Identification of an Immunodominant Region Recognized by Human ...

0013-7227/00/$03.00/0 Endocrinology Copyright © 2000 by The Endocrine Society

Vol. 141, No. 6 Printed in U.S.A.

Identification of an Immunodominant Region Recognized by Human Autoantibodies in a ThreeDimensional Model of Thyroid Peroxidase* PAUL HOBBY†, ANDRZEJ GARDAS†, ROMAN RADOMSKI, ALAN M. MCGREGOR, J. PAUL BANGA, AND BRIAN J. SUTTON The Randall Centre and Guy’s, King’s and St. Thomas’ School of Medicine (A.M.M., J.P.B.), King’s College London, London, United Kingdom SE1 1UL; and Medical Centre of Postgraduate Education (A.G.) and Department of Medical Informatics, Warsaw University Medical School (R.R.), 99 01-813 Warsaw, Poland ABSTRACT Autoimmune thyroid diseases (AITD) are characterized by the presence of autoantibodies to thyroid peroxidase (TPO). This response is dominated by autoantibodies to two conformational determinants, termed A and B, that have been defined by monoclonal antibodies but whose structures and location within TPO are unknown. We have modeled the three-dimensional structure of the extracellular region of TPO, raised antisera to prominent surface structures, and identi-

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UTOIMMUNE THYROID diseases (AITD) are among the most common human autoimmune disorders, affecting 2% of the adult female Caucasian population (1, 2). The presence of high titer autoantibodies to the microsomal antigen, now known to be thyroid peroxidase (TPO), is a hallmark of disease activity and is frequently used as a diagnostic indicator (3, 4). Autoantibodies to TPO are polyclonal, usually of the IgG1 and IgG4 subclass, and recognize conformational determinants on the molecule (4, 5), although serum reactivity with reduced and denatured TPO, recombinant fragments, and synthetic peptides has also been reported (6 –12). However, the major part of the autoantibody response to TPO is directed toward two dominant conformational determinants, which were initially defined with a panel of murine monoclonal antibodies (mabs) and termed A and B (13). Independent studies using human anti-TPO antibody Fabs derived from combinatorial Ig gene libraries from patients with thyroid autoimmunity also identified two, large neighboring determinants on TPO, which were also confusingly labeled as determinants A and B (14, 15). The relationship between these dominant determinants remained unresolved until recent competition studies (16, 17) showed that region A, recognized by murine mabs to TPO, was identical to region B, recognized by human antibody

Received November 17, 1999. Address all correspondence and requests for reprints to: Dr. B. J. Sutton, The Randall Centre, King’s College London, New Hunt’s House, Guy’s Campus, London Bridge, London, United Kingdom SE1 1UL. E-mail: [email protected]. * This work was supported by the Wellcome Trust, the Royal Society, the Polish Committee for Scientific Research (Grant 4PO5A), the British Council, and the Biotechnology and Biological Sciences Research Council, United Kingdom (to P.H.). † Both authors contributed equally to this work.

fied an epitope that we show to be a critical part of the B determinant. Antibodies to this epitope inhibit the binding to TPO of human autoantibodies in virtually all serum samples from 65 patients with AITD that were tested. This first description of a model of the threedimensional structure and location of a major autoantigenic determinant within the TPO molecule may provide structural clues for identifying causative agents or developing novel therapeutic strategies. (Endocrinology 141: 2018 –2026, 2000)

Fabs, and vice versa. In this communication we will use the original description of these determinants defined by murine mabs. A restricted gene usage has also been reported in the human autoantibody response to these two dominant determinants (15, 17, 18). TPO belongs to the family of mammalian peroxidases that include myeloperoxidase (MPO), eosinophil peroxidase, lactoperoxidase, and salivary peroxidase (19 –21). The crystal structure of only MPO is known, and it reveals a disulfidelinked dimer (22, 23). We have generated crystals of TPO, but they were unsuitable for x-ray diffraction analysis (24). More recently, diffracting crystals of TPO have been described, but they do not diffract to sufficiently high resolution for a structure determination (25). The degree of sequence similarity between TPO and MPO, however, is sufficient for homology modeling. We report here a model of the extracellular domains of TPO, from which we have selected peptides and generated antisera to accessible surface features of the molecule. Binding studies with these antipeptide antibodies and inhibition of patients’ autoantibodies identify a major autoreactive epitope in TPO. Furthermore, competition with murine mabs relates this epitope to the A and B determinants identified previously; thus, the three-dimensional structure and location of an immunodominant epitope of TPO is now defined. Materials and Methods Sequence alignment and modeling Proteins of known structure with sequence homology to TPO were identified using the Structural Classification of Proteins (SCOP) database (26), and all coordinates were taken from the Protein Data Bank (27). Three-dimensional models were constructed using HOMOLOGY (Molecular Simulations, Inc., Cambridge, UK) on Indigo and Indy work-

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AN IMMUNODOMINANT EPITOPE OF TPO stations (Silicon Graphics Ltd., Reading, UK), and model building and energy minimization were performed using INSIGHTII and DISCOVER (Molecular Simulations Inc.). The strategy adopted was as follows: 1) the structure with highest sequence homology to TPO, as judged by the SCOP score, was selected as template for each domain; 2) structurally conserved regions (SCR) were identified, and the intervening regions, generally surface loops and links between domains, were defined as variable regions (VR); 3) main chain coordinates for the SCRs were taken directly from the template structure; 4) main chain coordinates for VRs were generated using a conformational search algorithm (GENLOOPS within HOMOLOGY); conformers were considered if C␣-C␤ bonds of the overlapping residues at the splice points aligned well, and if main chain ␾ and ␸ angles were acceptable; minimal steric interaction with adjacent SCRs was the final criterion for selection; 5) side-chain coordinates for the TPO sequence, where different from the template, were generated using a rotamer library (within HOMOLOGY); 6) splice points between SCRs and VRs were refined, placing a torsion force to constrain the main chain ␻ angle to 180° during local energy minimization (splice repair subroutine of HOMOLOGY/DISCOVER); 7) energy minimization of the entire structure of each domain was performed (100 cycles of steepest descent followed by 900 cycles of conjugate gradient); and 8) structures were assessed using PROCHECK (28) to monitor stereochemistry. Solvent accessibilities of peptide segments within the model were calculated using NACCESS (S. J. Hubbard, Department of Biomolecular Sciences, University of Manchester).

Purification of human TPO (hTPO) Thyroid microsomes were prepared from pooled Graves’ thyroid tissue and human TPO (hTPO), purified as previously described (29).

Antibodies Serum from patients with thyroid autoimmune disease was obtained with permission from the Warsaw out-patient endocrine clinic. Diagnosis was made by standard clinical and biochemical criteria, identifying sera from patients with Graves’ disease (n ⫽ 45) and lymphocytic hypothyroid disease (n ⫽ 20). Pooled sera from normal healthy individuals (n ⫽ 20) were used a control. Autoantibody to TPO was measured by enzyme-linked immunosorbent assay (ELISA), standardized to the WHO/Medical Research Council International Standard 66/387 (29). Murine mabs to TPO, in the form of ascites of mabs 2, 9, 47, 15, 18, and 64, were provided by Dr. J. Ruf (13).

Rabbit antibody to synthetic peptides of TPO Peptides were synthesized by F-moc chemistry with C-terminal amides and purified by HPLC in trifluoroacetic acid/water and lyophilized. A cysteine residue was added to the N- or C-terminus of each peptide for coupling to carrier protein. All peptides were checked for purity by mass spectrometry. Peptides were conjugated to maleimideactivated keyhole limpet hemocyanin (1 mg peptide/1 mg keyhole limpet hemocyanin) and further purified by chromatography on Ultrogel AcA22 chromatography in PBS as described previously (29) for peptides 11–13. Either one or two New Zealand White rabbits per peptide were injected according to the schedule described previously (29).

RIA TPO was iodinated by the Iodogen method as previously described (30). Briefly, to an Eppendorf tube (Sigma-Aldrich, Poznan, Poland) coated with 25 ␮g Iodogen/tube, 10 ␮l containing 100 ␮Ci [125I]Na (Swierk, Warsaw, Poland), and 100 ␮g hTPO in PBS (100 ␮l) were added and incubated for 10 min. The contents were transferred to another tube containing 100 ␮l PBS/BSA and applied to a HPLC column (Superose 6 HR 10/30, Pharmacia Biotech, Uppsala, Sweden) and run in 0.1 m Tris-HCl buffer, pH 8.5, containing 0.1% deoxycholate and 0.5 m NaCl. Fractions containing protein and radioactivity were assayed for reactivity with rabbit anti-hTPO antibodies and pooled. Antisera were diluted from 1:100 to 1:5,000 in PBS, pH 8.0, containing 0.5% Triton X-100 and 3% polyethylene glycol 6000. Labeled TPO (25,000 cpm) was added to each tube, mixed, and incubated overnight at 8 C. Then, 100 ␮l goat antirabbit serum were added and mixed, and after 15

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min the precipitated complexes were microfuged at 10,000 gav for 10 min. The supernatant was carefully discarded, and the radioactivity of the pellet was measured in a ␥-counter (LKB Wallac, Inc., Turku, Finland). The amount of labeled TPO precipitated by rabbit polyclonal anti-hTPO antibody at 1:100 dilution was taken as 100%. When human sera were tested, the precipitating antibody was rabbit antihuman, instead of goat antirabbit serum. In all experiments the control was preimmune rabbit or human serum negative for TPO antibodies.

Inhibition of autoantibody binding to TPO and ELISA Microtiter plates (Nunc, Copenhagen, Denmark) were coated with purified hTPO (0.5 ␮g/ml), and 100 ␮l of 100-fold diluted rabbit antipeptide antiserum were added and incubated for 1 h at room temperature. After washing, patients’ sera that had been diluted to give 10 IU/ml TPO autoantibodies were added to the wells and incubated for 1 h at room temperature. After washing three times in PBS Tween (saline with 0.1% Tween-20), horseradish peroxidase-conjugated rabbit antihuman IgG (diluted 1:2000) was added and incubated for 1 h, followed by three washes with PBS Tween. The plates were developed with tetramethylbenzidine solution, and optical density was measured at 450 nm. Preimmune rabbit serum was used for controls, and wells without addition of human serum were considered blank. Inhibition of autoantibody binding was calculated in comparison to preimmune serum according to the formula: (A ⫺ B/T ⫺ B) ⫻ 100, where B is the OD of the background, T is the OD of preimmune serum, and A is the OD of antipeptide antiserum. Inhibition of binding of murine mabs to TPO was measured in the same way, but using rabbit antimouse IgG-horseradish peroxidase conjugate (diluted 1:2000). All ELISAs were performed as described previously (29).

Results Sequence alignment and modeling of the TPO structure

The only known crystal structures for the peroxidase family of enzymes are those of canine and human MPO (22, 23). These 2 structures have identical main chain conformations and provide the template for the principal domain within the extracellular sequence of TPO, residues 142–738 (sequence numbering according to human TPO; Fig. 1). Within this domain (overall sequence identity, 46%; overall sequence similarity, 71% between TPO and MPO) there are several extensive structurally conserved regions (SCRs; boxed regions in Fig. 1). These regions include conservation of the 13 cysteine residues that in MPO form 6 intramolecular disulfide bridges and 1 intermolecular bridge between subunits across the dimer interface. There is 1 additional cysteine residue in TPO (residue 146) in a 3-residue insertion located at the surface; this may bridge to a conserved cysteine (residue 133) in the N-terminal segment (residue 1–141) that is not present in mature MPO. All of the VRs (Fig. 1) correspond to surface loops. The first of the 2 large VRs (249 –256, SKAAFGGG) corresponds to the segment that in MPO is cleaved from the mature protein to generate the 2 polypeptide chains, and the second is an insertion of 10 residues (299 –308: GDQGALFGNL) in a surface location close to the dimer interface in the MPO structure. Model structures for these 2 loops were generated by conformational loop search (GENLOOPS) and are the only 2 regions where the modeled backbone conformation must be considered tentative. With the exception of a 3-residue insertion (376 –378), the other VRs involve only single residue insertions or deletions, and all are readily accommodated within the structure. The extensive SCRs and the surface location of all VRs are illustrated in the model of TPO shown in Fig. 2A. [PGH2 synthase-1 has sequence homology with MPO and a similar overall fold (31), but the mode of dimerization is quite different, and the

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FIG. 1. Sequence alignment of the TPO sequence with MPO (code MHL), factor H (FH), and fibrillin (FIBRIL). The boxed amino acid alignments indicate SCRs in which coordinates have been assigned from the template molecule to the TPO model. Other, VRs differ significantly from the template sequence; these all comprise surface loop structures (indicated with asterisks) that were modeled using the conformational search program GENLOOPS under INSIGHT II. Putative N-linked glycosylation sites are indicated with a filled circle under the appropriate asparagine residue.


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FIG. 2. A, An ␣-carbon trace of the modeled MPO-like domain of TPO (in black) associated as a dimer in the same fashion as MPO. The view is posterior to the active site. Loops that have been built by conformational search are indicated in purple, and the positions of the heme groups (green) and calcium ions (yellow) are shown. B, Ribbon diagram of the structure of TPO showing the MPO-like domain (cyan), CCP-like domain (orange), and EGF-like module (purple). Peptide sequences involved in this study are labeled. These include peptide 6 (yellow), peptide 14 (red), remainder of overlapping peptide 15 and peptides 16 –17 (yellow), and the peptide recognized by mab 47 (green). The inset depicts a space-filling model of the MPO-like domain of TPO in which the pronounced accessibility of peptide 14 (red) can be seen. The orientation of the TPO monomer in these pictures is virtually identical to that of the righthand domain of the dimer of TPO depicted in A above. The N-terminus of the MPO-like domain (residue 142) is indicated (N).

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long VRs of TPO have no counterparts in PGS to assist modeling.] The N-terminal residues 1–141 in TPO are not represented in the MPO crystal structures because this segment is cleaved in mature MPO. Comparison with sequences of proteins in the SCOP database (see Materials and Methods) revealed no significant alignments. This region cannot therefore be modeled in detail at present, although earlier sequence analysis (20) predicted four ␣-helical regions separated by turns, suggestive of a folded ␣-helical bundle motif, a result confirmed by fold recognition analysis (using the UCLA-DOE Fold Recognition server). To the C-terminal side of the MPO-like domain, between this domain and the predicted transmembrane region (Fig. 1), lies a sequence (residues 739 – 841) that corresponds precisely to the products of exons 13 and 14 (20). Comparison of this region with the SCOP database indicates a highly significant match with the 15th domain of complement factor H over the entire sequence corresponding to exon 13 (residues 739 –795; Fig. 1; 21% identity, 48% similarity between the two sequences). This domain of factor H is a typical complement control protein (CCP) module (32), and the alignment of four of the five cysteine residues in the exon 13 sequence of TPO with the only four cysteine residues in the CCP domain of factor H (where they form two disulfide bridges) strongly implicates this fold in TPO. Only one deletion of a five-residue loop from the factor H structure and a single residue insertion are required to model the main chain conformation of this domain (Figs. 1 and 2B). A strikingly similar match was obtained for the entire sequence corresponding to exon 14 (residues 796 – 841), which aligns with several epidermal growth factor (EGF)like domains, as noted previously (20). The highest score was with the EGF-like domain of fibrillin (33) (36% identity, 62% similarity between the two sequences). Again, all six cysteine residues of the exon 14 sequence (Fig. 1) could be aligned with the six cysteine residues of the EGF-like domain (where they formed three disulfide bridges), strongly implicating this fold in TPO. Deletion of only one extended loop region of the EGF-like structure was required to provide a model for the main chain conformation of this domain (Figs. 1 and 2B).

Predicted quaternary structure of TPO

If the TPO model is superimposed upon the MPO dimer, there are no steric clashes between TPO subunits, and most of the residues at the dimer interface of MPO are either identical, including the bridging cysteine residue 296, or conservatively substituted in TPO. The 10-residue loop insertion in TPO (residues 299 –308; Figs. 1 and 2A) has been modeled in such a way so as not to interfere with dimerization, but could conceivably adopt a conformation that would clash with the second subunit. Similarly, the location of the N-terminal domain (residues 1–141) is not known and might prevent dimerization, but could equally well be accommodated within a dimeric model of TPO, for which there is experimental evidence (34). (Note the location of the Nterminus of the MPO-like domain indicated in Fig. 2B.) The relative disposition of the three modeled domains (Fig. 2B) cannot be predicted, but is constrained by the fact that there are only five residues between the C-terminal end of the MPO-like domain fold and the start of the CCP domain, and there is no linker sequence between the CCP and EGF-like domains, which must therefore be closely packed together. Seven residues connect the C-terminal end of the EGF-like domain to the predicted transmembrane region. Finally, four potential sites for N-linked glycosylation are found in the extracellular sequence of TPO, one in the structurally undefined N-terminal region and three within the MPO-like domain, all of which lie at the surface of the TPO model. Location of peptide epitopes

The peptides used in this study, which all correspond to sequences within the MPO-like domain, are listed in Table 1, together with the accessibility of each to solvent, as calculated from the model structure. The total solvent accessible surface area for each peptide is quoted together with the fraction (percentage) that this represents of the theoretical maximum surface area for that particular sequence as an isolated peptide in an extended conformation. The location of the surface accessible peptides may be seen in Fig. 2B, in which the domain is viewed in virtually the same orientation

TABLE 1. Peptide sequences and their accessibility in the three-dimensional model of TPO Peptide no.

TPO sequence no.

Amino acid sequence

Total accessible surface area (Å2)a

% Theoretical max. surface areab

6 14 15 16 17 1 11 12 13

503–516 599 – 617 608 – 626 188 –203 175–186 225–242 574 –587 549 –563 567–581

RLDASFQEHPDLPGC-CONH2 GLPRLETPADLSTAIASRSC-CONH2 CDLSTAIASRSVADKILDLY-CONH2 CPRGWNPGFLYNGFPL-CONH2 CRWLPPVYEDGFS-CONH2 RYSDLLMAWGQYIDHDIA-CONH2 CDLASINLQRGRDHG-CONH2 LQVQDQLMNEELTERC-CONH2 LSNSSTLDLASINLQC-CONH2

933 1125 860 840 (785) 575 (273) 390 102 373 453

46 43 31 40 31 12 4 15 22

All peptides were used for generating rabbit antisera. Peptides 6, 14, 15, 16, and 17 are exposed on the surface, whereas peptides 1, 11, 12, and 13 are mainly buried. (Note the additional N- or C-terminal cysteine residues for coupling to carrier.) a Solvent-accessible surface area was calculated using NACCESS (see Materials and Methods). The accessibility values are the same in the monomer and dimer models, except for peptides 16 and 17 (values for dimer in parentheses). b Fraction (percentage) of the theoretical maximum surface area for the isolated peptide in an extended conformation.


as in Fig. 2A, i.e. from the side opposite that of the active site. Peptide 6 consists of a surface loop with little or no secondary structure and is furthest from the putative dimer interface (Fig. 2A). Peptide 14 is highly exposed and consists of ␤-strand/turn/␣-helix/turn, overlapping with peptide 15, ␣-helix/turn/␣-helix. Peptide 16 is a very exposed surface loop with turns, and lies close to the putative dimer interface. Peptide 17 is an extended surface peptide in the monomeric domain model, but would form part of the interface in a TPO dimer (compare accessibility values for monomer and dimer in Table 1). A number of buried peptides were selected as controls (not shown in Fig. 2B); peptide 11 is totally buried, whereas peptides 1, 12, and 13 include a few exposed residues. All of the peptides, both exposed and buried, correspond to SCRs in TPO (Fig. 1) and are therefore modeled with confidence. Analysis of anti-TPO peptide antisera

All rabbit antisera to TPO peptides were assayed for specific antibody by ELISA, which showed that antibodies were produced to the immunizing peptide and to human TPO (Table 2). Binding to TPO was also evaluated by RIA. Antibodies to peptide 14 from two different immunized rabbits precipitated the highest amounts of labeled TPO (60 –75% of the total labeled TPO at dilutions between 1:100 to 1:1000; Table 2), and antibodies to peptide 6 gave moderate precipitation of TPO (6 –22%), but antibodies to peptides 15, 16, and 17 showed negligible precipitation. As RIA involves protein modification (tyrosine iodination), inhibition ELISA with native soluble TPO was also performed. These data show that all of the antipeptide 14 antibodies react with native TPO (complete inhibition; Fig. 3), which correlates well with the fact that peptide 14 exhibits the greatest accessible surface area in the structure (Table 1). The other antipeptide antisera (peptides 6, 15, 16, and 17) do not show complete inhibition by native TPO, but this is consistent with their lower accessibility values. Antibodies to three buried sequences in the TPO model (peptides 1, 11, and 13) showed negligible bindTABLE 2. Titers of rabbit antipeptide antisera reactivity (reciprocal dilutions) with the immunizing peptide and hTPO by ELISA and by RIA using 125I-labeled hTPO at serum dilutions of 1:100, 1:250, and 1:1000, expressed as a percentage of labeled TPO precipitation (refer to Materials and Methods) Peptide no.

6 14 15 16 17 1 11 12 13 a

ELISAa

RIA

Rabbit

Peptide

hTPO

100

250

1000

A B A B A A A A B A B A B A B

51,200 25,600 256,000 64,000 102,000 102,400 102,400 256,000 102,000 256,000 256,000 64,000 64,000 64,000 32,000

6,400 12,800 256,000 128,000 1,600 3,200 100 512,000 512,000 128,000 12,800 256,000 256,000 128,000 64,000

22 13 75 61 2 8 0 5 2 4 0 36 14 0 5

13 12 69 61 0 6 0 1.8 1.2 3 0 12 12 0 1

6 6 60 70 0 0 0 0 0 0 0 8 6 0 0

Reciprocal values.

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ing to native TPO as expected, although antisera to a fourth buried peptide sequence (peptide 12) showed moderate reactivity with native TPO (Fig. 3) and precipitation of labeled TPO in RIA (Table 2), presumably due to reactivity with the few residues that are accessible. Mapping of peptide 14 sequence to the immunodominant region of TPO recognized by murine mabs

The relationship between the epitope recognized by antibodies to peptide 14 and the two major immunodominant regions on TPO was investigated with murine mabs recognizing the A and B determinants (13). The binding to TPO of mabs 15, 18, and 64, which recognize the B determinant, was completely inhibited by the antipeptide 14 antisera, but there was no inhibition for the second group of mabs 2, 9, and 47, which recognize the A determinant (13) (Fig. 4). The remaining antipeptide antibodies (Table 1) did not show any inhibition of binding of any of the murine mabs (data not shown). Inhibition of binding of patients’ autoantibodies to TPO by anti-peptide 14 antiserum

Sera from 65 patients with autoimmune thyroid disease and positive for autoantibodies to TPO were selected for this study. Almost all of the patients’ sera showed inhibition with antipeptide 14 antibodies, whereas all other antipeptide antibodies showed no inhibition of binding above that obtained using normal rabbit serum (Fig. 5). The mean level of inhibition with antipeptide 14 antibodies was 41% (sd ⫽ 16%), whereas the mean level for all other antipeptide antisera was 5.8% (sd ⫽ 4.4%). Discussion

The autoimmune response to TPO is focused to two dominant conformational determinants (reviewed in Ref. 4), but attempts to map the critical residues using chimeras of recombinant TPO and MPO have not led to the structural identification of these immunodominant regions (35–37). Our approach has involved building a model of TPO based upon the three-dimensional structure of MPO (22, 23) to identify and test peptides that may represent potential epitopes. In addition to the MPO-like domain, we modeled the membrane proximal, C-terminal region of TPO encoded by exons 13 and 14 (20), which consist of a closely linked CCP module and an EGF-like domain. In the MPO-like domain, all of the insertions and deletions in the TPO sequence were found to occur at the surface of the molecule, and with only two exceptions (the longer loops 249 –256 and 299 –308) could be modeled with considerable confidence. Biochemical studies indicate that membranebound TPO in thyroid cells exists as a disulfide-linked dimer (34), and conservation of many of the residues at the putative dimer interface, including the essential cysteine residue that in MPO covalently links the two subunits, supports a dimeric structure. Dimerization of the MPO-like domain at the membrane surface could be accommodated by flexibility at the link region between this domain and the CCP module. Similarity between the enzyme mechanisms of TPO and MPO is suggested by conservation of the critical residues surround-

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FIG. 3. Inhibition by soluble human TPO of rabbit antipeptide antibody binding to TPO coated on ELISA plates. All antipeptide antisera were used at a dilution that gave an OD of 1.5 by ELISA. Similar data were obtained with antisera for each peptide from two different immunized rabbits in each case.

FIG. 4. Inhibition of binding of murine mabs to TPO by rabbit antipeptide 14 antiserum. The mabs were used at a dilution that gave an OD of 1.3–1.6 by ELISA. Similar data were obtained with antipeptide 14 antiserum from a second rabbit. None of the antisera raised to any of the other peptides inhibited binding of any of the mabs (data not shown).

ing the heme group, including the proximal and distal histidines (residues 494 and 239) and an adjacent arginine (residue 396) (22, 23, 38). Residues that bind a Ca2⫹ ion in the MPO structure (Asp240, Thr321, Asp325, and Ser327) are also conserved, implying that TPO also binds Ca2⫹. Based upon this model, peptides corresponding to accessible surface features were synthesized, and antisera were raised in rabbits to probe for autoreactive epitopes, using buried peptides as controls. ELISA measurements showed that the antipeptide antisera cross-reacted well with TPO, but this was the case for both the surface peptides and the buried peptides, indicating that denaturation of the TPO may have occurred upon coating of the plates (39). RIA measurements showed minimal reactivity with radiolabeled TPO for three of the four antisera directed to buried peptides, but three of the five antisera to accessible peptides did not react either, perhaps because labeling affects the conformation of TPO.

Inhibition ELISAs were therefore carried out with native TPO in solution, and these data showed that all the antisera to peptides predicted to be accessible do indeed react with native TPO; furthermore, there is an approximate correlation between the degree of inhibition (i.e. extent of reactivity with native TPO) and the accessibility of the peptides predicted from the model. Antipeptide 14 antisera are clearly the most reactive, and it is this peptide that in the model is the most accessible and protrusive of the peptides tested. The relationship between the epitopes recognized by these antipeptide antisera and the previously identified immunodominant determinants A and B was investigated by competition with the murine mabs originally used to define them. The antipeptide 14 antisera inhibited TPO binding for all three mabs associated with determinant B, whereas none of the other antisera inhibited any of the mabs associated with either determinant. Thus, peptide 14 is either close to or part


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FIG. 5. Inhibition of autoantibody binding from patients with thyroid autoimmune disease to human TPO by preincubation with rabbit antipeptide antibodies. Sera from 65 patients positive for anti-TPO autoantibodies were used (10 IU/ml; OD, 1.3–1.5). Preimmune rabbit serum, collected before the immunization scheme, was used as a control. The mean values (percent inhibition) and SD (in parentheses) are as follows: normal rabbit control, 3.66 (4.1); rabbit antipeptide P1, 3.45 (3.73); P6, 7.39 (4.07); P11, 9.1 (2.28); P12, 9.64 (4.27); P13, 4.25 (4.13); P14, 41.1 (15.76); P15, 5.33 (3.62); P16, 5.57 (4.38); P17, 4.43 (3.9). Serum samples above the line (3 SD) denote positives.

of one of the dominant epitopes on TPO, and the fact that the antipeptide 14 antisera inhibited all three mabs that define the B determinant suggests that it may be a central feature of this epitope. Its prominent, exposed location distant from both the membrane, putative dimer interface and active site is shown in Fig. 2B (inset). The relevance of the epitope defined by peptide 14 to the human autoantibody response was then investigated, with the striking result that antipeptide 14 antisera inhibited to some degree all human patient sera tested, whereas none of the other antipeptide antisera tested showed any inhibition above that of control serum. It is known that 80 –90% of autoantibodies from patients with thyroid disease recognize the A and B determinants (13), and that the fraction directed to each varies among patients’ sera (15, 40, 41). Our results confirm that determinant B is indeed a dominant epitope and that peptide 14 represents a critical structural feature, the location of which is now mapped onto the structure of TPO. An earlier study (12) identified autoantigenic determinants within a recombinant fragment of TPO comprising amino acids 589 – 633, which includes the sequence of peptide 14 (residues 599 – 617). However, antibodies to determinants within this region recognized only denatured TPO, indicating that these determinants were not accessible in the native molecule, in contrast to our findings that antibodies to peptide 14 not only inhibit patients’ autoantibodies but also bind to native TPO. Furthermore, the highly accessible structure adopted by residues 599 – 617 in native TPO is quite different from the ␤-turn structure predicted for the isolated peptide of residues 592– 613 by Arscott and colleagues (12). In another study (42), a peptide fragment of residues 742– 848 displayed reactivity with patients’ sera, and the conformational nature of these epitopes is consistent with the fact that

the peptide corresponds almost exactly to the CCP and EGFlike domains together. It is striking that peptide 14, although a linear sequence, defines, by virtue of its conformation in the folded structure, a surface accessible epitope of about the size recognized by an antibody. This may in part account for the high level of reactivity of antipeptide 14 antibodies with native TPO, and their efficacy in inhibiting the binding of patients’ sera, although clearly antibodies to peptide 14 would be expected to block access to immediately adjacent segments should they also be important determinants. Nevertheless, peptide 14 appears to be a critical feature of the B determinant, the full extent of which can now be mapped, for example by single site mutagenesis. The negative results with antisera to peptides 15–17, however, do define the limits on one side. A clue to the location of the A determinant is provided by the fact that the only murine mab from the panel to be mapped to a sequence is mab 47, which binds to both denatured and native TPO (41) and recognizes residues 713–721 (8). In the model, as shown in Fig. 2B, this peptide is highly accessible and extended, consistent with this cross-reactivity. As mab 47 only weakly inhibits human autoantibody binding (13), the full extent of the A determinant must also now be mapped onto the structure of TPO. This study has defined for the first time the structure and location within the TPO molecule of a major part of the B determinant and confirmed that this structure is indeed a dominant epitope in the human autoantibody response in thyroid disease. Knowledge of the structures of such epitopes may provide clues to the origin of this focused response, and the identity of agents that, perhaps through molecular mimicry (43), may trigger autoreactivity.

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AN IMMUNODOMINANT EPITOPE OF TPO Acknowledgment

We are grateful to Dr. Jean Ruf for the provision of the murine mabs to TPO.

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