Inhibition of a-Ketoglutarate Dehydrogenase Activity by a Distinct Population of Autoantibodies Recognizing Dihydrolipoamide Succinyltransferase in Primary Biliary Cirrhosis DAVIDR. FREGEAU,' THOMAS PRINDMLLE,2 ROSS L. COPPEL,3 MARSHALL KAPLAN,4 E. ROLLAND DICKSON5 AND M. ERICGERSHWIN' Divisions of 'Clinical Immunology and 'Gastroenterology, Department of Internal Medicine, University of California at Davis, Davis, California 9561 6; 'Walter and Eliza Hall Institute for Medical Research, Royal Melbourne Hospital, Melbourne, Victoria, 3050, Australia; 4Division of Gastroenterology, Department of Medicine, New England Medical Center Hospital, Tufts University, Boston, Massachusetts 02111; and 5Department of Gastroenterology and Internal Medicine, Mayo Clinic, Rochester, Minnesota 55905

Sera from patients with primary biliary cirrhosis contain autoantibodies that recognize mitochondrial proteins. Five of the target autoantigens have now been identified as enzymes of three related multienzyme complexes: the pyruvate dehydrogenase complex, the branched chain a-ketoacid dehydrogenase complex and the a-ketoglutarate dehydrogenase complex. Each complex consists of component enzymes designated El, E2 and E3. In this report, we confirm that primary biliary cirrhosis sera react with dihydrolipoamide succinyltransferase, the E2 component of a-ketoglutarate dehydrogenase complex. Seventy-three of 188 (39%) primary biliary cirrhosis sera reacted with a-ketoglutarate dehydrogenase complex-E2 when immunoblotted against purified a-ketoglutarate dehydrogenase complex; one of these sera also reacted with the E l component. In addition, primary biliary cirrhosis sera possessing a-ketoglutarate dehydrogenase complexE2 reactivity specifically inhibited enzyme function of a-ketoglutarate dehydrogenase complex. Enzyme activity was not affected by primary biliary cirrhosis sera that contained autoantibodies to pyruvate dehydrogenase complex-E2 and/or branched chain a-ketoacid dehydrogenase complex-E2, which lacked a-ketoglutarate dehydrogenase complex-E2 reactivity. Furthermore, affinity-purified primary biliary cirrhosis sera against a-ketoglutarate dehydrogenase complex-E2 inhibited only a-ketoglutarate

Received November 13, 1989;accepted January 17, 1990. This work was supported by NIH grant DK 39588. David R. Fregeau was supported by a Morton Levitt Research Award from the School of Medicine, University of California at Davis. Address reprint requests to: M. Eric Gershwin, M.D., Division of Rheumatology, Allergy and Clinical Immunology, University of California, TB 192, Davis, CA 95616. 3111120982

dehydrogenase complex activity but did not alter enzyme activity of either pyruvate dehydrogenase complex or branched chain a-ketoacid dehydrogenase complex. Finally, a-ketoglutarate dehydrogenase complex-E2 specific affinity-purified antisera did not react on immunoblot with any component enzymes of pyruvate dehydrogenase complex or branched chain a-ketoacid dehydrogenase complex. These data demonstrate that the E2 component of a-ketoglutarate dehydrogenase complex is recognized by a distinct population of autoantibodies separate from autoantibodies that recognize pyruvate dehydrogenase complex-E2 or branched chain a-ketoacid dehydrogenase complex-E2. Our data further suggest that these autoantibodies are directed toward a functional 1990;11:975-981.) domain of this enzyme. (HEPATOLOGY

The major serological abnormality in patients with primary biliary cirrhosis (PBC) is the presence of autoantibodies that recognize three to five inner mitochondrial membrane proteins (1-6). These antigens have now been identified as enzymes of the pyruvate dehydrogenase complex (PDC), the branched chain aketoacid dehydrogenase complex (BCKDC) and the a-ketoglutarate dehydrogenase complex (KGDC) (7-16). These multienzyme complexes are functionally similar in that they each oxidatively decarboxylate their substrate and are structurally homologous in that each is composed of component enzymes designated E l , E2 and E3 (17). Three of the identified antigens recognized by antimitochondrial antibodies (AMA)are enzymes of PDC. The 74 kD E2 component (dihydrolipoamide acetyltransferase) has been identified as the major PBC autoantigen because it is recognized by more than 90% of PBC patient sera (7-12). The E2 component contains a lipoic acid cofactor that is covalently bound to a lysine

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A

TABLE 1. Reactivity of sera with the purified a-KGDC“

B C

No. positivebc/total Sera

-El

5848 --

-E3 -E2

PBC Afiinity sera against KGDC-EZd PSC CAH SLE Healthy volunteers

Dilution

E l (113 kD)

E2 (48 kD)

1: 1000 1:200 1: 10

11188 11188

19/188 731188

015

5f 5

1: 100 1: 100 1: 100 1: 100

0115 0115 0115 0115

0115 0115 Of15 0115

”Reactivity assessed by immunoblotting as described in “Materials and Methods.” bReactivityafter 12 to 14 h r autoradiographical exposure. ‘None of the sera reacted with the E3 component of KGDC. dAMinity-purified antisera were prepared with nitrocelluloseimmobilized antigens as described in “Materials and Methods.” PSC = progressive sclerosing cholangitis, SLE = systemic lupus erythematosus.

we confirm this finding and demonstrate that these KGDC-E2-specific AMA are a distinct population of autoantibodies that show no cross-reactivity with any of the other identified mitochondrial autoantigens. In addition, we also demonstrate specific inhibition of KGDC by KGDC-E2-specific autoantibodies. FIG.1.Resolution of the a-ketoglutarate dehydrogenase complex by SDS-PAGE. In lane A, four protein bands are detected by Coomassie blue staining. KGDC-El at 96 kD, KGDC-EZ at 48 kD and KGDC-E3 at 58 kD;the 90-kD protein is believed to be a breakdown product of KGDC-El. In lane B, the complex is probed with the single PBC serum that reacts with both the E l and E2 components of KGDC. In lane C, the complex is probed with a PBC serum that reacts with KGDC-E2 prepared with 2-mercaptoethanol (C).This same serum demonstrates markedly diminished reactivity (lane D ) when probed as against purified a-ketoglutarate dehydrogenase complex prepared without 2-mercaptoethanol (laneD).All samples were resolved on a 12-cm 10% resolving gel.

residue; AMA that recognize PDC-E2 are directed toward the lipoate-binding domain of this enzyme (9, 18). A corollary to this finding is the observation that sera from patients with PBC inhibit PDC enzyme function (10). All sera that react with PDC-E2 also recognize a second PDC component, protein X, because protein X and PDC-E2 share cross-reactive epitopeb) (15). More recently, we have demonstrated that PBC sera also contain a population of A M A that recognize the a chain of PDC-El and have shown that these autoantibodies also inhibit enzyme activity of PDC (16). A fourth autoantigen recognized by AMA in PBC sera is dihydrolipoamide acyltransferase, the E2 component of BCKDC (12,13).We have shown that these BCKDCE2-specific AMA are a separate population of autoantibodies that do not cross-react with PDC-E2 and are able to inhibit the enzyme function of BCKDC (14). Finally, the E2 component of KGDC (dihydrolipoamide succinyltransferase) has been reported to be an autoantigen recognized by PBC sera (12). In this report

MATERLALS AND METHODS Sem.Serum samples from 188 PBC patients (100 patients from the Mayo Clinic, 22 patients from the Walter and Eliza Hall Institute for Medical Research, 11 patients from the University of California and 55 patients from Tufts University) were used in this study. All patients had wellestablished clinical and laboratory diagnoses of PBC (19) and represented all histological stages of PBC (stage I, 10 patients; stage 11, 42 patients; stage 111, 48 patients and stage IV,88 patients). As controls, sera from 15 patients with progressive sclerosing cholangitis (PSC), 15 patients with CAH, 15 patients with systemic lupus erythematosus (SLE) and 15 healthy volunteers were used. Purification of the a-hk-toacid Dehydrogenate Complexes.

KGDC and PDC were isolated from bovine kidney cortex according to the method of Roche and Cate (20). Mitochondria were isolated by differential centrifugation followed by polyethylene glycol precipitation of PDC and KGDC. KGDC was separated from PDC by a modified polyethylene glycol fractionation and then pelleted through a three-step sucrose gradient. BCKDC was also prepared from bovine kidney cortex by standard methods (21). PDC and BCKDC used in this study are the same preparations described in recent reports from our laboratory (14, 16). Absorption a n d m n i t y Purification of KGDCE2Antibodies from PBC Sera. Absorption and affinity purification were

performed as described (14). Purified KGDC was resolved by SDS-PAGE (1mg KGDCI12 cm lane) on a 10%gel. SDS-PAGE was performed on a resolving gel 27 cm long to adequately resolve KGDC-E2 from any contaminating protein X because they have similar molecular weights. After blotting to nitrocellulose, the protein bands were visualized with Ponceau S stain and the KGDC-E2 band was excised. The strips were blocked with 1%(wt/vol) BSA in PBS and incubated with 1 ml of five selected PBC sera diluted 1: 100 in PBS-Tween for 1

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hr at room temperature. Each strip was washed with 2L of PBS-Tween (5 x 400 ml) followed by elution of bound AMA with 0.7 ml of 0.1 moVL glycine-HC1 (pH 2.5), 20 mmol/L MgCl,, 50 mmoVL KC1 and 0.5% Tween 20 for 15 rnin at room temperature. The eluates were immediately adjusted to pH 7.4 by addition of 0.3 ml 0.5 moVL Na,PO,. This procedure was repeated five times for each serum. The five eluates from each serum were pooled and concentrated to 50 p.1 with an Amicon 30 concentrator (Amicon Corporation, Danvers, MA) and brought to a final volume of 0.5 ml with PBS. SDS-PAGE and Zmmunoblotting. SDS-PAGE was performed on gel slabs 1.5 mm thick with a 4.75% stacking gel and a 10% resolving gel (22). Purified a-ketoacid dehydrogenase complexes (2 kg/lane) and beef heart mitochondria (40p.g/gel lane) were diluted in sample buffer (125 mmol/L Tris-HC1, pH 6.8, containing 4% SDS, 20% glycerol) with or without 2-mercaptoethanol and boiled for 5 min. Gels were run a t 30 mA at room temperature. Separated proteins were either stained for protein with Coomassie Brilliant Blue R or transferred electrophoretically to nitrocellulose sheets at 30 V for 12 t.o 16 hr (23). Immunoblotting was performed at room temperature and serum dilutions were made in 3% milk powder in PBS, pH 7.4,and all washes were performed with PBS and 0.5% Tween 20 (PBS-Tween). Nitrocellulose strips were blocked in PBS for 15 min and then probed for 1hr with the appropriate serum. Affinity-purified sera to KGDC-E2 were diluted 1: 10, control sera were diluted 1 : 100, and PBC sera were diluted 1: 1,000. After three 10-min washes, the strips were incubated for 1 hr with 1 ml of '261-sheepantihuman Ig (0.1 pCi/ml; Amersham, Arlington Heights, IL). After five washes with PBS-Tween, the strips were air-dried and exposed to x-ray film for 12-16 hr. Enzyme Inhibition Assay. Enzymatic activity of the aketoacid dehydrogenase complexes was assayed by monitoring the reduction of NAD' at 340 nm (24).Inhibition of enzyme activity was assayed using 18 PBC sera, eight control sera (three CAH sera, three SLE sera and two normal sera) and five affinity-purified PBC sera specific for the E2 component of KGDC. Ten microliters of purified enzyme preparation (containing 1.7 pg PDC, 4 pg KGDC or 10 p.g BCKDC) was incubated with 100 p.1 of serum diluted 1:lOO in PBS and incubated at 30" C for 5 min. Affinity-purified sera were used undiluted. One hundred microliters of this mixture was then added to 900 p1 of reaction mixture that had been preequilibrated t o 30" C so that the final mixture contained 0.5 mmol/L coenzyme A, 0.13 mmol/L cysteine hydrochloride, 0.1 mmol/L MgCl,, 1 mmol/L NAD', 0.2 mmoVL thiamin pyrophosphate and 0.1 mmol/L of the appropriate substrate in 50 mmoVL potassium phosphate buffer. The increase in absorbance was measured over 1min after a lag time of 10 sec, and the initial reaction rate over 20 sec was expressed as percent values of the controls (no serum) to compare enzyme activities. The amount of enzyme used was adjusted to give a rate of approximately 0.1 dA/min. Isotype Determination of KGDC-Esspecifie AMA. Isotype determination was performed using procedures and reagents as previously described from our laboratory (25). Briefly, purified a-KGDC was resolved by SDS-PAGE, transferred to nitrocellulose and then probed with various PBC sera. ANIA isotypes were detected using murine monoclonal antibodies followed by '261-labeled antimurine antibodies. After exposure to x-ray film for 12 hr, the bands were scanned with a laser densitometer and peak heights were used to determine the relative percentages of KGDC-E2-specific AMA isotypes. This method of laser density quantitation is more

A B C D E

-74

-i3

_._

-48 -41

FIG. 2. In lane A, a PBC serum diluted 1:1,000 reacts with KGDC-E2 when probed against purified KGDC. In lane B, the same serum shows reactivity with PDC-EQ(74 kD) and protein X (54 kD) and also reacts with a 48-kD protein when probed against bovine heart mitochondria. After absorption with nitrocellulose-immobilized KGDC-E2, reactivity against the 48-kD protein of bovine heart mitochondriais diminished, whereas reactivity with PDC-E2 (74 kD) and protein X (54 kD) is virtually unchanged (lane C). In lane 0, a PBC serum diluted 1:200 reacts with KGDC-E2 when probed against purified KGDC, but no corresponding protein band is detected when this serum is probed against bovine heart mitochondria (lane E ) . The 52-kDprotein and 41-kDprotein in lane E are identifiedas BCKDC-E2 and PDC-Ela respectively. Samples were resolved on a 27-cm 10% resolving gel.

sensitive with less variance than previous attempts at sera titration (25).

RESULTS Zmmunoblotting. Nineteen of the 188 PBC sera diluted 1 :1,000 reacted with the 48 kD dihydrolipoamide succinyltransferase when probed against purified aKGDC (Table 1). One of these sera also showed reactivity with the E 1component (Table 1, Fig. 1).These 19 sera also showed reactivity t o a 48-kD band when probed against beef heart mitochondria (Fig. 2). In contrast, when purified KGDC was probed with PBC sera at a lower dilution (1:ZOO), an additional 54 sera reacted with KGDC-E2 (Table 1). Reactivity t o a 48-kD band using purified beef heart mitochondria could n o t be demonstrated with these 54 sera (Fig. 2). None of the 188 PBC sera reacted with the E3 component of KGDC. In addition, immunoblot reactivity was decreased when the purified KGDC was not treated with

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FREGEAU E T AL.

HEPATOLOGY

TABLE2. Inhibition of KGDC activity by sera from patients with PBC Reactivity PDC-E2

PDC-Elu

BCKDC-E2

+ + + + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + + +

+ + + + +

6‘ 24‘ 36 71‘ 109 115 122 129 131 13Ef 1668 173 174 1768 213 215 W24 W27

%

-

Inhibitiond

KGDC E2

0 0 64.5 0 46.2 86.3 79.2 34.3 65.1 0 0 100 92.8 0 34.5 20.0 95.8 100

-

-

+ + + + + + + + +

~

_

~

~

_

~

“Sera were diluted 1:100. *Control sera (CAH, SLE, and healthy volunteers) did not inhibit KGDC enzyme activity. ‘Reactivity as assessed by immunoblotting against purified a-ketoacid dehydrogenase enzyme complexes. dThe percent inhibition was correlated with the titer of KGDC-E2-specific AMA as assessed by immunoblotting and is similar to our previous report regarding BCKDC inhibition by PBC sera (14). ‘These sera previously shown to inhibit (no. 6, 62.5%; no. 24, 29.2%; no. 71, 100%)PDC enzyme function (10). P h i s serum previously shown to inhibit BCKDC enzyme function by 24.1% (14). ‘These sera previously shown to inhibit (no. 166, 31.9%;no. 176, 40.1%) PDC enzyme activity by AMA populations recognizing the E2 and E l a components of PDC (16).

TABLE3. Inhibition of KGDC activity but not BCKDC or PDC activity by affinity-purified PBC sera specific for KGDC-E2

TABLE 4. Isotype distribution of KGDC-E2-specific A M A

Percent inhibitionb Serum“ 109 115 173 174 W24

KGDC

BCKDC

PDC

37.3 24.6 56.2 47.5 31.8

0 0 0 0 0

0 0 0 0 0

“Control sera (CAH, SLE and healthy volunteers) did not inhibit enzyme activity of any of the complexes. *Mean of duplicate determinations that did not differ by more than 10%.

2-mercaptoethanol before electrophoresis (Fig. 1). Finally, none of the 60 control sera demonstrated reactivity to any of the KGDC component enzymes, even at a 1: 100 dilution. Absorption and m n i t y Purification. PBC sera absorbed with KGDC-E2 immobilized to nitrocellulose showed diminished reactivity to KGDC-E2 as assessed by immunoblotting against beef heart mitochondria (Fig. 2). Reactivity to the remaining mitochondrial antigens was virtually unchanged. Affinity-purified PBC sera specific for KGDC-E2 reacted only with a 48-kD protein when probed against beef heart mitochondria

36 109 115 122 129 131 173 174 W24 w27

27 5 7 50 38 60 19 21 20 28

26 0 0 1 0 12 19 17 17 8

17 6 1 9 0 0 20 13 10 1

5 45 56 8 0 0 18 15 19 31

19 44 29 31 62 28 19 17 18 31

6 0 7 1 0 0 5 17 16 1

“Relative percentages determined by densitometry scanning of immunoblots as described in “Materials and Methods.”

and did not react with any of the component enzymes of PDC or BCKDC when probed against the purified complexes (Fig. 3). Enzyme Inhibition. All (12 of 12) PBC sera that reacted with KGDC-E2 were able to inhibit KGDC enzyme activity (Table 2). Six PBC sera with no KGDC-E2 reactivity did not inhibit enzyme activity; these same sera were known to be capable of inhibiting either PDC or BCKDC enzyme activity (Table 2) (10,14, 16). Control sera (CAH, SLE, healthy volunteers) were not able to inhibit enzyme activity of any of the or-ketoacid complexes. All (five of five) affinity-purified

AUTOANTIBODIES TO KGDC-EP IN PBC

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FIG.3. An affinity-purified PBC serum specific for KGDC-E2 reacts with only the E2 component of purified KGDC (lane A) but does not react with any of the component enzymes of purified PDC (lane B)or purified BCKDC (lane C). The affinity-purifiedserum reacts with a 48-kD protein when probed against bovine heart mitochondria (lane

D).

antisera specific for KGDC-E2 inhibited KGDC enzyme activity but did not affect activity of either PDC or BCKDC (Table 3). Isotype Distribution of KGDC-E2-Specific AMA. All PBC sera tested contained IgG3 and IgM AMA specific for KGDC-E2 (Table 4). Three of 10 sera (no. 122, 129 and 131) contained predominantly IgM and IgG3 AMA while one serum (W27) contained IgG2 AMA in addition to IgG3 and IgM AMA.Two of 10 sera (no. 109 and 115) possessed almost exclusively IgG2 and IgG3 AMA. The remaining four sera exhibited fairly equal AMA distribution among all isotypes examined. DISCUSSION

Mammalian KGDC is a multienzyme complex located within the mitochondrion and catalyzes the oxidative decarboxylation of a-ketoglutarate to succinyl CoA within the tricarboqlic acid cycle. KGDC is structurally and functionally similar to two other mitochondrial enzyme complexes, PDC and BCKDC (17,26). All three complexes are composed of component enzymes designated E l , E2 and E3. The E2 components of all three complexes have been shown to be autoantigens recognized by AMA in PBC sera. Our data confirm that the E2 component of KGDC, dihydrolipoamide succinyltransferase, is an autoantigen of PBC. Ten percent (19 of 188)

979

of PBC sera showed reactivity to KGDC-E2 when probed against bovine heart mitochondria. However, when PBC sera were probed against purified KGDC, an additional 54 sera with low-titer KGDC-E2 reactivity were detected, demonstrating the increased sensitivity when purified enzyme preparations are used. Overall, 39% of PBC sera reacted with KGDC-E2, which is lower than the 72% of PBC sera that reacted with KGDC-E2 noted by Fussey et al. (12). Reactivity to KGDC-E2 was greatly diminished in the absence of 2-mercaptoethanol. This phenomenon has previously been described in connection with the mitochondrial autoantigens (27-29). The most reasonable explanation for this observation is that the AMA epitope is only exposed by the reduction of cystine. The E2 components of PDC, BCKDC and KGDC, share significant amino acid homology (12). Interestingly, the present experiments, combined with previous data reported from our laboratory (10, 14, 161, demonstrate that the autoantibodies directed against the E2 enzymes are distinct AMA populations that recognize nonhomologous determinantb). Enzyme function of the a-ketoacid dehydrogenase complexes is inhibited only by PBC sera that contain A M A that recognize the E2 component of the respective complex. In this report, inhibition of KGDC activity was demonstrated with PBC sera containing KGDC-E2 AMA. Other PBC sera that lacked reactivity to KGDC-E2 did not affect the enzyme function of KGDC, despite the fact, as we previously reported, that these sera inhibited either PDC or BCKDC function (10, 14). In addition, affinity-purified PBC sera specific for each of the E2 enzymes do not exhibit cross-reactivity among all three E2 enzymes. Thus, in the present study, KGDC-E2-specific antisera did not react with either PDC-E2 or BCKDC-E2, nor did they affect enzyme function of PDC or BCKDC. We have reported similar findings using BCKDC-E2-specific affinity antisera (14). Finally, the most compelling evidence that the E2 enzymes are recognized by separate populations of AMA is the fact that many PBC sera do not react with all three E2 components (12-14, 16, 18). Rather, there is a variety of reactivities, with some sera reacting with either one, two or all three acyltransferases. We have previously reported a predominance of IgM and IgG3 AMA against the E2 components of PBC and BCKDC (25). Although all the PBC sera tested in this study contained IgG3 and IgM autoantibodies specific for KGDC-E2, the predominance of these two isotypes was only evident in 3 of 10 sera. The remaining seven PBC sera showed various distributions of AMA isotypes. The significance of this data is not clear. The development of AMA isotypes other than IgM and IgG3 may perhaps signal the progression of disease. Additional prospective studies are needed to address this issue. Although our data demonstrate that the E2 enzymes of PDC, BCKDC and KGDC are recognized by distinct populations of AMA, others have reported that the M2 autoantigens are recognized by cross-reacting AMA (27). Most recently, Flannery and colleagues (30) reported

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FREGEAU ET AL.

cross-reactivity between three M2 antigens with molecular weights of 70, 50 and 40 kD. The 70-kD antigen was identified as PDC-E2, whereas the 50-kD antigen was identified as protein X, an enzyme unique to PDC. We and others have previously reported that PBC sera that recognize PDC-E2 also recognize protein X, and we have shown that these two enzymes share cross-reactive epitope(s) (8, 15). In Flannery’s report, the 40-kD protein, although reported to be an M2 antigen, was not further identified. We believe, based on our data, that this antigen is neither BCKDC-E2 nor KGDC-E2. We have recently identified a protein of similar molecular weight as an autoantigen of PBC, namely the 41-kD a-chain of PDC-El. However, our experimental evidence indicates that this antigen is not cross-reactive with the E2 enzymes (16). The interlaboratory variation in the reported molecular weights of the mitochondrial autoantigens makes it difficult to compare results and speculate on the identity of the 40-kD protein. This is a continuing problem and we believe it is imperative that, in future reports, the mitochondrial autoantigens not be identified solely on the basis of molecular weight or the now-obsolete M2 nomenclature. Rather, we would suggest that reactivity to specific enzymes be identified by immunoblotting against either purified enzyme preparations or recombinant mitochondrial proteins. The role of the mitochondrial autoantigens, A M A or both, in the pathogenesis or pathologicalfindings of PBC is yet to be defined. Why AMA are directed toward enzymes of three structurally related macromolecular complexes also remains unanswered. All three aketoacid dehydrogenase complexes share an identical component enzyme, dihydrolipoamide dehydrogenase (E3) (17). We have previously hypothesized that chronic infection with a pathogen bearing E3 on its membrane may cause activation of T cell clones that, in turn, could activate B cells specific for other components of these multimeric complexes, namely the E2 enzymes (14,15). Given the homology of the E2 components (and possibly protein X), it is also possible that there may be a T cell epitope common to these mitochondrial autoantigens. Activation of these T cell clones may be due to immune dysregulation; alternatively exposure to exogenous antigen mimicking this epitope could also cause stimulation of E2-reactive B cells. The three acyltransferases and protein X use a covalently bound lipoic acid moiety as a necessary cofactor. We have previously shown that the lipoic domain of PDC contains the epitope(s) recognized by AMA (9) and, given the inhibition data in this present study and in previous reports (10, 14), we predict that the AMA epitope of BCKDC-E2, KGDC-E2 and protein X will also be localized to this region. Lipoic acid has been shown to augment, both in uitro and in viuo, antibody response (31,321, and it has, therefore, been suggested that lipoic acid may play a role in the immunogenicity of these autoantigens (33). However, it has been reported that PBC sera do not react with the H protein of the glycine cleavage system (181, the only other protein known to contain lipoic acid. In addition, we have recently demonstrated that PBC sera contain

HEPATOLOGY

AMA that recognize the a-chain of PDC-El, an enzyme that does not contain lipoic acid (16). Clearly, factors other than the bound lipoate must be involved in the immunogenicity of the mitochondrial antigens. The lipoate-binding domains of the E2 enzymes are exposed, hydrophilic and highly mobile, and these are characteristics that are believed to contribute to antigenicity (34). Interestingly, the information being accumulated on the mitochondrial autoantigens is similar to data reported for other autoimmune diseases such as polymyositis, scleroderma and SLE (35).Many of the autoantigens are also component enzymes of large aggregates, whereas the autoantibodies directed against these intracellular enzymes appear to recognize functional domains because they are able to inhibit enzyme function. Acknowledgments: We thank Dr. Thomas E. Roche for providing the purified KGDC, Janet Harrison for the use of her laboratory and equipment, Nikki Rojo for her preparation of this manuscript and Dr. Paul Davis for his helpful comments. REFERENCES 1. Walker JG, Doniach D, Roitt IM, Sherlock S.Serological tests in diagnosis of primary biliary cirrhosis. Lancet 1965;1:827-831. 2. Berg PA, Doniach D, Roitt IM. Mitochondrial antibodies in primary biliary cirrhosis. I. Localization of the antigen to mitochondrial membranes. J Exp Med 1967;126:277-290. 3. Berg PA, Muscatello U, Horne RW, Roitt IM, Doniach D. Mitochondrial antibodies in primary billary cirrhosis. 11. The complement fixing antigen as a component of mitochondrial inner membranes. Br J Exp Pathol 1969;50:200-208. 4. Lindenborn-Fotinos J, Baum H, Berg PA. Mitochondrial autoantibodies in primary biliary cirrhosis: species and nonspecies specific 1985;5:763-769. determinants of the M2 antigen. HEPATOLOGY 5. Ishii H, Saifuku K, Namihisa T. Multiplicity of mitochondrial inner membrane antigens from beef heart with antimitochondrial antibodies in sera of patients with primary biliary cirrhosis. Immunol Lett 1985;9:325-330. 6. Frazer IH, Mackay IR, Jordan TW, Whittingham S, Marzuki S. Reactivity of anti-mitochondrial autoantibodies in primary biliary cirrhosis: definition of two novel mitochondrial polypeptide autoantigens. J Immunol 1985;135:1739-1745. 7. Gershwin ME, Ahmed A, Danner D, Fregeau D, Van de Water J , h u n g P, Coppel R. Identification of the mitochondrial enzymes that react with autoantibodies from patients with primary biliary cirrhosis (PBC) [Abstractl. FASEB J 1988;2(3334):A869. 8. Yeaman SJ,Danner DJ, Mutimer DJ, Fussey SPM, James OFW, Bassendine MF. Primary biliarg cirrhosis: identification of two w ’ o r M2 mitochondrial autoantigens. Lancet 1988;1:1067-1070. 9. Van de Water J , Gershwin ME, h u n g P, Ansari A, Coppel RL. The autoepitope of the 74-kD mitochondrial autoantigen of primary biliary cirrhosis corresponds to the functional site of dihydrolipoamide acetyltransferase. J Exp Med 1988;167:1791-1799. 10. Van de Water J , Fregeau D, Davis P, Ansari A, Danner D, Leung P, Coppel R, et al. Autoantibodies of primary biliary cirrhosis recognize dihydrolipoamide acetyltransferase and inhibit enzyme function. J Immunol 1988;141:2321-2324. 11. Coppel RL, McNeilage U, Surh CD, Van de Water J, Spithiil TW, Whittingham S,Gershwin ME. Primary structure of the human M.2 mitochondrial autoantigen of primary biliary cirrhosis: dihydrolipoamide acetyltransferase. Proc Natl Acad Sci USA 1988;85: 7317-7321. 12. Fussey SPM, Guest JR,James QFW, Bassendine MF, Yeaman SJ. Identification and analysis of the -or M2 autoantigens in primary biliary cirrhosis. Proc Natl Acad Sci USA 1988;85:86548658. 13. Surh CD, Danner DJ, Ahemd A, Coppel RL, Mackay IR, Dickson

Vol. 11, No. 6, 1990

AUTOANTIBODIES TO KGDC-E2 IN PBC

R, Gershwin ME. Reactivity of PBC sera with a human fetal liver cDNA clone of branched chain alpha-keto acid dehydrogenase (BCKD) dihydrolipoamide acyltransferase, the 52 kD mitochondrial autoantigen. HEPATOLOGY 1988;9:63-68. 14. Fregeau DR, Davis PA, Danner DJ, Ansari A, Coppel RL, Dickson ER, Gershwin ME. Antimitochondrial antibodies of primary biliary cirrhosis recognize dihydrolipoamide acyltransferase and inhibit enzyme function of the branched chain a-ketoacid dehydrogenase complex. J Immunol 1989;142:3815-3820. 15. Surh CD, Toche TE, Danner DJ, Ansari A, Coppel RL, Dickson R, Gershwin ME. Antimitochondrial autoantibodies in primary biliary cirrhosis recognize cross-reactive epitope(s) on protein X and dihydrolipoamide acetyltransferase of pyruvate dehydrogenase complex. HEPATOLOGY 1989;lO:127-133. 16. Fregeau DR, Roche TE, Davis PA, Gershwin ME. Primary biliary cirrhosis: inhibition of pyruvate dehydrogenase complex activity by autoantibodies specific for the Elcv component, a non-lipoic acid containing mitochondrial enzyme. J Immunol 1990;144:16711676. 17. Yeaman SJ.The mammalian 2-oxoacid dehydrogenases: a complex family. Trends Biochem Sci 1986;11:293-297. 18. Fussey SPM, Bassendine MF, James OFW, Yeaman SJ. Characterization of the reactivity of autoantibodies in primary biliary cirrhosis. FEBS Lett 1989;246:49-53. 19. Williamson JM, Chalmers DM, Clayden AD, Dixon M-F, Ruddell WS, Losowosky MS. Primary biliary cirrhosis and chronic active hepatitis: an examination of clinical, biochemical, and histopathological features in differential diagnosis. J Clin Pathol 1985;38: 1007.1012. 20. Roche TE, Cate RL. Purification of porcine liver pyruvate dehydrogenase complex and characterization of its catalytic and regulatory properties. Arch Biochem Biophys 1977;183:664-677. 21. Lawson RKG, Cook KG, Yeaman SJ. Rapid purification of bovine kidney branched chain 2-oxoacid dehydrogenase complex containing endogenous kinase activity. FEBS Lett 1983;157:54-58. 22. Laemmli UK. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 1970;227:680-685. 23. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 1979;76: 4350-4354. 24. Perham RN, Lowe PN. Isolation and properties of the branchedchain 2-keto acid and pyruvate dehydrogenase multienzyme

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