(Received for publication, October 20, 1995; and in revised form, December 21, 1995)
From the
To define the autoantibody epitopes in amino acids 513-633 of thyroid peroxidase (TPO), a region frequently recognized in thyroiditis, cDNA sequences coding for peptide fragments of this region were amplified and ligated into pMalcRI and pGEX vectors for expression as recombinant fusion proteins. Western blots and enzyme-linked immunosorbent assay were then used to examine the reactivity in sera from 45 Hashimoto's and 47 Graves' disease patients. Two autoantibody epitopes within TPO amino acids 589-633 were identified; 16 of 35 patients reactive to TPO513-633 recognized the epitope of TPO592-613, while 6 patients recognized the epitope of TPO607-633. Eleven other patients with thyroiditis and two with Graves' disease recognized only the whole 589-633 fragment, and this response accounted for the Hashimoto's disease specificity. An amino acid sequence comparison of TPO592-613 with analogous regions of other peroxidase enzymes revealed significant differences in this area, and the substitution of even a single amino acid in one of the epitopes markedly decreased the binding affinity of autoantibodies. Additionally, the exclusive recognition by patients of only one of the epitopes within this region suggests a genetic restriction of the autoantibody response.
Thyroid peroxidase (TPO) ()is an autoantigen that is
recognized by autoantibodies from patients with either
Hashimoto's thyroiditis or Graves' disease(1) .
However, the basis of immune responses to this antigen in autoimmune
thyroid disease (AITD) is not clear(2, 3) . Studies
have indicated that TPO is a complex autoantigen having at least two
conformational and several localized autoantibody
epitopes(4, 5, 6) . Two TPO regions, amino
acids 513-633 and 710-740, have been identified as
containing autoantibody binding sites(6, 7) , and we
have attempted to correlate autoantibodies to these regions with
manifestations of AITD(8) . No difference was observed in the
overall serologic response to either native or denatured TPO in
Graves' disease and Hashimoto's thyroiditis(8) .
However, autoantibodies against TPO amino acids 513-633 were
identified more commonly in Hashimoto's thyroiditis patients than
in Graves' disease patients(8) . This has focused our
current studies on the antigenic characterization of this region of
TPO.
The exact nature of the autoantibody epitope or epitopes in TPO amino acids 513-633 is not clear. The location and conformational dependence of epitopes in this region may be important because this area contains a cysteine which may be involved in intra- or interchain disulfide linkages in the TPO enzyme complex(9) . A histidine residue proposed to be involved in tethering the heme coenzyme is also located within this site, suggesting a role in enzymatic function(10) . This region of TPO shares significant structural homology with myeloperoxidase (11) and lactoperoxidase(12) , suggesting the possibility of antigenic cross-reactivity or molecular mimicry(13, 14) . This suggests that there may be unique structural and immunologic aspects to this area of TPO. Therefore, characterizing the antigenicity of this region may lead to an understanding of the potential role of this activity in disease pathogenesis(15) .
In order to better understand the antigenicity of this region, we employed recombinant fusion proteins and synthetic peptides to characterize the autoantibody epitopes within the region of TPO amino acids 513-633. The structural nature and specificity of these sites were clarified, and the associations with different forms of autoimmune thyroid disease were analyzed. The results suggest that the autoantibody responses to this region in patients with AITD are heterogeneous in nature and may be genetically restricted.
Figure 1: Recombinant proteins were used to map a TPO epitope within the region of amino acids 513-633. The DNA coding sequence for TPO amino acids 589-633 and the primers used in various PCR amplifications is shown. The PCR primers were constructed to produce 5` EcoRI and 3` XbaI restriction sites (underlined) for ligation of the PCR product into the pMalcRI vector. Primer sequence complementary to TPO sequence (in bold) is spaced in triplets to show the reading frame after insertion into the vector. A stop codon in-frame at the 3` end of the PCR product was also engineered into the 3` primer to ensure the appropriate termination of the fusion protein.
Figure 3: The amino acid sequence for TPO592-613 is compared to the analogous sequences from MPO and LPO. Substitutions from the TPO sequence are indicated. The glutamic acids at positions 593 and 596 of TPO are underlined to identify the substitutions made in several of the TPO fusion proteins used to test binding specificity. The amino acids after position 601 (in TPO) show great heterogeneity between the three proteins.
Figure 2: Patient reactivity to specific recombinant TPO fragments was determined by Western blot analysis of bacterial lysates containing MBP-TPO fusion proteins. Western blots were performed using an anti-TPO513-633 reactive Hashimoto's patient serum at 1:400 (right lanes) to identify TPO-specific reactivity or an anti-MBP monoclonal antibody (MAb) to identify the presence of MBP fusion proteins (left lanes). A, the Hashimoto's patient serum does not bind to MBP-TPO589-607 (lane 1, arrow) but is reactive with the MBP-TPO589-613 (lane 2, 40-kDa band) and the MBP-TPO513-633 (lane 3, 56-kDa band) control proteins. The anti-MBP monoclonal antibody blot as well as the Coomassie Blue-stained gel (B) demonstrate that large amounts of all of the fusion proteins are present. C, the patient serum is reactive with MBP-TPO592-613 (lane 1, arrow) and MBP-TPO589-613 control proteins (lanes 2 and 3). D, TPO fragments were also expressed as GST fusion proteins using the pGEX vector. Some GST-TPO fusion proteins are shown on a gel stained with Coomassie Blue (bottom section, 28-31 kDa), and TPO reactivity is confirmed by Western blot using the same patient serum (top section).
To determine whether patients reacted to more than a single epitope within the larger region of amino acids 513-633, lysates of the various MBP-TPO fusion proteins were used to inhibit binding to MBP-TPO513-633 on Western blot (Fig. 4). Serum from 5 positive patients and the anti-MBP monoclonal antibody were preincubated with either 1% bovine serum albumin (``+'' strips), MBP-TPO592-613 lysate (A strips), MBP-TPO607-633 lysate (C strips), or purified MBP-TPO513-633 (B strips), then incubated with strips from a Western blot of MBP-TPO513-633. Control strips incubated with anti-MBP monoclonal antibody identify the 56-kDa MBP-TPO513-633 fusion protein and confirm that preincubation with the other MBP fusion proteins would inhibit antibody binding. Sixteen patients in all demonstrated binding to the TPO592-613 fragment and had the reactivity to the TPO513-633 fusion protein inhibited by preincubation with this fragment (``A'' strips, demonstrated by the blots from patients H1, H2, and G1). Six other patients had binding to TPO513-633 inhibited only by preincubation with the MBP-TPO607-633 fusion protein (C strips, demonstrated by the blot from patient H4), while in 13 other patients, binding to the TPO513-633 fusion protein was not inhibited by either or both smaller fusion proteins (demonstrated by the blot from patient H3). GST-TPO fusion proteins containing the same TPO amino acid sequences (Fig. 2D) demonstrated identical patterns of reactivity to what was observed with the MBP-TPO fusion proteins, indicating that the fusion protein or linker portion of the construct did not alter the binding to this region of TPO and was not involved in the inhibition. The ability of autoantibodies against TPO513-633 to bind whole native TPO was assessed by Western blot using affinity-purified antibody. Affinity-purified antibody to 513-633 recognized only the reduced form of native TPO (data not shown).
Figure 4: Inhibition of patient serum binding to MBP-TPO513-633 by preincubation with different MBP-TPO fusion proteins indicates the presence of an epitope in amino acids 592-633 other than either 592-613 or 607-633. Western blots are shown using serum from four Hashimoto's thyroiditis patients and one Graves' disease patient, as well as a positive control blot made with an anti-MBP monoclonal antibody. Each serum was preincubated with 0.2 mg of either MBP-TPO592-613 lysate (A strips), MBP-TPO513-633 (B strips), or MBP-TPO607-633 lysate (C strips) for 1 h at 37 °C. A control strip, preincubated in 1% bovine serum albumin (+ lanes), shows the uninhibited binding of each patient to the MBP-TPO513-633 band on the blot. Some patients demonstrated complete inhibition with either the MBP-TPO592-613 (H1, H2) or the MBP-TPO607-633 (H4) fusion protein. However, binding of serum from another patient is only partially inhibited (G1) or not inhibited (H3) by the MBP-TPO592-613 and the MBP-TPO607-633 proteins.
Figure 5: A MAP peptide ELISA was used to quantify autoantibody responses to TPO amino acids 592-613. Dilution curves of serum from six positive and five negative patients are shown as examples. Serum was serially diluted 1:200-1:25,600 and added to ELISA plates coated with 25 µg/ml MAP-TPO592-613. Titers for positive patients ranged from 1:200 to 1:25,600, while the negative patients (those with absorbance less than 0.2 at 1:200 dilution) show no reactivity at any sera dilution. The shaded line indicates the 95% confidence interval of normal sera.
ELISA employing GST fusion proteins containing TPO589-633 or TPO607-633 were used to examine the patients who reacted with other epitopes within TPO513-633. 4 of 31 Hashimoto's thyroiditis patients and 2 of 25 Graves' disease patients tested were positive to the 607-633 epitope. Thirteen sera that bound TPO513-633 also reacted with the whole 589-633 region, but not to either the 592-613 or the 607-633 epitope, reinforcing the finding of the inhibition study that this whole fragment appeared to be a single epitope in some patients. Patients reactive to TPO589-633 but not to TPO592-613 also did not recognize TPO589-613. 34 of the 35 patients with autoantibodies reactive to the TPO589-633 region exclusively recognized one of the defined epitopes (Table 1).
Figure 6:
Comparative affinity of autoantibodies for
the TPO 592-613 epitope was determined by the inhibition of serum
binding in the TPO592-613 peptide ELISA. Sera from positive
patients were preincubated with decreasing concentrations of GST-TPO
fusion proteins containing the sequence of amino acids 592-613 or
607-633 as a negative control. The preincubated serum was then
used in the TPO592-613 peptide ELISA. Binding in the ELISA was
inhibited completely by 10M or
10
M of all constructs containing amino
acids 592-613. The larger constructs, containing 589-633,
demonstrated a higher affinity of about 10
about
10-80 times greater than either of the smaller constructs of
589-613 or 592-613. The 607-633 fusion protein served
as a control and demonstrated minimal inhibition capability (<15%)
at 10
M which disappeared completely at
10
M. Similar results were obtained using
MBP-fusion proteins (not shown). The relative affinities for 8 patients (inset graph) show high affinity binding in 5 of these
patients.
Figure 7: The specificity of autoantibody binding to TPO592-613 was demonstrated by the inhibition of the TPO592-613 peptide ELISA with GST-TPO fusion proteins containing altered amino acids within the region of amino acids 589-613. Constructs were made which replaced the glutamic acids at amino acids 593 and 596 of TPO with serine at 593 and glycine at 596 or with alanine at 593 and arginine at 596. An additional construct contained the single substitution of glycine for glutamic acid at amino acid 596 in TPO589-633. The mutated TPO fusion proteins were used to inhibit positive serum binding in the TPO592-513 peptide ELISA and were compared to inhibition with the native sequences. None of the altered constructs demonstrated 50% inhibition of binding at concentrations nearly 100 times greater than that which achieved this level of inhibition in the native constructs. Similar results were obtained using MBP-fusion proteins (not shown).
Figure 8: Computer modeling of the 592-613 epitope of TPO. Note the tight loop without tertiary sheet or helical structure. The two glutamic acids, substituted in some of the fusion proteins, are shown with space-filling balls.
No correlation between reactivity to 592-613 and measures of thyroid function such as FT4, FT3, TSH levels, iodine uptake, or presence of goiter was observed for either Hashimoto's thyroiditis or Graves' disease patients. However, among the thyroiditis patients, hypothyroidism was significantly more common in those that recognized TPO592-613 (7 of 8, 88%) as compared to individuals recognizing the whole 589-633 epitope (5/11, 45%, p < 0.05).
These studies identify three autoantibody epitopes in the
region defined by TPO amino acids 589-633; two containing only
portions of the amino acid sequence, as well as the whole region which
was also recognized as a single epitope. Computer modeling of this area
revealed neither helix nor
sheet structure, but instead the
region appears to be a loop held in conformation by prolines at
positions 601 and 606. A cysteine at residue 598, just before the two
prolines and near the tip of the loop, suggests that the region may be
involved in disulfide linkages within the TPO molecule. This is in
accordance with the structure of TPO, which is currently believed to be
a disulfide-linked dimer of two 105-kDa chains containing both
intrachain and interchain disulfide
bonds(9, 10, 21, 22) .
Autoantibodies against the 589-633 region are unable to bind to
TPO unless it is reduced, indicating that the epitopes are inaccessible
in the native molecule. Together, this information indicates that the
epitopes within the 589-633 region of TPO may be neotopes, or
sites that are not normally exposed to the immune system(23) .
The serologic response to this particular region of TPO provides a number of insights into immune mechanisms underlying autoimmune thyroid disease. The autoantibody response to TPO is heterogeneous with different individuals recognizing unique epitopes. This has also been observed with the immune response to conformational epitopes in TPO, and it has been hypothesized to occur due to differences in immunoglobulin variable-region genes that might allow interaction with specific autoantibody binding sites(24) . In contrast to the response to conformational epitopes, the immune response to epitopes in the 589-633 region is restricted to a single site in almost all the individuals examined. The basis of this mutually exclusive epitope recognition is not clear, but it is possible that several of the binding sites within this region are initially recognized, and the immune response then evolves to a single, strongest epitope that excludes the other responses. This possibility is difficult to evaluate since the patients we investigated had mature autoimmune disease and had already evolved their autoantibody response. This restricted recognition could also be based on a genetic restriction, not the result of evolutionary changes in the immune response, and the evaluation of TPO epitope recognition in relatives of these patients may help to clarify this issue.
While portions of the 589-633 region demonstrate substantial conservation in amino acid sequence with analogous areas of other peroxidase enzymes, including myeloperoxidase and lactoperoxidase(11, 12) , areas of the amino acid sequences of the autoantibody epitopes within this region appear to be unique to TPO. Particularly, the charged residues and the prolines are unique to TPO and appear necessary to maintain antigenicity. Substitution of one of the charged amino acids with the corresponding sequence of LPO or MPO markedly decreases antibody affinity for the most dominant epitope in this region. This suggests that TPO autoantibodies develop from recognition of the autoantigen and not molecular mimicry from LPO or other antigens. However, a cross-reactive response to another antigen, such as LPO, that has subsequently affinity matured specifically to the autoantigen, cannot be excluded entirely since these patients have well-established autoimmune disease.
The importance of antibodies binding to epitopes within the 589-633 region in the diagnosis and treatment of AITD is of interest. The titer of antibodies directed against this particular region of TPO varies between different individuals and does not correlate with the overall anti-TPO titer. As a result, some patients have high titer antibody against this region but relatively modest titers of anti-TPO antibodies in conventional assays. This has the practical implication that anti-TPO titers in some patients may be underestimated with serologic assays that measure antibodies against native TPO. The mechanisms underlying the recognition of this site in AITD is difficult to ascertain. It is possible that these epitopes are recognized only after inflammation has disrupted thyroid follicular cells and released unfolded or reduced TPO, a form of epitope spreading. Recognition of the whole region, which is restricted to Hashimoto's disease patients, would seem likely to be the result of this process. However, the presence of autoantibodies against the local epitopes did not correlate with overall anti-TPO titers, a putative marker of glandular inflammation and release of TPO. This makes it less likely that the response to these epitope is based purely on this process. The presence of antibodies to the 592-613 epitope in patients with Graves' disease, which is not associated with extensive thyroid follicular cell disruption, would also seem to argue against this. However, this latter finding might be explained by an overlap syndrome, where thyroiditis and Graves' disease exist concurrently. Prospective studies screening for TPO epitope autoantibodies in Graves' disease may therefore identify individuals who have concurrent thyroiditis and might not need thyroid ablative therapy.
Other autoimmune diseases have identified
heterogeneous responses to autoantigens, particularly in inflammatory
processes. The autoantibody response to the acetylcholine receptor in
myasthenia gravis is similarly diverse, with the recognition of both
localized and conformational epitopes on the chain of this
protein(25) . The recognition of glutamic acid decarboxylase in
insulin-dependent diabetes mellitus is also heterogeneous with
recognition of both localized and conformational binding sites (26) . It has been difficult in both of these situations to
associate any particular manifestation of the disease with variations
in autoantibody epitope recognition, so the lack of an association in
this case is not surprising. Graves' disease has been suggested
to be associated with several types of autoantibodies to the TSH
receptor protein(27) . However, responses to particular
epitopes in the TSH receptor have not been entirely
characterized(28) , and mutually exclusive epitope recognition,
in a manner similar to the response against the 589-633 region of
TPO, has not been described. Thus, the findings of the present work on
TPO in AITD support and extend what has been observed in other
autoimmune diseases.
In conclusion, these studies demonstrate conclusively that the autoimmune response to TPO in autoimmune thyroid disease is heterogeneous and appears restricted in most patients and indicate that the initial events of TPO recognition involve the autoantigen itself. However, multiple factors are likely to be involved in initiating the autoimmune response in thyroiditis and Graves' disease, and some of these may be related to environmental antigens. These variables, together with the patient's own genetic background, might best account for the heterogeneity in immune responses that are seen in this protein in these diseases. Monitoring the response to the epitopes of TPO in the 589-633 area over time in patients with autoimmune thyroid diseases may clarify the evolution of the autoantibody response in these disorders and provide a better understanding of how the immune response to this protein evolves and contributes to thyroid disease pathogenesis.