New Insights into the Conformational Dominant Epitopes on Thyroid Peroxidase Recognized by Human Autoantibodies
http://www.100md.com
内分泌学杂志 2005年第6期
Centre National de la Recherche Scientifique Unité Mixte de Recherche 5160 (D.B., S.A.R., B.N., S.P.-R.), Centre de Pharmacologie et Biotechnologie pour la Santé, Faculté de Pharmacie, BP 14491, 34093 Montpellier Cedex 5, France; Medical Centre of Postgraduate Education (A.G.), 01-813 Warsaw, Poland; and Division of Medicine (J.P.B.), Guy’s, King’s, and St. Thomas’ School of Medicine, King’s College London, London, United Kingdom SE1 9RT
Address all correspondence and requests for reprints to: Dr. Sylvie Péraldi-Roux, Centre National de la Recherche Scientifique UMR 5160, Centre de Pharmacologie et Biotechnologie pour la Santé, 15 avenue Charles Flahault, BP 14491, 34093 Montpellier, Cedex 5, France. E-mail: sylvie.roux@ibph.pharma.univ-montp1.fr.
Abstract
Human anti-thyroperoxidase (TPO) autoantibodies (aAbs) are a major hallmark of autoimmune thyroid diseases. Their epitopes are discontinuous and mainly restricted to an immunodominant region (IDR) consisting of two overlapping regions (IDR/A and B). To shed light on the relationship between these regions, we first performed competitive studies using all available reference anti-TPO antibodies. Interestingly, we showed that human IDR/A- and B-specific anti-TPO aAbs recognized essentially the same regions on the TPO molecule. However, our data also indicated that IDR/A-specific human aAbs strongly recognized the region containing residues 599–617, whereas the IDR/B-specific aAbs bind to several regions as well as region 599–617. Next, we scanned this key region to identify the residues involved in the immunodominant autoepitope. Using peptide spot technology together with competitive ELISA experiments, we demonstrated that residues 604ETP-DL609 play a major role in the anti-peptide P14 epitope and that IDR/A-specific human anti-TPO aAbs, either expressed as recombinant Fab or obtained from Graves’ disease patients, specifically recognize the sequences 597FCGLPRLE604 and 611TAIASRSV618. All together our data emphasize that both the IDRs involve the same surface area on human TPO, but the differential usage of one or the other regions leads to different inhibition patterns in competitive experiments. In conclusion, our data help to resolve the long-sought issue on the molecular immunology of the two IDRs on TPO and provide new clues to design efficient peptides that may be part of a combinatorial treatment aiming at delaying development of autoimmune thyroiditis when used prophylactically.
Introduction
THYROID PEROXIDASE (TPO) is a major autoantigen involved in autoimmune thyroid diseases (AITD). High amounts of anti-TPO autoantibodies (aAbs) are frequently present in the sera of patients suffering from AITD (1). Anti-TPO aAbs are thought to be involved in thyroid cell destruction through cytotoxic mechanisms mediated by effector cells (2) and/or complement activation (3). More recently it has also been shown that the complement pathway may be activated via direct binding of the C4 complement component to human TPO (hTPO) without mediation by immunoglobulin (4). More importantly, anti-TPO aAbs have been found to be involved in antigen presentation to autoaggressive T cells (5) and may favor the exacerbation of AITD. Consequently, information on how the TPO molecule is seen by the immune system could lead to a greater understanding of why and how immune tolerance is lost during onset of AITD. Such information would also be of great importance to design, using a rational approach, therapeutic components (as peptides) able to interfere with those mechanisms. These peptides could be used in combination with other immunotherapies such as systemic antibody treatment, antigen-specific immunizations, or others generating antigen-specific regulatory T cells capable to block, at least for a period of time, an ongoing autoimmune process and may synergize to delay hypothyroiditis.
TPO is a large and highly complex molecule comprising three distinct domains: a myeloperoxidase (MPO)-like, a complement control protein (CCP)-like, and an epidermal growth factor-like domain, from the N- to the C-terminal extremities. Although the three domains were separately modeled (6, 7, 8, 9), the full three-dimensional structure of TPO has to be yet elucidated. It is well recognized that TPO is a highly heterogeneous protein with alternative splicing of some exons, different N-terminal start sites, and endoproteolytic sites contributing to the diversity of the polypeptide chain resulting in the well-known doublet and that a flexible molecule, all of which have made it difficult to obtain high resolution diffracting crystals for three-dimensional structural analysis (10, 11, 12).
It is well known that the majority of the anti-TPO aAbs are dependent on the three-dimensional tertiary structure of the molecule and interact with a limited surface region termed the immunodominant region (IDR). By using competitive studies between human anti-TPO aAbs and a panel of mouse anti-TPO monoclonal antibodies (mAbs), this IDR has been divided into two domains named A (IDR/A) and B (IDR/B), containing different but adjacent surface epitopes (13). Subsequent characterization of recombinant human anti-TPO antibody fragments (Fabs) derived from combinatorial libraries constructed with intrathyroidal B cells purified from Graves’ disease patients also allowed the identification of two large determinants on TPO (14, 15, 16), defining the A and B domains with an inverted nomenclature. Competitive experiments between human anti-TPO aAbs from patients’ sera and recombinant anti-TPO Abs (human Fabs or mouse mAbs) led to the definition of global contours of the IDR/A and B domains on the TPO molecule. Several data emerged showing that these two domains, although different, overlap in part.
To date, the major region (599–617) belonging to the IDR/A and five regions (210–225, 353–363, 549–563, 713–720, and 766–775) structuring the IDR/B have been identified by different groups (12, 17, 18). In an attempt to improve the knowledge on immunodominant amino acid residues recognized by human anti-TPO aAbs, several groups ex pressed recombinant human TPO mutants with single amino acid replacements (7, 17, 18, 19, 20). This strategy led to the identification of a number of key residues (R225, Y772, and K713 to D717) involved in the IDR/B-specific human anti-TPO aAbs epitopes (7, 17, 19, 21). On the other hand, only one amino acid residue (K627) has been clearly assigned to the IDR/A-specific human anti-TPO aAbs epitopes (20).
Despite the growing number of data available for delimiting each domain of the IDR, several questions still remain. First, even if the existence of two domains (A and B) forming the entire IDR is now well documented, it is unclear whether these domains form two separate clusters recognized by either IDR/A- or B-specific human anti-TPO aAbs or are structurally similar (involving the same surface area on the TPO molecule). Second, although region 599–617 represents a major region strongly recognized by the IDR/A-specific aAbs, none of its amino acid residues have been shown to be directly involved in their binding to human TPO.
To address these questions, we used for the first time all the reference anti-TPO Abs (recombinant human Fabs, mouse mAbs, and rabbit polyclonal antipeptides) produced and characterized by different groups to study the relationship between IDR/A and B domains. We show that human IDR/A- and B-specific anti-TPO aAbs recognize essentially the same region, on the surface of the TPO molecule. The main characteristic found is a stronger binding in region 599–617 for the IDR/A-specific aAbs, leading to different inhibition patterns in competitive experiments. Furthermore, due to the key role of region 599–617, we searched the residues in this region participating in the interaction with rabbit antipeptide P14 (immunoreactive with the sequence 599–617) and human aAbs. For this purpose, we prepared a series of alanine scanning mutations by the Spot method and synthesized six punctually mutated peptides. For the first time, we demonstrated that residues 604ETP-DL609 play a major role in the antipeptide P14 epitope (reference polyclonal for the IDR/A) and that IDR/A-specific human anti-TPO aAbs, expressed as recombinant Fab or obtained from Graves’ disease patients, specifically recognize the sequences 597FCGLPRLE607 and 611TAIASRSV618.
Materials and Methods
Patients’ sera
Sera from patients with Graves’ disease were obtained from Dr. L. Baldet and Dr. A.-M. Puech (Lapeyronie Hospital, Montpellier, France). The anti-TPO aAbs titers were determined by RIA using the TPO-AB-CT kit (CIS bio, Gif sur Yvette, France). IgG from patients’ sera were purified on a protein A column.
Human TPO and anti-TPO antibodies
The hTPO, purified (greater than 95% pure) from thyroid glands, was obtained from HyTest Ltd. (Turku, Finland). Polyclonal antipeptide sera were produced by immunizing rabbits with peptides derived from the primary sequence of human TPO (Table 1). The antipeptides P1, P12, P14, P18, P24, P43, P50, and P760–779 (rabbit reference polyclonal Abs) were produced by the groups of Gardas and colleagues (9, 22). S10 was produced in our laboratory. The IDR/A- (15, 18, 59, and 64) and B-specific (2, 9, 47, and 60) mouse anti-TPO mAbs (human nomenclature) (mouse reference Abs) were generously provided by Ruf et al. (13). Polyclonal antipeptides antibodies and mouse mAbs were purified on a protein G column. Recombinant human anti-TPO Fabs TR1.9, TR1.8, SP1.4, and WR1.7 (human reference Abs) were kindly provided by McLachlan and colleagues (15).
TABLE 1. Inhibition of human Fab or mouse monoclonal antibody binding inhibition to TPO by rabbit polyclonal antipeptide antisera
Binding to TPO of purified rabbit polyclonal antipeptides, mouse anti-TPO mAbs, and human anti-TPO Fabs by ELISA
Microtiter plates were coated with 1 μg/ml human (h)TPO in 100 mM NaHCO3 (pH 9.6) overnight at 4 C. Plates were washed with 0.05% Tween 20 in PBS (PBS-T) (pH 7.3) and blocked with 2% nonfat dry milk in PBS-T (saturation buffer) for 1 h at 37 C. After three washes, polyclonal antipeptides, anti-TPO mAbs, or human anti-TPO Fabs were incubated with 1% nonfat dry milk in PBS-T (incubation buffer) for 2 h at 37 C, serial dilutions were performed as indicated in Fig. 1, A–C. A horseradish peroxidase (HRP)-conjugated antirabbit IgG, antimouse IgG, or antihuman F(ab')2 (diluted at 1:2,000; in the incubation buffer; Sigma, Meylan, France) was added, and the plates were incubated for 1 h at 37 C. The reactivity was detected with a 4 mg/ml 2-phenylenediamine solution containing 0.03% (vol/vol) hydrogen peroxide in 0.1 M citrate buffer (pH 9.5). The reaction was stopped by adding 50 μl of 2 M H2SO4 to each well, and the resulting absorbance was measured at 490 nm.
FIG. 1. Binding of anti-TPO antibodies (human Fabs, rabbit antipeptides, and mouse mAbs) to human TPO. ELISA experiments were performed by coating human TPO at 1 μg/ml. A, Human anti-TPO Fabs; B, purified rabbit polyclonal antisera directed against TPO peptides (as described in Table 1); and C, mouse anti-TPO mAbs (IDR/A- or B-specific as indicated) were incubated in serial dilutions on hTPO as stated on the figure.
Blocking ELISA experiments
The wells were coated with 1 μg/ml hTPO (overnight at 4 C) and then washed and blocked as described above. Rabbit polyclonal antipeptides (P1, P12, P14, P18, P24, P50, P43, S10, and P760–779) were incubated in the incubation buffer for 2 h at 37 C. After three washings, human anti-TPO Fabs (SP1.4, WR1.7, TR1.8, TR1.9, and T13) or mouse anti-TPO mAbs (2, 9, 15, 18, 47, 59, 60, and 64) were incubated for 1 h at 37 C at a concentration giving an absorbance (A) of 1.5 at 490 nm. An HRP-conjugated antihuman F(ab')2 or antimouse IgG (Sigma), incubated for 1 h at 37 C, was used to detect the binding of human anti-TPO Fabs or mouse mAb to TPO, respectively. Three washes were performed, and then the reactivity was revealed as described above. The percent inhibition was calculated by comparing the binding of human Fabs and mouse mAbs with or without inhibitor.
Peptide synthesis and immunoassay on cellulose membrane-bound peptides
The general protocol for Spot parallel peptide synthesis was described previously (23). Two different cellulose membrane-bound peptides were synthesized. The first one was synthesized with a set of 25-mer synthetic peptides frame shifted by two residues, corresponding to the entire sequence of hTPO and the second with three sets of 8-mer synthetic peptides (regions 597–604, 604–611, 611–618, and their 18 alanine analogs), corresponding to sequences including the antipeptide P14 epitope. The membrane-bound peptides were probed with rabbit antipeptide (1 μg/ml), human anti-TPO TR1.8 Fab (0.1 μg/ml), or purified IgG from patients’ sera (1 μg/ml), coincubated with the specific HRP-conjugated anti-species IgG (antirabbit dilution 1:15,000, antihuman F(ab')2 or antihuman Fc-specific antibody, respectively, diluted 1:2000), for 1 h 30 at 37 C.
Competition studies were performed by preincubation of human anti-TPO TR1.8 Fab for 10 min on the membrane-bound peptides followed by coincubation with rabbit antipeptide P14 and HRP-conjugated antirabbit IgG (same concentration as previously mentioned). After three washes, the complex (polyclonal P14/peptides) was revealed using the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech) on a sensitive film. Membranes were reused after a regeneration cycle. Importantly, to make sure that the regeneration cycles were efficient, membranes were always incubated with enhanced chemiluminescence directly after regeneration. As a control, all HRP-conjugated Abs were incubated alone and showed no reactivity with the spots.
Synthesis of soluble peptides derived from the sequence 599–617 of hTPO
A series of soluble peptides derived from the sequence 599–617 of hTPO (amino acid sequence 599GLPRLETPADLSTAIASRS617) was synthesized on a Multipep synthesizer (Intavis AG, Cologne, Germany) by Fmoc chemistry. The wild-type (wt) sequence and six mutated peptides were generated: five peptide with a single mutation (residues were replaced by an alanine) at positions Glu604 (mut E604), Thr605 (mut T605), Pro606 (mut P606), Asp608 (mut D608), Leu609 (mut L609), and one peptide with a triple mutation in which residues Thr605, Asp608, and Leu609 were replaced by Ala605, Gln608, and Ala609 (mut TDL). The peptides released from the resin by trifluoroacetic acid treatment in the presence of appropriate scavengers. They were lyophilized and their purity assessed by HPLC (all peptides showed a purity greater than 85%)
Competitive ELISA experiments with soluble peptides derived from the sequence 599–617 of hTPO
Rabbit polyclonal antipeptide P14 (7 ng/ml) was preincubated with wt or mutated soluble peptides derived from the 599–617 amino acid sequence of hTPO at increasing concentrations overnight at 4 C. At the same time, microtiter plates were coated with hTPO at 1 μg/ml in 100 mM NaHCO3 (pH 9.6) overnight at 4 C. After washings, the plates were blocked with saturation buffer for 1 h at 37 C. Saturation buffer was removed and the complex, composed of the antipeptide P14 preloaded with the synthetic peptides, was incubated with coated hTPO for 2 h at 37 C. Rabbit antipeptide P14 bound on hTPO was detected by adding a peroxidase-conjugated antirabbit IgG (Sigma, dilution 1:2000) for 1 h at 37 C. The complex was revealed by adding 100 μl of a 4 mg/ml 2-phenylenediamine solution containing 0.03% (vol/vol) hydrogen peroxide in 0.1 M citrate buffer (pH 9.5). The reaction was stopped by addition of 50 μl/well of 2 M H2SO4, and the resulting absorbance was measured at 490 nm.
Statistical analysis
All inhibition data were expressed as the mean ± SD. The statistical significance of the difference between means was determined using the two-tailed Student’s t test. Differences were considered significant at P < 0.05.
Results
IDR/A- and B-specific human anti-TPO Fabs recognize the same regions on hTPO
To obtain new information on the relationship between IDR/A and B, we decided to use all the available reference anti-TPO Abs and analyzed the profile of reactivity of the human anti-TPO Fabs and mouse anti-TPO mAbs in competition with rabbit polyclonal antipeptide Abs (all blocking ELISA experiments were performed using the same protocol). First, we assessed the binding of all reference anti-TPO Abs on hTPO by ELISA. Figure 1 shows the reactivities of each Ab with the coated hTPO.
Next, the five human TPO-specific Fabs, two IDR/A- (SP1.4 and WR1.7), and three IDR/B-specific (TR1.8, TR1.9, and T13) Fabs (12, 16) were used for competition experiments with a panel of antipeptide Abs at a concentration giving an A490 of 1.5 (as calculated by using the data in Fig. 1A). The resulting inhibition profiles are shown in Fig. 2. At high concentration (0.7 μM), antipeptides P14, P24, P43, and P50, directed against the hTPO sequences 599–617, 375–387, 702–721, and 353–372, respectively (Table 1), inhibited the binding of Fabs T13 and TR1.9 between 12 and 24%, whereas antipeptides P1 and P18 were unable to inhibit these Fabs by more than 9%. Surprisingly, antipeptides P12 and P760–779 interfered only with the binding of Fab TR1.9 on hTPO (inhibition ranging between 18 and 20%) but not with FabT13. This indicates that even if these Fabs recognize the IDR/B on hTPO, their contact points are probably slightly different. These first results are in agreement with our previous studies, which demonstrated that regions 353–363, 377–386, and 713–720 from the MPO-like domain and region 766–775 from the CCP-like domain are a part of the IDR/B (12). More interestingly, the significant inhibition (20–24%) observed with the human IDR/B-specific Fabs (TR1.9 and T13) in competition with the antipeptide P14 strongly suggests that region 599–617 of hTPO also constitutes a part of IDR/B. However, its contribution to the IDR/B epitope is much less, compared with the IDR/A epitope.
FIG. 2. Inhibition of human Fabs binding to TPO by rabbit polyclonal antipeptide antibodies. The binding of human anti-TPO-specific Fabs (T13, TR1.9, TR1.8, SP1.4, and WR1.7) were inhibited by rabbit antipeptide antibodies (P1, P12, P14, P18, P24, P43, P50, P760-779, and S10) as described in Materials and Methods. The human Fabs were used at a concentration that gave an absorbance of 1.5 by ELISA at 490 nm. The rabbit polyclonal antibodies were tested at two different concentrations (inhibitor concentration shown in this figure). The higher concentrations were used according to the data shown in Fig. 1B, for saturating the binding sites on coated TPO. The results represent the mean ± SD of the duplicate determinations. Any inhibition over 10% (at least three times the lowest inhibition observed for each Fab) was considered as significant. One antipeptide for each human Fab, showing an inhibition around 10% with the highest concentration, was taken as reference for the statistical analyses (antipeptide P18 for the Fabs T13, TR1.9, and WR1.7; antipeptide P43 for the Fab SP1.4 and antipeptide S10 for the Fab TR1.8). These inhibitions of reference were used to determine the statistical significance of each inhibition using the two-tailed Student’s t test. *, P < 0.05 (data significantly different). The data shown are representative results from two independent experiments.
Indeed, the binding of the IDR/A-specific Fabs, SP1.4 and WR1.7, was strongly hampered (inhibition from 79 to 89%) by the antipeptide P14, clearly demonstrating that amino acid residues in position 599–617 constitute the major anchor for the IDR/A-specific aAbs on hTPO (Fig. 2). Unexpectedly, the binding of Fab TR1.8 on hTPO, previously described as IDR/B-specific (16), was inhibited up to 89% by rabbit antipeptide P14 and consequently showed a reactivity profile similar to that of the IDR/A-specific Fabs in our inhibition format. Interestingly, the IDR/A-specific Fabs also competed with several other antipeptides but weakly. As observed with the IDR/B-specific Fabs, a particular antipeptide may compete differently with one or another IDR/A-specific Fabs. For example, P12 and P43 clearly inhibited the Fabs WR1.7 and TR1.8 (more than 15%) but were not considered to be competitive with SP1.4 (less than 10% inhibition). On the contrary, P50 was shown to displace the binding of SP1.4, WR1.7, and TR1.8 to hTPO (from 14 to 23% inhibition).
Globally, our data indicate that not only IDR/A-specific human Fabs (SP1.4, WR1.7) but also TR1.8 interact with the same regions previously shown to be involved in the IDR/B-specific human aAbs (present data and Ref. 12).
Inhibition of mouse mAb binding to TPO by rabbit polyclonal antipeptide Abs
As indicated previously, all the reference mouse mAbs but mAb47 bind conformational epitopes. Thus, to localize more precisely their epitopes and compare the regions recognized by the reference human Fabs and the mouse mAbs, we tested the mouse anti-TPO-specific mAbs [15, 18, 59, and 64 (IDR/A-specific) and 2, 9, 47, and 60 (IDR/B-specific)] in a competitive ELISA experiment (same protocol as that used for the human Fabs) with rabbit polyclonal antipeptides (Fig. 3). The binding of mAbs 15, 64, and 18 on hTPO was inhibited 72, 65, and 54%, respectively, by rabbit antiserum P14. Therefore, these data confirm that mAbs 15, 64, and 18 are IDR/A specific. On the other hand, the binding of mAb 59 was not affected by rabbit antiserum P14; nevertheless, an unambiguous inhibition (26%) was obtained by using the rabbit antiserum P43. These observations suggests that mAb 59, as well as mAb47, which showed a strong inhibition (80%) with the polyclonal antipeptide P43, is categorized as IDR/B specific. The mAb 2 was moderately inhibited by both antipeptides P43 (10%) and P14 (25%). This inhibition profile, involving regions 599–617 and 713–720, confirms its IDR/B specificity. Finally, the inhibition profiles observed in the competitive ELISA experiments for both mAbs 60 and 9, described as IDR/B specific, did not allow us to determine their IDR specificity.
FIG. 3. Inhibition of mouse mAbs binding to TPO by rabbit polyclonal antipeptides. The binding of mouse anti-TPO-specific mAbs [15, 18, 59, and 64 (IDR/A specific) or 2, 9, 47, and 60 (IDR/B–specific)] were inhibited by the antipeptide polyclonal antibodies (1, 12, 14, 18, 24, 43, 50, S10, and P760-779) as described in Materials and Methods. The mouse mAbs were used at a concentration that gave an absorbance of 1.5 by ELISA at 490 nm and the polyclonal antipeptides were used at a concentration required to saturate the binding sites on coated TPO as defined in Fig. 1B. The results represent the mean ± SD of the duplicate determinations. Any inhibition over 10% (at least three times the lowest inhibition observed for each Fab) was considered as significant.
The inhibitions observed with human Fabs and mAbs are summarized in Table 1. Taken together, these results confirm the involvement of several regions at the surface of the TPO in the binding of human aAbs (some of them have already been identified by using site-directed mutations) and demonstrate for the first time that IDR/A- and B-specific human Fab epitopes are built around the same regions on hTPO but in fact differ in their ability to use the region 599–617 as the main anchor point. A faint but reproducible binding was also observed for almost all human Fabs in the regions 353–372 and 702–721 (delimited by the antipeptides P50 and P43, respectively). These regions possess the amino acid sequences 353–363 and 713–720 we previously described as contact points for the human Fab T13 (12).
Is steric hindrance a major factor in the inhibition experiments? Epitope analysis of rabbit antipeptides P43 and S10
One could argue that the degree of inhibition we observed by using these competitive ELISA experiments could be the result of a strong steric hindrance and not direct competition for the same epitopes. To evaluate the importance of steric hindrance, we compared the ability of rabbit antisera P43 and S10 to compete with mAb 47 for the binding on hTPO. Antipeptides P43 and S10 were obtained by immunizing rabbits, respectively, with peptides corresponding to amino acid residues 702–721 and 716–731 of hTPO (Table 1). Peptide P43 (702–721) entirely encompasses the mAb47 epitope (713–717), whereas S10 (716–731) possesses at one extremity only two residues involved in the mAb47 epitope, among their, residue D717 has been shown to weakly participate in the interaction with hTPO (19). First, rabbit antiserum P43 and S10 epitopes were studied by using overlapping peptides derived from the sequence of the hTPO (Fig. 4A). Twelve overlapping 25-mer peptides surrounding residues Leu691 to Pro737 of hTPO were reactive with rabbit antipeptide P43, indicating that the residues KFP713–715 are central for the binding of this antipeptide. Reactivity of the rabbit antiserum S10 on the Spot membrane shows up with eight overlapping peptides [from residues Arg703 to Lys741], revealing that peptide 717DFESCDSIPGM727 plays a crucial role in the binding of rabbit antisera S10 to hTPO. These observations confirmed that the S10 epitope does not contain all the main residues recognized by mAb 47 [713KFPED717], but the P43 epitope does. The S10 epitope, however partially overlaps with the mAb 47 epitope and thus could interfere with mAb 47 binding to TPO by steric hindrance. This possibility has been evaluated by ELISA (Fig. 4B). Interestingly, the binding of mAb 47 was inhibited in a dose-dependent fashion by the antipeptide P43 (inhibition by up to 80%), but only a maximum of 10% inhibition was observed with S10. These results emphasize that steric hindrance is not a major contributor to the inhibitions observed in our competitive ELISA experiments.
FIG. 4. Delineation of rabbit antiserum P43 and S10 epitopes. A, Immunoreactivity of peptides derived from the primary sequence of human TPO with rabbit antiserum was studied by the Spot technology. Reactivities of rabbit antiserum P43 and S10 on the Spot membrane are shown on the left and right side of the figure, respectively. The amino acid sequences of TPO corresponding to the immunoreactive spots are surrounded on the figure (solid lines for P43 and dashed lines for S10). B, Competitive ELISA experiments were performed with rabbit antipeptide P43 and S10 as inhibitor for the binding of mAb 47 (used at a fixed concentration giving an OD of 1.5 at 490 nm) to hTPO (see Materials and Methods).
Epitopic dissection of polyclonal antipeptide P14
The present study and previous data (9) point toward the importance of the rabbit antipeptide P14 epitope in the binding of human anti-TPO aAbs on their cognate antigen. Thus, it seems obvious that a precise localization of its epitope would be of great interest. To this end, we first used the Spot technology to dissect this epitope. In region 581–633, 11 overlapping peptides were found to be reactive (Fig. 5A). A common amino acid sequence TPADL (residues 605–609 of hTPO) appears to constitute the critical residues in interaction with the antipeptide P14. To precisely identify which residues play a major role in the binding of rabbit antipeptide P14 to hTPO, we synthesized on a spot membrane three 8-mer peptides covering the sequence 599–617 [597FCGLPRLE604, 604ETPADLST611, and 611TAIASRSV618] and their alanine analogs. The results in Fig. 5B show that only peptides 597FCGLPRLE604 and 604ETPADLST611 were immunoreactive and hence involved in the binding of rabbit antipeptide P14 on hTPO. Alanine replacements of residues C598 and E604 in the peptide 597FCGLPRLE604 and E604, T605, P606, D608, and L609 in the peptide 604ETPADLST611 reduced or totally abrogated the binding of P14. To be certain that these binding inhibitions were specific, we decided to synthesize soluble peptides corresponding to the sequence 599–617 of hTPO (wt) and six mutants in positions E604, T605, P606, D608, L609, and T-D-L605–609. These soluble peptides were then tested, by using a competitive ELISA, for their capacity to block the binding of rabbit antiserum P14 to hTPO (Fig. 5C). Although the wt peptide was able to block the binding of antisera P14 to coated hTPO in a dose-dependent manner (95% inhibition at 10 nM), all replacements impaired the ability of the mutated peptides to efficiently compete with antipeptide P14. More precisely, the punctual mutations showed an average of 50% inhibition at 10 nM, whereas the triple mutant T-D-L605–609 was far less efficient (20% inhibition at 10 nM). Taken together, the alanine scanning and competitive ELISA experiments demonstrated that residues T605, P606, D608, and L609 are critical for the binding of antipeptide P14 to hTPO.
FIG. 5. Determination of critical amino acid residues recognized by rabbit antiserum P14. A, The binding of rabbit antiserum P14 onto membrane-bound peptides covering the entire primary sequence of the hTPO was determined by Spot technology. Here, only the immunoreactive peptides (boxed with solid lines) and the four surrounding peptides are shown. B, Contributor residues from the sequence 599-617 of hTPO to rabbit antiserum P14 epitope were determined by Spot with an alanine-scanning approach. The region 597-618 of hTPO has been truncated into three shorter peptides [597FCGLPRLE604 (Seq1), 604ETPADLST611 (Seq2), and 611TAIASRSV618 (Seq3)] to improve the analyses. Then, a series of alanine analogs of each short peptide sequence was synthesized in parallel with the wild-type sequence. Finally, the binding of the polyclonal anti-peptide P14 to each peptide was analyzed by the spot technology. Underlined amino acids represent the critical residues observed for each peptide. C, The binding of rabbit antipeptide P14 to hTPO (coated at 1 μg/ml in a 96-well plate) was inhibited by free synthetic peptides (wt or a series of mutated peptides derived from the amino acid sequence 599GLPRLETPADLSTAIASRS617) in a competitive ELISA experiment as described in Materials and Methods.
Binding of human anti-TPO aAbs to their cognate antigen involves residues 597FCGLPRLE604 and 611TAIASRSV618
Because polyclonal P14 showed epitopic cross-reactivity in the region 599–617 with all the recombinant human anti-TPO Fabs tested, we decided to set up an experiment with the soluble peptides we designed previously to dissect the P14 epitope. Competitive ELISA experiments were performed with recombinant human anti-TPO Fabs (IDR/A and B specific) to determine whether the same residues in the region 599–617 are involved in their epitopes. Unfortunately, the mutated peptides were able to displace the binding of human Fabs to TPO as efficiently as the wt sequence (data not shown). This suggests that residues E604, T605, P606, D608, and L609, which are central for the interaction of rabbit antiserum P14 with hTPO, are not strongly involved in the epitopes recognized by the recombinant human anti-TPO Fabs. Consequently, we chose to use the recombinant human anti-TPO Fab TR1.8 [showing the strongest binding on hTPO (Fig. 1A) and using the region 599–617 as the main anchor point (Fig. 2)] to test its ability to interact with the spot membrane carrying the three wt 8-mer peptides [597FCGLPRLE604, 604ETPADLST611, 611TAIASRSV618] and their alanine analogs (Fig. 6). Interestingly, the high avidity due to the large number of peptides synthesized at the individual spots by this technology together with the high affinity of the TR1.8 Fab allowed us to detect a strong and specific binding on the wt sequences 597FCGLPRLE604 and 611TAIASRSV618 (Fig. 6B).
FIG. 6. Analysis of IDR/A epitope recognized by human anti-TPO aAbs by the Spot technology. Binding of antibodies (antipeptide P14, human anti-TPO Fabs, and purified IgG from Grave’s patients’ sera) was performed by using the Spot technology with an alanine-scanning membrane as described in Materials and Methods. A, Immunoreactivity of peptides with rabbit antiserum P14 was studied by the Spot technology on the three peptides (FCGLPRLE, ETPADLST, and TAIASRSV) and a series of alanine analogs of each peptide sequences surrounding the immunoreactive sequence 599-617. Rabbit antiserum P14 reactivity on the Spot membrane is represented at the top of the figure, and the binding of antipeptide P14 to wt peptide and each analog is also represented by histograms. The color intensity of each spot was determined using ScionImage software. Binding of (B) human anti-TPO Fab TR1.8 and (D) purified IgG from three Grave’s patients (patient 1, ; 2, ; and 3, ) onto the spot membrane is represented by the intensity of each spot. C, Similarly, the competitive Spot experiment between antipeptide P14 and human anti-TPO Fab TR1.8 is shown. Competition study was performed by coincubating rabbit antipeptide P14 with TR1.8, then binding of P14 on the membrane was revealed. As control, no reactivity was observed on the membrane with the HRP-conjugated antibodies used alone.
By comparison, antipeptide P14 showed reactivity with the wt sequences 597FCGLPRLE604 and 604ETPADLST611 but not with611TAIASRSV618 (Fig. 6A), explaining why the binding of P14, but not TR1.8, to the soluble peptide 599–617 was affected by the mutations in region 604ETPADLST611. Importantly, two mutated residues, P601 and R616, caused a decrease in the binding of TR1.8 and thus might participate in the epitope recognition (Fig. 6B). To verify that P14 and TR1.8 specifically competed in the region 597FCGLPRLE604 for their binding to hTPO, we performed a competition between these Abs. Figure 6C shows that after incubation with TR1.8, antipeptide P14 was no longer able to bind region 597–604 but retained its ability to react with region 604–611. These results argue for a strong contribution of residues 597–604 in the epitopes recognized by the IDR/A-specific aAbs. To validate this hypothesis, we analyzed the binding of purified IgG obtained from three Graves’ disease patients to the same spot membrane. Recently the group of Gardas and colleagues (24) reported that a majority of anti-TPO aAbs from patients’ sera affected by AITD is directed against IDR/A (approximately 53%). Thus, the binding of aAbs from patients’ sera should reflect the binding of the IDR/A-specific components. As shown in Fig. 6D, the reactivity patterns seen with these three patients were greatly similar to that seen with TR1.8. Interestingly, mutations in regions 597–604 and 611–618 displayed two different inhibition profiles. Punctual mutations in region 597–604 did not seem to induce an effective loss in the binding of aAbs from patients’ sera. On the other hand, certain mutations in region 611–618 markedly weakened the binding of the sera, namely residues R616, S617, and V618 (an average of 22, 40, and 30% inhibition, respectively, taking the intensity of the spot for the wt sequence as 100%). All together, these findings shed light on two regions, 597–604 and 611–618, strongly involved in the binding of the IDR/A-specific human aAbs.
Discussion
Anti-TPO aAbs predominantly recognize conformational epitopes. Previous studies clearly demonstrated that most of the human anti-TPO aAbs epitopes are located inside an IDR that has been divided into two overlapping but distinct regions known as A and B (13, 15, 16). These regions are thought to be formed by different amino acid sequences at the surface of the hTPO. For example, the sequence 599–617, defined by the polyclonal antipeptide P14, was directly assigned to IDR/A, whereas sequence 713–720 was shown to belong to IDR/B (12, 19, 21). However, the diversity in the protocols used for the competitive experiments (RIA vs. ELISA or coated vs. soluble TPO) as well as the growing number of anti-TPO Abs produced by all the groups working in the field has increased the available data but at the same time complicated the localization of IDR/A and B on the TPO molecule.
Even if the region 599–617 of hTPO is the main component in the IDR/A-specific aAb epitopes, only one amino acid residue, K627, curiously outside the sequence 599–617, has been identified as being involved in the IDR/A autoepitope (20). On the other hand, localization of the IDR/B at the surface of hTPO has been more intensively investigated, and numerous contributing amino acids have been identified (7, 17, 19, 20, 21). Up till now, these data suggested that the IDR/A epitope is much more restricted than the IDR/B epitope at the surface of hTPO. However, it is unclear whether these domains form two separate clusters recognized by either IDR/A- or B-specific human anti-TPO aAb or are structurally similar (involving the same surface area on the TPO molecule).
To answer these questions, we used for the first time all the reference anti-TPO Abs (human Fabs, mouse mAbs, and rabbit antipeptides) to evaluate, using a uniform approach, the relationship between the IDR/A and B epitopes. A series of purified rabbit antipeptides directed against short TPO sequences allowed us to demonstrate that epitopes recognized by recombinant human IDR/A-specific Fabs are characterized by a strong inhibition by the antipeptide P14 (epitope 599–617) but are also directed against other regions on hTPO (amino acid residues 353–372, 702–720, 375–387, and 549–63). Interestingly, the same regions were found to be involved in human IDR/B-specific Fab epitopes (present data and Refs. 12 , 17 , 19), but the strength of binding in region 599–617 was markedly lower. The present data corroborate previous results suggesting that the sequence recognized by the antipeptide P14 is an essential component of IDR/A (17) but also demonstrate that this region of hTPO is also recognized by the IDR/B-specific human aAbs. These findings allowed us to establish, for the first time that IDR/A and B anti-TPO aAbs interact with the same regions on the TPO molecule.
Whereas the binding patterns clearly identify human anti-TPO Fabs WR1.7 or SP1.4 as IDR/A specific, and TR1.9 or T13 as IDR/B specific, a doubt subsists concerning Fab TR1.8. Indeed, this Fab was previously thought to belong to IDR/B (16) but revealed an IDR/A inhibition pattern in the present study (Table 1). The discrepancy observed between these results is probably due to the experimental protocol used for these competition studies. Here we performed the experiments by using coated TPO and purified rabbit antipeptides, previously obtained by immunizing rabbits with relatively short linear peptide, accessible on the surface of TPO (9), thus recognizing linear epitopes on the hTPO. In contrast, early experiments by Chazenbalk et al. (16), carried out by using soluble TPO and a competition format between pairs of human Fabs, showed that both recognized conformational epitopes. However, in the same study, coated IDR/A-specific Fabs (WR1.7 and SP1.5) were able to compete in a dose-dependent manner with TR1.8 but not with TR1.9, confirming that TR1.8 is not a pure IDR/B. We argue that TR1.8 binds strongly in the region 599–617 but also shares several amino acid residues with the TR1.9 epitope in regions 353–372, 549–563, 702–720, and 760–779 (Table 1), thus explaining the mixed IDR/A-B epitope.
Such observations emphasize the high complexity of the IDR on the TPO molecule. Moreover, the IDR is stretched along the MPO- and CCP-like domains of hTPO, and some regions might in fact be too far to compose a single epitope, even discontinuous. We argue, however, that until now the only three-dimensional structure of hTPO known is a computer model, and the actual structure of hTPO might be more compact than the modeled one. Some observations support this argument. First, we and others described flexibility in the hinge region between the MPO-, CCP-, and epidermal growth factor-like domains, enabling the formation of a more closely folded molecule (7, 12). Second, because the membrane-bound TPO, at the surface of thyrocytes as well as Chinese hamster ovary cells, exists as a disulfide-linked dimer (25), this quaternary structure favors a spatial gathering of the IDR. Because anti-TPO aAb epitopes are largely restricted to one facet of the native molecule (present data and Refs. 12 , 16), this allows the formation of a dimer on the other side, as previously modeled by homology with the crystal structure of the MPO molecule (9).
To complete our study, we examined, by using the same ELISA format, all the reference mouse anti-TPO mAbs from the group of Ruf and colleagues, which was the first group to use anti-TPO Abs to study the epitopic profile of human anti-TPO aAbs and reveal the presence of two IDR regions (A and B, with an inverted nomenclature) on their cognate antigen (13). Distinct inhibition patterns were observed, and, similar to that we observed with human Fabs, IDR/A-specific mouse mAbs strongly interact with region 599–617 (mAbs 15, 64, and 18), whereas the IDR/B-specific epitopes are mainly outside this region. But as exemplified again with mAb 2, an IDR/B-specific Ab is able to bind to several regions, namely 599–617, 549–563, 353–372, and 702–721. This observation explains why addition of rabbit antisera P43 to the mixture of antiserum P12 and P14 leads to a substantial increase in the inhibition of mAb 2, as previously reported (20). Even if human anti-TPO Fabs and mAbs interact with the same IDR on TPO, by using rabbit antipeptide Abs, we observed that the inhibition profiles are more heterogeneous with human anti-TPO Fabs than with mouse mAbs. One could explain these data by the fact that mouse and human anti-TPO antibody repertoires are not similar. An illustration comes from the mutation of amino acid residue N642 leading to a clear inhibition in the binding of mAb 15 but not in the binding of human Fabs (17).
Several reports from our laboratory and others already pointed out numerous residues on hTPO as being part of the IDR/B; however, only one residue (K627), outside the major region 599–617, has been identified so far as being recognized by IDR/A aAbs. Thus, we used Spot analysis and rational mutagenesis on synthetic peptides to highlight amino acid residues in the region 599–617, part of the IDR/A epitope. These results allowed to restrict the epitope of polyclonal P14 on hTPO to the region 599–611 and outlined five residues (E604, T605, P606, D608, and L609) critical for binding. However, we have to keep in mind that antipeptide P14 is a polyclonal Ab; thus, although these five residues are surely involved in the binding of polyclonal P14, other residues might be involved in the epitope recognized by one or another individual Ab forming the polyclonal. More interestingly, we found that human anti-TPO aAbs (not only recombinant IDR/A-specific Fab TR1.8 but also purified IgG from patients’ sera) recognized only the extremities of the region 599–617. Precisely we described here regions 597FCGLPRLE604 and 611TAIASRSV618 as being part of their conformational autoepitopes (Fig. 6A). This gap in the middle of the region 599–617 point toward a major difference in the epitopes seen by polyclonal P14 and human aAbs. Indeed, it now seems obvious that antipeptide P14 competes only in the region 597FCGLPRLE604 with human aAbs for binding on hTPO. Furthermore, these findings are in agreement with previous studies demonstrating that mutation of residue D608 abrogates the binding of anti-peptide to hTPO, whereas the binding of IDR/A- and B-specific mAb (mAb 64 and 2) is not modified (20). In fact, residue D608 is not involved in the human IDR but only in the P14 epitope, as shown in the present study. On the contrary, amino acid residues R616, S617, and V618 were found to be reactive with the anti-TPO aAbs from patients’ sera (Fig. 6D).
Finally, why and how a human anti-TPO aAb will be IDR/A and B specific and what will make the difference to orientate such recognition remain unknown. Interestingly, the group of Gardas and colleagues (24) showed that a majority of the human anti-TPO aAbs from patients’ sera is directed against IDR/A (53 vs. 24% for IDR/B). We hypothesize that IDR/A may be primarily recognized in patients because it might be the first immunodominant epitope generated during the development of AITD. Indeed, it is well known that the epitope-spreading phenomenon, outside the IDR, does not occur in AITD patients, even with high-titer anti-TPO aAbs like during Hashimoto thyroiditis (1). But we do not know whether such a phenomenon could occur inside the IDR, and thus we suppose that before onset of AITD, the Ab repertoire is mainly directed against the region 599–617 and also more lightly against other regions, thus producing aAbs with an IDR/A-specific pattern. Later in the process, the repertoire may spread to other regions and/or amino acid residues inside or close to the IDR/A, generating a second aAb wave known as IDR/B specific but using similar regions for their epitope recognition. This two-wave hypothesis is already under investigation in our laboratory.
Taken together, our data demonstrate that human IDR/A- or B-specific anti-TPO aAbs recognize the same regions on the surface of the TPO molecule. However, IDR/A-specific aAbs use much the region 599–617 more forcefully as the anchor point for their binding to hTPO. We provide here some evidence showing that the two IDRs at the surface of hTPO are very closely related. Thus, our findings are greatly encouraging to design potent therapeutic peptides aiming at deviating both IDR/A- and B-specific aAbs from their targets during the development of AITD. Such strategy might decrease antigen presentation by TPO-specific B cells to autoaggressive T cells. Obviously, such an approach cannot be powerful enough to prevent or block alone an ongoing autoimmune process; however, combined with other more efficient immunotherapies, before or during recent onset, it might be possible to synergize and delay organ-specific destruction in patients with hypothyroiditis.
Acknowledgments
The authors thank Dr. S. L. Salhi for carefully reading the manuscript. We are indebted to Drs. J. Ruf and P. Carayon for providing us with the mouse mAbs to TPO and Drs. S. M. McLachlan and B. Rapoport for the human anti-TPO Fabs (TR1.8, TR1.9, WR1.7, and SP1.4). We also thank Drs. L. Baldet and A. M. Puech for providing the patients’ sera. We are also grateful to Dr. S. Villard for Spot synthesis and helpful discussions and C. Nguyen for peptide synthesis.
References
McLachlan SM, Rapoport B 2000 Autoimmune response to the thyroid in humans: thyroid peroxidase—the common autoantigenic denominator. Int Rev Immunol 19:587–618
Rodien P, Madec AM, Ruf J, Rajas F, Bornet H, Carayon P, Orgiazzi J 1996 Antibody-dependent cell-mediated cytotoxicity in autoimmune thyroid disease: relationship to antithyroperoxidase antibodies. J Clin Endocrinol Metab 81:2595–2600
Wadeleux P, Winand-Devigne J, Ruf J, Carayon P, Winand R 1989 Cytotoxic assay of circulating thyroid peroxidase antibodies. Autoimmunity 4:247–254
Blanchin S, Estienne V, Durand-Gorde JM, Carayon P, Ruf J 2003 Complement activation by direct C4 binding to thyroperoxidase in Hashimoto’s thyroiditis. Endocrinology 144:5422–5429
Guo J, Wang Y, Rapoport B, McLachlan SM 2000 Evidence for antigen presentation to sensitized T cells by thyroid peroxidase (TPO)-specific B cells in mice injected with fibroblasts co-expressing TPO and MHC class II. Clin Exp Immunol 119:38–46
Estienne V, Blanchet C, Niccoli-Sire P, Duthoit C, Durand-Gorde JM, Geourjon C, Baty D, Carayon P, Ruf J 1999 Molecular model, calcium sensitivity, and disease specificity of a conformational thyroperoxidase B-cell epitope. J Biol Chem 274:35313–35317
Estienne V, Duthoit C, Blanchin S, Montserret R, Durand-Gorde JM, Chartier M, Baty D, Carayon P, Ruf J 2002 Analysis of a conformational B cell epitope of human thyroid peroxidase: identification of a tyrosine residue at a strategic location for immunodominance. Int Immunol 14:359–366
Arscott PL, Koenig RJ, Kaplan MM, Glick GD, Baker JR 1996 Unique autoantibody epitopes in an immunodominant region of thyroid peroxidase. J Biol Chem 271:4966–4973
Hobby P, Gardas A, Radomski R, McGregor AM, Banga JP, Sutton BJ 2000 Identification of an immunodominant region recognized by human autoantibodies in a three-dimensional model of thyroid peroxidase. Endocrinology 141:2018–2026
Gardas A, Sohi MK, Sutton BJ, McGregor AM, Banga JP 1997 Purification and crystallisation of the autoantigen thyroid peroxidase from human Graves’ thyroid tissue. Biochem Biophys Res Commun 234:366–370
Hendry E, Taylor G, Ziemnicka K, Grennan Jones F, Furmaniak J, Rees Smith B 1999 Recombinant human thyroid peroxidase expressed in insect cells is soluble at high concentrations and forms diffracting crystals. J Endocrinol 160:R13–R15
Bresson D, Cerutti M, Devauchelle G, Pugniere M, Roquet F, Bes C, Bossard C, Chardes T, Peraldi-Roux S 2003 Localization of the discontinuous immunodominant region recognized by human anti-thyroperoxidase autoantibodies in autoimmune thyroid diseases. J Biol Chem 278:9560–9569
Ruf J, Toubert ME, Czarnocka B, Durand-Gorde JM, Ferrand M, Carayon P 1989 Relationship between immunological structure and biochemical properties of human thyroid peroxidase. Endocrinology 125:1211–1218
McLachlan SM, Rapoport B 1995 Genetic and epitopic analysis of thyroid peroxidase (TPO) autoantibodies: markers of the human thyroid autoimmune response. Clin Exp Immunol 101:200–206
Portolano S, Chazenbalk GD, Seto P, Hutchison JS, Rapoport B, McLachlan SM 1992 Recognition by recombinant autoimmune thyroid disease-derived Fab fragments of a dominant conformational epitope on human thyroid peroxidase. J Clin Invest 90:720–726
Chazenbalk GD, Costante G, Portolano S, McLachlan SM, Rapoport B 1993 The immunodominant region on human thyroid peroxidase recognized by autoantibodies does not contain the monoclonal antibody 47/c21 linear epitope. J Clin Endocrinol Metab 77:1715–1718
Gora M, Gardas A, Wiktorowicz W, Hobby P, Watson PF, Weetman AP, Sutton BJ, Banga JP 2004 Evaluation of conformational epitopes on thyroid peroxidase by antipeptide antibody binding and mutagenesis. Clin Exp Immunol 136:137–144
Libert F, Ludgate M, Dinsart C, Vassart G 1991 Thyroperoxidase, but not the thyrotropin receptor, contains sequential epitopes recognized by autoantibodies in recombinant peptides expressed in the pUEX vector. J Clin Endocrinol Metab 73:857–860
Bresson D, Pugniere M, Roquet F, Rebuffat SA, N-Guyen B, Cerutti M, Guo J, McLachlan SM, Rapoport B, Estienne V, Ruf J, Chardes T, Peraldi-Roux S 2004 Directed mutagenesis in region 713–720 of human thyroperoxidase assigns 713KFPED717 residues as being involved in the B domain of the discontinuous immunodominant region recognized by human autoantibodies. J Biol Chem 279:39058–39067
Gora M, Gardas A, Watson PF, Hobby P, Weetman AP, Sutton BJ, Banga JP 2004 Key residues contributing to dominant conformational autoantigenic epitopes on thyroid peroxidase identified by mutagenesis. Biochem Biophys Res Commun 320:795–801
Guo J, Yan XM, McLachlan SM, Rapoport B 2001 Search for the autoantibody immunodominant region on thyroid peroxidase: epitopic footprinting with a human monoclonal autoantibody locates a facet on the native antigen containing a highly conformational epitope. J Immunol 166:1327–1333
Gardas A, Watson PF, Hobby P, Smith A, Weetman AP, Sutton BJ, Banga JP 2000 Human thyroid peroxidase: mapping of autoantibodies, conformational epitopes to the enzyme surface. Redox Rep 5:237–241
Molina F, Laune D, Gougat C, Pau B, Granier C 1996 Improved performances of spot multiple peptide synthesis. Pept Res 9:151–155
Jastrzebska-Bohaterewicz E, Gardas A 2004 Proportion of antibodies to the A and B immunodominant regions of thyroid peroxidase in Graves and Hashimoto disease. Autoimmunity 37:211–216
Baker JR, Arscott P, Johnson J 1994 An analysis of the structure and antigenicity of different forms of human thyroid peroxidase. Thyroid 4:173–178(Damien Bresson, Sandra A.)
Address all correspondence and requests for reprints to: Dr. Sylvie Péraldi-Roux, Centre National de la Recherche Scientifique UMR 5160, Centre de Pharmacologie et Biotechnologie pour la Santé, 15 avenue Charles Flahault, BP 14491, 34093 Montpellier, Cedex 5, France. E-mail: sylvie.roux@ibph.pharma.univ-montp1.fr.
Abstract
Human anti-thyroperoxidase (TPO) autoantibodies (aAbs) are a major hallmark of autoimmune thyroid diseases. Their epitopes are discontinuous and mainly restricted to an immunodominant region (IDR) consisting of two overlapping regions (IDR/A and B). To shed light on the relationship between these regions, we first performed competitive studies using all available reference anti-TPO antibodies. Interestingly, we showed that human IDR/A- and B-specific anti-TPO aAbs recognized essentially the same regions on the TPO molecule. However, our data also indicated that IDR/A-specific human aAbs strongly recognized the region containing residues 599–617, whereas the IDR/B-specific aAbs bind to several regions as well as region 599–617. Next, we scanned this key region to identify the residues involved in the immunodominant autoepitope. Using peptide spot technology together with competitive ELISA experiments, we demonstrated that residues 604ETP-DL609 play a major role in the anti-peptide P14 epitope and that IDR/A-specific human anti-TPO aAbs, either expressed as recombinant Fab or obtained from Graves’ disease patients, specifically recognize the sequences 597FCGLPRLE604 and 611TAIASRSV618. All together our data emphasize that both the IDRs involve the same surface area on human TPO, but the differential usage of one or the other regions leads to different inhibition patterns in competitive experiments. In conclusion, our data help to resolve the long-sought issue on the molecular immunology of the two IDRs on TPO and provide new clues to design efficient peptides that may be part of a combinatorial treatment aiming at delaying development of autoimmune thyroiditis when used prophylactically.
Introduction
THYROID PEROXIDASE (TPO) is a major autoantigen involved in autoimmune thyroid diseases (AITD). High amounts of anti-TPO autoantibodies (aAbs) are frequently present in the sera of patients suffering from AITD (1). Anti-TPO aAbs are thought to be involved in thyroid cell destruction through cytotoxic mechanisms mediated by effector cells (2) and/or complement activation (3). More recently it has also been shown that the complement pathway may be activated via direct binding of the C4 complement component to human TPO (hTPO) without mediation by immunoglobulin (4). More importantly, anti-TPO aAbs have been found to be involved in antigen presentation to autoaggressive T cells (5) and may favor the exacerbation of AITD. Consequently, information on how the TPO molecule is seen by the immune system could lead to a greater understanding of why and how immune tolerance is lost during onset of AITD. Such information would also be of great importance to design, using a rational approach, therapeutic components (as peptides) able to interfere with those mechanisms. These peptides could be used in combination with other immunotherapies such as systemic antibody treatment, antigen-specific immunizations, or others generating antigen-specific regulatory T cells capable to block, at least for a period of time, an ongoing autoimmune process and may synergize to delay hypothyroiditis.
TPO is a large and highly complex molecule comprising three distinct domains: a myeloperoxidase (MPO)-like, a complement control protein (CCP)-like, and an epidermal growth factor-like domain, from the N- to the C-terminal extremities. Although the three domains were separately modeled (6, 7, 8, 9), the full three-dimensional structure of TPO has to be yet elucidated. It is well recognized that TPO is a highly heterogeneous protein with alternative splicing of some exons, different N-terminal start sites, and endoproteolytic sites contributing to the diversity of the polypeptide chain resulting in the well-known doublet and that a flexible molecule, all of which have made it difficult to obtain high resolution diffracting crystals for three-dimensional structural analysis (10, 11, 12).
It is well known that the majority of the anti-TPO aAbs are dependent on the three-dimensional tertiary structure of the molecule and interact with a limited surface region termed the immunodominant region (IDR). By using competitive studies between human anti-TPO aAbs and a panel of mouse anti-TPO monoclonal antibodies (mAbs), this IDR has been divided into two domains named A (IDR/A) and B (IDR/B), containing different but adjacent surface epitopes (13). Subsequent characterization of recombinant human anti-TPO antibody fragments (Fabs) derived from combinatorial libraries constructed with intrathyroidal B cells purified from Graves’ disease patients also allowed the identification of two large determinants on TPO (14, 15, 16), defining the A and B domains with an inverted nomenclature. Competitive experiments between human anti-TPO aAbs from patients’ sera and recombinant anti-TPO Abs (human Fabs or mouse mAbs) led to the definition of global contours of the IDR/A and B domains on the TPO molecule. Several data emerged showing that these two domains, although different, overlap in part.
To date, the major region (599–617) belonging to the IDR/A and five regions (210–225, 353–363, 549–563, 713–720, and 766–775) structuring the IDR/B have been identified by different groups (12, 17, 18). In an attempt to improve the knowledge on immunodominant amino acid residues recognized by human anti-TPO aAbs, several groups ex pressed recombinant human TPO mutants with single amino acid replacements (7, 17, 18, 19, 20). This strategy led to the identification of a number of key residues (R225, Y772, and K713 to D717) involved in the IDR/B-specific human anti-TPO aAbs epitopes (7, 17, 19, 21). On the other hand, only one amino acid residue (K627) has been clearly assigned to the IDR/A-specific human anti-TPO aAbs epitopes (20).
Despite the growing number of data available for delimiting each domain of the IDR, several questions still remain. First, even if the existence of two domains (A and B) forming the entire IDR is now well documented, it is unclear whether these domains form two separate clusters recognized by either IDR/A- or B-specific human anti-TPO aAbs or are structurally similar (involving the same surface area on the TPO molecule). Second, although region 599–617 represents a major region strongly recognized by the IDR/A-specific aAbs, none of its amino acid residues have been shown to be directly involved in their binding to human TPO.
To address these questions, we used for the first time all the reference anti-TPO Abs (recombinant human Fabs, mouse mAbs, and rabbit polyclonal antipeptides) produced and characterized by different groups to study the relationship between IDR/A and B domains. We show that human IDR/A- and B-specific anti-TPO aAbs recognize essentially the same region, on the surface of the TPO molecule. The main characteristic found is a stronger binding in region 599–617 for the IDR/A-specific aAbs, leading to different inhibition patterns in competitive experiments. Furthermore, due to the key role of region 599–617, we searched the residues in this region participating in the interaction with rabbit antipeptide P14 (immunoreactive with the sequence 599–617) and human aAbs. For this purpose, we prepared a series of alanine scanning mutations by the Spot method and synthesized six punctually mutated peptides. For the first time, we demonstrated that residues 604ETP-DL609 play a major role in the antipeptide P14 epitope (reference polyclonal for the IDR/A) and that IDR/A-specific human anti-TPO aAbs, expressed as recombinant Fab or obtained from Graves’ disease patients, specifically recognize the sequences 597FCGLPRLE607 and 611TAIASRSV618.
Materials and Methods
Patients’ sera
Sera from patients with Graves’ disease were obtained from Dr. L. Baldet and Dr. A.-M. Puech (Lapeyronie Hospital, Montpellier, France). The anti-TPO aAbs titers were determined by RIA using the TPO-AB-CT kit (CIS bio, Gif sur Yvette, France). IgG from patients’ sera were purified on a protein A column.
Human TPO and anti-TPO antibodies
The hTPO, purified (greater than 95% pure) from thyroid glands, was obtained from HyTest Ltd. (Turku, Finland). Polyclonal antipeptide sera were produced by immunizing rabbits with peptides derived from the primary sequence of human TPO (Table 1). The antipeptides P1, P12, P14, P18, P24, P43, P50, and P760–779 (rabbit reference polyclonal Abs) were produced by the groups of Gardas and colleagues (9, 22). S10 was produced in our laboratory. The IDR/A- (15, 18, 59, and 64) and B-specific (2, 9, 47, and 60) mouse anti-TPO mAbs (human nomenclature) (mouse reference Abs) were generously provided by Ruf et al. (13). Polyclonal antipeptides antibodies and mouse mAbs were purified on a protein G column. Recombinant human anti-TPO Fabs TR1.9, TR1.8, SP1.4, and WR1.7 (human reference Abs) were kindly provided by McLachlan and colleagues (15).
TABLE 1. Inhibition of human Fab or mouse monoclonal antibody binding inhibition to TPO by rabbit polyclonal antipeptide antisera
Binding to TPO of purified rabbit polyclonal antipeptides, mouse anti-TPO mAbs, and human anti-TPO Fabs by ELISA
Microtiter plates were coated with 1 μg/ml human (h)TPO in 100 mM NaHCO3 (pH 9.6) overnight at 4 C. Plates were washed with 0.05% Tween 20 in PBS (PBS-T) (pH 7.3) and blocked with 2% nonfat dry milk in PBS-T (saturation buffer) for 1 h at 37 C. After three washes, polyclonal antipeptides, anti-TPO mAbs, or human anti-TPO Fabs were incubated with 1% nonfat dry milk in PBS-T (incubation buffer) for 2 h at 37 C, serial dilutions were performed as indicated in Fig. 1, A–C. A horseradish peroxidase (HRP)-conjugated antirabbit IgG, antimouse IgG, or antihuman F(ab')2 (diluted at 1:2,000; in the incubation buffer; Sigma, Meylan, France) was added, and the plates were incubated for 1 h at 37 C. The reactivity was detected with a 4 mg/ml 2-phenylenediamine solution containing 0.03% (vol/vol) hydrogen peroxide in 0.1 M citrate buffer (pH 9.5). The reaction was stopped by adding 50 μl of 2 M H2SO4 to each well, and the resulting absorbance was measured at 490 nm.
FIG. 1. Binding of anti-TPO antibodies (human Fabs, rabbit antipeptides, and mouse mAbs) to human TPO. ELISA experiments were performed by coating human TPO at 1 μg/ml. A, Human anti-TPO Fabs; B, purified rabbit polyclonal antisera directed against TPO peptides (as described in Table 1); and C, mouse anti-TPO mAbs (IDR/A- or B-specific as indicated) were incubated in serial dilutions on hTPO as stated on the figure.
Blocking ELISA experiments
The wells were coated with 1 μg/ml hTPO (overnight at 4 C) and then washed and blocked as described above. Rabbit polyclonal antipeptides (P1, P12, P14, P18, P24, P50, P43, S10, and P760–779) were incubated in the incubation buffer for 2 h at 37 C. After three washings, human anti-TPO Fabs (SP1.4, WR1.7, TR1.8, TR1.9, and T13) or mouse anti-TPO mAbs (2, 9, 15, 18, 47, 59, 60, and 64) were incubated for 1 h at 37 C at a concentration giving an absorbance (A) of 1.5 at 490 nm. An HRP-conjugated antihuman F(ab')2 or antimouse IgG (Sigma), incubated for 1 h at 37 C, was used to detect the binding of human anti-TPO Fabs or mouse mAb to TPO, respectively. Three washes were performed, and then the reactivity was revealed as described above. The percent inhibition was calculated by comparing the binding of human Fabs and mouse mAbs with or without inhibitor.
Peptide synthesis and immunoassay on cellulose membrane-bound peptides
The general protocol for Spot parallel peptide synthesis was described previously (23). Two different cellulose membrane-bound peptides were synthesized. The first one was synthesized with a set of 25-mer synthetic peptides frame shifted by two residues, corresponding to the entire sequence of hTPO and the second with three sets of 8-mer synthetic peptides (regions 597–604, 604–611, 611–618, and their 18 alanine analogs), corresponding to sequences including the antipeptide P14 epitope. The membrane-bound peptides were probed with rabbit antipeptide (1 μg/ml), human anti-TPO TR1.8 Fab (0.1 μg/ml), or purified IgG from patients’ sera (1 μg/ml), coincubated with the specific HRP-conjugated anti-species IgG (antirabbit dilution 1:15,000, antihuman F(ab')2 or antihuman Fc-specific antibody, respectively, diluted 1:2000), for 1 h 30 at 37 C.
Competition studies were performed by preincubation of human anti-TPO TR1.8 Fab for 10 min on the membrane-bound peptides followed by coincubation with rabbit antipeptide P14 and HRP-conjugated antirabbit IgG (same concentration as previously mentioned). After three washes, the complex (polyclonal P14/peptides) was revealed using the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech) on a sensitive film. Membranes were reused after a regeneration cycle. Importantly, to make sure that the regeneration cycles were efficient, membranes were always incubated with enhanced chemiluminescence directly after regeneration. As a control, all HRP-conjugated Abs were incubated alone and showed no reactivity with the spots.
Synthesis of soluble peptides derived from the sequence 599–617 of hTPO
A series of soluble peptides derived from the sequence 599–617 of hTPO (amino acid sequence 599GLPRLETPADLSTAIASRS617) was synthesized on a Multipep synthesizer (Intavis AG, Cologne, Germany) by Fmoc chemistry. The wild-type (wt) sequence and six mutated peptides were generated: five peptide with a single mutation (residues were replaced by an alanine) at positions Glu604 (mut E604), Thr605 (mut T605), Pro606 (mut P606), Asp608 (mut D608), Leu609 (mut L609), and one peptide with a triple mutation in which residues Thr605, Asp608, and Leu609 were replaced by Ala605, Gln608, and Ala609 (mut TDL). The peptides released from the resin by trifluoroacetic acid treatment in the presence of appropriate scavengers. They were lyophilized and their purity assessed by HPLC (all peptides showed a purity greater than 85%)
Competitive ELISA experiments with soluble peptides derived from the sequence 599–617 of hTPO
Rabbit polyclonal antipeptide P14 (7 ng/ml) was preincubated with wt or mutated soluble peptides derived from the 599–617 amino acid sequence of hTPO at increasing concentrations overnight at 4 C. At the same time, microtiter plates were coated with hTPO at 1 μg/ml in 100 mM NaHCO3 (pH 9.6) overnight at 4 C. After washings, the plates were blocked with saturation buffer for 1 h at 37 C. Saturation buffer was removed and the complex, composed of the antipeptide P14 preloaded with the synthetic peptides, was incubated with coated hTPO for 2 h at 37 C. Rabbit antipeptide P14 bound on hTPO was detected by adding a peroxidase-conjugated antirabbit IgG (Sigma, dilution 1:2000) for 1 h at 37 C. The complex was revealed by adding 100 μl of a 4 mg/ml 2-phenylenediamine solution containing 0.03% (vol/vol) hydrogen peroxide in 0.1 M citrate buffer (pH 9.5). The reaction was stopped by addition of 50 μl/well of 2 M H2SO4, and the resulting absorbance was measured at 490 nm.
Statistical analysis
All inhibition data were expressed as the mean ± SD. The statistical significance of the difference between means was determined using the two-tailed Student’s t test. Differences were considered significant at P < 0.05.
Results
IDR/A- and B-specific human anti-TPO Fabs recognize the same regions on hTPO
To obtain new information on the relationship between IDR/A and B, we decided to use all the available reference anti-TPO Abs and analyzed the profile of reactivity of the human anti-TPO Fabs and mouse anti-TPO mAbs in competition with rabbit polyclonal antipeptide Abs (all blocking ELISA experiments were performed using the same protocol). First, we assessed the binding of all reference anti-TPO Abs on hTPO by ELISA. Figure 1 shows the reactivities of each Ab with the coated hTPO.
Next, the five human TPO-specific Fabs, two IDR/A- (SP1.4 and WR1.7), and three IDR/B-specific (TR1.8, TR1.9, and T13) Fabs (12, 16) were used for competition experiments with a panel of antipeptide Abs at a concentration giving an A490 of 1.5 (as calculated by using the data in Fig. 1A). The resulting inhibition profiles are shown in Fig. 2. At high concentration (0.7 μM), antipeptides P14, P24, P43, and P50, directed against the hTPO sequences 599–617, 375–387, 702–721, and 353–372, respectively (Table 1), inhibited the binding of Fabs T13 and TR1.9 between 12 and 24%, whereas antipeptides P1 and P18 were unable to inhibit these Fabs by more than 9%. Surprisingly, antipeptides P12 and P760–779 interfered only with the binding of Fab TR1.9 on hTPO (inhibition ranging between 18 and 20%) but not with FabT13. This indicates that even if these Fabs recognize the IDR/B on hTPO, their contact points are probably slightly different. These first results are in agreement with our previous studies, which demonstrated that regions 353–363, 377–386, and 713–720 from the MPO-like domain and region 766–775 from the CCP-like domain are a part of the IDR/B (12). More interestingly, the significant inhibition (20–24%) observed with the human IDR/B-specific Fabs (TR1.9 and T13) in competition with the antipeptide P14 strongly suggests that region 599–617 of hTPO also constitutes a part of IDR/B. However, its contribution to the IDR/B epitope is much less, compared with the IDR/A epitope.
FIG. 2. Inhibition of human Fabs binding to TPO by rabbit polyclonal antipeptide antibodies. The binding of human anti-TPO-specific Fabs (T13, TR1.9, TR1.8, SP1.4, and WR1.7) were inhibited by rabbit antipeptide antibodies (P1, P12, P14, P18, P24, P43, P50, P760-779, and S10) as described in Materials and Methods. The human Fabs were used at a concentration that gave an absorbance of 1.5 by ELISA at 490 nm. The rabbit polyclonal antibodies were tested at two different concentrations (inhibitor concentration shown in this figure). The higher concentrations were used according to the data shown in Fig. 1B, for saturating the binding sites on coated TPO. The results represent the mean ± SD of the duplicate determinations. Any inhibition over 10% (at least three times the lowest inhibition observed for each Fab) was considered as significant. One antipeptide for each human Fab, showing an inhibition around 10% with the highest concentration, was taken as reference for the statistical analyses (antipeptide P18 for the Fabs T13, TR1.9, and WR1.7; antipeptide P43 for the Fab SP1.4 and antipeptide S10 for the Fab TR1.8). These inhibitions of reference were used to determine the statistical significance of each inhibition using the two-tailed Student’s t test. *, P < 0.05 (data significantly different). The data shown are representative results from two independent experiments.
Indeed, the binding of the IDR/A-specific Fabs, SP1.4 and WR1.7, was strongly hampered (inhibition from 79 to 89%) by the antipeptide P14, clearly demonstrating that amino acid residues in position 599–617 constitute the major anchor for the IDR/A-specific aAbs on hTPO (Fig. 2). Unexpectedly, the binding of Fab TR1.8 on hTPO, previously described as IDR/B-specific (16), was inhibited up to 89% by rabbit antipeptide P14 and consequently showed a reactivity profile similar to that of the IDR/A-specific Fabs in our inhibition format. Interestingly, the IDR/A-specific Fabs also competed with several other antipeptides but weakly. As observed with the IDR/B-specific Fabs, a particular antipeptide may compete differently with one or another IDR/A-specific Fabs. For example, P12 and P43 clearly inhibited the Fabs WR1.7 and TR1.8 (more than 15%) but were not considered to be competitive with SP1.4 (less than 10% inhibition). On the contrary, P50 was shown to displace the binding of SP1.4, WR1.7, and TR1.8 to hTPO (from 14 to 23% inhibition).
Globally, our data indicate that not only IDR/A-specific human Fabs (SP1.4, WR1.7) but also TR1.8 interact with the same regions previously shown to be involved in the IDR/B-specific human aAbs (present data and Ref. 12).
Inhibition of mouse mAb binding to TPO by rabbit polyclonal antipeptide Abs
As indicated previously, all the reference mouse mAbs but mAb47 bind conformational epitopes. Thus, to localize more precisely their epitopes and compare the regions recognized by the reference human Fabs and the mouse mAbs, we tested the mouse anti-TPO-specific mAbs [15, 18, 59, and 64 (IDR/A-specific) and 2, 9, 47, and 60 (IDR/B-specific)] in a competitive ELISA experiment (same protocol as that used for the human Fabs) with rabbit polyclonal antipeptides (Fig. 3). The binding of mAbs 15, 64, and 18 on hTPO was inhibited 72, 65, and 54%, respectively, by rabbit antiserum P14. Therefore, these data confirm that mAbs 15, 64, and 18 are IDR/A specific. On the other hand, the binding of mAb 59 was not affected by rabbit antiserum P14; nevertheless, an unambiguous inhibition (26%) was obtained by using the rabbit antiserum P43. These observations suggests that mAb 59, as well as mAb47, which showed a strong inhibition (80%) with the polyclonal antipeptide P43, is categorized as IDR/B specific. The mAb 2 was moderately inhibited by both antipeptides P43 (10%) and P14 (25%). This inhibition profile, involving regions 599–617 and 713–720, confirms its IDR/B specificity. Finally, the inhibition profiles observed in the competitive ELISA experiments for both mAbs 60 and 9, described as IDR/B specific, did not allow us to determine their IDR specificity.
FIG. 3. Inhibition of mouse mAbs binding to TPO by rabbit polyclonal antipeptides. The binding of mouse anti-TPO-specific mAbs [15, 18, 59, and 64 (IDR/A specific) or 2, 9, 47, and 60 (IDR/B–specific)] were inhibited by the antipeptide polyclonal antibodies (1, 12, 14, 18, 24, 43, 50, S10, and P760-779) as described in Materials and Methods. The mouse mAbs were used at a concentration that gave an absorbance of 1.5 by ELISA at 490 nm and the polyclonal antipeptides were used at a concentration required to saturate the binding sites on coated TPO as defined in Fig. 1B. The results represent the mean ± SD of the duplicate determinations. Any inhibition over 10% (at least three times the lowest inhibition observed for each Fab) was considered as significant.
The inhibitions observed with human Fabs and mAbs are summarized in Table 1. Taken together, these results confirm the involvement of several regions at the surface of the TPO in the binding of human aAbs (some of them have already been identified by using site-directed mutations) and demonstrate for the first time that IDR/A- and B-specific human Fab epitopes are built around the same regions on hTPO but in fact differ in their ability to use the region 599–617 as the main anchor point. A faint but reproducible binding was also observed for almost all human Fabs in the regions 353–372 and 702–721 (delimited by the antipeptides P50 and P43, respectively). These regions possess the amino acid sequences 353–363 and 713–720 we previously described as contact points for the human Fab T13 (12).
Is steric hindrance a major factor in the inhibition experiments? Epitope analysis of rabbit antipeptides P43 and S10
One could argue that the degree of inhibition we observed by using these competitive ELISA experiments could be the result of a strong steric hindrance and not direct competition for the same epitopes. To evaluate the importance of steric hindrance, we compared the ability of rabbit antisera P43 and S10 to compete with mAb 47 for the binding on hTPO. Antipeptides P43 and S10 were obtained by immunizing rabbits, respectively, with peptides corresponding to amino acid residues 702–721 and 716–731 of hTPO (Table 1). Peptide P43 (702–721) entirely encompasses the mAb47 epitope (713–717), whereas S10 (716–731) possesses at one extremity only two residues involved in the mAb47 epitope, among their, residue D717 has been shown to weakly participate in the interaction with hTPO (19). First, rabbit antiserum P43 and S10 epitopes were studied by using overlapping peptides derived from the sequence of the hTPO (Fig. 4A). Twelve overlapping 25-mer peptides surrounding residues Leu691 to Pro737 of hTPO were reactive with rabbit antipeptide P43, indicating that the residues KFP713–715 are central for the binding of this antipeptide. Reactivity of the rabbit antiserum S10 on the Spot membrane shows up with eight overlapping peptides [from residues Arg703 to Lys741], revealing that peptide 717DFESCDSIPGM727 plays a crucial role in the binding of rabbit antisera S10 to hTPO. These observations confirmed that the S10 epitope does not contain all the main residues recognized by mAb 47 [713KFPED717], but the P43 epitope does. The S10 epitope, however partially overlaps with the mAb 47 epitope and thus could interfere with mAb 47 binding to TPO by steric hindrance. This possibility has been evaluated by ELISA (Fig. 4B). Interestingly, the binding of mAb 47 was inhibited in a dose-dependent fashion by the antipeptide P43 (inhibition by up to 80%), but only a maximum of 10% inhibition was observed with S10. These results emphasize that steric hindrance is not a major contributor to the inhibitions observed in our competitive ELISA experiments.
FIG. 4. Delineation of rabbit antiserum P43 and S10 epitopes. A, Immunoreactivity of peptides derived from the primary sequence of human TPO with rabbit antiserum was studied by the Spot technology. Reactivities of rabbit antiserum P43 and S10 on the Spot membrane are shown on the left and right side of the figure, respectively. The amino acid sequences of TPO corresponding to the immunoreactive spots are surrounded on the figure (solid lines for P43 and dashed lines for S10). B, Competitive ELISA experiments were performed with rabbit antipeptide P43 and S10 as inhibitor for the binding of mAb 47 (used at a fixed concentration giving an OD of 1.5 at 490 nm) to hTPO (see Materials and Methods).
Epitopic dissection of polyclonal antipeptide P14
The present study and previous data (9) point toward the importance of the rabbit antipeptide P14 epitope in the binding of human anti-TPO aAbs on their cognate antigen. Thus, it seems obvious that a precise localization of its epitope would be of great interest. To this end, we first used the Spot technology to dissect this epitope. In region 581–633, 11 overlapping peptides were found to be reactive (Fig. 5A). A common amino acid sequence TPADL (residues 605–609 of hTPO) appears to constitute the critical residues in interaction with the antipeptide P14. To precisely identify which residues play a major role in the binding of rabbit antipeptide P14 to hTPO, we synthesized on a spot membrane three 8-mer peptides covering the sequence 599–617 [597FCGLPRLE604, 604ETPADLST611, and 611TAIASRSV618] and their alanine analogs. The results in Fig. 5B show that only peptides 597FCGLPRLE604 and 604ETPADLST611 were immunoreactive and hence involved in the binding of rabbit antipeptide P14 on hTPO. Alanine replacements of residues C598 and E604 in the peptide 597FCGLPRLE604 and E604, T605, P606, D608, and L609 in the peptide 604ETPADLST611 reduced or totally abrogated the binding of P14. To be certain that these binding inhibitions were specific, we decided to synthesize soluble peptides corresponding to the sequence 599–617 of hTPO (wt) and six mutants in positions E604, T605, P606, D608, L609, and T-D-L605–609. These soluble peptides were then tested, by using a competitive ELISA, for their capacity to block the binding of rabbit antiserum P14 to hTPO (Fig. 5C). Although the wt peptide was able to block the binding of antisera P14 to coated hTPO in a dose-dependent manner (95% inhibition at 10 nM), all replacements impaired the ability of the mutated peptides to efficiently compete with antipeptide P14. More precisely, the punctual mutations showed an average of 50% inhibition at 10 nM, whereas the triple mutant T-D-L605–609 was far less efficient (20% inhibition at 10 nM). Taken together, the alanine scanning and competitive ELISA experiments demonstrated that residues T605, P606, D608, and L609 are critical for the binding of antipeptide P14 to hTPO.
FIG. 5. Determination of critical amino acid residues recognized by rabbit antiserum P14. A, The binding of rabbit antiserum P14 onto membrane-bound peptides covering the entire primary sequence of the hTPO was determined by Spot technology. Here, only the immunoreactive peptides (boxed with solid lines) and the four surrounding peptides are shown. B, Contributor residues from the sequence 599-617 of hTPO to rabbit antiserum P14 epitope were determined by Spot with an alanine-scanning approach. The region 597-618 of hTPO has been truncated into three shorter peptides [597FCGLPRLE604 (Seq1), 604ETPADLST611 (Seq2), and 611TAIASRSV618 (Seq3)] to improve the analyses. Then, a series of alanine analogs of each short peptide sequence was synthesized in parallel with the wild-type sequence. Finally, the binding of the polyclonal anti-peptide P14 to each peptide was analyzed by the spot technology. Underlined amino acids represent the critical residues observed for each peptide. C, The binding of rabbit antipeptide P14 to hTPO (coated at 1 μg/ml in a 96-well plate) was inhibited by free synthetic peptides (wt or a series of mutated peptides derived from the amino acid sequence 599GLPRLETPADLSTAIASRS617) in a competitive ELISA experiment as described in Materials and Methods.
Binding of human anti-TPO aAbs to their cognate antigen involves residues 597FCGLPRLE604 and 611TAIASRSV618
Because polyclonal P14 showed epitopic cross-reactivity in the region 599–617 with all the recombinant human anti-TPO Fabs tested, we decided to set up an experiment with the soluble peptides we designed previously to dissect the P14 epitope. Competitive ELISA experiments were performed with recombinant human anti-TPO Fabs (IDR/A and B specific) to determine whether the same residues in the region 599–617 are involved in their epitopes. Unfortunately, the mutated peptides were able to displace the binding of human Fabs to TPO as efficiently as the wt sequence (data not shown). This suggests that residues E604, T605, P606, D608, and L609, which are central for the interaction of rabbit antiserum P14 with hTPO, are not strongly involved in the epitopes recognized by the recombinant human anti-TPO Fabs. Consequently, we chose to use the recombinant human anti-TPO Fab TR1.8 [showing the strongest binding on hTPO (Fig. 1A) and using the region 599–617 as the main anchor point (Fig. 2)] to test its ability to interact with the spot membrane carrying the three wt 8-mer peptides [597FCGLPRLE604, 604ETPADLST611, 611TAIASRSV618] and their alanine analogs (Fig. 6). Interestingly, the high avidity due to the large number of peptides synthesized at the individual spots by this technology together with the high affinity of the TR1.8 Fab allowed us to detect a strong and specific binding on the wt sequences 597FCGLPRLE604 and 611TAIASRSV618 (Fig. 6B).
FIG. 6. Analysis of IDR/A epitope recognized by human anti-TPO aAbs by the Spot technology. Binding of antibodies (antipeptide P14, human anti-TPO Fabs, and purified IgG from Grave’s patients’ sera) was performed by using the Spot technology with an alanine-scanning membrane as described in Materials and Methods. A, Immunoreactivity of peptides with rabbit antiserum P14 was studied by the Spot technology on the three peptides (FCGLPRLE, ETPADLST, and TAIASRSV) and a series of alanine analogs of each peptide sequences surrounding the immunoreactive sequence 599-617. Rabbit antiserum P14 reactivity on the Spot membrane is represented at the top of the figure, and the binding of antipeptide P14 to wt peptide and each analog is also represented by histograms. The color intensity of each spot was determined using ScionImage software. Binding of (B) human anti-TPO Fab TR1.8 and (D) purified IgG from three Grave’s patients (patient 1, ; 2, ; and 3, ) onto the spot membrane is represented by the intensity of each spot. C, Similarly, the competitive Spot experiment between antipeptide P14 and human anti-TPO Fab TR1.8 is shown. Competition study was performed by coincubating rabbit antipeptide P14 with TR1.8, then binding of P14 on the membrane was revealed. As control, no reactivity was observed on the membrane with the HRP-conjugated antibodies used alone.
By comparison, antipeptide P14 showed reactivity with the wt sequences 597FCGLPRLE604 and 604ETPADLST611 but not with611TAIASRSV618 (Fig. 6A), explaining why the binding of P14, but not TR1.8, to the soluble peptide 599–617 was affected by the mutations in region 604ETPADLST611. Importantly, two mutated residues, P601 and R616, caused a decrease in the binding of TR1.8 and thus might participate in the epitope recognition (Fig. 6B). To verify that P14 and TR1.8 specifically competed in the region 597FCGLPRLE604 for their binding to hTPO, we performed a competition between these Abs. Figure 6C shows that after incubation with TR1.8, antipeptide P14 was no longer able to bind region 597–604 but retained its ability to react with region 604–611. These results argue for a strong contribution of residues 597–604 in the epitopes recognized by the IDR/A-specific aAbs. To validate this hypothesis, we analyzed the binding of purified IgG obtained from three Graves’ disease patients to the same spot membrane. Recently the group of Gardas and colleagues (24) reported that a majority of anti-TPO aAbs from patients’ sera affected by AITD is directed against IDR/A (approximately 53%). Thus, the binding of aAbs from patients’ sera should reflect the binding of the IDR/A-specific components. As shown in Fig. 6D, the reactivity patterns seen with these three patients were greatly similar to that seen with TR1.8. Interestingly, mutations in regions 597–604 and 611–618 displayed two different inhibition profiles. Punctual mutations in region 597–604 did not seem to induce an effective loss in the binding of aAbs from patients’ sera. On the other hand, certain mutations in region 611–618 markedly weakened the binding of the sera, namely residues R616, S617, and V618 (an average of 22, 40, and 30% inhibition, respectively, taking the intensity of the spot for the wt sequence as 100%). All together, these findings shed light on two regions, 597–604 and 611–618, strongly involved in the binding of the IDR/A-specific human aAbs.
Discussion
Anti-TPO aAbs predominantly recognize conformational epitopes. Previous studies clearly demonstrated that most of the human anti-TPO aAbs epitopes are located inside an IDR that has been divided into two overlapping but distinct regions known as A and B (13, 15, 16). These regions are thought to be formed by different amino acid sequences at the surface of the hTPO. For example, the sequence 599–617, defined by the polyclonal antipeptide P14, was directly assigned to IDR/A, whereas sequence 713–720 was shown to belong to IDR/B (12, 19, 21). However, the diversity in the protocols used for the competitive experiments (RIA vs. ELISA or coated vs. soluble TPO) as well as the growing number of anti-TPO Abs produced by all the groups working in the field has increased the available data but at the same time complicated the localization of IDR/A and B on the TPO molecule.
Even if the region 599–617 of hTPO is the main component in the IDR/A-specific aAb epitopes, only one amino acid residue, K627, curiously outside the sequence 599–617, has been identified as being involved in the IDR/A autoepitope (20). On the other hand, localization of the IDR/B at the surface of hTPO has been more intensively investigated, and numerous contributing amino acids have been identified (7, 17, 19, 20, 21). Up till now, these data suggested that the IDR/A epitope is much more restricted than the IDR/B epitope at the surface of hTPO. However, it is unclear whether these domains form two separate clusters recognized by either IDR/A- or B-specific human anti-TPO aAb or are structurally similar (involving the same surface area on the TPO molecule).
To answer these questions, we used for the first time all the reference anti-TPO Abs (human Fabs, mouse mAbs, and rabbit antipeptides) to evaluate, using a uniform approach, the relationship between the IDR/A and B epitopes. A series of purified rabbit antipeptides directed against short TPO sequences allowed us to demonstrate that epitopes recognized by recombinant human IDR/A-specific Fabs are characterized by a strong inhibition by the antipeptide P14 (epitope 599–617) but are also directed against other regions on hTPO (amino acid residues 353–372, 702–720, 375–387, and 549–63). Interestingly, the same regions were found to be involved in human IDR/B-specific Fab epitopes (present data and Refs. 12 , 17 , 19), but the strength of binding in region 599–617 was markedly lower. The present data corroborate previous results suggesting that the sequence recognized by the antipeptide P14 is an essential component of IDR/A (17) but also demonstrate that this region of hTPO is also recognized by the IDR/B-specific human aAbs. These findings allowed us to establish, for the first time that IDR/A and B anti-TPO aAbs interact with the same regions on the TPO molecule.
Whereas the binding patterns clearly identify human anti-TPO Fabs WR1.7 or SP1.4 as IDR/A specific, and TR1.9 or T13 as IDR/B specific, a doubt subsists concerning Fab TR1.8. Indeed, this Fab was previously thought to belong to IDR/B (16) but revealed an IDR/A inhibition pattern in the present study (Table 1). The discrepancy observed between these results is probably due to the experimental protocol used for these competition studies. Here we performed the experiments by using coated TPO and purified rabbit antipeptides, previously obtained by immunizing rabbits with relatively short linear peptide, accessible on the surface of TPO (9), thus recognizing linear epitopes on the hTPO. In contrast, early experiments by Chazenbalk et al. (16), carried out by using soluble TPO and a competition format between pairs of human Fabs, showed that both recognized conformational epitopes. However, in the same study, coated IDR/A-specific Fabs (WR1.7 and SP1.5) were able to compete in a dose-dependent manner with TR1.8 but not with TR1.9, confirming that TR1.8 is not a pure IDR/B. We argue that TR1.8 binds strongly in the region 599–617 but also shares several amino acid residues with the TR1.9 epitope in regions 353–372, 549–563, 702–720, and 760–779 (Table 1), thus explaining the mixed IDR/A-B epitope.
Such observations emphasize the high complexity of the IDR on the TPO molecule. Moreover, the IDR is stretched along the MPO- and CCP-like domains of hTPO, and some regions might in fact be too far to compose a single epitope, even discontinuous. We argue, however, that until now the only three-dimensional structure of hTPO known is a computer model, and the actual structure of hTPO might be more compact than the modeled one. Some observations support this argument. First, we and others described flexibility in the hinge region between the MPO-, CCP-, and epidermal growth factor-like domains, enabling the formation of a more closely folded molecule (7, 12). Second, because the membrane-bound TPO, at the surface of thyrocytes as well as Chinese hamster ovary cells, exists as a disulfide-linked dimer (25), this quaternary structure favors a spatial gathering of the IDR. Because anti-TPO aAb epitopes are largely restricted to one facet of the native molecule (present data and Refs. 12 , 16), this allows the formation of a dimer on the other side, as previously modeled by homology with the crystal structure of the MPO molecule (9).
To complete our study, we examined, by using the same ELISA format, all the reference mouse anti-TPO mAbs from the group of Ruf and colleagues, which was the first group to use anti-TPO Abs to study the epitopic profile of human anti-TPO aAbs and reveal the presence of two IDR regions (A and B, with an inverted nomenclature) on their cognate antigen (13). Distinct inhibition patterns were observed, and, similar to that we observed with human Fabs, IDR/A-specific mouse mAbs strongly interact with region 599–617 (mAbs 15, 64, and 18), whereas the IDR/B-specific epitopes are mainly outside this region. But as exemplified again with mAb 2, an IDR/B-specific Ab is able to bind to several regions, namely 599–617, 549–563, 353–372, and 702–721. This observation explains why addition of rabbit antisera P43 to the mixture of antiserum P12 and P14 leads to a substantial increase in the inhibition of mAb 2, as previously reported (20). Even if human anti-TPO Fabs and mAbs interact with the same IDR on TPO, by using rabbit antipeptide Abs, we observed that the inhibition profiles are more heterogeneous with human anti-TPO Fabs than with mouse mAbs. One could explain these data by the fact that mouse and human anti-TPO antibody repertoires are not similar. An illustration comes from the mutation of amino acid residue N642 leading to a clear inhibition in the binding of mAb 15 but not in the binding of human Fabs (17).
Several reports from our laboratory and others already pointed out numerous residues on hTPO as being part of the IDR/B; however, only one residue (K627), outside the major region 599–617, has been identified so far as being recognized by IDR/A aAbs. Thus, we used Spot analysis and rational mutagenesis on synthetic peptides to highlight amino acid residues in the region 599–617, part of the IDR/A epitope. These results allowed to restrict the epitope of polyclonal P14 on hTPO to the region 599–611 and outlined five residues (E604, T605, P606, D608, and L609) critical for binding. However, we have to keep in mind that antipeptide P14 is a polyclonal Ab; thus, although these five residues are surely involved in the binding of polyclonal P14, other residues might be involved in the epitope recognized by one or another individual Ab forming the polyclonal. More interestingly, we found that human anti-TPO aAbs (not only recombinant IDR/A-specific Fab TR1.8 but also purified IgG from patients’ sera) recognized only the extremities of the region 599–617. Precisely we described here regions 597FCGLPRLE604 and 611TAIASRSV618 as being part of their conformational autoepitopes (Fig. 6A). This gap in the middle of the region 599–617 point toward a major difference in the epitopes seen by polyclonal P14 and human aAbs. Indeed, it now seems obvious that antipeptide P14 competes only in the region 597FCGLPRLE604 with human aAbs for binding on hTPO. Furthermore, these findings are in agreement with previous studies demonstrating that mutation of residue D608 abrogates the binding of anti-peptide to hTPO, whereas the binding of IDR/A- and B-specific mAb (mAb 64 and 2) is not modified (20). In fact, residue D608 is not involved in the human IDR but only in the P14 epitope, as shown in the present study. On the contrary, amino acid residues R616, S617, and V618 were found to be reactive with the anti-TPO aAbs from patients’ sera (Fig. 6D).
Finally, why and how a human anti-TPO aAb will be IDR/A and B specific and what will make the difference to orientate such recognition remain unknown. Interestingly, the group of Gardas and colleagues (24) showed that a majority of the human anti-TPO aAbs from patients’ sera is directed against IDR/A (53 vs. 24% for IDR/B). We hypothesize that IDR/A may be primarily recognized in patients because it might be the first immunodominant epitope generated during the development of AITD. Indeed, it is well known that the epitope-spreading phenomenon, outside the IDR, does not occur in AITD patients, even with high-titer anti-TPO aAbs like during Hashimoto thyroiditis (1). But we do not know whether such a phenomenon could occur inside the IDR, and thus we suppose that before onset of AITD, the Ab repertoire is mainly directed against the region 599–617 and also more lightly against other regions, thus producing aAbs with an IDR/A-specific pattern. Later in the process, the repertoire may spread to other regions and/or amino acid residues inside or close to the IDR/A, generating a second aAb wave known as IDR/B specific but using similar regions for their epitope recognition. This two-wave hypothesis is already under investigation in our laboratory.
Taken together, our data demonstrate that human IDR/A- or B-specific anti-TPO aAbs recognize the same regions on the surface of the TPO molecule. However, IDR/A-specific aAbs use much the region 599–617 more forcefully as the anchor point for their binding to hTPO. We provide here some evidence showing that the two IDRs at the surface of hTPO are very closely related. Thus, our findings are greatly encouraging to design potent therapeutic peptides aiming at deviating both IDR/A- and B-specific aAbs from their targets during the development of AITD. Such strategy might decrease antigen presentation by TPO-specific B cells to autoaggressive T cells. Obviously, such an approach cannot be powerful enough to prevent or block alone an ongoing autoimmune process; however, combined with other more efficient immunotherapies, before or during recent onset, it might be possible to synergize and delay organ-specific destruction in patients with hypothyroiditis.
Acknowledgments
The authors thank Dr. S. L. Salhi for carefully reading the manuscript. We are indebted to Drs. J. Ruf and P. Carayon for providing us with the mouse mAbs to TPO and Drs. S. M. McLachlan and B. Rapoport for the human anti-TPO Fabs (TR1.8, TR1.9, WR1.7, and SP1.4). We also thank Drs. L. Baldet and A. M. Puech for providing the patients’ sera. We are also grateful to Dr. S. Villard for Spot synthesis and helpful discussions and C. Nguyen for peptide synthesis.
References
McLachlan SM, Rapoport B 2000 Autoimmune response to the thyroid in humans: thyroid peroxidase—the common autoantigenic denominator. Int Rev Immunol 19:587–618
Rodien P, Madec AM, Ruf J, Rajas F, Bornet H, Carayon P, Orgiazzi J 1996 Antibody-dependent cell-mediated cytotoxicity in autoimmune thyroid disease: relationship to antithyroperoxidase antibodies. J Clin Endocrinol Metab 81:2595–2600
Wadeleux P, Winand-Devigne J, Ruf J, Carayon P, Winand R 1989 Cytotoxic assay of circulating thyroid peroxidase antibodies. Autoimmunity 4:247–254
Blanchin S, Estienne V, Durand-Gorde JM, Carayon P, Ruf J 2003 Complement activation by direct C4 binding to thyroperoxidase in Hashimoto’s thyroiditis. Endocrinology 144:5422–5429
Guo J, Wang Y, Rapoport B, McLachlan SM 2000 Evidence for antigen presentation to sensitized T cells by thyroid peroxidase (TPO)-specific B cells in mice injected with fibroblasts co-expressing TPO and MHC class II. Clin Exp Immunol 119:38–46
Estienne V, Blanchet C, Niccoli-Sire P, Duthoit C, Durand-Gorde JM, Geourjon C, Baty D, Carayon P, Ruf J 1999 Molecular model, calcium sensitivity, and disease specificity of a conformational thyroperoxidase B-cell epitope. J Biol Chem 274:35313–35317
Estienne V, Duthoit C, Blanchin S, Montserret R, Durand-Gorde JM, Chartier M, Baty D, Carayon P, Ruf J 2002 Analysis of a conformational B cell epitope of human thyroid peroxidase: identification of a tyrosine residue at a strategic location for immunodominance. Int Immunol 14:359–366
Arscott PL, Koenig RJ, Kaplan MM, Glick GD, Baker JR 1996 Unique autoantibody epitopes in an immunodominant region of thyroid peroxidase. J Biol Chem 271:4966–4973
Hobby P, Gardas A, Radomski R, McGregor AM, Banga JP, Sutton BJ 2000 Identification of an immunodominant region recognized by human autoantibodies in a three-dimensional model of thyroid peroxidase. Endocrinology 141:2018–2026
Gardas A, Sohi MK, Sutton BJ, McGregor AM, Banga JP 1997 Purification and crystallisation of the autoantigen thyroid peroxidase from human Graves’ thyroid tissue. Biochem Biophys Res Commun 234:366–370
Hendry E, Taylor G, Ziemnicka K, Grennan Jones F, Furmaniak J, Rees Smith B 1999 Recombinant human thyroid peroxidase expressed in insect cells is soluble at high concentrations and forms diffracting crystals. J Endocrinol 160:R13–R15
Bresson D, Cerutti M, Devauchelle G, Pugniere M, Roquet F, Bes C, Bossard C, Chardes T, Peraldi-Roux S 2003 Localization of the discontinuous immunodominant region recognized by human anti-thyroperoxidase autoantibodies in autoimmune thyroid diseases. J Biol Chem 278:9560–9569
Ruf J, Toubert ME, Czarnocka B, Durand-Gorde JM, Ferrand M, Carayon P 1989 Relationship between immunological structure and biochemical properties of human thyroid peroxidase. Endocrinology 125:1211–1218
McLachlan SM, Rapoport B 1995 Genetic and epitopic analysis of thyroid peroxidase (TPO) autoantibodies: markers of the human thyroid autoimmune response. Clin Exp Immunol 101:200–206
Portolano S, Chazenbalk GD, Seto P, Hutchison JS, Rapoport B, McLachlan SM 1992 Recognition by recombinant autoimmune thyroid disease-derived Fab fragments of a dominant conformational epitope on human thyroid peroxidase. J Clin Invest 90:720–726
Chazenbalk GD, Costante G, Portolano S, McLachlan SM, Rapoport B 1993 The immunodominant region on human thyroid peroxidase recognized by autoantibodies does not contain the monoclonal antibody 47/c21 linear epitope. J Clin Endocrinol Metab 77:1715–1718
Gora M, Gardas A, Wiktorowicz W, Hobby P, Watson PF, Weetman AP, Sutton BJ, Banga JP 2004 Evaluation of conformational epitopes on thyroid peroxidase by antipeptide antibody binding and mutagenesis. Clin Exp Immunol 136:137–144
Libert F, Ludgate M, Dinsart C, Vassart G 1991 Thyroperoxidase, but not the thyrotropin receptor, contains sequential epitopes recognized by autoantibodies in recombinant peptides expressed in the pUEX vector. J Clin Endocrinol Metab 73:857–860
Bresson D, Pugniere M, Roquet F, Rebuffat SA, N-Guyen B, Cerutti M, Guo J, McLachlan SM, Rapoport B, Estienne V, Ruf J, Chardes T, Peraldi-Roux S 2004 Directed mutagenesis in region 713–720 of human thyroperoxidase assigns 713KFPED717 residues as being involved in the B domain of the discontinuous immunodominant region recognized by human autoantibodies. J Biol Chem 279:39058–39067
Gora M, Gardas A, Watson PF, Hobby P, Weetman AP, Sutton BJ, Banga JP 2004 Key residues contributing to dominant conformational autoantigenic epitopes on thyroid peroxidase identified by mutagenesis. Biochem Biophys Res Commun 320:795–801
Guo J, Yan XM, McLachlan SM, Rapoport B 2001 Search for the autoantibody immunodominant region on thyroid peroxidase: epitopic footprinting with a human monoclonal autoantibody locates a facet on the native antigen containing a highly conformational epitope. J Immunol 166:1327–1333
Gardas A, Watson PF, Hobby P, Smith A, Weetman AP, Sutton BJ, Banga JP 2000 Human thyroid peroxidase: mapping of autoantibodies, conformational epitopes to the enzyme surface. Redox Rep 5:237–241
Molina F, Laune D, Gougat C, Pau B, Granier C 1996 Improved performances of spot multiple peptide synthesis. Pept Res 9:151–155
Jastrzebska-Bohaterewicz E, Gardas A 2004 Proportion of antibodies to the A and B immunodominant regions of thyroid peroxidase in Graves and Hashimoto disease. Autoimmunity 37:211–216
Baker JR, Arscott P, Johnson J 1994 An analysis of the structure and antigenicity of different forms of human thyroid peroxidase. Thyroid 4:173–178(Damien Bresson, Sandra A.)