from a series of other control sera utilized including sera from AMA-negative patients with PBC, PSC, SLE or healthy individuals. This finding is very intriguing because the major function of PDC-E2 is to catalytically transfer an acetyl group from pyruvate to coenzyme A (CoA) to produce acetyl-CoA. This function relies on the lipoic acid molecule on PDC-E2 which, during this process, opens the catalytic disulfide and transfers the acetyl group from the E1 component to CoA by using one of the arms of this open disulfide (). This function is crucial during the synthesis of adenosine triphosphate (ATP) and will be discussed below with regards to its importance in the modification of the disulfide of lipoic acid by the xenobiotics.
Schematic representation showing the physiological function of lipoylated PDC-E2 during ATP synthesis.
In this study, we found that among all the modified compounds used in our current experiments, three of them (SAc, OASAc, and SCOEt) had very high levels of reactivity against sera from AMA-positive PBC patients that were even higher than the reactivity against lipoic-acid-conjugated PDC-E2 peptide which is a functional form of native PDC-E2. Furthermore, Pearson product-moment correlation coefficient analysis of our data showed that there were significant correlations (p < 0.05) among Ig reactivities against SAc, OASAc, SCOEt, SCOPh, and 2OA from AMA-positive PBC sera. In other words, the probability to bind to one of the xenobiotic-modified peptides was higher in sera binding to other xenobiotic-modified peptides.
These findings prompted us to investigate the similarity between these three compounds in efforts to determine the potential structural similarities in the nature of the compounds that imparted such higher levels of reactivity. First, efforts were made to determine whether these compounds and/or structurally similar compounds are found in nature, in the laboratory or industry. An examination of the available literature on structurally identical and/or similar compounds failed to show similar compounds. However, a closer study of the nature of these compounds led us to the conclusion that these three compounds are in fact quite similar to what happens naturally during physiological reactions. These thioester modified analogs are known to arise from acylation of reduced lipoic acid. That is, these compounds contain a reduced lipoic acid with the addition of one (OASAc) or two (SAc) acetyl groups or the addition of two propionyl groups (SCOEt). Since there are many electrophilic agents that can chemically modify proteins, for example aspirin (ASA) and acetaminophen (APAP), we speculate that this modified disulfide of lipoic acid may prevent the lipoic-acid-conjugated PDC-E2 from functioning properly as we discuss above. We reason that such modifications are likely to result in the intracellular disruption of ATP synthesis and are thus likely to lead to cell death. Therefore, such chemical reactions may lead to the release of the xenobiotics-modified PDC-E2 and the exposure of this modified self-protein to the immune system of genetically susceptible individuals [28
] leading in turn to the breakdown of self-tolerance to native PDC-E2 itself by molecular mimicry and epitope spreading mechanism () [30
]. In fact, our laboratory has previously shown that transient AMAs were present in approximately 35% of patients with acute liver failure caused by APAP toxicity [31
]. However, ASA or APAP induced animal models of PBC have not been reported in the literature.
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 (more ...)
APAP metabolism primarily occurs in the liver. Roughly eighty five percent of the metabolites of APAP are conjugations of the aromatic ring to sulfate or glucoronic acid. These inert products are then excreted through the kidneys. The remaining fifteen percent is converted into N
-benzoquinoneimine (NAPQI) through isozymes of cytochrome p450. NAPQI is a highly-electrophilic metabolite, and is readily intercepted by glutathione. These reactions of glutathione either involve Michael addition to the aromatic ring, or reduction of NAPQI back to APAP. The predominant method of NAPQI detoxification occurs through the former mechanism, resulting in depletion of glutathione molecules from within a cell [32
In the presence of excess APAP, glutathione depletion occurs. The resulting decrease in cellular glutathione allows for the accumulation of the reactive NAPQI metabolite. NAPQI will rapidly undergo addition reactions that preferentially target the thiol groups of proteins and related cofactors [33
]. Previous data [35
] have suggested that glutathiolation would decrease the antigenicity of PDC-E2. However, due to cellular depletion of glutathione, very little glutathione would be available for such covalent protection of PDC-E2. This could lead to possible modification of native PDC-E2 by high levels of reactive NAPQI or other electrophilic agents.
Interestingly, the oxidative states of lipoic acid have been shown to affect the immunogenicity of PDC-E2 [36
]. The lipoic-acid-conjugated PDC-E2 is more immunogenic if it is in a reduced form, which corresponds to the ring-open form of lipoic acid. This observation supports our findings that select modifications of the disulfide of lipoic-acid-conjugated PDC-E2 moiety makes it more immunogenic. However, in nature this reduced and more immunogenic form of PDC-E2 is less stable than the oxidized and less immunogenic form of PDC-E2, which corresponds to the closed ring form of lipoic acid. Hence, the modification which we found in this study may not only make the PDC-E2 more immunogenic but also maintains it in an immunogenic form because the disulfide bond cannot re-form. This modification could enhance the potential for a breakdown in self-tolerance to PDC-E2.
One important question that remains un-answered with regards to the pathogenic mechanisms of PBC concerns the specific targeting of biliary epithelial cells (BECs). The results from the studies reported herein provide some clues to this enigma. According to earlier data [36
], BECs are different from other cell types in that they have higher levels of glutathione, an anti-oxidant, inside the cells. When cell lineages other than BECs undergo apoptosis or oxidative stress, the GSSG (oxidized form of glutathione):GSH (reduced form of glutathione) ratio increases. Subsequently, the oxidized form of glutathione binds to the lipoic acid on PDC-E2 at the disulfide bond position. It has been hypothesized that this binding masks the PDC-E2 epitope and thus prevents the immune response to PDC-E2 [35
]. However, in BECs since there is a relatively higher level of intracellular glutathione, the GSSG:GSH ratio does not increase as much as within other cell types during apoptosis or oxidative stress. Therefore, the masking effect of the oxidized form of glutathione to PDC-E2 cannot occur efficiently. This remaining unmasked PDC-E2 thus becomes prone to immune recognition and subsequent activation of the immune system. Our findings support this notion. That is, if the lipoic-acid-conjugated PDC-E2 is modified at the disulfide bond position, such as in SAc, OASAc, and SCOEt, this modification will further prevent the binding of PDC-E2 to glutathione in BECs. Such modification will increase the likelihood of the exposure of this unmasked form of PDC-E2 in BECs to the immune system and aggravate the breaking of self-tolerance to PDC-E2.
It is also important to note that our laboratory has recently shown that when BECs, but not control cells, undergo apoptosis, PDC-E2 remains immunologically intact [37
]. More importantly, when monocyte-derived macrophages from PBC patients were incubated with apoptotic bodies from BECs in the presence of AMAs, there was intense pro-inflammatory cytokine production. This production was inhibited by anti-CD16, an antibody against the Fc (fragment crystallizable) receptor. Our data suggest that the triad of AMAs, biliary apotopes, and innate immune response plays an important role in disease pathogenesis [38
]. These recent findings emphasize a unique role of AMAs. Notably, AMAs are recognized as a serologic marker of PBC; they can be detected in individuals long before the manifestation of liver pathology and remain at high titer in patients with PBC even after liver transplantation [41
]. Based on the data from this study and our working hypothesis, it is likely that Ig reactivities to modified disulfide forms of lipoic acid, such as OASAc, SAc, SCOEt and SCOPh, could also be present in the individuals who are genetically susceptible to PBC. Lastly, it is interesting to note that the IgG reactivities were specifically reactive against these xenobiotic-modified PDC-E2 peptides and correlated with the levels of AMA more so than the IgM counterpart. These findings may be explained by the affinity maturation and isotype switching process [43
]. Hence, our study not only suggests the potential trigger of the breakdown of self-tolerance to PDC-E2 but also reveals the footsteps or the initial process of how AMAs might develop.
Our data suggest that direct alteration of the lipoyl ring – i.e., disruption of the S-S linkage – renders the lipoic acid “activated” and receptive for xenobiotic modification and subsequent AMA recognition. This is of significance in light of the biochemical function of the lipoyl moiety in electron transport that constantly cycles opening and closing of the disulfide. Thus, in genetically susceptible individuals, the prolonged exposure to electrophilic agents, such as acetaminophen and NSAIDs, may initiate and/or enhance the breakdown of self-tolerance to PDC-E2 and eventually lead to PBC.