Analysis of a conformational B cell epitope of human thyroid peroxidase: identification of a tyrosine residue at a strategic location for immunodominance
Valérie Estienne1,
Christine Duthoit1,
Stéphanie Blanchin1,
Roland Montserret2,
Josée-Martine Durand-Gorde1,
Martine Chartier3,
Daniel Baty3,
Pierre Carayon1 and
Jean Ruf1
1 U555 INSERM/Laboratoire de Biochimie Endocrinienne et Métabolique, Faculté de Médecine, Université de la Méditerranée, 13385 Marseille Cedex 05, France 2 Pôle de BioInformatique Lyonnais, IBCP, UMR 5086 CNRS, 69367 Lyon Cedex 07, France 3 Laboratoire dIngénierie des Systèmes Macromoléculaires, IBSM, UPR 9027 CNRS, 13402 Marseille Cedex 20, France
Correspondence to: J. Ruf; E-mail: jean.ruf{at}medecine.univ-mrs.fr
Transmitting editor: E. Möller
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Abstract
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Thyroid peroxidase (TPO) is involved in autoimmune thyroid diseases and high titers of TPO autoantibodies directed to various conformational B cell epitopes are frequently present in patients sera. Deciphering these epitopes is a difficult task, but can give insight into the structural basis of autoimmune recognition. TPO is a membrane-bound enzyme with the extracellular part organized in three protein domains, but of unknown three-dimensional structure. We previously localized a TPO B cell epitope within amino acid residues 742848, a region encompassing the two C-terminal, extracellular domains of the protein. We found that at least one of the three tyrosine residues of the peptide 742848 might be involved in autoantibody binding. In this study, we show by site-directed mutagenesis that the autoepitope contains tyrosine 772 located near the hinge area between the two protein domains, suggesting they are both involved in the epitope structure. The B cell epitopes of TPO are clustered in two overlapping immunodominant regions. To map the newly localized epitope with respect of these regions, competition experiments were performed using a reference panel of TPO mAb and a further mAb previously found to be specific for the TPO peptide 742848 at variance with all the other ones. Here, we show that the tyrosine 772-bearing epitope in the peptide 742848 maps in a region that partly overlaps the reported two immunodominant regions. These results are suggestive of a complex TPO folding that involves all the three TPO protein domains to form a highly conformational immunodominant region.
Keywords: autoimmunity, B cell epitope, circular dichroism, thyroid peroxidase, thyroperoxidase
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Introduction
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Like thyroglobulin and the thyrotropin receptor, thyroid peroxidase (TPO) is an autoantigen involved in autoimmune thyroid diseases (AITD). High amounts of TPO autoantibodies (aAb) are frequently present in the sera of patients with AITD (1). TPO aAb are involved in thyroid cell destruction through cytotoxic mechanisms mediated by the complement (2) and the killer cells (3). They also play a direct role in TPO processing (4,5). TPO is a 933-amino-acid long, type I integral membrane protein, which catalyzes the thyroid hormone synthesis (6). It contains a large extracellular region extending from amino acids 1 to 848 which consists of a N-terminal myeloperoxidase (MPO)-like domain followed by complement control protein (CCP)-like and epidermal growth factor (EGF)-like domains. The structure of these protein domains has been independently reported, but their exact organization in the three-dimensional structure of the TPO remains unknown (7). Noteworthy, TPO acts at the apical membrane of thyroid cells facing a closed colloid space, making this protein a sequestered autoantigen. The mechanism by which TPO aAb arise remains a mystery (8). Thus, characterization of structural requirements for a TPO B cell epitope would add to the knowledge of the autoimmune process.
It is generally agreed that the TPO B cell aAb repertoire is restricted. The concept of two overlapping immunodominant regions on the surface of the TPO molecule containing different but adjacent epitopes has been confirmed (9) in a previous study using our panel of mouse mAb (10) and recombinant human Fab from the RapoportMcLachlan (11) and Weetman (12) laboratories. Most of the TPO B cell autoepitopes are conformational and their structural characterization is difficult, explaining why, despite intensive investigations, none of them have yet been solved. Recently, numerous efforts have been made to more finely characterize them by biochemical and molecular methods (1317). Using large proteolytic fragments, we localized a conformational B cell autoepitope involving (a) tyrosine residue(s) of the C-terminal extracellular region of TPO (amino acids 742848) just before the transmembrane segment (13). Of major interest, this epitope appeared as the first positive probe for Hashimotos thyroiditis, an AITD with a diagnosis mainly based on histological examination (14). The region 742848 of TPO encompasses the CCP-like and the EGF-like domains, and a structural model of it has been proposed (14). In this model, the two domains have a flexible linkage. The EGF-like domain contains a calcium-binding site and it was shown that calcium binding decreases the flexibility of the neighboring inter-domain region (1820). In this context, we previously found that the flexible arrangement is important for antibody binding, showing that the two domains are closely linked and suggesting an important role for such an inter-domain region in the three-dimensional structure of a TPO B cell epitope (14).
Disclosing a B cell TPO autoepitope may provide clues for further understanding the mechanisms of thyroid autoimmunity and open new avenues for the use of recombinant peptides in therapeutic intervention aimed at blocking or removing toxic aAb. We have previously shown that iodination of the C-terminal peptide from human TPO (amino acids 742933) led to a loss of its immune recognition by TPO aAb from patients with AITD (13). It was therefore suggested that at least one of the three tyrosine residues of the peptide was part of the aAb binding site. All these tyrosine residues were located in the extracellular part of this peptide (amino acids 742848). To further locate the B cell epitope present in this peptide, we used the TPO recombinant peptide 742933 (r-pep) (14) to perform site-directed mutagenesis on the tyrosine residues. We also mapped this epitope relative to the two TPO immunodominant regions A and B previously described, using a reference panel of mAb which bind to TPO but not to r-pep and a further one directed against the r-pep.
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Methods
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Site-directed mutagenesis
The cDNA encoding for the TPO peptide 742848 was cloned into HindIII and XbaI sites of the transfer vector pcDNA3 (14). The PCR-based overlap extension method (21) was used for site-directed mutagenesis on each tyrosine residue. Two overlapping fragments were amplified in the initial PCR on
TPO-pcDNA3. One of them bore the point mutation which was introduced by the 3' primers, which in the case of tyrosine 766 gave 3'-A CCC GTG CCG GCA GGA AGA CAC CAG CAC GCG CC-5', for tyrosine 772 3'-C CCG GCC TTG GAG CTC AGA CCC GTG CCG GCA GG-5' and for tyrosine 829 3'-GGT TCT CCC ATC GTC TCC TAA CTC AGA GGG GTC CGC GCA GAG-5'. In these primers the triplet TAT/C coding for the tyrosine residue was changed to TCT to obtain the serine residue and they were used with the external primer 5'-ACT ACC GCT CGA GCG ACG ACA AGT GTG GCT TCC-3' to obtain this first fragment. For the second fragment, the primers 5'-TCC TGC CGG CAC GGG-3', 5'-GAG CTC CAA GGC CGG G-3' and 5'-TA GGA GAC GAT GGG AGA ACC-3' for the tyrosine residues 766, 772 and 829 respectively were used with the external primer 3'-GAT TTA CCC GTG TTG GG-5'. The second PCR resulted in the complete mutated PCR product. The primers were synthesized by the molecular biology facility of the CNRS (Marseille, France). The cDNA of the various mutants was sequenced to make sure that the mutants of
TPO-pcDNA3 constructs were correctly engineered and exclude any further mutations.
Transfection into Chinese hamster ovary (CHO) cells
The mutated
TPO-pcDNA3 constructs were transfected into CHO cells using the lipofectAMINE reagent according to the manufacturers protocol (Life Technologies, Gaithersburg, MD). The CHO cells were kept in Hams F-12 medium supplemented with 10% FCS, penicillin (100 IU/ml) and streptomycin (0.1 mg/ml) in a humidified atmosphere under 7.5% CO2 at 37°C. Stable transfectants were selected with 400 µg/ml geneticin G418 sulfate for 6 weeks. Single cell clones were established by limiting dilution and grown in vitro in the presence of 10 mM Na butyrate to enhance the transcription of TPO r-pep mutants.
Immunodetection of the TPO r-pep mutants
CHO cells transfected with the wild-type and mutagenized
TPO-pcDNA3 cDNA were grown in 100 x 20-mm tissue culture dishes and washed 3 times with PBS, pH 7.3. Confluent cells from each dish were lysed by adding 800 µl TrisHCl, pH 6.8, containing 30% glycerol, 1% SDS and 0.02% G-250 Coomassie brilliant blue to the tissue culture dishes. The cells were scraped off and homogenized by vortexing and sonication. After being centrifuged at 10,000 g for 10 min, the supernatant was analyzed using the TricineSDSPAGE method (22). Aliquots of 10 µl of supernatant per lane were electrophoresed under non-reducing conditions on a 16.5% acrylamide, 80 x 100-mm minigel (0.5-mm thick) and directly electrotransferred onto a 0.2 µm Trans-blot PVDF membrane (BioRad, Hercules, CA). Unspecific antibody binding was blocked with 3% BSA in PBS. Membranes were incubated overnight at 4°C with a pool of patients sera (250 µl/lane) or TPO mAb 54 (50 µg/lane) in 5 ml PBS with 3% BSA. The membrane was then washed 3 times for 10 min in PBS. The membrane was incubated with anti-human or anti-mouse secondary antibodies labeled with horseradish peroxidase for 2 h in PBS, 3% BSA at room temperature with constant shaking. After several additional washes, the blots were developed with 4-chloro 1-naphthol as the substrate.
Continuous elution electrophoresis
Wild-type and mutant Y772S r-pep were purified with the model 491 Prep Cell from BioRad. The procedure consisted of separating the proteins from cell lysates, by SDSPAGE, prepared in a 28-mm diameter gel tube as previously described by Duthoit et al. (23). Briefly, CHO cells transfected with wild-type or mutagenized
TPO-pcDNA3 cDNA were lysed, scraped off and homogenized as above. After being centrifuged at 10,000 g for 10 min, the supernatant was loaded onto a 15% acrylamide gel and the separation was run with Laemmli buffer. Fractions from the elution flow were collected, and aliquots were analyzed by SDSPAGE and Western blotting experiments. Fractions containing the eluted 20-kDa peptide were then pooled, concentrated and dialyzed against 2 mM sodium phosphate buffer, pH 7.3.
Circular dichroism (CD) measurements
CD spectra were recorded on a Jobin-Yvon CD6 spectrometer calibrated with (+)10-camphorsulfonic acid. Measurements were done at room temperature in 0.2-mm path length quartz cells (Hellma, Müllheim, Germany) with proteins concentrations ranging from 17.7 (mutant Y772S) to 27.5 (wild-type) µM in 2 mM sodium phosphate buffer, pH 7.3. Spectra were recorded in the 190250 nm wavelength range with 0.2 nm increments and 2 s integration time. The spectra calculations were performed using the CD6 processing software as detailed previously (24).
TPO mAb
A panel of eight TPO mAb that bind to various epitopes clustered in two previously defined immunodominant regions (A and B) of the TPO molecule was used (10). Because none of the mAb bound to the r-pep, we also used another TPO mAb (mAb 54) which was previously found to recognize the r-pep in Western blotting experiments (14).
TPO radiolabeling
Human TPO was prepared as previously described (13). Purified TPO (10 µg) were labeled with 250 µCi carrier-free 125I-Na and 2.5 µg chloramine-T in 30 µl 0.2 M sodium phosphate buffer, pH 7.0. The reaction was stopped after 60 s by adding 5 µg sodium metabisulfite in sodium phosphate buffer. Labeled TPO was further purified and separated from the free iodide by gel filtration on BioGel A 1.5 m equilibrated in PBS containing 1% BSA and 0.1% Tween 20. The fractions containing the labeled TPO were pooled and adjusted to 2 µCi/ml.
TPO binding assay
Wells of Falcon flexible assay plates (Becton Dickinson, Oxnard, CA) were coated overnight at 4°C with 100 µl PBS containing 5 µg/ml of either mAb 54 or each of the eight mAb from cluster A and B. The wells were washed and then unspecific binding sites were blocked with PBS containing 1% BSA. Aliquots of 50 µl labeled TPO were mixed with increasing amounts of competitor mAb in a final volume of 50 µl and added to the antibody coated wells; i.e. for the wells which are coated with mAb 54, the eight mAb were used as competitor antibodies and vice versa. Coated and uncoated wells were included to evaluate the maximum TPO binding and non-specific TPO binding respectively. After a 3 h incubation at 37°C, the wells were washed thoroughly, cut into individual tubes and the radioactivity was measured in a
-counter.
Protein concentration
The protein content of the various reagents used in this study was determined by performing the micro BCA assay (Pierce, Oud Beijerland, The Netherlands) with BSA as standard.
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Results
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Site-directed mutagenesis
Using the PCR-based overlap extension method (Fig. 1), we obtained tyrosine to serine mutations for the three tyrosine residues of the r-pep at position 766, 772 and 829. We stably transfected cDNA mutants of the
TPO-pcDNA3 construct into CHO cells and we obtained the three r-pep mutants Y766S, Y772S and Y829S. Several clones of transfected CHO cells were obtained for each cDNA mutant. To avoid any structural problem in protein folding, the cDNA constructs were not tagged. Consequently, transfection efficiency and peptide production were monitored by SDSPAGE and Western blotting experiments performed on lysates of transfected CHO cells. Our reference pool of 40 patients sera previously found to be reactive with the native (13) and recombinant (14) TPO peptide 742933 was used. Fitting the qualitative and quantitative criteria for TPO aAb heterogeneity, this pool was considered as a valuable reagent to test epitopes from the TPO immunodominant region. As shown in Fig. 2(A), only the r-pep mutant Y772S lost aAb binding and the corresponding cell lysate gave a pattern of staining similar to that obtained with untransfected cells. These results suggested that tyrosine 772 was an essential residue for TPO aAb binding to the r-pep epitope. TPO mAb 54 has been previously reported to bind to the r-pep and cross-react with TPO aAb (14). Effectively, mAb 54 recognized the wild-type r-pep and we noted that none of the tyrosine to serine substitutions tested was deleterious to the mAb 54 epitope (Fig. 2B). These results showed that although mAb 54 and patients TPO aAb were found cross-reactive, and consequently similar to each other, there existed a marked difference between the epitopic structures of the r-pep which was recognized by the mouse and the human antibodies. These results further suggested that while affecting one of these two adjacent epitope structures, tyrosine to serine 772 substitution did not change the overall three-dimensional structure of the peptide.

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Fig. 1. Schematic diagram of the mutagenesis procedure used. The PCR-based overlap extension method was used (21). Two intermediate products were obtained in the first PCR; one of them containing the point mutation. These PCR products were purified and used as templates in the second PCR to obtain a complete fragment with the point mutation.
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Fig. 2. Immunoreactivity of the various TPO r-pep. Western blotting experiments were performed on lysates of untransfected CHO cells (lane 0) and CHO cells producing mutants (lane 1, Y766S; lane 2, Y772S; lane 3, Y829S) and wild-type (lane 4) TPO r-pep. Immunodetection was performed using aAb from pooled sera of patients (A) and mAb 54 (B). Prestained electrophoresis standards were run with the samples. Their respective mol. wt (kDa) are given on the left of the figure. Arrows indicate the immunoreactive TPO r-pep.
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CD
CD is a sensitive method used in determining whether a protein is folded by characterizing its secondary structure (25). It was particularly useful to evaluate conformational changes induced by amino acid sequence alteration in site-directed mutagenesis. Changes in the secondary structure of proteins were found to alter both the shape and the amplitude of the resulting CD spectrum (26). The wild-type and mutated r-pep were similarly purified by preparative electrophoresis. Figure 3 shows the elution profiles of the total proteins and the purified r-pep respectively obtained from lysate of CHO cells transfected with the wild-type
TPO-pcDNA3 construct. Using mAb 54 in Western blotting experiments, we found that the wild-type r-pep eluted in fractions 1215. In a similar experiment, we found that the r-pep mutant Y772S recovered into the same fractions and we further ascertained that purified wild-type but not mutant Y772S remained immunoreactive for TPO aAb (not shown). The far UV CD spectrum of the purified wild-type r-pep showed two negative bands at 208 and 222 nm, and a positive band around 192 nm (Fig. 4). This CD pattern indicated that the r-pep folded mainly in the
-helical conformation. Moreover, the spectra of the purified wild-type and mutated r-pep exactly overlapped, both displaying minimum values at the same wavelengths. The change of amplitude we observed between the two spectra was only linked to the concentrations of the respective r-pep in the CD quartz cells. This result stressed that the secondary structure of the r-pep was not affected by the Y772S mutation and further indicated that its tertiary structure remained unchanged.

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Fig. 3. Elution profile of proteins from lysate of transfected CHO cells. After running on continuous elution electrophoresis, aliquots from one to three eluted fractions were electrophoresed and the proteins from the gels were either directly stained with G-250 Coomassie brilliant blue (A) or electrotransferred on PVDF membrane, and then tested in Western blotting experiments with TPO mAb 54 (B). The purified r-pep was present in the starting material (lysate) and eluted from fractions 12 to 15 (see the arrow). The mol. wt (MW) of the standards are indicated on the left.
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Fig. 4. Far UV CD spectra for wild-type and mutant Y772S r-pep. The protein concentrations were 27.5 and 17.7 µM respectively. Measurements were performed at room temperature in 2 mM Na phosphate buffer, pH 7.3.
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Epitope mapping of the TPO r-pep
We previously described two immunodominant regions on TPO, named A and B, which include the epitopes recognized by TPO aAb from patients with AITD (10). The same immunodominant regions achieved a reciprocal nomenclature with the human recombinant Fab panels (11,12). To investigate whether the r-pep localized close or distant to A and B immunodominant regions, we used the reference method initially designed to map the antigenic surface of TPO (10). Since we showed by site-directed mutagenesis that, at variance with the TPO aAb, any tyrosine residue of the r-pep was part of the mAb 54 TPO epitope, we could safely use the radiolabeled TPO to perform the epitope mapping of the r-pep. Effectively, we found that mAb 54, like all the other TPO mAb, bound to labeled TPO, and then we criss-crossed it with all the TPO mAb from clusters A and B to determine their binding behavior to labeled TPO. The binding of coated mAb 54 to labeled TPO was inhibited by the homologous mAb. The TPO mAb from clusters A and B behaved similarly as inhibitors with the exception of mAb 9, which was found to highly potentiate mAb 54 binding to TPO at all doses tested (Fig. 5A). In the reverse experiment, however, mAb 54 inhibited only TPO binding of mAb 47 from cluster A and mAb 64, 18 and 59 from cluster B (Fig. 5B).

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Fig. 5. Epitope mapping of TPO r-pep. Criss-cross competitive experiments were performed with a set of TPO mAb to test the mAb 54 binding to the r-pep region of labeled TPO. Soluble mAb 54 and TPO mAb from clusters A and B competed with coated mAb 54 (A), and soluble mAb 54 competed with the various coated mAb from clusters A and B (B) for binding to labeled TPO. Results are expressed as percentages of labeled TPO bound in the presence of competitor with respect to those bound without competitor, after subtracting the non-specific TPO binding.
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Discussion
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We previously observed that mAb 54 and patients aAb cross-reacted with a conformational epitope on the r-pep (14), suggesting that the mAb 54 epitope might be similar, if not identical, to the human B cell autoepitope. However, the mutation Y772S in the r-pep completely abolished the immunoreactivity towards TPO aAb but not towards the mAb 54. Our data showed that mAb 54 recognized an epitope adjacent to but different from the human autoepitope, suggesting that site-directed mutagenesis introduced minor structural changes pointing to only one of the two adjacent epitopes. The total binding inhibition of a heterogeneous population of TPO aAb to the Y772S mutant strongly suggested that only one autoepitope was present within the amino acid 742848 region of the TPO molecule and that only one of the three tyrosine residues of the peptide was involved in this epitope. However, the absence of a loss in TPO aAb immunoreactivity for the other mutants did not exclude the possibility that minor aAb directed to epitopes involving tyrosine other than tyrosine 772 were present in some individuals from the pool. Furthermore, the total loss of the aAb binding to the r-pep mutant Y772S showed that tyrosine 772 was crucial for aAb binding. This result agrees with the molecular model we previously described which showed that among the three candidates, tyrosine 772 was the most exposed at the surface of the molecule and was therefore probably affected by the iodination process (14). As monitored by CD spectroscopy, the secondary structures of the wild-type and r-pep mutant Y772S were found to be similar. These results further demonstrated that no significant change in the tertiary structure of the r-pep mutant occurred. The extracellular region of the r-pep (amino acids 742848) encompasses the CCP-like and the EGF-like domains of TPO. Figure 6 adds further insights in our understanding, showing that it is not possible to superimpose these domains together. This structural characteristic suggests that these two domains link in a highly flexible arrangement. Tyrosine 772 is in the CCP-like domain of the r-pep. This type of domain folds in a small hydrophobic core enveloped by eight ß strands (27), six of them being major and stabilized by four conserved cysteines forming disulfide bridges arranged in a 13,24 Sushi pattern (28). In this organization tyrosine 772 localizes in the third major ß strand which forms a surface patch located close to the C-terminal extremity of the module (28). This residue is therefore located in the flexible hinge area between the CCP-like and the EGF-like domains. Being in a ß strand structure, tyrosine 772 is taken into account in CD spectroscopy. Similar CD profiles were obtained with wild-type and r-pep mutants. This further indicates that the tyrosine to serine change brought by the site-directed mutagenesis did not have substantial effects on the overall conformation of this part of the molecule but that tyrosine772 was directly involved in the epitope recognized by TPO aAb. We thus suggest that the aAb epitope is indeed located around the flexible hinge area spanning the two constitutive protein domains of the r-pep. Our findings agree with the characteristics of a B cell epitope structure that is commonly part of hyper-flexible regions in the target protein (29). Molecular flexibility contributes to the activity and biochemical properties of macromolecules [for a recent example, see (30)]. For aAb binding, the flexibility may lead to an atomic mobility that allows molecular transconformations to occur in the antigen during aAb binding. Transconformation can then reinforce the binding between the antigen and aAb (31,32).

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Fig. 6. Molecular flexibility of the domain interface between the N-terminal CCP-like and the C-terminal EGF-like domains. We generated various three-dimensional models for the TPO peptide 742848 containing this domain association (14). Here, either the CCP-like domains in black (A) or the EGF-like domains in grey (B) are superimposed. When the CCP-like domains are superimposed, the linked EGF-like domains are not and vice versa.
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The exact location of the conformational TPO B cell epitopes has not been yet identified. The r-pep was found to contain a B cell epitope recognized in Western blot analysis by only one mAb (mAb 54) out of a panel of 66 mouse TPO mAb we previously produced (14). To determine the relationship between the r-pep epitope recognized by mAb 54 and the previously identified TPO immunodominant regions A and B, competition experiments were performed using labeled TPO and the reference TPO mAb from clusters A and B (10). The mAb 54 criss-crossed for TPO binding with various mAb from clusters A and B, providing evidence that mAb 54 recognized an epitope structure in a region which partly overlaps both the A and B immunodominant regions. These results explain why mAb 54 and patients aAb cross-reacted, and further prove that TPO aAb and mAb 54 epitopes of the r-pep co-localize in the TPO immunodominant region. The TPO B cell autoepitopes are generally conformational (3335), i.e. they consist of various segments dispersed along the whole molecule, but gathered closely together by the three-dimensional folding structure of the native molecule. Interestingly, although the majority of the TPO mAb, including mAb 54, recognize conformational epitopes, mAb 47 from cluster A recognizes a known linear epitope (amino acids 713721) not topologically distant from the r-pep (36). A possible explanation for the pattern of cross-reactivity shown between mAb 47 and mAb 54 is that the immunodominant region A of TPO consists partly of the segment of mAb 47 epitope and of amino acids residues from r-pep. Furthermore, mAb 54 was found to cross-react with mAb 64, 59 and, to a lesser extent, with mAb 18 from cluster B. This feature is also characteristic of mAb 47 and was described in our initial report (10). We thus conclude that the tyrosine 772-bearing epitope becomes localized at the junction of A and B immunodominant regions of TPO 7(Fig.7). Our conclusion is further supported by the behavior of mAb 9, which since it was bound to TPO, significantly increased the mAb 54 binding to TPO. It was previously found that mAb 9 from cluster A was directed toward the major antigenic region targeted by 8090% of the TPO aAb from individual patients (37). The mAb 9-mediated effect on mAb 54 binding to TPO probably resulted from structural changes occurring in related epitopic structures during the transconformational process described above. That structural transconformation might occur further highlights the flexible characteristic of the immunodominant region of TPO which can adapt its structure for aAb binding. However, the fact that we did not observe the reverse effect from mAb 54 to mAb 9 binding to TPO confirms some differences between the respective epitopes recognized by the mAb.

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Fig. 7. Schematic representation of A and B immunodominant regions on the surface of the TPO molecule. Epitopes from A and B regions are shown as black and white circles respectively. The circles contain the appropriate mAb number and are grouped according to the initial mapping (10). The Y772-bearing autoepitope and the atypical mAb 54 epitope are shown as grey circles in the AB overlapping region. The Y772-bearing autoepitope is in a central position and in close proximity to various mAb epitopes.
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Taken as a whole, we clearly identified for the first time the tyrosine 772 of the human TPO as part of an autoepitope recognized by aAb from patients with an AITD. This epitope is different but not so far from those recognized by mAb 54 and consequently it maps in the same immunodominant region. The epitope location around tyrosine 772 is consistent with recent data on the analysis of TPO aAb epitopes showing that some amino acids from TPO regions 631772 and 514772 are involved in aAb binding (15). Both regions include tyrosine 772, which we found crucial for the aAb binding. In contrast, tyrosine 772 is not located in the MPO-like domain of TPO believed to contain in part the immunodominant region of TPO (16,17). However, based on a putative three-dimensional model of TPO, lysine 713 was recently identified in the TPO immunodominant region and would lie in the vicinity of tyrosine 772 (17). The spatial organization of the three extracellular domains of TPO remains to be determined. Tyrosine 772 is at the intersection between the CCP-like and the EGF-like domains, and it localizes close to lysine 713 in the MPO-like domain. We therefore suggest that the TPO immunodominant region, at the molecular level, is formed by the spatial arrangement of amino acids residues from the three independent protein domains brought into close proximity with each other. This structural organization would add complexity to the epitope structures for TPO aAb, thus explaining why it is so difficult to decipher them.
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Acknowledgements
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We thank Dr P.-J. Lejeune for providing patients sera with positive TPO aAb titers and Dr C. Blanchet for performing graphic displays of three-dimensional model superimpositions.
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Abbreviations
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aAbautoantibodies
AITDautoimmune thyroid disease
CCPcomplement control protein
CDcircular dichroism
CHOChinese hamster ovary
EGFepidermal growth factor
MPOmyeloperoxidase
r-peprecombinant peptide
TPOthyroid peroxidase
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