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The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 2 443-447
Copyright © 1998 by The Endocrine Society

Original Studies

The 64-Kilodalton Eye Muscle Protein Is the Flavoprotein Subunit of Mitochondrial Succinate Dehydrogenase: The Corresponding Serum Antibodies Are Good Markers of an Immune-Mediated Damage to the Eye Muscle in Patients with Graves’ Hyperthyroidism1

S. Kubota2, K. Gunji2, B. A. C. Ackrell, B. Cochran, C. Stolarski, S. Wengrowicz, J. S. Kennerdell, Y. Hiromatsu and J. Wall

Departments of Medicine and Ophthalmology, Allegheny University of the Health Sciences, Pittsburgh, Pennsylvania 15212; the Department of Veterans Affairs Medical Center and Department of Biochemistry and Biophysics, University of California (B.A.C.A., B.C.), San Francisco, California 94121; the Endocrine Research Laboratory, Hospital de Sant Pau, Autonomous University of Barcelona (S.W.), Barcelona, Spain, and Division of Endocrinology (Y.H.), Kurume University School of Medicine, Kurume, Japan 830

Address all correspondence and requests for reprints to: Dr. J. Wall, Departments of Medicine and Ophthalmology, Allegheny University of the Health Sciences, Pittsburgh, Pennsylvania 15212. E-mail: wall{at}pgh.auhs.edu


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Thyroid-associated ophthalmopathy (TAO) is a progressive eye disorder associated with thyroid autoimmunity, particularly Graves’ hyperthyroidism, which is generally considered to have an autoimmune etiology. Eye muscle membrane proteins reportedly of 55 and 64 kDa are the best markers of the ophthalmopathy. The main focus of our recent studies has been to purify the pertinent proteins from porcine eye muscle membranes and characterize them. The 64-kDa protein is now shown from a partial sequence and by Western blotting using specific antibody probes to be the flavoprotein (Fp) subunit of succinate dehydrogenase and to have a correct molecular mass of 67 kDa. The protein was purified and cleaved with cyanogen bromide, and the N-terminal region of an immunoreactive partial peptide was determined. The 20-amino acid porcine sequence so obtained matched one within the Fp subunits of human and bovine succinate dehydrogenases in 20 and 18 of these positions, respectively. Succinate dehydrogenase is both a citric acid cycle enzyme and a component (complex II) of the mitochondrial respiratory chain. It is thus essential for aerobic energy production and is highly conserved. The mature human and bovine Fp subunits are 92% homologous and have a molecular mass of ~67 kDa, the same as our redetermined value for the 64-kDa marker protein. Sera from patients with TAO and from those with Graves’ hyperthyroidism without evident ophthalmopathy highlighted the 64-kDa marker protein in crude porcine eye muscle membranes and the Fp subunit of highly purified bovine succinate dehydrogenase at the identical position on Western blots. Anti-beef Fp antibodies were detected in sera from 67% of patients with active TAO of more than 1-yr duration, in 30% with stable TAO of more than 3-yr duration, and in 30% of patients with Graves’ hyperthyroidism without ophthalmopathy, but in only 7% of age- and sex-matched normal subjects. As succinate dehydrogenase is bound to the matrix (inside) surface of the mitochondrial inner membrane, it is unlikely to be accessible to circulating autoantibodies. We would postulate that eye muscle damage in ophthalmopathy is probably caused by cytotoxic antibodies or CD+ T lymphocytes targeting a cell membrane antigen, such as the thyroid and eye muscle shared protein G2s, and that presentation of succinate dehydrogenase is secondary. On the other hand, an autoantibody response to succinate dehydrogenase may be a good marker of immune-mediated damage to the eye muscle fiber and may support the idea that the extraocular muscles are targets of the autoimmune reactions of TAO.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
THYROID-ASSOCIATED ophthalmopathy (TAO) is a progressive eye disorder associated with thyroid autoimmunity, particularly Graves’ hyperthyroidism, in which visual impairment and exophthalmos occur as a result of swelling of the orbital contents (1). Although the disorder is generally considered to have an autoimmune etiology (2, 3), the identity and nature of the principal target antigens, the mechanism for the close association of ophthalmopathy with thyroid autoimmunity, and the basis for the localization of a muscle reaction mainly in the orbit are unclear. There are two main hypotheses concerning the pathogenesis of the orbital reactions of TAO. Firstly, some workers believe that the orbital connective tissue and fat are the sites of the primary inflammatory reaction and that eye muscle damage is secondary to this (4, 5). No candidate antigens have been identified in this compartment, and although the TSH receptor is thought by some (6, 7) to be the sought after thyroid and orbit shared antigen, recent studies by Bahn and colleagues (8) suggest that it is not expressed in the orbit. Secondly, we (9) and others (10, 11) have postulated that the primary autoimmune reaction in TAO is directed against eye muscle antigens, in particular those eye muscle membrane proteins of 55 and 64 kDa identified by immunoblotting, with secondary stimulation of the orbital fibroblasts. Although it has been difficult to convincingly demonstrate eye muscle fiber damage in the early stages of the eye disorder (12, 13), this may reflect the fact that eye muscle tissue from patients with Graves’ hyperthyroidism without eye signs but with eye muscle swelling on orbital imaging is usually unavailable for examination.

We have addressed the nature and significance of eye muscle antigens identified by SDS-PAGE and Western blotting using TAO patients’ sera. Eye muscle membrane proteins reportedly of 55 and 64 kDa appear to be the best markers of ophthalmopathy in patients with thyroid autoimmunity (reviewed in Refs. 3 and 9). Antibodies reactive with a 64-kDa protein are most closely associated with progressive ophthalmopathy (14, 15, 16), whereas those reactive with a 55-kDa eye muscle protein may be the first produced in patients with Graves’ hyperthyroidism who developed the eye disorder (17). These findings have been confirmed by several other groups (10, 11, 18, 19, 20). Although some researchers (21, 22, 23) have not been able to show the specificity and sensitivity that we have reported, this can be explained by technical differences and the fact that there are at least three proteins of 63–67 kDa that are targeted by serum antibodies in TAO (24).

We now report the partial sequencing of the 64-kDa protein, which is identified as the flavoprotein (Fp) subunit of the mitochondrial enzyme succinate dehydrogenase, and show that antibodies in sera from 67% of patients with active TAO, but from only 7% of normal subjects, react against purified Fp in Western blotting.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Clinical subjects

Sera from the following groups of patients were studied.

TAO with active disease of less than 1-yr duration. Eleven women and 4 men, aged 20–74 yr (mean age, 45 yr), all of whom had Graves’ disease, were studied. Three patients were hyperthyroid, and 12 were euthyroid after treatment with antithyroid drugs or radioactive iodine. None of the patients was being treated with corticosteroids at the time of study.

TAO with inactive disease of more than 3-yr duration. Fourteen women and six men, aged 21–60 yr (mean age, 34 yr), all of whom had associated Graves’ hyperthyroidism, were studied. All patients were euthyroid after treatment with antithyroid drugs or radioactive iodine. None of the patients was being treated with corticosteroids at the time of study.

Clinical assessment and characterization of the ophthalmopathy as active or inactive in patients with TAO was made following American Thyroid Association guidelines, using an activity index of 0–7, where one point is assigned to each of seven signs or symptoms of orbital inflammation, with inactive eye disease being taken as a score of 0 (25).

Graves’ hyperthyroidism. Fifteen women and 5 men, aged 22–68 yr (mean age, 46 yr), were studied. At the time of the study, 6 were hyperthyroid, and 14 were euthyroid following treatment. No patient had evidence of ophthalmopathy.

Normal subjects. Ten women and four men, aged 22–52 yr (mean age, 39 yr), with no personal or family history of thyroid disease, ophthalmopathy, or other autoimmune disease were recruited from ancillary hospital and laboratory staff of Allegheny General Hospital.

Informed written consent was obtained from all patients and normal subjects studied.

SDS-PAGE and Western blotting

Antibodies reactive with porcine eye muscle membrane proteins and purified beef heart succinate dehydrogenase Fp subunit were detected after standard Laemmli SDS-PAGE (26) using an 8.5% separating gel and a 4% stacking gel in a minigel apparatus, as reported previously (14, 15, 16). Primary antibodies were patients’ sera diluted 1:50 or a rabbit anti-Fp subunit antiserum diluted 1:2000, and secondary antibody was an alkaline phosphatase-conjugated antihuman IgG ({gamma}-chain specific) antiserum diluted 1:2000 for patients’ sera or antirabbit IgG (whole molecule) antiserum diluted 1:2000 for the anti-Fp subunit antiserum. Tests were read by two observers, and results were expressed as band density. A band density of + or greater was taken as a positive test.

Isolation of purified beef succinate dehydrogenase

Succinate dehydrogenase was solubilized by perchlorate treatment (27) of succinate:coenzyme Q oxidoreductase (complex II of the respiratory chain) that had been isolated from beef heart mitochondria by the method of Baginsky and Hatefi (28). The enzyme preparation was more than 90% pure based on gel analysis and content of covalently bound flavin adenine dinucleotide. Pure Fp subunit was excised from SDS-polyacrylamide gels according to the method of Merli et al. (29) and used as antigen in Western blotting.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
The 64-kDa protein was purified from solubilized porcine eye muscle membranes, prepared as described above, as follows; eye muscle membrane was run on standard SDS-PAGE. An aliquot was blotted onto polyvinylidene difluoride (PVDF) paper, and the 64-kDa protein was probed using a positive TAO patient serum. From our earlier studies (24) we knew that the 64-kDa protein had a corrected mol wt of 67 kDa. The rest of the membrane preparation was run on an SDS gel. Proteins of approximately 67 kDa were carefully cut out from the gel. The protein(s) was then digested with cyanogen bromide according to the Promega Probe-Design Peptide Separation Technical Manual (Promega, Madison, WI). The digested material was run on a peptide gel, blotted onto PVDF paper, and incubated with a TAO patient’s serum. The most reactive peptide (indicated by the lower arrow in Fig. 1Go), which had a mol wt of 10 kDa, was eluted from the gel using peptide elution solvent and sent to Kendrick Laboratories (Madison, WI), where an additional one-dimensional SDS-PAGE separation was performed on a large gel. After final confirmation that the peptide was recognized by antibodies in a TAO patient’s serum, the peptide was sent to the Medical College of Wisconsin Protein and Nucleic Acid Shared Facility for microsequencing. A Beckman/Porton model LF 3000 instrument (Beckman Instruments, Palo Alto, CA), which can determine a sequence on 1–10 pmol purified peptide, was used, and a 20-amino acid sequence of the 64-kDa protein molecule was obtained, namely: L C A L Q T I Y G A E A R K E S R G A H. The 20-amino acid porcine sequence so obtained matched one within the Fp subunits of human and bovine succinate dehydrogenases in 20 and 18 of these positions, respectively.



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  		Figure 1. Western blotting of peptides of a 67-kDa protein with serum from a patient with TAO and identification of reactive peptides. The 67-kDa protein was purified from porcine eye muscle membrane, cut from the gel, and digested with cyanogen bromide. The resulting peptides were separated by electrophoresis using a high percentage gel to resolve small peptide fragments and then transferred to sequencing grade Immobilon-PSQ PVDF sequencing membrane. The gel was stained with Coomassie (lane 1) and destained or was used for immunoblotting with TAO patient serum, in which case Immobilon-P was used instead of the sequencing grade PVDF (lane 2). The lower, more reactive peptide (~10 kDa) was sequenced at the Medical College of Wisconsin Protein and Nucleic Acid Shared Facility and identified as a fragment of human succinate dehydrogenase flavoprotein subunit. MW, Molecular mass standards.

 
Next, we tested selected sera from patients with TAO and Graves’ hyperthyroidism and from normal subjects for reactivity against purified beef succinate dehydrogenase Fp subunit and porcine eye muscle membranes in parallel immunoblot assays. The object of this experiment was to confirm that the 67-kDa protein in porcine eye muscle membranes and the Fp subunit of succinate dehydrogenase were the same protein. Sera from patients with active or inactive TAO and Graves’ hyperthyroidism without ophthalmopathy, which had been tested previously for reactivity against porcine eye muscle membrane in SDS-PAGE and comprised both positive and negative sera, as well as nonreactive sera from normal subjects were selected. In preliminary experiments we determined that optimal reactivity against purified succinate dehydrogenase Fp subunit was found with 0.75 µg/mL native enzyme, a serum dilution of 1:50, and a second antibody dilution of 1:1000. The concentration of porcine eye muscle membrane used was 10 µg/mL. As a positive control we used a polyclonal antibody against succinate dehydrogenase Fp subunit. The results are shown in Fig. 2Go. In Fig. 2AGo, reactivity against succinate dehydrogenase Fp subunit, at 67 kDa, is observed with sera from patients with TAO or Graves’ hyperthyroidism. In Fig. 2BGo, reactivity with many different porcine eye muscle membrane proteins, including a 67-kDa protein (indicated by the arrow), is noted. By comparing reactivity with a 67-kDa protein in Fig. 2BGo and reactivity with purified succinate dehydrogenase Fp subunit in Fig. 2AGo, it is apparent that all sera demonstrated the same reactivity (positive or negative) and band density (+/-, +, ++, and +++) with both preparations, confirming that the Fp subunit of succinate dehydrogenase and the 67-kDa porcine membrane protein are the same protein. We then tested representative sera from patients with TAO and from normal subjects for reactivity against purified succinate dehydrogenase Fp in Western blotting. Tests were positive in sera from 10 of 15 (67%) patients with active TAO of less than 1-yr duration, in 6 of 20 (30%) with stable TAO of more than 3-yr duration, and in 6 of 20 (30%) patients with Graves’ hyperthyroidism without ophthalmopathy, but in only 1 of 14 (7%) normal subjects (results not shown). Interestingly, three normal sera that were previously reactive with a 67-kDa protein in porcine eye muscle membrane (30) were negative when tested with purified succinate dehydrogenase (results not shown). The differences between patient groups and normal subjects were significant for TAO of less than 1-yr duration (by {chi}2 test, P < 0.001), but not for the other 2 groups. The prevalences of antibodies against purified succinate dehydrogenase Fp in patients with other thyroid disorders, other skeletal muscle disorders, and other autoimmune disorders, are being determined and will be reported separately (Gunji, K, Kubota S, Swanson J, Tamai H, Kennerdell JS, Wall JR, unpublished data).



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  		Figure 2. SDS-PAGE of purified succinate dehydrogenase Fp subunit (A) or porcine eye muscle membranes (B) and Western blotting with selected sera from patients with thyroid-associated ophthalmopathy, Graves’ hyperthyroidism, and normal subjects. Succinate dehydrogenase Fp subunit (A) or porcine eye muscle membrane (B) was applied to SDS-PAGE, immunoblotted onto PVDF paper, and incubated with Tris-buffered saline (lane 2), an antisuccinate dehydrogenase Fp polyclonal antibody (lane 3), serum from patients with active TAO (lanes 3–8), serum from patients with stable (inactive) TAO (lanes 9–11), serum from patients with Graves’ hyperthyroidism without ophthalmopathy (lanes 12 and 13), or serum from normal subjects (lanes 14–17) in both A and B. Reactivity with succinate dehydrogenase Fp is seen at 67 kDa (indicated by an arrow in B). Lane 1 is Coomassie-stained purified succinate dehydrogenase Fp (A) or porcine eye muscle membranes (B). MW, Molecular mass standards.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
The main focus of our recent studies has been to characterize the principal eye muscle autoantigens and to develop antibody tests for clinical use. We have attempted to purify the 55- and 64-kDa proteins from solubilized porcine eye muscle membranes to obtain microsequence information as the basis for their cloning from an eye muscle expression library, beginning with the 64-kDa protein. It proved difficult to purify, sequence, and characterize the 64-kDa protein for several reasons, including 1) the protein appeared to be of low abundance in the membrane fraction of the eye muscle fiber, and we were unable to obtain the amount of purified protein needed for sequencing; 2) as prepared by SDS-PAGE and isoelectric focusing, the 64-kDa protein was blocked at the N-terminus; and 3) preparations of the 64-kDa protein were often contaminated with the highly abundant albumin, which has a similar molecular mass and isoelectric point. During the course of these experiments we sequenced and cloned calsequestrin, a 63-kDa muscle protein that is recognized by sera from 40% of patients with active TAO and 5% of normal subjects (31). When we carried out SDS-PAGE of porcine eye muscle membrane using a monoclonal antibody probe for calsequestrin in Western blotting, we were surprised to find that the majority of TAO patients’ sera, previously considered reactive with the 64-kDa protein, recognized an antigen that did not correspond to calsequestrin, which has an estimated molecular mass of 63 kDa, but corresponded to a slightly larger protein of 67 kDa (24). As reported here we have purified and sequenced part of this latter protein, which is identified as succinate dehydrogenase Fp subunit. Calsequestrin and the flavoprotein do not share any significant homologies. Serum autoantibodies reactive with purified succinate dehydrogenase Fp were detected in 67% of patients with active TAO, 30% with more chronic, stable, disease, and 30% of patients with Graves’ hyperthyroidism without ophthalmopathy, but in only 7% of normal subjects. Because sera from approximately 20% of the normal subjects were positive when tested against porcine eye muscle membranes in another study (30) but negative in all but one subject when tested for reactivity against purified Fp in this study, we presume that sera from some normal subjects contain antibodies recognizing other proteins of 65–67 kDa, such as albumin. As the Fp subunit is highly conserved, it is possible that the one control with "autoantibodies" in the present study had an infection, and the antibodies were generated against a bacterial or yeast Fp.

Succinate dehydrogenase is a flavo enzyme consisting of a Fp subunit that contains the active site and the FAD cofactor of the enzyme and an iron sulfur subunit containing three nonidentical iron-sulfur clusters that catalyzes oxidation/reduction reactions. Its specific action is to oxidize succinate to fumarate in the mitochondrial matrix and pass the electrons directly into the ubiquinone pool of the respiratory chain. The enzyme is anchored to the inner mitochondrial membrane by two small hydrophobic and membrane-spanning polypeptides; indeed, succinate dehydrogenase is the only citric acid cycle enzyme that is membrane bound. Succinate dehydrogenase is expressed in the mitochondria as a 664-amino acid protein comprising a presequence of 43 amino acids and a mature protein of 621 amino acids. The latter corresponds to a molecule of 67 kDa, which is close to the observed mol wt of the 64-kDa protein (which has a corrected mol wt of 67 kDa as determined from SDS-PAGE). The small presequence is required for targeting and is removed in the mitochondria. The iron-sulfur protein subunit of the enzyme is highly conserved in evolution. DNA sequences have been published for human heart succinate dehydrogenase Fp subunit (32) and for the human liver iron sulfur protein (33, 34). The enzyme is most highly expressed in skeletal muscle, cardiac muscle, and liver, where it is involved in energy production. The small presequence is required for targeting and is removed in the mitochondria.

What is the significance of autoantibodies in TAO? It is unclear whether a globular antibody molecule directed against an intracellular protein would be able to penetrate the intact cell and subcellular membranes to bind to its target antigen, although recent evidence supports the idea that some autoantibodies do enter human cells and produce disease (35). If anti-Fp antibodies can get into the cytoplasm, they might react with newly synthesized (precursor) Fp, which is nuclear encoded and synthesized on cytoplasmic ribosomes before being imported into the mitochondrion for assembly into the active enzyme. It is also possible that cytoplasmic Fp precursor might be presented at the muscle cell surface without a need to disrupt the cell. In the so-called mitochondrial respiratory chain syndromes, a variety of muscular and neurological symptoms result from deficiency of individual mitochondrial enzymes. Many patients present with ptosis, ophthalmoplegia and myopathy, decreased muscle fiber succinate dehydrogenase activity and concentration, and associated mitochondrial abnormalities (36). Interestingly, we have demonstrated similar mitochondrial abnormalities on electron microscopic examination of eye muscle fibers in patients with TAO (3). The most frequent finding was an alteration in the number, size, and morphology of the mitochondria. In some cases the mitochondria were larger than normal, with clear ballooned spaces between their cristae. In other cases, the intercristal spaces were widened. Although the electron microscopic findings in TAO are consistent with a direct effect of antibodies reacting with succinate dehydrogenase, the observed changes could also reflect a nonspecific result of muscle fiber necrosis.

To summarize, although it is most likely that eye muscle damage in ophthalmopathy is mediated by cytotoxic antibodies or CD+ T lymphocytes targeting a cell membrane antigen, such as the thyroid and eye muscle shared protein G2s that we have recently cloned (37), and that presentation of succinate dehydrogenase is secondary, sensitization to succinate dehydrogenase Fp subunit may be a good marker of immune-mediated damage to the eye muscle fiber. Moreover, the findings support the hypothesis that the eye muscle is a target of the autoimmune reactions of TAO (3). The utility of antisuccinate dehydrogenase Fp antibody measurement as a clinical test for ophthalmopathy is being addressed by studying a cohort of newly diagnosed patients with Graves’ hyperthyroidism after treatment as well as patients with other thyroid and skeletal muscle disorders.


   Footnotes
 
1 This work was supported by research grants from Toray-Fuji Bionics (Japan) and the Allegheny-Singer Research Institute and NIH Grant HL-16251 (to B.A.C.A.). Back

2 S.K. and K.G. contributed equally to this work. Back

Received August 20, 1997.

Revised October 17, 1997.

Accepted October 28, 1997.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. Burch H, Wartofsky L. 1993 Graves’ ophthalmopathy: current concepts regarding pathogenesis and management. Endocr Rev. 14:747–793.[Abstract]
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K. Gunji, A. De Bellis, S. Kubota, J. Swanson, S. Wengrowicz, B. Cochran, B. A. C. Ackrell, M. Salvi, A. Bellastella, A. Bizzarro, et al.
Serum Antibodies against the Flavoprotein Subunit of Succinate Dehydrogenase Are Sensitive Markers of Eye Muscle Autoimmunity in Patients with Graves' Hyperthyroidism
J. Clin. Endocrinol. Metab., April 1, 1999; 84(4): 1255 - 1262.
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A. Kromminga, C. Hagel, R. Arndt, and F. Schuppert
Serological Reactivity of Recombinant 1D Autoantigen and Its Expression in Human Thyroid and Eye Muscle Tissue: A Possible Autoantigenic Link in Graves' Patients
J. Clin. Endocrinol. Metab., August 1, 1998; 83(8): 2817 - 2823.
[Abstract] [Full Text]

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