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Divisions of
*
Rheumatology, Allergy, and Clinical Immunology and
Hematology/Oncology and
Department of Chemistry, University of California School of Medicine, Davis, CA 95616;
Lawrence Livermore National Laboratory, Livermore, CA 94550;
¶ Department of Microbiology, Monash University, Victoria, Australia;
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Department of Pathology, Emory University, Atlanta, GA 30322
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Xenobiotics are foreign compounds that may either alter or complex to defined self proteins, inducing a change in the molecular structure of the native protein sufficient to induce an immune response. Such immune responses may then result in the recognition of not only the modified or altered protein, but also the unmodified native protein (4, 5). The chronic presence of the self protein serves to perpetuate the immune response initiated by the xenobiotic-induced adduct and leads to autoimmunity (6, 7). Many xenobiotics are metabolized in the liver, thereby increasing the potential for liver-specific alteration of proteins (8). In fact, a liver-specific autoimmune disease can be observed in some patients exposed to chlorofluorohydrocarbon anesthetics (9, 10). Previous work has reported that immunization with halothane, whose trifluoroacetyl (TFA) metabolite covalently links to lysine on cytochrome p450 2E1 (11), induces the formation of Abs that cross-react not only with the haptenated (TFA) immunogen, but also to the lipoated E2 component of pyruvate hydrogenase (PDC-E2), the major autoantigen of PBC (12, 13). This finding has important implications in the pathogenic mechanisms associated with PBC, an autoimmune disease marked by the presence of AMAs (14, 15). The target of AMAs are the E2 components of the 2-oxo acid dehydrogenase pathway, particularly PDC-E2 (14), and the primary B cell epitope of PDC-E2 recognized by AMAs includes a lipoated lysine residue (16, 17).
We hypothesize that the lipoic acid residue of PDC-E2 serves as a xenobiotic target that, following the modification of the lipoyl lysine residue, becomes immunogenic and initiates or perpetuates an AMA response. We further hypothesize that the AMA response is induced by a modified self protein and that the Ab specificities present in such sera include those that recognize the xenobiotic modification. Herein, we took advantage of a microbead system for which peptide synthesis, derivatization, and determination of Ab reactivity can all be performed on the same solid support (18). Our data reflect, for the first time, that organic chemicals, some of which could occur after exposure to common environmental agents, can serve as a more effective epitopes than the native autoantigen. These results may have significant implication for the pathogenesis of PBC.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Approximately 500 beads with the appropriate peptide organic mimeotope bound via a polyethylene glycol linker were incubated in a polypropylene column (PerkinElmer Wallac, Gaithersburg, MD) containing 90 µg TentaGel resin at 0.27 mmol/g (Rapp Polymere, Tübingen, Germany) as previously described (18). Each bead has 100 pmol peptide conjugate (19). Columns containing beads were first blocked with 1% BSA in PBS for 30 min. The 1% BSA was removed, and the beads were incubated with 300–500 µl of sera diluted 1/1000 at room temperature for 1–3 h. After incubation with sera, the columns were washed four times with PBS and 1% Tween 20; Ab binding was quantitated by the addition of HRP-labeled secondary anti-human IgG, IgA, and IgM Abs diluted 1/2000 (BioSource International, Camarillo, CA). After 30 min, the beads were washed, ABTS substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD) added, and the OD determined at 405 nm.
Affinity purification of sera
Briefly, 100 µg purified recombinant human PDC-E2 (20) was separated on a 10% SDS-PAGE and transferred to nitrocellulose. The separated recombinant protein was cut from the nitrocellulose and blocked with 1% milk for 30 min before incubation with sera diluted 1/5. The strips were extensively washed and then subjected to elution with 0.1 M glycine-HCl, 20 mM MgCl2, and 50 mM KCl at pH 2. The eluate was immediately adjusted to pH 7.0 with 1 M Tris. The affinity-purified sera were tested for specificity by ELISA using native PDC-E2 (21, 22).
Synthesis of peptides and mimeotopes
Peptide synthesis was performed on 90 µ TentaGel resin
(
0.27 mmol/g) in a 5–10 polypropylene column (PerkinElmer Wallac).
The resin was first swollen in N,N-dimethyl
formamide (DMF). Four-fold excess of F-moc amino acids were added,
followed by equal amounts of 1-hydroxybenzotrizole hydrate solution and
N,N-diisopropylcarbodiimide. After gentle shaking
for 1–2 h, 20% piperidine in DMF was added to deprotect the Fmoc
group. Coupling-deprotection steps were repeated after each amino acid
addition for the entire peptide sequence. The N
-terminal amino group
was acylated with acetic anhydride and
N,N-diisopropylethylamine in DMF. In addition,
lipoic acid was coupled to the lysine residue of the peptide sequences
by N-hydroxysuccinimide (NHS) ester conjugation after
side-chain deprotection. The Dde side-chain-protecting group on Lys was
deprotected with 2% NH2NH2
in DMF (5 min). A Kaiser test was performed to ensure complete
deprotection before the addition of the chosen NHS esters.
The 18 compounds included NHS esters from carboxylic acids (compounds
1–3, 5–7, 9–10, and 12–17), which were synthesized as follows: The
carboxylic acid derivative (2.5 mmol) was placed in a round-bottom
flask under nitrogen and NHS (288 mg, 2.5 mmol), and 1,
2-dimethoxyethane (8 ml) were added. After stirring at room temperature
and cooling to 0°C, a solution of dicyclohexylcarbodimide (620 mg,
3.0 mmol) in 1,2-dimethoxyethane (1 ml) was added. The mixture was
stirred at 0°C for 15 min and placed in a freezer overnight. The
precipitate (dicyclohexylurea) was removed by Celite filtration. The
crude solid was obtained by removal of the solvent and recrystallized
with isopropanol to give pure NHS ester. Compound purity was verified
by proton nuclear magnetic resonance (NMR) and the presence of
only one spot on TLC. 1H NMR indicated four
additional protons (
2.8–3.0) in the products (evidence of NHS
incorporation). Yields typically ranged from 50 to 90%. Also included
were NHS esters from acid chloride (compounds 4 and 8). Pyridine (0.24
ml, 3.0 mmol) and NHS (288 mg, 2.5 mmol) were dissolved in 10 ml
CH2Cl2, placed under a
nitrogen atmosphere, and treated as described above. The solution was
washed with 10% HCl, saturated NaHCO3, water,
saturated NaCl, and once again with water. The organic phase was dried,
evaporated, and purified by recrystallization from isopropanol.
Compound purity was verified as described above. Yields were 60%
for both compounds. Finally, compound 11 NaH (60% in mineral oil,
2 g, 50 mmol) was washed with anhydrous pentane three times under
nitrogen. Next CF3CH2OH
(3.64 ml, 50 mmol) and 75 ml tetrahydrofuran were added. The solution
was heated and stirred at reflux (65°C) for 1 h, at which time
HOCOCH2I was added to the resulting solution and
stirred overnight. Addition of HCl to pH 4 produced a white
precipitate. The organic phase was separated and washed with saturated
Na2S2O3.
The desired product (0.65 g, 41% yield) was obtained by evaporating
the solvent. The preparation of NHS esters from carboxylic acids was
used to obtain the corresponding product (55% yield). Compound 18,
3-(trifluoromethyl)cinnamic acid (0.43g, 2 mmol) was hydrogenated in 10
ml ethylacetate with catalyst (10% palladium-activated
charcoal, 200 mg) under 3 atm H2. A yellow
liquid (0.42g, 96% yield) was obtained by evaporating the solvent:
1H NMR (CDCl3)
2.67–2.74 (t, 2H, CH2),
2.99–3.04 (t, 2H, CH2),
7.40–7.41 (d, 3H, CH), 7.47 (s,
1H, CH).
Computational methods
All lysine substituents shown in Fig. 1
, except for compound 6, were optimized
by ab initio quantum chemical methods, with a hydrogen atom substituted
for the lysine side chain. The optimizations were done in the gas phase
using a density functional method (B3LYP) and a 6–31G(d,p) basis set
(23, 24). Solvent energy calculations were performed on
the optimized structures using the conductor-like solvent model
(25) at the B3LYP/6–31++G(d,p) level and the Gaussian 98
program (26). Compound 6 was not optimized due to
the presence of iodine, which cannot be accurately modeled without
including relativistic corrections.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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First, to verify the epitope most commonly shared by PBC patients,
sera from 10 patients were assayed against select peptides within the
inner lipoyl domain of PDC-E2 (17, 21, 22). Because the
vast majority of PBC patients possess AMAs that react with a linear
epitope, conformational epitopes were not addressed in this study
(14). We prepared varying lengths (7-, 9-, 10-, and
12-mers) of peptides encompassing the previously defined inner lipoyl
domain of PDC-E2. These included lipoated and nonlipoated 7-residue
amino acid 171–177 (ETDKATI), 9-residue amino acid 170–178
(IETDKATIG), 10-residue amino acid 170–179 (IETDKATIGE), and
12-residue amino acid 173–184 (DKATIGFEVQEE) peptides. We chose
the N terminus acylated, lipoated 12-mer 173–184 because, in an ELISA,
the mean OD of binding in sera from 10 patients with PBC was highest
against this peptide and >3 SD above the mean of the control group
(data not shown). Subsequently, we used this 12-mer as the
representative PDC-E2 autoepitope to analyze PBC patient sera affinity
purified against full-length rPDC-E2 (27). Representative
affinity-purified sera from 10 PBC patients and 10 healthy control
individuals were tested by ELISA for reactivity against nonlipoated and
lipoated forms of the 12-mer PDC-E2 peptide. The OD for PBC sera
against the lipoated native peptide was 0.49 ± 0.07 compared with
0.1 ± 0.04 for the nonlipoated native peptide (Fig. 1
). OD values
of the control sera were all in the background range. The data clearly
indicate that lipoic acid is necessary for binding of the AMA to this
peptide. Because the dominant epitopic region of PDC-E2 that
encompasses both the T and B cell epitopes includes an amino acid that
is lipoated (17, 21, 22), we reasoned that the lipoate
molecule has the highest potential to serve as a target for
modification by a xenobiotic agent.
Select halogenated conjugates create mimeotopes
To determine whether there is evidence for a xenobiotic-induced
modification of this region of PDC-E2, we replaced the lipoated lysine
on the PDC-E2 peptide with one of 18 lysine modifications
(KM) (Fig. 1). These compounds were selected to
offer a range of steric features and functional groups, with many of
them designed to act as a cyclic scaffold for a halogenated compound.
The compounds can be divided into four groups: group I includes rigid,
short structures; group II includes linear, flexible structures; group
III includes rigid, extended aromatic compounds; and group IV includes
an example of a flexible, extended aromatic compound. We reasoned that
structures resembling the initiating xenobiotically modified epitope
would bind autoantibodies with a higher affinity than lipoic acid. The
KM peptides were assayed by the microbead ELISA
using PBC and control sera. Data are represented as experimental values
subtracted from background data (i.e., data from control sera) for each
compound tested. A variety of binding strengths were observed (Fig. 1).
Of interest is that peptides 13, 14, 15, and 18 showed significantly
higher binding with aliquots of the same PBC sera as compared with
reactivity against the same amount of the native protein. This result
fits our hypothesis that these modified peptides would be more similar
to the xenobiotically modified substrate. Overall, the group I
benzamides have the lowest binding, the group II alkanamides are more
reactive, and the groups III and IV cinnamides are the most
reactive.
Structure function relationships
The structural features of the peptide lysine substituents (the
"M" groups attached to the lysine side chain of the
KM peptides) were further analyzed by ab initio
quantum chemistry. Within group III, there is little chemical
difference between the meta- and
para-trifluoromethyl substituents or between the
para-trifluorotrifluoromethylated and
para-chlorinated substituents, and the immunological
activities of the corresponding KM peptides are
all similar. However, two KM peptides exhibit
considerably less activity, one with an
ortho-trifluoromethylated substituent,
KM peptide 13, and the other with a
meta-methylated substituent, KM
peptide 16. This contrast could arise from two interrelated effects:
the steric fit of the KM peptide within the
binding site and the binding free energy thereof. The former depends
primarily on the size and flexibility of the compound, the latter on
the electrostatic and solvation properties. Although calculated
solvation energies of the substituents failed to correspond to the OD,
the distance measured between the amidal nitrogen atom and the farthest
halogen correlates directly, if weakly, with binding activity, as
measured by OD (Fig. 1, N-X; R2 = 0.61, excluding
nonhalogenated compounds). However, when we considered the distance
from N to the farthest atomic surface of all the molecules, we found no
correlation, except perhaps a minimum size criterion (Fig. 1, N-A).
Putting all the data together, we can propose a qualitative list of
features that correlate with high binding affinity to the Ab pocket:
the pocket favors KM peptides with a halide or
methyl halide in the meta or para position on the
phenyl ring of the lysine substituent, KM
peptides that do not contain highly polar groups on the phenyl ring of
the lysine substituents, and KM peptides with a
relatively low barrier to rotation about a linkage bond in the lysine
substituent.
AMAs require the peptide backbone of the native modified proteins
Other compounds are of interest because of their unique relationship to lipoic acid. Interestingly, octanoic acid (10, group II), a precursor of lipoic acid, fails to react well with AMA. In addition, Abs reactive against malondialdehyde, a byproduct of arachidonic acid metabolism, and 4-hydroxy-2-nonenal also failed to react with KM peptides (data not shown) (28, 29, 30).
To ascertain features of the molecule that may be involved in tolerance
breakdown, we investigated whether a high degree of reactivity requires
the specific peptide sequence of the inner lipoyl domain of PDC-E2
(15). Six representative sera were affinity purified
against native lipoated PDC-E2 and screened for reactivity (Fig. 2). Altered peptides included peptide 1
with a D173A mutation, peptide 2 with a double K174E and E183K
mutation, and an albumin peptide. AMA failed to react with peptides in
which the amino acids flanking the lysine residue were altered, moved,
or replaced by an irrelevant amino acid/residue. Thus, in addition to
the chemical substituents, the native PDC-E2 peptide backbone is
required for reactivity.
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Collectively, AMAs of multiple PBC patients cross-reacted with
both the native lipoated peptide and select KM
peptides. However, individual patients have a heterogeneous population
of AMAs that react with multiple KM peptides, as
shown in Table I by the responses
of aliquots of a single serum from individual patients. For example,
patient 1 responded to all four of the selected peptides although at
different levels. Moreover, the pattern of reactivity is relatively
different for each individual patient as seen by comparing patients 6
and 8. In addition, reactivity of each compound produces varied levels
of AMA as shown by AMA dilutions for each patient, which fail to
correspond to the overall level of reactivity for each individual
patient tested.
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In light of the data shown above, we propose that
KM peptides may not only effectively mimic lipoic
acid in vitro, but that exposure to environmental organics similar to
halothane may also lead to modification in vivo of lipoic acid (Fig. 3). To test this theory, we synthesized
one of the possible products of halothane metabolism,
trifluroacetaldehyde, and coupled this with the reduced bis-mercaptan
form of lipoic acid to give
R/S-5-(2-trifluoromethyl-[1,3]dithian-4-yl)pentanoic acid. We found
the KM peptide of this compound to be highly AMA
reactive (Fig. 4), meaning this compound,
with a triflouromethyl group, fits the quantitative structure-activity
relationships profile of long, flexible xenobiotically
halogenated agent. Therefore, it supports our etiological hypothesis
that patient AMAs showed a greater affinity to the
KX peptide than lipoic acid and an even
greater reactivity than that of all KM peptides
tested previously (refer to Fig. 3).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several interesting elements were revealed in this study, including our observation that affinity purified autoantisera reacted with multiple modified organic structures. The fact that reactivity was significantly greater than that of the native peptide in several cases (groups III and IV) raises two important points. More than one form of a xenobiotic may be able to generate an autoantigenic determinant that would culminate in the production of anti-PDC-E2 Abs. Also, these results provide strong evidence for a structural requirement of PDC-E2 recognition with respect to the lysine-bound side-chain moiety.
The computational studies have revealed properties that both correlate with activity, namely size and flexibility (see below), and that do not correlate with activity, most notably hydrophobicity. In the absence of structural and biochemical observation of the interactions between the substituent, the peptide and the Ab, the structure vs activity relationships of the substituents can provide important clues toward those interactions. By quantum chemical calculations, we have pursued the idea that the rather surprising difference in binding between the ortho-trifluoromethyl-substituted KM peptide 13 and the meta-methyl substituted KM peptide 16—an apparent dependence on halogens for xenobiotic influence—could be due to an electron-withdrawing effect of the halogens in the lysine substituent of KM peptide 13 that allows greater torsional freedom in the C-C single bond linking the phenyl ring to the alkene by reducing the degree of resonance-induced double-bond character in this bond. This idea is reinforced by quantum chemical geometry optimization of the lysine substituent of the most highly active KM peptide 18, where the linkage contains no double bond and where, unlike all of the alkenes, the ring of the lysine substituent optimized to an orientation perpendicular to the amidal group. Thus, we calculated the torsional barriers of the C-C bond nearest the ring; in solvent, the barrier was indeed lowered slightly by the presence of the halogens, by 0.5 (substituent on KM peptide 13) and 3.6 kcal/mol (substituent on KM peptide 14), relative to that of substituent 16, which showed the highest torsion barrier (5.73 kcal/mol). Finally, we noted that although substituent 17 is both long (N-A = 10.4) and flexible (with a torsion barrier of 3.3 kcal/mol lower than substituent 16), the KM peptide shows poor binding. Because the NO2 group of KM peptide 17 is much more polar than the CF3 and CH3 groups, we suggest that unfavorable electrostatics in the binding pocket rule out KM peptide 17. Therefore, the recognition of the modified E2 peptides required at least three physical criteria be met: 1) a halide or methyl halide be present in the meta or para position on the phenyl ring; 2) phenyl substituents must have low polarity; and 3) there must be a relatively low barrier to rotation around the phenyl linkage. However, it is not necessary for this side chain to be a fatty acid such as lipoic acid. Moreover, it is of great interest to note that substitution of the lipoic acid side chain with a phenyl ring containing a halide actually increased Ab binding. Therefore, one must consider the possibility that a structure similar to that found in groups III and IV initiated the response to PDC-E2 in patients.
Our working model of the chain of events that lead to breakdown of
self-tolerance is outlined in Fig. 3, in which the lipoyl moiety of
PDC-E2 reacts via its reduced bis-mercaptan form with long, flexible,
halogenated xenobiotic agents to yield the KX
peptide conjugate. In vivo, both oxidative and reductive metabolism of
halothane can lead to active xenobiotics that we and others postulate
form cyclic KX adducts (see Fig. 3)
(33). Determinant spreading and affinity maturation may
lead to change in the Ab repertoire. The observation that sera showed
as much or greater reactivity with the xenobiotically modified peptide
as with the native lipoyl domain has provided strong evidence that
xenobiotic modification could be the initiating event of PBC.
The liver is an important organ for metabolism/degradation for xenobiotics and an altered immune response (34). A large number of chemicals, including halogenated compounds, are detoxified through the liver and secreted in the bile. Hence, exposure to an agent that would uniquely modify the mitochondrial Ags within biliary epithelium could lead to a breakdown of tolerance and induction of a self-reactive response that is target specific. Moreover, there is evidence based on in vivo studies in guinea pigs exposed to halothane, that Kupffer cells carry trifluoroacetylated protein adducts (35); these protein adducts are not found in other organs, including hilar lymph nodes. This provides evidence that the generation of autoreactivity to the protein adducts is likely a local liver response. We should also note the possibility that xenobiotics have other immunotoxic potential, including a selective stimulation or inhibition of components of the immune system (6). Such effects, of course, would be independent of any modification of autoantigens.
We postulate that people genetically predisposed to PBC have inherited such predisposition based on either the cytochrome p450 pathway or another metabolic process responsible for degrading halogenated compounds. A large number of common pharmaceuticals, such as diuretic agents, are halogenated structures. In fact, halogens are common substituents in pharmaceuticals that modulate binding activity and metabolism. In addition, there are large numbers of detergents commonly used at home and commercially that are rich in halogenated derivatives. Estrogens have already been shown to modulate the expression of many liver-metabolic pathways and may explain the preponderance of women with PBC. Finally, the presence of primarily small bile duct destruction may be reflective of the local mucosal immune response, which is more prominent on epithelial surfaces (36). Indeed, PBC is often referred to as an epithelitis with involvement not only of bile ducts, but also of salivary glands.
Finally, one should discuss the role of xenobiotics in the induction of AMA and their relevance in the disease process. The destructive phase of PBC appears to be mediated locally by an intense mucosal immune response. High titer IgA PDC-E2-specific autoantibodies have been found in the bile, saliva, and even the urine of patients with PBC (37, 38). Moreover, there is unique staining of the cell surface of both bile duct and salivary gland epithelial cells with mAbs to PDC-E2 that colocalizes with IgA transcytosing the cell, suggesting that these may be IgA-PDC-E2 complexes (38, 39). Furthermore, the precursor frequency of PDC-E2-specific CD4 T cells is 100- to 150-fold higher in liver than in the peripheral blood of patients with PBC (17). In addition, we have recently observed the presence of PDC-E2 peptide-specific MHC class I-restricted CD8 T cells only in patients with PBC. Chronic intense PDC-E2-specific responses, either individually or in concert, contribute to the overall pathology. It is ironic that the liver, known to contribute to the induction of tolerance, is precisely the organ that is the target of a central breakdown in tolerance. Toward that end, the influence of these xenobiotics on cellular immune responses in PBC is currently underway. Defining the precise sequence and the molecular basis by which xenobiotics initiate the cascade of autoimmune responses is the next challenge for understanding the etiology and pathogenesis of PBC.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. M. Eric Gershwin, Division of Rheumatology, Allergy, and Clinical Immunology, University of California School of Medicine, TB 192, 1 Shields Avenue, Davis, CA 95616. E-mail address: megershwin{at}ucdavis.edu
3 Abbreviations used in this paper: PBC, primary biliary cirrhosis; AMA, antimitochondrial Ab; TFA, trifluoracetyl; PDC-E2, E2 component of pyruvate dehydrogenase; DMF, N, N-dimethyl formamide; NHS, N-hydroxysuccinimide; NMR, nuclear magnetic resonance; KM, modified lysine.
Received for publication May 15, 2001. Accepted for publication July 5, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-ketoacid dehydrogenase complex. J. Immunol. 142:3815.[Abstract]
-ketoglutarate dehydrogenase and pyruvate dehydrogenase: reaction of lipoic acid with 4-hydroxy-2-nonenal. Biochemistry 37:15835.[Medline]
-Lipoic acid in liver metabolism and disease. Free Radical Biol. Med. 24:1023.[Medline]
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) delivers a cyclic
adduct (KX).
