Download Clarification of the identity of the major M2

Clinical Science (1991) 80, 451-455
451
Clarification of the identity of the major M2 autoantigen in
primary biliary cirrhosis
SHELLEY P. M. FUSSEY~, SHAUNA M. WEST2, J. GORDON UNDSAy2, C. IAN RAGAN 3,
OUYER F. W. JAMES, MARGARET F. BASSENDINE4 AND STEPHEN J. YEAMAN!
I Department of Biochemistry and Genetics and "Department of Medicine, The Medical School University of Newcastle upon Tyne
Newcastle upon Tyne, UK., 2Department of Biochemistry, University of Glasgow, Glasgow, UK:, and 3Neuroscience Research Centre
'
Merck Sharp and Dohme, Harlow, Essex, UK.
(Received 10 December 1990; accepted 15 January 1991)
SUMMARY
1. In primary biliary cirrhosis, the major M2 autoantigen, reacting with antimitochondrial antibodies in sera
from > 90% of patients, has been identified as the E2
component of the pyruvate dehydrogenase complex.
However, two recent reports suggest that alternative polypeptides may be major autoantigens.
2. The evidence that a 75 kDa subunit of complex I of
the respiratory chain is a major autoantigen (Frostell,
Mendel-Hartvig, Nelson, Totterman, Bjorkland & Ragan,
Scand. J. Immunol. 1988; 28, 157-65) is refuted. The
findings of Frostell et al. can be explained by contamination of complex I with the pyruvate dehydrogenase
complex, evidence for which is presented here.
3. Inspection of the partial amino acid sequence of
an unidentified mitochondrial autoantigen (Muno,
Kominami, Ishii, Usui, Saituku, Sakakibara & Narnihisa
Hepatology 1990; 11, 16-23) shows that it is the E1 f3~
subunit of the pyruvate dehydrogenase complex, previously identified as a major auto antigen, and not a 'new'
alternative major autoantigen.
4. These findings substantiate previous work showing
that the mitochondrial M2 auto antigens identified so far
in primary biliary cirrhosis are all polypeptide components of the pyruvate dehydrogenase complex or the
other related 2-oxo acid dehydrogenase complexes.
Key words: antimitochondrial antibodies, autoantigens,
M2 autoantigens, primary biliary cirrhosis.
Correspondence: Professor S. J. Yeaman, Department of
Biochemistry and Genetics, The Medical School, University of
Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, U.K.
Abbreviations: AMA, antimitochondrial antibodies'
BCOAOC, branched-chain 2-oxo acid dehydrogenas~
complex; OGDC, 2-oxoglutarate dehydrogenase complex; PBC, primary biliary cirrhosis; POC, pyruvate
dehydrogenase complex.
INTRODUCTION
Primary biliary cirrhosis (PBC) is a chronic cholestatic
liver disease in which progressive destruction of the intrahepatic bile ducts leads to cirrhosis and liver failure [1].
PBC is a well-characterized example of an autoimmune
disorder, the presence of PBC-specific autoantibodies in
patients' sera being noted as early as 1965 [2]. The
antigens were subsequently localized to the inner mitochondrial membrane [3], hence the term antimitochondrial antibodies (AMA). PBC-specific antigens were
termed 'M2', and the presence of 'M2' AMA was found to
be a marker for the serological diagnosis ofPBC [4].
By immunoblotting PBC sera against mitochondrial
extracts, several groups showed that the 'M2' antigen contained distinct polypeptides [5-7]. They were classified as
M2 'a-e', according to their apparent molecular mass on
SOSjPAOE [8]. The predominant antigen is the M2 'a'
antigen, a 70-75 kDa polypeptide that is recognized by
antibodies in the sera of approximately 95% of patients
with PBC [5]. This antigen has now been cloned,
sequenced [9] and identified as the E2 component of PDC
[10, 11]. PDC is one ofthree related 2-oxo acid dehydrogenase complexes found within mammalian mitochondria, the others being the 2-oxoglutarate
dehydrogenase complex (OOOC) and the branched-chain
2-oxo-acid dehydrogenase complex (BCOADC). Each
complex consists of three component enzymes: E 1, E2
452
S. P. M. Fussey et al.
and E3 [12]. Five additional components of the 2-oxo
acid dehydrogenase complexes have now been identified
as M2 autoantigens, namely the E2 components of
OGDC and BCOADC, and protein X and the E 1 a- and
El p-polypeptides of PDC [10, 13-17]. It has been
demonstrated that the major immunoreactive determinants within PDC E2 are situated in the lipoyl domains
[18, 19] and involve the covalently linked lipoyl cofactor
[20].
Although there is general agreement that the E2 component of PDC is the M2 'a' autoantigen in PBC, there
have been two recent suggestions that this may not be the
case and which require further clarification. Frostell et at.
[21] have reported that the M2 'a' antigen is the 75 kDa
subunit of complex I (NADH-CoQ reductase) of the
respiratory chain. Muno et at. [22] have recently purified
and determined the N-terminal amino acid sequence of a
36 kDa tryptic fragment apparently derived from the 70
kDa M2 'a' antigen in PBe. Inspection of the reported Nterminal sequence indicates that this protein fragment is
unrelated to the E2 polypeptide. In this communication
we report new data clarifying the identity of the major
autoantigens in PBe.
METHODS
Complex I and PDC were purified as described in [23]
and [24], respectively. Specific antisera to the 75 kDa subunit of complex I and the E2 subunit of PDC were raised
in rabbits, as previously described [25, 26]. The antiserum
against complex I was the same preparation as that used
by Frostell et at. [21].
Proteins were subjected to SDS/PAGE on 10% (w/v)
gels [27]. Unless otherwise stated, before electrophoresis
samples were boiled for 5 min at 100°C in the presence of
2% (v/v) p-mercaptoethanol. After SDS/PAGE, samples
were electrophoretically transferred to nitrocellulose [28]
and either stained-with Amido Black or immunoblotted
using the following protocol [10]. After blocking with 1%
(w/v) skimmed milk powder and washing, the nitrocellulose was incubated with either PBC sera (at dilutions
of between 1:250 and 1:2000) or specific rabbit antisera
(at a dilution of 1:1000). Detection of bound antibodies
was by use of secondary goat anti-human IgG (or antirabbit immunoglobulin) peroxidase-conjugated antibodies (Sigma) with 4-chloro-l-naphthol as substrate.
Sera from 10 individual PBC patients (three at histological stage I, three at stage II/III, four at stage IV) and
pooled PBC sera (equal portions of the sera from the 129
patients described in [29]) were analysed in this study.
Searching of protein sequence data for sequence
identity was carried out by using the lEI Pustell Sequence
Analysis Program to search the NBRF protein database.
RESULTS AND DISCUSSION
Is the 7S kDa subunit of complex I an M2 'a' antigen?
Highly purified complex I from bovine heart was
found, as expected, to be composed of multiple subunits
with a major protein component of molecular mass
approximately 75 kDa (Fig. 1, lane 2). Immunoblotting of
this complex against sera from patients with PBC indicated that a major antigenic band of molecular mass
70-75 kDa was present in this preparation (Fig. 1, lane 4).
However, close inspection of the gels indicates that the
immunogenic band recognized by PBC sera has slightly
increased mobility on SOS/PAGE compared with the 75
kOa polypeptide of complex I. On immunoblotting of
PBC sera from 10 different patients against the complex I
preparation, a positive response was not observed against
the 75 kDa subunit of complex I in any instance. However, in all cases there was recognition of the slightly
smaller polypeptide.
One possible explanation of this finding is that the
preparation of complex I is contaminated by PDC, or at
least by its E2 polypeptide. As seen in Fig. 1 (lanes 1 and
4), the apparent molecular mass of POC E2 on SOS/
PAGE is identical to that of the immunogenic polypeptide
in the preparation of complex I. Immunoblotting complex
I against POC E2-specific rabbit antisera (Fig. 1, lane 5)
indicated that the preparation was indeed contaminated
to a significant extent with POe. This conclusion is
further supported by comparing the immunoblots of PBC
sera against POC and complex I (Fig. 1, lanes 3 and 4),
both of which show an additional immunoreactive polypeptide of molecular mass approximately 50 kOa, corresponding to protein X, another auto antigen present in
Complex I
75 kOa
subunit
POC E2
POC E2
POCX
2
3
4
5
Fig. 1. Immunoblotting analysis of PDC and complex I
with PBC sera. Samples were subjected to SOS/PAGE,
transferred to nitrocellulose and either stained with
Amido Black (lanes 1 and 2) or immunoblotted (lanes
3-5), as described in the Methods section. Lane 1, POC
(15 ,ug); lane 2, complex I (18 ,ug); lane 3, PDC E2X (1
J.1g) blotted against pooled PBC sera at a dilution of
1:1000; lanes 4 and 5, complex I (each lane 9 ,ug) blotted
against pooled PBC sera diluted 1: 1000 (lane 4) and antiE2-specific rabbit antisera diluted 1: 1000 (lane 5).
Identical results were obtained on immunoblotting complex I against pooled PBC sera and sera from 10
individual patients with PBC at dilutions of between
I :250 and 1:2000, using anti-human IgG (y-chain
specific) or anti-human IgM (,u-chain specific) peroxidase-conjugated secondary antisera; in no case was reactivity to the distinct 75 kDa subunit observed.
The major M2 autoantigen in primary biliary cirrhosis
POC [10]. It is possible to quantify the contamination of
complex I with POC by immunoblotting increasing
amounts of complex I and POC against anti-E2 serum
and analysing the intensity of the reaction by using 1251_
labelled protein A and autoradiography [26]. Using this
technique, it is estimated that 40 Jtg of complex I contains
approximately 1 Jtg of POC (results not shown). Such a
level of contamination would not allow the POC polypeptides in the preparation of complex I to be visualized
by protein staining under normal loading conditions. In
this context, however, it is also relevant that PBC sera are
still reactive with the E2 polypeptide at dilutions of up to
lOs- 10 8 in e.l.i.s.a. [30]; thus even very minor contamination of complex I by POC could create confusion in
immunoblotting experiments.
A characteristic feature of the immunoblotting experiments in the work of Frostell et al. [21, 31] was that the
mobility and antigenicity of the reactive polypeptide was
markedly influenced by the method of preparing the
antigen for SOSjPAGE. We have also found that the
apparent mobility of POC E2 on SOSjPAGE is influenced by the method of preparation. Thus its reactivity
with autoantibodies is slightly enhanced by the presence
of f:l-mercaptoethanol in the sample buffer, and the
preparation of the sample in SOS at 3T'C instead of boiling results in the appearance of an additional reactive
species with a slightly greater mobility (lower molecular
mass) than the single component normally observed (not
shown). It is evident that, under our conditions for SOSj
PAGE, the E2 polypeptide of POC is resolved from the
large (75 kOa) subunit of complex I. However, the two
polypeptides may not have been resolved by the gradient
electrophoresis system used by Frostell et al. [21]. Similarly, the two-dimensional gels used in the study described
in [31] may not have resolved the two polypeptides as
they have similar isoelectric points.
Subunit-specific antiserum to the large subunit of complex I was used extensively in the original characterization
of this polypeptide as a major autoantigen in PBC. The
specificity of this antibody (identical to that used in the
present work) was further investigated, since it is possible
that the polypeptide excised from SOSjpolyacrylamide
gels for antibody production could also contain appreciable amounts of the highly immunogenic E2 polypeptide
on POc. It was found, however, that that antibody to the
large subunit of complex I reacts exclusively with its
parent antigen and fails to recognize the E2 polypeptide
of POC (results not shown). Frostell et al. [21] purified the
major PBC-specific antigen from Zwittergent-solubilized
beef heart mitochondria by affinity chromatography using
immobilized immunoglobulin from the sera of patients
with PBC, and subsequently showed that this affinitypurified antigen exhibits a strong cross-reaction when
probed with the antibody to the complex I 75 kDa subunit. Why does this antibody react with affinity-purified
autoantigen? One explanation of this anomaly could be
that the affinity-purified antigen contains both complex I
and POc. This possibility is suggested by data showing a
strong and specific interaction, at least in vitro, between
several mitochondrial dehydrogenases and complex I.
453
Specifically, it has been demonstrated, by chromatography on columns of immobilized POC, that complex I is
retained on such a column [32]. This is supported by the
present data confirming the presence of POC in purified
complex I preparations. Thus, during the affinity purification described by Frostell et at. [21], it is possible that
AMA in PBC sera recognized POC E2 as the primary
antigen but also absorbed out associated components
including complex I.
Is the M2 'a'-derived antigen unrelated to PDC E2?
Muno et at. [22] recently reported the purification and
N-terminal amino acid sequencing of a 36 kDa tryptic
fragment of the major PBC-specific antigen isolated from
mitochondria by affinity purification with sera from
patients with PBC. The published sequence does not
show any identity with the known sequences of rat or
human POC E2 [9, 33]. The E2 subunit, the most proteolytically sensitive subunit of POC, is known to yield a
stable 36 kDa lipoyl-containing fragment on tryptic
digestion [34]. This has been shown to contain the main
immunogenic region of the E2 protein [18]. However,
analysis of the amino acid sequence reported by Muno et
at. [22] indicates that this in fact corresponds to the Nterminal sequence of the intact POC El f:l-subunit (Fig.
2), which also has a molecular mass of 36 kDa, and is a
minor auto antigen in PBC [16].
There are two possible explanations for the findings of
Muno et al. [22]. One is that the PBC sera used in their
study contained antibodies directed against the E 1 13subunit. This polypeptide has previously been identified
as an autoantigen, but is recognized by sera from only a
small percentage of patients with PBC [16]. A second
possible explanation is that the purified antigen used for
sequencing contained mainly El f:l-subunit, but that it was
contaminated with small amounts of lipoyl fragments
(apparent molecular mass on SOSjPAGE, 36 kOa [18])
which conferred its reactivity towards PBC sera.
Concluding remarks
In conclusion, we emphasize that both of the above
cases of confusion over the identity of the major autoantigens in PBC are salient reminders of the problems in
assuming that the major immunoreactive species in an
(a)
L Q V T V Q E A I N Q G MOE X L X V 0 E K V F L
*
(~
IIII
*
I1111111
I
1\11\1
VQVTVRDAINQGMDEELERDEKVFL
Fig. 2. Comparison of the N-terminal sequences of (a)
the tryptic fragment of PBC antigen from rat liver [22] and
(b) the El f:l-subunit of human POC [35],. deri~ed from
the nucleotide sequence of cONA. Vertical lmes and
asterisks indicate identical and conserved residues,
respectively. The one-letter symbols for amino acids are
used.
454
S. P. M. Fussey et al.
immunoblot corresponds to the prominent polypeptide
observed in that region of an SDS gel stained with
Coomassie Blue. In view of the sensitivity of the procedures employed in immunological analysis, it may be the
case that a positive response is elicited by a minor
immunogenic contaminant co-migrating with a more
abundant polypeptide. The clarification of the nature of
the antigens is important for diagnostic purposes and
furthermore confirms the identification of the M2
antigens in PBC as components of the 2-oxo acid
dehydrogenase complexes.
ACKNOWLEDGEMENTS
S.P.M.E held a studentship from the Science and Engineering Research Council (U.K.). This work was
supported in part by the Medical Research Council (U.K.)
and the Wellcome Trust. We thank Miss Janet Harrison
for carrying out the computer analysis of the protein
sequences, and Mrs Dorothy Fittes for excellent technical
assistance.
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