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© Borgis - Postępy Nauk Medycznych 11/2011, s. 942-949
Marlena Godlewska, Alicja Bauer, *Barbara Czarnocka, Andrzej Gardas
Application of peptide antibodies to studies on the immunodominant conformation dependent epitopes of human thyroid peroxidase**
Zastosowanie przeciwciał peptydowych w badaniach nad konformacyjnymi epitopami immunodominującymi w ludzkiej peroksydazie tarczycowej
Department of Biochemistry and Molecular Biology, Medical Centre of Postgraduate Education, Warsaw
Head of Department: prof. dr hab. Barbara Czarnocka
Streszczenie
Lokalizacja nieciągłych regionów immunodominujących (IDR) rozpoznawanych przez autoprzeciwciała skierowane przeciwko peroksydazie tarczycowej (TPO) nie została w pełni poznana. W prezentowanej pracy zbadaliśmy lokalizację nieciągłych regionów immunodominujących (IDR) poprzez wytworzenie króliczych przeciwciał skierowanych przeciwko peptydom TPO oraz kompetycyjne eksperymenty z monoklonalnymi przeciwciałami (moabs) specyficznie wiążącymi się z regionami IDR. Sprawdziliśmy, czy potwierdzi się wcześniej przez nas zaproponowana lokalizacja regionów IDR-A i IDR-B. Określiliśmy specyficzność, reaktywność z natywną TPO, reaktywność krzyżową z homologicznymi białkami, efekt zawady sterycznej i potencjalną możliwość zaistnienia zmian w konformacji TPO indukowanych przyłączaniem się przeciwciał specyficznie wiążących peptydy. Inhibicja wiązania z TPO monoklonalnych przeciwciał specyficznych dla IDR-B i autoprzeciwciał przez pojedyncze przeciwciała anty-peptydowe lub ich mieszaniny osiągała poziom 90%. To pozwala sądzić, że przynajmniej część badanych sekwencji aminokwasowych peptydów wchodzi w skład struktury regionów IDR-A i IDR-B.
Summary
The discontinuous immunodominant regions (IDRs) recognized by autoantibodies directed to thyroid peroxidase (TPO) have not been unequivocally localized. We have explored the location of the IDRs by generation rabbit anti-TPO peptide antibodies and competition experiments with monoclonal antibodies (moabs) specific for those IDRs. Previously we suggested the localization of IDR-A and IDR-B and here we tested the validity of our conclusions. The specificity, reactivity with native TPO, cross reactivity with homologous proteins, the effect of steric hindrance, and the possibility of conformational changes induced in TPO by peptide antibody binding have been explored. The inhibition of IDR-B specific moabs and autoantibodies binding to TPO approaching 90% by peptide antibodies or their mixture call for an explanation and we think that at least part of those peptide amino acid sequences could be involved in building the IDR-A and IDR-B regions.



INTRODUCTION
Thyroid peroxidase (TPO) is responsible for the thyroid hormone biosynthesis (1). TPO is also one of the autoantigens in disorders such as Hashimoto’s thyroiditis and Graves’ disease, which are the most common human autoimmune diseases (2, 3). Autoantibodies to TPO are polyclonal and recognise discontinuous immunodominant regions (IDRs) on the molecule (4, 5). The major part of the autoantibody response to TPO is directed towards two regions, which were defined with a panel of murine monoclonal antibodies (moabs), termed IDR-A and -B (4). These IDR regions of TPO have not been unequivocally identified so far. Several techniques have been used in elucidating the location of IDR on the TPO molecule. Chimeric molecules of TPO-myeloperoxidase (6, 7) allowed exclusion of two major areas of TPO from involvement in IDRs of TPO. Studies on large recombinant fragments of TPO suggested the involvement of amino acids 590-622 and 709-721 (8). Deletion of large segment of TPO suggested the importance of sequence 386-652 (9). Studies on proteolytic fragments of TPO suggested an involvement of C-terminal amino acids (742-848) in building up the IDRs (10, 11). This finding have been questioned by others (12) as the recombinant TPO with truncated C-terminal (1-741) was precipitated by four monoclonal antibodies as well as TPO 1-771 and the full TPO ectodomain. The participation of the EGF-like domain (796-841) has been excluded as being a part of the IDR (13), while some evidences has been presented for the involvement of the CCP-like domain (739-795). Footprinting experiments suggested the participation of Lys 713, but it location at the fringe of IDR is possible (14). Other studies with recombinant TPO fragments suggested the involvement of a junction region between the MPO- and CCP-like domain of TPO (13, 15). An earlier work using recombinant TPO fragments (16) suggested the involvement of amino acid sequence 513-633 in building up the IDR, the importance of the sequence 589-633 was underlined. We have described that anti-TPO peptide antibodies to part of the sequence (599-617) described by Arscott et al. (16) strongly inhibit autoantibodies and IDR-B specific moabs binding to TPO (17). Using the model of TPO and the known localization of one of the IDR-A specific moab 47, we obtained antibodies to peptides covering the whole surface between and around the moab 47 and the sequence 599-617 (18), and found that a mixture of rabbit antibodies to this region inhibit binding of IDR-A and -B specific recombinant fab fragments and autoantibodies to TPO up to 90%. Bresson and co-workers (19) in an elegant paper described that four regions are taking part in building up the IDR for one human monoclonal antibody to TPO (moab T13), by replacement of 8 to 10 amino acids sequence. These results are in apparent conflict with our results as they do not involve the sequences described by us (17, 18). Strong inhibition of autoantibodies, moabs, and recombinant fabs binding to TPO by rabbit peptide antibodies are clear cut, however, interpretation of these results might be more complicated and we have addressed these questions in the present work.
MATERIALS AND METHODS
Synthesis of peptides and modeling of TPO structure
The molecular model of TPO, based upon the homologous structure of MPO, has been described previously (17). All the synthetic peptide sequences used in this study (tab. 1) correspond to sequences in the MPO- and CCP-like domain of TPO. The location and solvent accessibility of some of these peptides such as P6, P14, P15, P16, and P17 has been described (17). The other peptides were selected by inspection of the model to cover the TPO surface around and between the epitope for moab 47 (713-721) and our peptide P14 (599-617). All peptides were synthesized by F-moc chemistry with C-terminal amides and a cysteine residue at the N- or C-terminus for coupling to carrier protein as described earlier (17). All peptides were checked for purity by mass spectrometry.
Table 1. Rabbit anti-peptide antibodies.
Peptide numberTPO sequenceTiter with peptideTiter with TPOTiter with MPO*Titer with LPO*Inhibition by native TPO** (%)
P1225-242-C102.40051.2004001.600 (60%)8
P2403-421-C12.8002.00040080016
P4451-469-C12.80051.2000042
P5489-507-C6.4008.000000
P6503-516-C25.4008.000000
P8618-636-C12.80032.000000
P9662-680-C12.8002.00040000
P10679-696-C3.2002.000008
P11574-587-C256.000128.000016.000 (80%)48
P12549-563-C64.000256.000800063
P13567-581-C128.000128.0000045
P14599-617-C256.000256.000020095
P15610-622-C128.00032.0000020
P16189-201-C320.000320.00040040036
P17179-190-C640.00040.00020020038
P18C-210-22564.00032.0008008000
P19468-477-C256.00064.000000
P20C-721-728256.00010000046
P21249-256-C128.00064.000040023
P22536-546-C256.00032.0003.200 (60%)16.000 (88%)17
P23C-425-438128.0001.6000010
P24C-375-387320.000320.0000061
P25C-389-398640.00016.000000
P26607-617-C128.00064.000000
P27C-697-710256.000640.0001001.600 (40%)38
P28222-229-C32.00064.00002000
P31C-461-476256.000128.00020020052
P32273-286-C256.00064.0000022
P35642-650-C64.0001000068
P37C-756-76764.0008.00004000
P39C-604-612256.0001.024.0000068
P41C-742-755128.00016.0000025
P42599-607-C128.00064000015
P43702-721-C256.000256.000020073
P46603-617-C32.00016.0000045
P50353-372-C256.00016.000020027
P51321-340-C256.00064.0003.200 (70%)16.000 (81%)51
P53287-296-C128.0002.000040021
P54760-779128.000128.000020056
P55599-611-C64.00064.0000042
P56599-609-C64.00064.0000032
*Percent of homology with myeloperoxidase (MPO) or lactoperoxidase (LPO) in brackets.
**Inhibition of antibodies binding to TPO coated on polystyrene plates by the same TPO preparation in solution.
Antibodies
Mouse moabs to TPO were obtained from Dr. J. Ruf (4). Serum from patients with thyroid autoimmune disease was obtained from the Warsaw Outpatient Endocrine Clinic. Pooled serum from normal healthy individuals (n = 20) was used as a control. Pooled sera from 20 patients with thyroid autoimmune disease, positive for TPO antibodies, were used as positive control for experiments with human sera. Autoantibodies to TPO were measured by ELISA, standardized to the WHO/MRC international standard 66/387 (17). Peptides were conjugated to maleimide activated keyhole limpet hemocyanin (KLH) (1 mg peptide/1 mg KLH) and further purified by chromatography on Sephadex G-100 in PBS (17). At least two New Zealand White rabbits per peptide were injected according to the described schedule (17). All antisera were tested for reactivity to human proteins (albumin, IgG, thyroglobulin), bovine albumin, and egg albumin. All antisera were also tested for reactivity with human myeloperoxidase and lactoperoxidase.
All experiments with animals were approved by the Warsaw Ethical Committee for Experiments on Animals no. 55/01.
Purification of hTPO
Human TPO was prepared from pooled Graves’ thyroid tissue as described (20). TPO preparations used for ELISA were further purified by affinity chromatography on protein L-Sepharose. A column containing 2 ml of protein L-Sepharose (Actigen) was washed with 20ml of Tris-buffered saline (TBS) pH 8.0 containing 0.05% deoxycholate (DOC) followed by TPO solubilisation in the same buffer. The column was incubated for 1 h at room temperature, washed with TBS containing 0.05% DOC and the non-retained fraction collected and concentrated for use in the ELISA experiments. This step removes almost all IgG contamination from the TPO preparation.
ELISA and inhibition of antibodies binding to TPO
All ELISA tests were performed as described previously (17). In short, microtitre plates (Nunc) were coated with purified human TPO (or other protein at 1 μg/ml), 100 μl of diluted rabbit anti-peptide serum added and incubated for 1 h at room temperature. After washing three times with PBST, HRP-conjugated goat anti-rabbit autoantibodies were added to the wells and incubated for 1 h at room temperature. The plates were developed with TMB solution and the optical density (OD) was measured at 450 nm.

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Piśmiennictwo
1. Taurog AM: Hormone synthesis: thyroid iodine metabolism. [In:] Braverman LE, Utiger RD, editors. The Thyroid. 8th ed. Philadelphia: Lippincott Williams & Wilkins 2000; p. 61-85.
2. Banga JP: Developments in our understanding of the structure of thyroid peroxidase and the relevance of these findings to autoimmunity. Current Opinion in Endocrinology & Diabetes 1998; 5: 275-281.
3. Rapoport B, McLachlan SM: Thyroid autoimmunity. J Clin Invest 2001; 108: 1253-9
4. Ruf J, Toubert ME, Czarnocka B et al.: Relationship between immunological structures and biochemical properties of human thyroid peroxidase. Endocrinology 1989; 125: 1211-8.
5. Portolano S, Chazenbalk GD, Seto P et al.: Recognition by recombinant autoimmune thyroid disease-derived Fab fragments of a dominant conformational epitope on human thyroid peroxidase. J Clin Invest 1992; 90: 720-6.
6. Nishikawa T, Nagayama Y, Seto P et al.: Human thyroid peroxidase-myeloperoxidase chimeric molecules: tools for the study of antigen recognition by thyroid peroxidase autoantibodies. Endocrinology 1993; 133: 2496-501.
7. Nishikawa T, Rapoport B, McLachlan SM: Exclusion of two major areas on thyroid peroxidase from the immunodominant region containing the conformational epitopes recognized by human autoantibodies. J Clin Endocrinol Metab 1994; 79: 1648-54.
8. Tonacchera M, Cetani F, Costagliola S et al.: Mapping thyroid peroxidase epitopes using recombinant protein fragments. Eur J Endocrinol 1995; 132: 53-61.
9. Grennan Jones F, Ziemnicka K, Sanders J et al.: Analysis of autoantibody epitopes on human thyroid peroxidase. Autoimmunity 1999; 30: 157-69.
10. Estienne V, Duthoit C, Vinet L et al.: A conformational B-cell epitope on the C-terminal end of the extracellular part of human thyroid peroxidase. J Biol Chem 1998; 273: 8056-62.
11. Estienne V, Duthoit C, Blanchin S et al.: Analysis of a conformational B cell epitope of human thyroid peroxidase: identification of a tyrosine residue at a strategic location for immunodominance. Int Immunol 2002; 14: 359-66.
12. Xiong Z, Farilla L, Guo J et al.: Does the autoantibody immunodominant region on thyroid peroxidase include amino acid residues 742-771? Thyroid 2001; 11: 227-31.
13. Guo J, McLachlan SM, Rapoport B: Localization of the thyroid peroxidase autoantibody immunodominant region to a junctional region containing portions of the domains homologous to complement control protein and myeloperoxidase. J Biol Chem 2002; 277: 40189-95.
14. Guo J, Yan XM, McLachlan SM et al.: 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 2001; 166: 1327-33.
15. Blanchin S, Estienne V, Guo J et al.: Human thyroperoxidase folds in one complex B-cell immunodominant region. Biochem Biophys Res Commun 2002; 295: 1118-24.
16. Arscott PL, Koenig RJ, Kaplan MM et al.: Unique autoantibody epitopes in an immunodominant region of thyroid peroxidase. J Biol Chem 1996; 271: 4966-73.
17. Hobby P, Gardas A, Radomski R et al.: Identification of an immunodominant region recognized by human autoantibodies in a three-dimensional model of thyroid peroxidase. Endocrinology 2000; 141: 2018-26.
18. Gardas A, Watson PF, Hobby P et al.: Human thyroid peroxidase: mapping of autoantibodies, conformational epitopes to the enzyme surface. Redox Rep 2000; 5: 237-41.
19. Bresson D, Cerutti M, Devauchelle G et al.: Localization of the discontinuous immunodominant region recognized by human anti-thyroperoxidase autoantibodies in autoimmune thyroid diseases. J Biol Chem 2003; 278: 9560-9.
20. Gardas A, Lewartowska A, Sutton BJ et al.: Human thyroid peroxidase (TPO) isoforms, TPO-1 and TPO-2: analysis of protein expression in Graves’ thyroid tissue. J Clin Endocrinol Metab 1997; 82: 3752-7.
21. Chersi A, Di Modugno F, Rosano L: Aims and limitations in the use of antipeptide antibodies in molecular biology. Biol Chem 1997;378: 635-40.
22. Ewins DL, Barnett PS, Tomlinson RW et al.: Mapping epitope specificities of monoclonal antibodies to thyroid peroxidase using recombinant antigen preparations. Autoimmunity 1992; 11: 141-9.
23. van Regenmortel MH: The recognition of proteins and peptides by antibodies. J Immunoassay 2000; 21: 85-108.
24. Finke R, Seto P, Ruf J et a.: Determination at the molecular level of a B-cell epitope on thyroid peroxidase likely to be associated with autoimmune thyroid disease. J Clin Endocrinol Metab 1991; 73: 919-21.
25. Jemmerson R: Antigenicity and native structure of globular proteins: low frequency of peptide reactive antibodies. Proc Natl Acad Sci U S A 1987; 84: 9180-4.
26. Spangler BD: Binding to native proteins by antipeptide monoclonal antibodies. J Immunol 1991; 146: 1591-5.
27. Dyson HJ, Lerner RA, Wright PE: The physical basis for induction of protein-reactive antipeptide antibodies. Annu Rev Biophys Biophys Chem 1988; 17: 305-24.
28. Smyth MS, Trudgett A, Hoey EM et al.: Characterization of neutralizing antibodies to bovine enterovirus elicited by synthetic peptides. Arch Virol 1992; 126: 21-33.
29. Choppin J, Metzger JJ, Bouillot M et al.: Recognition of HLA class I molecules by antisera directed to synthetic peptides corresponding to different regions of the HLA-B7 heavy chain. J Immunol 1986; 136: 1738-44.
30. Leatherbarrow RJ, Stedman M, Wells TN: Structure of immunoglobulin G by scanning tunnelling microscopy. J Mol Biol 1991; 221: 361-5.
otrzymano: 2011-09-12
zaakceptowano do druku: 2011-10-17

Adres do korespondencji:
*Barbara Czarnocka
Zakład Biochemii i Biologii Molekularnej Centrum Medyczne Kształcenia Podyplomowego
ul. Marymoncka 99, 01-813 Warszawa
tel.: (22) 569-38-10
e-mail: barbarac@cmkp.edu.pl

Postępy Nauk Medycznych 11/2011
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