Serial changes of serum anti-M2 proteins in patients with primary biliary cirrhosis: a follow-up study by immunoblotting

doi:10.1016/S1386-6346(98)00088-6

Hepatology Research

Volume 13, Issue 2, January 1999, Pages 143-152

https://doi.org/10.1016/S1386-6346(98)00088-6 Get rights and content

Abstract

To investigate the pathogenic role of M2 components in primary biliary cirrhosis (PBC), we studied the serial changes of serum anti-M2 protein profiles by immunoblotting in 21 patients with PBC who were observed for at least 3 years. Immunoblotting was done using bovine heart mitochondrial fraction as the antigen. First, we confirmed the antigen specificity by inhibition tests using recombinant proteins associated with two types of major M2 proteins. At the initial examination, 15 patients showed positivity for the 74-kDa protein corresponding to anti-PDC-E2, and 19 patients showed the 50-kDa protein corresponding to anti-BCOADC-E2. In subsequent examinations, four of the 21 patients continuously did not show the 74-kDa protein and only one patient showed the 50-kDa protein although it was not detected at the initial examination. The anti-M2s other than 74-kDa protein, especially 50-kDa protein, showed almost no changes from the initial examinations. These data indicate that part of the onset and development of PBC is highly associated with M2 proteins other than PDC-E2.

Introduction

Primary biliary cirrhosis (PBC) is classified as an autoimmune liver disease because anti-mitochondrial antibody (AMA) is present in the sera of most PBC patients 1, 2. The specific AMA in PBC is anti-M2 [3]. The autoantigens that react to anti-M2 are pyruvate dehydrogenase complex (PDC)-E2 4, 5, protein X of PDC-E2 [6], branched chain 2-oxo acid dehydrogenase complex (BCOADC)-E2 7, 8, and oxo-glutarate dehydrogenase complex (OGDC)-E2 [9]. Of the anti-M2 proteins, anti-PDC-E2 is most commonly recognized by serum antibodies of patients with PBC, and much of the research regarding PBC has focused on determining the reactivity of anti-PDC-E2 with AMA. For the serodiagnosis of PBC, immunofluorescence for AMA [10]and an enzyme-linked immunosorbent assay (ELISA) for anti-M2 11, 12have already been established. However, an adequate M2 protein analysis cannot be done with these assays. Immunoblotting 13, 14is the most sensitive means of detecting anti-M2, because it allows a precise indication of mitochondrial proteins that are specific for antibodies from patients with PBC [15]. The role of these M2 autoantigens in the pathogenesis of PBC has been controversial. To analyze the relationship between the pathogenesis and serum anti-M2 proteins in PBC, we studied the changes of serum anti-M2 proteins by immunoblotting in patients with PBC who were followed for at least 3 years.

Section snippets

Patients

Of 42 patients with PBC who had been admitted to our hospital during the period from January 1992 to December 1997, 21 patients who had been followed-up for at least 36 months (mean observation period: 60.7 months, range: 36–132 months) were the subjects of the present study. All of the patients fulfilled the diagnostic criteria of PBC proposed by Wiesner et al. [16]. The profiles of these patients are shown in Table 1. A total of 17 of the patients had been treated with ursodeoxycholic acid

Antigen-specificity of immunoblotting

To assess the specificity of the immunoblotting, we performed inhibition tests. With a pre-incubation of recombinant PDC-E2 protein, recombinant BCOADC-E2 protein, and porcine heart OGDC, 74-, 50-, and 48-kDa protein bands were deduced, respectively (Fig. 1). In contrast, with a pre-incubation of PBS alone (control), these bands were not inhibited. These data confirmed the antigen-specificity of immunoblotting.

Serial changes of serum anti-PDC-E2 proteins in 21 patients with PBC

The serial changes of the 74-kDa band corresponding to anti-PDC-E2 shown by

Discussion

AMA was once believed to be only a serological diagnostic marker for PBC, because this autoantibody was non-organ-specific [10]. However, with advances in gene technology, major autoantigens of anti-M2 detected in sera from PBC patients were identified to be PDC-E2, BCOADC-E2, and OGDC-E2 4, 5, 6, 7, 8, 9. In recent immunohistochemistry studies using the monoclonal antibody, it was found that the PDC-E2 molecule was stained on bile duct epithelial cells in PBC patients 19, 20. These data

References (22)

J.G Walker et al.
Serological test in diagnosis of primary biliary cirrhosis
Lancet
(1965)
S Moteki et al.
Epitope mapping and reactivity of autoantibodies to the E2 component of 2-oxoglutarate dehydrogenase complex in primary biliary cirrhosis using recombinant 2-oxoglutarate dehydrogenase complex
Hepatology
(1996)
R.H Wiesner et al.
Comparison of the clinicopathologic features in primary sclerosing cholangitis and primary biliary cirrhosis
Gastroenterology
(1985)
Y Nakanuma et al.
Biliary epithelial expression of pyruvate dehydrogenase complex in primary biliary cirrhosis: an immunohistochemical and immunoelectron microscopic study
Hum Pathol
(1995)
I.R Mackay
Primary biliary cirrhosis showing a high titer of autoantibody
N Engl J Med
(1958)
P.A Berg et al.
Heterogeneity of antimitochondrial antibodies
Semin Liver Dis
(1989)
R.L Coppel et al.
Primary structure of the human M2 mitochondrial autoantigen of primary biliary cirrhosis: dihydrolipoamide acetyltransferase
Proc Natl Acad Sci USA
(1988)
M.E Gershwin et al.
Identification and specificity of a cDNA encoding the 70-kDa mitochondrial antigen recognized in primary biliary cirrhosis
J Immunol
(1987)
O DeMarcucci et al.
Component X - an immunologically distinct polypeptide associated with mammalian pyruvate dehydrogenase multienzyme complex
Eur J Biochem
(1985)
K.S Lau et al.
Conservation of primary structure in the lipoyl-bearing and dihydrolipoyl dehydrogenase binding domains of mammalian branched-chain alpha-keto acid dehydrogenase complex: molecular cloning of human and bovine transacylase (E2) cDNAs
Biochemistry
(1988)

C.D Surh et al.

Reactivity of primary biliary cirrhosis sera with a human fetal liver cDNA clone of branched-chain a-keto acid dehydrogenase dihydrolipoamide acetyltransferase, 52-kDa mitochondrial autoantigen

Hepatology

(1989)

Cited by (17)

Immunoreactivity to pyruvate dehydrogenase complex-E2 in well-defined patients with autoimmune hepatitis: Western blot analysis
2003, Hepatology Research
Anti-mitochondrial antibodies (AMA) are frequently detected in sera from patients with primary biliary cirrhosis (PBC). Major autoantigens for AMA have been identified as members of the 2-oxoacid dehydrogenase enzyme complex family, with pyruvate dehydrogenase complex (PDC)-E2 showing strongest reactivity to AMA in PBC patients. Recently, anti-PDC-E2 has been found in patients with other diseases. Since frequency and significance of anti-PDC-E2 in patients with autoimmune hepatitis (AIH) remain obscure, we measured anti-PDC-E2 in sera from well-defined AIH cases by Western blotting using bovine heart mitochondrial protein and recombinant PDC-E2 protein as antigen sources. All 55 enrolled patients fulfilled the international diagnostic criteria for definite or probable AIH. Anti-PDC-E2 positivity showed concordance between native and recombinant antigens. Anti-PDC-E2 was detected in nine of 55 sera from AIH patients (16%). Variables including alkaline phosphatase (ALP) and IgM concentrations, effects of prednisolone, and pathologic findings concerning bile ducts showed no significant differences between anti-PDC-E2-positive and anti-PDC-E2-negative AIH patients. These data indicate that detection of anti-PDC-E2 is not rare in defined AIH, but anti-PDC-E2-positive AIH does not represent an intermediate entity in a clinical spectrum between AIH and PBC.
Anti-mitochondrial autoantibodies
2002, Clinical and Applied Immunology Reviews
Anti-mitochondrial antibodies (AMA) are a serological hallmark of primary biliary cirrhosis (PBC). The major autoantibody targets are located in the inner mitochondrial membrane, are encoded by nuclear genes and are components of the 2-oxo acid dehydrogenase complexes. Greater than 90% of PBC patients react with one or more of these autoantigens. The major epitopes have been mapped to the lipoic acid binding domain. The apparent hepatic targets of immune destruction, the apical biliary epithelial cells, express proteins that mimic these epitopes. Nevertheless, the role of AMA in biliary pathology is not clear and observed abnormalities may be due to T-cells that respond to similar epitopes. AMAs are often accompanied by autoantibodies to other intracellular components such as the nuclear pore complex, centromeres/kinetochores, and other nuclear antigens. The origin or inciting agent(s) of AMA is not known but epidemiological and immunological evidence implicates environmental agents.
Anti-pyruvate dehydrogenase complex-E2 antibody immunoglobulin class switch from IgM to IgG in long-term follow-up patients with primary biliary cirrhosis
2002, Hepatology Research
Antibody immunoglobulin class switching from IgM to IgG is usually observed in acute viral infections. However, in autoimmune diseases, the autoantibody immunoglobulin class switch from IgM to IgG has been observed only rarely, and the clinical relevance of this immune phenomenon remains unclear. In this report, anti-pyruvate dehydrogenase complex (PDC)-E2 antibody immunoglobulin class switching was followed in two patients with primary biliary cirrhosis (PBC). IgG and IgM anti-PDC-E2 antibodies were examined by an originally enzyme-linked immunosorbent assay and by immunoblot using human recombinant PDC-E2 protein. In both patients, serum IgG and IgM anti-PDC-E2 antibodies could not be detected at initial admission. However, IgM antibody was subsequently detected, and IgG antibody appeared several years thereafter.
Detection of antimitochondrial autoantibodies in immunofluorescent AMA-negative patients with primary biliary cirrhosis using recombinant autoantigens
2001, Hepatology
Antimitochondrial antibodies (AMA) are the serologic hallmark of primary biliary cirrhosis (PBC). However, depending on the clinical laboratory, from 5% to 17% of PBC patients are consistently AMA-negative, using native mitochondrial antigens and a variety of conventional assays including immunofluorescence (IMF) and enzyme-linked immunosorbent assay (ELISA). The major immunoreactive mitochondrial autoantigens are the E2 members of the 2-oxo-acid dehydrogenase complex family, including pyruvate dehydrogenase complex-E2 (PDC-E2), branched chain 2-oxo acid dehydrogenase complex-E2 (BCOADC-E2), and oxo-glutarate dehydrogenase complex-E2 (OGDC-E2); cDNAs of these proteins have now been cloned, sequenced, and their B-cell epitopes defined. In the present study, we cloned cDNAs encoding these proteins from human, not bovine, sources, and expressed the recombinant proteins in a newly developed ELISA that employs a unique Escherichia coli buffer, and compared the data with previous assays using both AMA-positive and -negative patients. Using this new assay and our criteria for positive as an optical density (OD) greater than 10 SD above the mean of control sera, the AMA-positive rate of 191 PBC sera was 94% (179 of 191) compared with 84% (161 of 191) by IMF. None of the 316 control sera were reactive. Using our recombinant assays, we focused attention on the 30 IMF-AMA–negative patients. Twenty-two of 30 (73%) of these patients were positive using this new ELISA. The group of 30 IMF-AMA–negative/ELISA-positive patients did not differ significantly from a comparable population of IMF-AMA–positive patients with respect to age, sex distribution, liver function tests, elevation of serum IgM, or pathologic stage. (HEPATOLOGY 2001;34:243-248.)
Definition of antigen specificity for antimitochondrial proteins detected by Western blotting using native mitochondrial proteins in primary biliary cirrhosis
2001, Hepatology Research
The major autoantigens to anti-mitochondrial antibody (AMA) in primary biliary cirrhosis (PBC) have previously been identified to be PDC-E2, BCOADC-E2, and OGDC-E2. However, analysis of these autoantigens to AMA cannot be examined using the two routine assays; immmunofluorescence and ELISA. Moreover, there are some problems in specificity and sensitivity in these routine assays. So, analysis with Western blotting using native mitochondrial protein as the antigen is required; it allows the identification of the molecular weights for the proteins which react with AMA in patients' sera. However, since the antigen-proteins used are not unified, molecular weights of AMA corresponding proteins vary among laboratories. In the present study, as the first step to help address this issue, we investigated the antigen specificity of protein bands detected by Western blotting using our in-house bovine and porcine heart mitochondrial proteins. Three major recombinant mitochondrial proteins were prepared. The antigen specificity was examined by the absorption tests preincubated with the three recombinant mitochondrial proteins. The molecular weights of developing our bovine and porcine heart mitochondrial proteins using SDS-PAGE were multiple protein bands including 74, 52, 50, and 43 kDa protein bands. Of them, the 74, 50, and 43 kDa protein bands were absorbed with preincubations of recombinant PDC-E2, BCOADC-E2, and OGDC-E2 protein, respectively. AMA specificity of these three major proteins with our Western blotting was confirmed.
Analysis of two major anti-M2 antibodies (anti-PDC-E2/BCOADC-E2) in primary biliary cirrhosis: Relationship to titers of immunofluorescent anti-mitochondrial antibody
2000, Hepatology Research
To analyze anti-M2 components in primary biliary cirrhosis (PBC) we measured two major anti-M2 antibodies (anti-PDC-E2 and anti-BCOADC-E2) by immunoblotting and ELISA, and compared the results between 38 immunofluorescent anti-mitochondrial antibody (AMA)-negative PBC patients (group A) and 39 strongly AMA-positive PBC patients (group B) with titers of 1:640. Using bovine heart mitochondrial fraction as antigen, the immunoblot positivity rate of anti-PDC-E2 in group B was significantly higher than that in group A, whereas the positivity rate of anti-BCOADC-E2 was not significantly different between the two groups. This result was similar to that obtained by ELISA using recombinant fusion proteins. In group A there was a significant inverse correlation between ELISA optical density values of anti-PDC-E2 and of anti-BCOADC-E2, but in group B there was no correlation between the two values. Only three patients from group A and 21 from group B were positive for both antibodies. Taken together these results appear to indicate that the detection of anti-BCOADC-E2 is critical for the accurate serological diagnosis of AMA-negative PBC patients. The detection of anti-BCOADC-E2 may also help to distinguish between AMA-negative PBC and autoimmune cholangitis patients.

View all citing articles on Scopus

View full text

Serial changes of serum anti-M2 proteins in patients with primary biliary cirrhosis: a follow-up study by immunoblotting

Abstract

Introduction

Section snippets

Patients

Antigen-specificity of immunoblotting

Serial changes of serum anti-PDC-E2 proteins in 21 patients with PBC

Discussion

Lancet

Hepatology

Gastroenterology

Hum Pathol

Primary biliary cirrhosis showing a high titer of autoantibody

N Engl J Med

Heterogeneity of antimitochondrial antibodies

Semin Liver Dis

Primary structure of the human M2 mitochondrial autoantigen of primary biliary cirrhosis: dihydrolipoamide acetyltransferase

Proc Natl Acad Sci USA

Identification and specificity of a cDNA encoding the 70-kDa mitochondrial antigen recognized in primary biliary cirrhosis

J Immunol

Component X - an immunologically distinct polypeptide associated with mammalian pyruvate dehydrogenase multienzyme complex

Eur J Biochem

Conservation of primary structure in the lipoyl-bearing and dihydrolipoyl dehydrogenase binding domains of mammalian branched-chain alpha-keto acid dehydrogenase complex: molecular cloning of human and bovine transacylase (E2) cDNAs

Biochemistry

Reactivity of primary biliary cirrhosis sera with a human fetal liver cDNA clone of branched-chain a-keto acid dehydrogenase dihydrolipoamide acetyltransferase, 52-kDa mitochondrial autoantigen

Hepatology