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• W2153157758 abstract "Autoantibodies (aAbs) to thyroid peroxidase (TPO), the hallmark of autoimmune thyroid disease (AITD), recognize conformational epitopes restricted to an immunodominant region (IDR), divided into two overlapping domains A and B. Despite numerous efforts aimed at localizing the IDR and identifying aAb-interacting residues on TPO, only two critical amino acids, Lys713 and Tyr772, have been characterized. Precise and complete delineation of the other residues involved in the IDR remains to be defined. By using a recombinant anti-TPO aAb T13, we demonstrated that four regions on TPO are part of the IDR/B; one of them, located between amino acids 713 and 720, is particularly important for the binding of sera from patients suffering from AITD. To precisely define critical residues implicated in the binding of aAb to human TPO, we used directed mutagenesis and expressed the mutants in stably transfected CHO cells. Then we assessed the kinetic parameters involved in the interactions between anti-TPO aAbs and mutants by real-time analysis. We identified (i) the minimal epitope 713-717 recognized by mAb 47 (a reference antibody) and (ii) the amino acids used as contact points for two IDR-specific human monoclonal aAbs TR1.9 (Pro715 and Asp717) and T13 (Lys713, Phe714, Pro715, and Glu716). Using a rational strategy to identify complex epitopes on proteins showing a highly convoluted architecture, this study definitively identifies the amino acids Lys713-Asp717 as being the key residues recognized by IDR/B-specific anti-TPO aAbs in AITD. Autoantibodies (aAbs) to thyroid peroxidase (TPO), the hallmark of autoimmune thyroid disease (AITD), recognize conformational epitopes restricted to an immunodominant region (IDR), divided into two overlapping domains A and B. Despite numerous efforts aimed at localizing the IDR and identifying aAb-interacting residues on TPO, only two critical amino acids, Lys713 and Tyr772, have been characterized. Precise and complete delineation of the other residues involved in the IDR remains to be defined. By using a recombinant anti-TPO aAb T13, we demonstrated that four regions on TPO are part of the IDR/B; one of them, located between amino acids 713 and 720, is particularly important for the binding of sera from patients suffering from AITD. To precisely define critical residues implicated in the binding of aAb to human TPO, we used directed mutagenesis and expressed the mutants in stably transfected CHO cells. Then we assessed the kinetic parameters involved in the interactions between anti-TPO aAbs and mutants by real-time analysis. We identified (i) the minimal epitope 713-717 recognized by mAb 47 (a reference antibody) and (ii) the amino acids used as contact points for two IDR-specific human monoclonal aAbs TR1.9 (Pro715 and Asp717) and T13 (Lys713, Phe714, Pro715, and Glu716). Using a rational strategy to identify complex epitopes on proteins showing a highly convoluted architecture, this study definitively identifies the amino acids Lys713-Asp717 as being the key residues recognized by IDR/B-specific anti-TPO aAbs in AITD. Thyroid peroxidase (TPO) 1The abbreviations used are: TPO, thyroid peroxidase; aAg, autoantigen; AITD, autoimmune thyroid diseases; MPO, myeloperoxidase; CCP, complement control protein; EGF, epidermal growth factor; aAb, autoantibody; IDR, immunodominant region; CHO, chinese hamster ovary; wt, wild type; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; RU, resonance unit; mAb, monoclonal antibody; FACS, fluorescent-activated cell sorting. is an essential membrane-bound enzyme involved in the biosynthesis of iodinated thyroid hormones. As well as thyroglobulin and the thyrotropin receptor, TPO is a major thyroid autoantigen (aAg) targeted by the immune system during autoimmune thyroid diseases (AITD). Human TPO (hTPO) is a 933 amino acid molecule and possesses a highly convoluted three-dimensional structure formed by three distinct modules, respectively, homologous with the myeloperoxidase (MPO), the complement control protein (CCP), and the epidermal growth factor (EGF) (1Hobby P. Gardas A. Radomski R. McGregor A.M. Banga J.P. Sutton B.J. Endocrinology. 2000; 141: 2018-2026Crossref PubMed Scopus (43) Google Scholar). This architectural complexity is a strong obstacle for producing high resolution crystals (2Hendry E. Taylor G. Ziemnicka K. Grennan Jones F. Furmaniak J. Rees Smith B. J. Endocrinol. 1999; 160: R13-R15Crossref PubMed Google Scholar, 3Gardas A. Sohi M.K. Sutton B.J. McGregor A.M. Banga J.P. Biochem. Biophys. Res. Commun. 1997; 234: 366-370Crossref PubMed Scopus (34) Google Scholar), explaining why the only available three-dimensional model has been generated by computer approaches (1Hobby P. Gardas A. Radomski R. McGregor A.M. Banga J.P. Sutton B.J. Endocrinology. 2000; 141: 2018-2026Crossref PubMed Scopus (43) Google Scholar). Autoantibodies (aAbs) against hTPO, strongly expressed in patients' sera, are sensitive and specific diagnostic markers for AITD and may play a role in the process leading to thyroid cellular dysfunction and destruction (4Chiovato L. Bassi P. Santini F. Mammoli C. Lapi P. Carayon P. Pinchera A. J. Clin. Endocrinol. Metab. 1993; 77: 1700-1705Crossref PubMed Scopus (89) Google Scholar, 5Parkes A.B. Othman S. Hall R. John R. Richards C.J. Lazarus J.H. J. Clin. Endocrinol. Metab. 1994; 79: 395-400Crossref PubMed Scopus (34) Google Scholar, 6Rodien P. Madec A.M. Ruf J. Rajas F. Bornet H. Carayon P. Orgiazzi J. J. Clin. Endocrinol. Metab. 1996; 81: 2595-2600Crossref PubMed Scopus (76) Google Scholar, 7Metcalfe R.A. Oh Y.S. Stroud C. Arnold K. Weetman A.P. Autoimmunity. 1997; 25: 65-72Crossref PubMed Scopus (21) Google Scholar, 8Guo J. Jaume J.C. Rapoport B. McLachlan S.M. J. Clin. Endocrinol. Metab. 1997; 82: 925-931Crossref PubMed Scopus (55) Google Scholar). More importantly, anti-TPO aAbs have been found to be involved in antigen presentation to autoaggressive T cells (9Guo J. Wang B. Rapoport B. McLachlan S.M. Clin. Exp. Immunol. 2000; 119: 38-46Crossref PubMed Scopus (30) Google Scholar) and may consequently enhance AITD as previously shown for anti-thyroglobulin aAbs (10Dai Y. Carayanniotis K.A. Eliades P. Lymberi P. Shepherd P. Kong Y. Carayanniotis G. J. Immunol. 1999; 162: 6987-6992PubMed Google Scholar). Therefore, a precise delineation of the amino acids constituting the epitopes recognized by human aAbs on TPO would be very helpful for understanding how this aAg is recognized during the onset of AITD, as well as for developing specific immune interventions to prevent or block such pathologies. A high proportion of anti-TPO aAbs from patients' sera recognizes conformational and discontinuous epitopes, restricted to an immunodominant region (IDR) composed of two overlapping domains called A and B (11Chazenbalk G.D. Costante G. Portolano S. McLachlan S.M. Rapoport B. J. Clin. Endocrinol. Metab. 1993; 77: 1715-1718PubMed Google Scholar-13Guo J. Mcintosh R.S. Czarnocka B. Weetman A.P. Rapoport B. McLachlan S.M. Clin. Exp. Immunol. 1998; 111: 408-414Crossref PubMed Scopus (43) Google Scholar). Several strategies have been used to localize these domains and, in particular, the critical amino acids involved in aAb interactions: (i) competition for TPO binding between mouse monoclonal antibodies (mAbs) or rabbit polyclonal antisera and human anti-TPO aAb (1Hobby P. Gardas A. Radomski R. McGregor A.M. Banga J.P. Sutton B.J. Endocrinology. 2000; 141: 2018-2026Crossref PubMed Scopus (43) Google Scholar, 11Chazenbalk G.D. Costante G. Portolano S. McLachlan S.M. Rapoport B. J. Clin. Endocrinol. Metab. 1993; 77: 1715-1718PubMed Google Scholar, 12Ruf J. Toubert M.E. Czarnocka B. Durand-Gorde J.M. Ferrand M. Carayon P. Endocrinology. 1989; 125: 1211-1218Crossref PubMed Scopus (155) Google Scholar, 14Gardas A. Watson P.F. Hobby P. Smith A. Weetman A.P. Sutton B.J. Banga J.P. Redox. Rep. 2000; 5: 237-241Crossref PubMed Scopus (19) Google Scholar), (ii) analysis of human anti-TPO aAb binding to recombinant and/or truncated antigen (15Blanchin S. Estienne V. Guo J. Rapoport B. McLachlan S.M. Carayon P. Ruf J. Biochem. Biophys. Res. Commun. 2002; 295: 1118-1124Crossref PubMed Scopus (11) Google Scholar, 16Arscott P.L. Koenig R.J. Kaplan M.M. Glick G.D. Baker J.R. J. Biol. Chem. 1996; 271: 4966-4973Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 17Zanelli E. Henry M. Malthiery Y. Clin. Exp. Immunol. 1992; 87: 80-86Crossref PubMed Scopus (25) Google Scholar, 18Zanelli E. Henry M. Malthiery Y. Cell. Mol. Biol. (Noisy-legrand). 1993; 39: 491-501PubMed Google Scholar, 19Grennan Jones F. Ziemnicka K. Sanders J. Wolstenholme A. Fiera R. Furmaniak J. Rees Smith B. Autoimmunity. 1999; 30: 157-169Crossref PubMed Scopus (26) Google Scholar, 20Ewins D.L. Barnett P.S. Tomlinson R.W. McGregor A.M. Banga J.P. Autoimmunity. 1992; 11: 141-149Crossref PubMed Scopus (30) Google Scholar), (iii) generation of TPO fragments by enzymatic hydrolysis followed by analysis of their binding to TPO aAb (21Estienne V. Duthoit C. Vinet L. Durand-Gorde J.M. Carayon P. Ruf J. J. Biol. Chem. 1998; 273: 8056-8062Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar), (iv) eukaryotic cell expression of TPO mutants obtained by directed mutagenesis and binding analysis to TPO aAb (22Estienne V. Duthoit C. Blanchin S. Montserret R. Durand-Gorde J.M. Chartier M. Baty D. Carayon P. Ruf J. Int. Immunol. 2002; 14: 359-366Crossref PubMed Scopus (23) Google Scholar, 23Nishikawa T. Nagayama Y. Seto P. Rapoport B. Endocrinology. 1993; 133: 2496-2501Crossref PubMed Scopus (23) Google Scholar, 24Nishikawa T. Rapoport B. McLachlan S.M. J. Clin. Endocrinol. Metab. 1994; 79: 1648-1654PubMed Google Scholar, 25Nishikawa T. Rapoport B. McLachlan S.M. Endocrinology. 1996; 137: 1000-1006Crossref PubMed Scopus (32) Google Scholar), and (v) epitopic footprinting (26Guo J. Yan X.M. McLachlan S.M. Rapoport B. J. Immunol. 2001; 166: 1327-1333Crossref PubMed Scopus (32) Google Scholar). Interestingly, different studies have pointed out the relationship between region 710 and 722 of hTPO and the B domain of the IDR, as defined with four recombinant human Fab molecules from McLachlan and Rapoport's group (nomenclature used in this article). Chronologically, in 1991, Libert et al. (27Libert F. Ludgate M. Dinsart C. Vassart G. J. Clin. Endocrinol. Metab. 1991; 73: 857-1860Crossref PubMed Scopus (75) Google Scholar) described a linear epitope, called C21, located between amino acid residues 710 and 722, that is able to interact with anti-TPO aAbs in the sera from patients suffering from AITD. During the same year, Finke et al. (28Finke R. Seto P. Rapoport B. J. Clin. Endocrinol. Metab. 1990; 71: 53-59Crossref PubMed Scopus (43) Google Scholar) characterized the mouse mAb 47 produced by Ruf et al. (12Ruf J. Toubert M.E. Czarnocka B. Durand-Gorde J.M. Ferrand M. Carayon P. Endocrinology. 1989; 125: 1211-1218Crossref PubMed Scopus (155) Google Scholar), as recognizing a linear determinant in the region 713-721. Further studies, performed by different groups, showed that mAb 47/C21 epitope (i) characterizes in part the B domain of the IDR (12Ruf J. Toubert M.E. Czarnocka B. Durand-Gorde J.M. Ferrand M. Carayon P. Endocrinology. 1989; 125: 1211-1218Crossref PubMed Scopus (155) Google Scholar, 13Guo J. Mcintosh R.S. Czarnocka B. Weetman A.P. Rapoport B. McLachlan S.M. Clin. Exp. Immunol. 1998; 111: 408-414Crossref PubMed Scopus (43) Google Scholar) and (ii) specifically competes with the IDR/B-specific recombinant human monoclonal anti-TPO aAbs TR1.9 and T13 for binding to their cognate antigen (13Guo J. Mcintosh R.S. Czarnocka B. Weetman A.P. Rapoport B. McLachlan S.M. Clin. Exp. Immunol. 1998; 111: 408-414Crossref PubMed Scopus (43) Google Scholar, 29Bresson D. Cerutti M. Devauchelle G. Pugniere M. Roquet F. Bes C. Bossard C. Chardes T. Peraldi-Roux S. J. Biol. Chem. 2003; 278: 9560-9569Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). These competitions have been explained, firstly, by the fact that Lys713, comprising part of the mAb 47 epitope, was shown to be a contact point for Fab TR1.9 on TPO (26Guo J. Yan X.M. McLachlan S.M. Rapoport B. J. Immunol. 2001; 166: 1327-1333Crossref PubMed Scopus (32) Google Scholar). Secondly, by using the combination of phage-displayed peptide technology followed by sequence alignment between mimotopes and primary sequence of hTPO, we recently localized the discontinuous immunodominant epitope of aAb T13 and found that it is partially composed of region 713-720 of hTPO (29Bresson D. Cerutti M. Devauchelle G. Pugniere M. Roquet F. Bes C. Bossard C. Chardes T. Peraldi-Roux S. J. Biol. Chem. 2003; 278: 9560-9569Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Importantly, in the same study, we revealed by directed mutagenesis that this region is one major component of the epitopes recognized by human anti-TPO aAbs from patients' sera. Despite the fact that all these data argue in favor of a critical role of region 713-720 inside the IDR, only one of the residues, Lys713, has been described as being a part of the IDR/B; thus, precise delineation of the critical residues from region 713-720 involved in IDR remains to be elucidated. By a guided mutagenesis study of this region followed by eukaryotic cell expression of each mutant, we identified: (i) the minimal epitope recognized by mAb 47 (amino acids 713-717) and (ii) the amino acids used as contact points for human aAbs TR1.9 (Pro715 and Asp717) and T13 (Lys713, Phe714, Pro715, and Glu716). By using a rational strategy to identify complex epitopes on proteins showing a highly convoluted architecture, the present study definitively assigns the amino acids from TPO region 713-717 as being the key residues recognized by IDR/B-specific anti-TPO aAbs in AITD. These data should be of great importance to rationally design therapeutic peptides able to block undesired autoimmune responses. TPO and anti-TPO Autoantibodies—The hTPO, purified (greater than 95% pure) from thyroid glands, was obtained from HyTest Ltd (Turku, Finland). Production and purification of the mouse mAb 47 (12Ruf J. Toubert M.E. Czarnocka B. Durand-Gorde J.M. Ferrand M. Carayon P. Endocrinology. 1989; 125: 1211-1218Crossref PubMed Scopus (155) Google Scholar) and the recombinant human Fab TR1.9 (29Bresson D. Cerutti M. Devauchelle G. Pugniere M. Roquet F. Bes C. Bossard C. Chardes T. Peraldi-Roux S. J. Biol. Chem. 2003; 278: 9560-9569Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) were previously described. Anti-TPO mAb 6H7 was purchased from Abcys S.A. (Paris, France). Rabbit polyclonal anti-TPO was prepared in the laboratory by injection of purified hTPO (HyTest Ltd.) in a New Zealand white rabbit, followed by two boosts. Each injection contained ∼600 μg of antigen in 1 ml of saline solution and 1 ml of Freund's incomplete adjuvant. The human recombinant aAb T13 was expressed as IgG1 or Fab by using the baculovirus/insect cell system as described (30Bresson D. Chardes T. Chapal N. Bes C. Cerutti M. Devauchelle G. Bouanani M. Mani J.C. Peraldi-Roux S. Hum. Antibodies. 2001; 10: 109-118Crossref PubMed Google Scholar). Human Fab T13 was purified on a protein G affinity column and the concentration determined by measuring the absorbance at 280 nm, E0.1% of 1.56. Human aAb T13 (0.5 μg), expressed as Fab or full IgG1, was electrophoresed under non-reducing conditions on 10% SDS-polyacrylamide gel and then transferred to a nitrocellulose membrane (Amersham Biosciences). The Fab T13 expression was checked by Western blotting, using a peroxidase-conjugated anti-human Fab- or Fc-specific Ab (Sigma, dilution 1:1,000) and the ECL detection system (Amersham Biosciences). The Fab T13 specificity was determined by ELISA. Wells were coated with 1 μg/ml of TPO in 100 mm NaHCO3, pH 9, 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 washing, aAb T13, expressed as Fab or the full IgG1 molecule, was incubated with 1% nonfat dry milk in PBS-T (incubation buffer) for 1 h 30 min at 37 °C. After washing, a peroxidase-conjugated anti-human Fab Ab (Sigma, diluted 1:1,000 in the incubation buffer) was added, and the plates were incubated for 1 h at 37 °C. After washing three times, the enzyme activity was detected with a 4 mg/ml 2-phenylenediamine solution containing 0.03% (v/v) hydrogen peroxide in 0.1 m citrate buffer, pH 5.0. After 20 min, the reaction was stopped by adding 50 μl of 2 m H2SO4 to each well, and the resulting absorbance was measured at 490 nm (A490). Kinetic Parameters of anti-TPO Autoantibody Binding Assessed by BIACORE Analysis—Surface plasmon resonance (SPR) analysis was performed at 25 °C with a 30 μl/min flow rate in HBS-EP (10 mm HEPES buffer, pH 7.4, 3 mm EDTA, 0.005% Biacore surfactant P20, 150 mm NaCl), using a BIACORE 3000 instrument (Biacore AB, Uppsala, Sweden). To measure the association and dissociation rate constants (ka and kd, respectively), and the dissociation equilibrium constant (KD = kd/ka) for the binding of mAb 47, IgG1 or Fab T13, and Fab TR1.9, human TPO was covalently immobilized at 1500 RU on the flow cell of a CM5 sensorchip by the amine coupling kit provided by the manufacturer. A second flow cell was subjected to the same chemical treatment without the protein and used as reference. Five concentrations of anti-TPO Ab were injected during 180 s over the two flow cells, followed by a dissociation step of 400 s and a regeneration step (10 μl of 40 mm HCl) between each concentration analysis. All the sensorgrams were corrected by subtracting the signal from the reference flow cell and were globally fitted to a 1:1 Langmuir binding isotherm using BIAevaluation version 3.2 software. Irrelevant mAb and Fab were used as analyte without giving any binding signal. Experiments at different flow rates showed an absence of mass transport and rebinding effects. Peptide Synthesis and Immunoassay on Cellulose Membrane-bound Peptides—The general protocol for Spot parallel peptide synthesis was described previously (31Laune D. Molina F. Ferrieres G. Mani J.C. Cohen P. Simon D. Bernardi T. Piechaczyk M. Pau B. Granier C. J. Biol. Chem. 1997; 272: 30937-30944Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). A set of 14-mer synthetic peptides corresponding to the region 709-722 of hTPO and its fourteen alanine analogs were synthesized by the Spot method. The membrane-bound peptides were probed by incubation of 2 μg/ml of mAb 47, Fabs T13, or TR1.9. Antibody binding was detected by using an alkaline phosphatase-conjugated anti-mouse Ig (Sigma, diluted 1:1,000), followed by addition of the phosphatase substrate 5-bromo-4-chloro-3-indoyl phosphate and thiazolyl blue tetrazolium (Sigma), which gives a blue precipitate on peptides having bound the antibody. The membrane was re-used after a regeneration cycle. The intensity of the spots was evaluated with the ScionImage software. Blocking ELISA Experiments—The wells were coated with 1 μg/ml TPO in 100 mm NaHCO3, pH 9.6, overnight at 4 °C. Plates were washed and blocked with saturation buffer for 1 h at 37 °C. After washing three times, mouse mAb 47 or 6H7 was incubated at two concentrations (50 and 5 μg/ml) in incubation buffer for 2 h at 37 °C. Washing was performed and then human Fab T13 or TR1.9 was incubated at a concentration giving an A490 of 1.5. After 1 h at 37 °C and washing, a horse-radish peroxidase-conjugated anti-human (Fab′)2, diluted 1:8,000 (Sigma), was used to detect the binding of human Fab on TPO by incubation 1 h at 37 °C. Washing (3×) was performed, and then the reactivity was revealed as described above. The percent inhibition was calculated by comparing the binding of human Fab with or without inhibitor. Guided Mutagenesis and Stable Expression of Wild-type and Mutated TPO Complementary DNA—The full-length wild type (wt) and mutated TPO in the region 713-720 (fully mutated TPO713-720) were previously cloned in the pcDNA5/FRT expression vector from the Flp-In™ system (29Bresson D. Cerutti M. Devauchelle G. Pugniere M. Roquet F. Bes C. Bossard C. Chardes T. Peraldi-Roux S. J. Biol. Chem. 2003; 278: 9560-9569Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The eight amino acids in the region 713-720 were replaced by leucine, and a triple mutant was constructed by replacing the residues Lys713, Pro715, and Glu716 by residues Ser713 and Arg716, derived from the myeloperoxidase sequence, and Ala715. All mutants were constructed by overlap extension PCR as described previously (32Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6833) Google Scholar). The final PCR products were cloned into the full-length TPO cDNA by using the unique restriction endonuclease sites ClaI and EcoNI. All sequences were verified by the dideoxynucleotide termination method (33Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52678) Google Scholar). Then, the Flp-In™ system was used to generate isogenic stable Chinese hamster ovary (CHO) cell lines expressing wt or mutated TPO as we previously described (29Bresson D. Cerutti M. Devauchelle G. Pugniere M. Roquet F. Bes C. Bossard C. Chardes T. Peraldi-Roux S. J. Biol. Chem. 2003; 278: 9560-9569Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Cloning and expression of TPO fully mutated in the regions 713-720 (TPO713-720) or 506-514 (TPO506-514) were previously described (29Bresson D. Cerutti M. Devauchelle G. Pugniere M. Roquet F. Bes C. Bossard C. Chardes T. Peraldi-Roux S. J. Biol. Chem. 2003; 278: 9560-9569Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Flow Cytometry Analysis of Wild-type or Mutated TPO Expressed on the Surface of Stably Transfected CHO Cells—Stably transfected CHO cells were scraped, rinsed, and pelleted (5 min, 900 rpm, 4 °C) in PBS supplemented with 2% heat-inactivated fetal calf serum (FACS buffer). The cells were resuspended and incubated with 200 μl of buffer containing 5 μg/ml of a rabbit polyclonal anti-TPO Ab or 2 μg/ml of mAb 47 for 45 min at 4 °C. The cells were washed three times and incubated in 200 μl of FACS buffer with 10 μg/ml of fluorescein-conjugated anti-rabbit (Sigma) or anti-mouse IgG (Rockland, Gilbertsville, PA) for 45 min at 4 °C in the dark. As control, the cells were incubated with second Ab alone. After washing three times with FACS buffer, the cells were analyzed (10,000 events) on an EPICS cytofluorometer (Beckman-Coulter, Fullerton, CA). Membrane Protein Extraction from CHO Cells—Stably transfected or wt CHO cells were washed three times with PBS and scraped at 4 °C. Membrane protein extraction was performed as previously described (29Bresson D. Cerutti M. Devauchelle G. Pugniere M. Roquet F. Bes C. Bossard C. Chardes T. Peraldi-Roux S. J. Biol. Chem. 2003; 278: 9560-9569Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). After centrifugation at 1,000 rpm for 5 min at 4 °C, membrane proteins were solubilized by adding 500 μl of cold lysis buffer (PBS containing 0.5% Triton X-114 and a protease inhibitor mixture tablet/10 ml of lysis buffer (Roche Applied Science), maintained at 4 °C) per 106 cells, and incubated for 30 min on ice (mixed by vortexing every 10 min). After centrifugation at 800 rpm for 8 min at 4 °C, the supernatants containing the membrane proteins were recovered and incubated for 5 min at 33 °C to allow the separation of the detergent from the aqueous phase. After centrifugation at 800 rpm for 8 min at 22 °C, the upper aqueous phase, containing soluble TPO, was removed and concentrated to ∼10 mg/ml using an Ultrafree®-4 Centrifugal Filter Unit (Millipore Corp., Bedford, MA). All protein concentrations were evaluated by the BCA protein assay reagent (Pierce). Binding of Solubilized Membrane Proteins Containing Wild-type or Mutated TPO to Human Fab Assessed by BIACORE Analysis—Human Fab T13 or TR1.9 were covalently immobilized at 9,600 RU or 3,600 RU, respectively, on flow cells 2 and 3, respectively, of a CM5 sensorchip-activated surface with EDC/NHS (amine coupling kit from Biacore AB). Flow cell 1 was used as the reference control surface. Each solubilized membrane protein extract, containing wt or mutated TPO, was diluted in HBS-EP buffer to the same final total protein concentration (1 mg/ml) and loaded onto flow cells 1, 2, and 3 in a single injection of 90 μl. After dissociation (400 s), eluent buffer (HBS-EP) at a flow rate of 30 μl/min was injected, and then the flow cell surfaces were regenerated with 10 μl of 5 mm HCl. To control the binding stability of the immobilized Fab T13 and TR1.9, each membrane protein analysis was preceded and followed by an injection of solubilized membrane protein extract containing wt TPO under the same conditions. As a negative control, a preparation of membrane proteins obtained from non-transfected CHO cells was diluted to the same final protein concentration and injected over the flow cells. Three independent experiments were performed. Production, Functionality Analysis, and Kinetic Binding Parameters of Human Fab T13—The anti-TPO aAb T13, previously produced as full IgG1 (30Bresson D. Chardes T. Chapal N. Bes C. Cerutti M. Devauchelle G. Bouanani M. Mani J.C. Peraldi-Roux S. Hum. Antibodies. 2001; 10: 109-118Crossref PubMed Google Scholar), was cloned and expressed as human Fab by using the baculovirus/insect cell system as described (34Bes C. Briant-Longuet L. Cerutti M. Heitz F. Troadec S. Pugniere M. Roquet F. Molina F. Casset F. Bresson D. Peraldi-Roux S. Devauchelle G. Devaux C. Granier C. Chardes T. J. Biol. Chem. 2003; 278: 14265-14273Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Human Fab T13 was purified by protein-G affinity chromatography (purity greater than 95%, data not shown) and then analyzed on 10% SDS-PAGE (Fig. 1A). We observed a 50-kDa band corresponding to human Fab T13, only detectable by the peroxidase-conjugated anti-human Fab Ab but not by the peroxidase-conjugated anti-human Fc-specific Ab (Fig. 1A, lane 3 versus lane 1). Human T13 expressed as full IgG1 could be detected by both secondary Abs (Fig. 1A, lane 2 versus lane 4). Approximately 0.5 mg of purified Fab T13 was obtained per liter of Sf9 insect cell culture supernatant. The functionality of Fab T13 was checked by ELISA. The results clearly show that human Fab T13 was able to bind to hTPO, in a dose-dependent manner, as was the full IgG1 T13 (Fig. 1B). Affinity Measurements of Human Fabs T13, TR1.9, and mAb 47 Binding to TPO—By using BIACORE technology (Fig. 2, A-D), we determined the binding kinetics for the interaction between Fab T13, full IgG1 T13, Fab TR1.9, or mAb 47 and hTPO immobilized on the sensorchip. The kinetic parameters of aAb T13 binding, produced as Fab (Fig. 2A) or full IgG1 (Fig. 2B), were measured after separate injections of five different concentrations of purified aAb on TPO. We found affinities (KD) for IgG1 and Fab T13 of 0.19 ± 0.01 nm and 0.80 ± 0.02 nm, respectively, using the same global fitting model, with a higher association rate constant for aAb T13 expressed as whole IgG1versus Fab and quite similar dissociation rate constants (Fig. 2E). The difference observed between the association rate constants is probably due to a stronger avidity of the full IgG1 (two paratopes) in comparison with the Fab T13 (one paratope). In a previous study (30Bresson D. Chardes T. Chapal N. Bes C. Cerutti M. Devauchelle G. Bouanani M. Mani J.C. Peraldi-Roux S. Hum. Antibodies. 2001; 10: 109-118Crossref PubMed Google Scholar), we reported an affinity value for aAb T13 immobilized by an anti-human IgG Fc antibody similar to that of Fab T13, avidity effects being avoided by this protocol. These data confirm the high affinities of human Fabs T13 and TR1.9 for TPO (KD = 0.80 ± 0.02 nm and 0.05 ± 0.01 nm, respectively) (26Guo J. Yan X.M. McLachlan S.M. Rapoport B. J. Immunol. 2001; 166: 1327-1333Crossref PubMed Scopus (32) Google Scholar). The stronger affinity for Fab TR1.9 could be explained by the remarkable association rate constant of human Fab TR1.9 (ka = 64.10 ± 0.02 × 105m-1 s-1) (Fig. 2E). In previous studies, using Scatchard analysis, the affinity of mAb 47 could not be calculated, probably because this mAb binds with a low affinity to TPO (KD less than 10-7m) (13Guo J. Mcintosh R.S. Czarnocka B. Weetman A.P. Rapoport B. McLachlan S.M. Clin. Exp. Immunol. 1998; 111: 408-414Crossref PubMed Scopus (43) Google Scholar). In our experiment (Fig. 2E), this mAb strongly interacted with TPO immobilized on the BIACORE sensor chip and showed high affinity binding with KD = 0.11 ± 0.001 nm, which is similar to that obtained with human Fabs T13 and TR1.9 using the same binding protocol. One explanation for such a difference in the mAb 47 affinity value could be that the Scatchard analysis was performed with soluble and highly convoluted TPO, whereas real-time analysis used a covalently immobilized TPO. Evidence That Human Fabs T13 and TR1.9, as Well as mAb 47, Interact with the Region 713-721 of hTPO—Mouse mAb 47 has been used as reference to describe the B domain of the IDR and often in competition with human anti-TPO aAb (13Guo J. Mcintosh R.S. Czarnocka B. Weetman A.P. Rapoport B. McLachlan S.M. Clin. Exp. Immunol. 1998; 111: 408-414Crossref PubMed Scopus (43) Google Scholar, 15Blanchin S. Estienne V. Guo J. Rapoport B. McLachlan S.M. Carayon P. Ruf J. Biochem. Biophys. Res. Commun. 2002; 295: 1118-1124Crossref PubMed Scopus (11) Google Scholar, 29Bresson D. Cerutti M. Devauchelle G. Pugniere M. Roquet F. Bes C. Bossard C. Chardes T. Peraldi-Roux S. J. Biol. Chem. 2003; 278: 9560-9569Abstract Full Text Full Text PDF PubMed Scop" @default.
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• W2153157758 title "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" @default.
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