Skip to main content
NCBI home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1989 Aug;84(2):394–403. doi: 10.1172/JCI114179

Generation of recombinant, enzymatically active human thyroid peroxidase and its recognition by antibodies in the sera of patients with Hashimoto's thyroiditis.

K D Kaufman 1, B Rapoport 1, P Seto 1, G D Chazenbalk 1, R P Magnusson 1
PMCID: PMC548896  PMID: 2474568

Abstract

A full-length cDNA clone for human thyroid peroxidase (TPO) inserted into the mammalian cell expression vector pECE was stably transfected into Chinese hamster ovary (CHO) cells. Clones were assayed for human TPO mRNA, TPO protein, and TPO enzymatic activity. One subclone, expressing the highest TPO enzymatic activity, was used in further studies. FACS analysis of these cells preincubated in Hashimoto's serum revealed approximately 100-fold greater fluorescence compared with controls, indicating that recombinant TPO is expressed on the cell surface. Particulate antigen was extracted from these cells and studied by Western blot analysis using a panel of Hashimoto's sera of known antimicrosomal antibody (anti-MSA) titer. Under nonreducing conditions a broad, immunoreactive band of approximately 200 kD was observed, as well as a doublet of approximately 110 kD. All of the 36 Hashimoto's sera tested reacted with these bands, most in proportion to their anti-MSA titer. Six normal sera tested against this antigen(s) were nonreactive, as were the Hashimoto's sera tested against nontransfected CHO cells. Western blots under reducing conditions revealed a considerably diminished signal, with some of the sera of lower anti-MSA titer becoming negative, the loss of the 200-kD broad band, and the apparent conversion of the 110-kD doublet into a single band. Preincubation of cells in tunicamycin revealed no decrease in TPO immunoreactivity. In conclusion, we expressed enzymatically active human TPO in nonthyroidal eukaryotic cells. Our data prove that functionally active TPO is a major component of the thyroid microsomal antigen.

Full text

PDF
394

Images in this article

397Image
on p.397
399Image
on p.399
400Image
on p.400
400Image
on p.400
401Image
on p.401
401Image
on p.401

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bogner U., Schleusener H., Wall J. R. Antibody-dependent cell mediated cytotoxicity against human thyroid cells in Hashimoto's thyroiditis but not Graves' disease. J Clin Endocrinol Metab. 1984 Oct;59(4):734–738. doi: 10.1210/jcem-59-4-734. [DOI] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  3. Chen C., Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987 Aug;7(8):2745–2752. doi: 10.1128/mcb.7.8.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  5. Czarnocka B., Ruf J., Ferrand M., Carayon P., Lissitzky S. Purification of the human thyroid peroxidase and its identification as the microsomal antigen involved in autoimmune thyroid diseases. FEBS Lett. 1985 Oct 7;190(1):147–152. doi: 10.1016/0014-5793(85)80446-4. [DOI] [PubMed] [Google Scholar]
  6. Davies T. F., Weber C. M., Wallack P., Platzer M. Restricted heterogeneity and T cell dependence of human thyroid autoantibody immunoglobulin G subclasses. J Clin Endocrinol Metab. 1986 May;62(5):945–949. doi: 10.1210/jcem-62-5-945. [DOI] [PubMed] [Google Scholar]
  7. Doniach D., Bottazzo G. F., Russell R. C. Goitrous autoimmune thyroiditis (Hashimoto's disease). Clin Endocrinol Metab. 1979 Mar;8(1):63–80. doi: 10.1016/s0300-595x(79)80010-9. [DOI] [PubMed] [Google Scholar]
  8. Ellis L., Clauser E., Morgan D. O., Edery M., Roth R. A., Rutter W. J. Replacement of insulin receptor tyrosine residues 1162 and 1163 compromises insulin-stimulated kinase activity and uptake of 2-deoxyglucose. Cell. 1986 Jun 6;45(5):721–732. doi: 10.1016/0092-8674(86)90786-5. [DOI] [PubMed] [Google Scholar]
  9. Gardas A., Domek H. The effect of sulphydryl reagents on the human thyroid microsomal antigen. J Endocrinol Invest. 1988 May;11(5):385–388. doi: 10.1007/BF03349061. [DOI] [PubMed] [Google Scholar]
  10. Gordin A., Maatela J., Miettinen A., Helenius T., Lamberg B. A. Serum thyrotrophin and circulating thyroglobulin and thyroid microsomal antibodies in a Finnish population. Acta Endocrinol (Copenh) 1979 Jan;90(1):33–42. doi: 10.1530/acta.0.0900033. [DOI] [PubMed] [Google Scholar]
  11. Gubler U., Hoffman B. J. A simple and very efficient method for generating cDNA libraries. Gene. 1983 Nov;25(2-3):263–269. doi: 10.1016/0378-1119(83)90230-5. [DOI] [PubMed] [Google Scholar]
  12. Hamada N., Grimm C., Mori H., DeGroot L. J. Identification of a thyroid microsomal antigen by Western blot and immunoprecipitation. J Clin Endocrinol Metab. 1985 Jul;61(1):120–128. doi: 10.1210/jcem-61-1-120. [DOI] [PubMed] [Google Scholar]
  13. Hamada N., Portmann L., DeGroot L. J. Characterization and isolation of thyroid microsomal antigen. J Clin Invest. 1987 Mar;79(3):819–825. doi: 10.1172/JCI112889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Han J. H., Stratowa C., Rutter W. J. Isolation of full-length putative rat lysophospholipase cDNA using improved methods for mRNA isolation and cDNA cloning. Biochemistry. 1987 Mar 24;26(6):1617–1625. doi: 10.1021/bi00380a020. [DOI] [PubMed] [Google Scholar]
  15. Hinds W. E., Takai N., Rapoport B., Filetti S., Clark O. H. Thyroid-stimulating immunoglobulin bioassay using cultured human thyroid cells. J Clin Endocrinol Metab. 1981 Jun;52(6):1204–1210. doi: 10.1210/jcem-52-6-1204. [DOI] [PubMed] [Google Scholar]
  16. Hirayu H., Seto P., Magnusson R. P., Filetti S., Rapoport B. Molecular cloning and partial characterization of a new autoimmune thyroid disease-related antigen. J Clin Endocrinol Metab. 1987 Mar;64(3):578–584. doi: 10.1210/jcem-64-3-578. [DOI] [PubMed] [Google Scholar]
  17. Hohlfeld R., Toyka K. V., Miner L. L., Walgrave S. L., Conti-Tronconi B. M. Amphipathic segment of the nicotinic receptor alpha subunit contains epitopes recognized by T lymphocytes in myasthenia gravis. J Clin Invest. 1988 Mar;81(3):657–660. doi: 10.1172/JCI113369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jansson R., Thompson P. M., Clark F., McLachlan S. M. Association between thyroid microsomal antibodies of subclass IgG-1 and hypothyroidism in autoimmune postpartum thyroiditis. Clin Exp Immunol. 1986 Jan;63(1):80–86. [PMC free article] [PubMed] [Google Scholar]
  19. Katakura M., Yamada T., Aizawa T., Hiramatsu K., Yukimura Y., Ishihara M., Takasu N., Maruyama K., Kameko M., Kanai M. Presence of antideoxyribonucleic acid antibody in patients with hyperthyroidism of Graves' disease. J Clin Endocrinol Metab. 1987 Mar;64(3):405–408. doi: 10.1210/jcem-64-3-405. [DOI] [PubMed] [Google Scholar]
  20. Khoury E. L., Hammond L., Bottazzo G. F., Doniach D. Presence of the organ-specific 'microsomal' autoantigen on the surface of human thyroid cells in culture: its involvement in complement-mediated cytotoxicity. Clin Exp Immunol. 1981 Aug;45(2):316–328. [PMC free article] [PubMed] [Google Scholar]
  21. Kimura S., Kotani T., McBride O. W., Umeki K., Hirai K., Nakayama T., Ohtaki S. Human thyroid peroxidase: complete cDNA and protein sequence, chromosome mapping, and identification of two alternately spliced mRNAs. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5555–5559. doi: 10.1073/pnas.84.16.5555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kotani T., Umeki K., Matsunaga S., Kato E., Ohtaki S. Detection of autoantibodies to thyroid peroxidase in autoimmune thyroid diseases by micro-ELISA and immunoblotting. J Clin Endocrinol Metab. 1986 May;62(5):928–933. doi: 10.1210/jcem-62-5-928. [DOI] [PubMed] [Google Scholar]
  23. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  24. Libert F., Ruel J., Ludgate M., Swillens S., Alexander N., Vassart G., Dinsart C. Thyroperoxidase, an auto-antigen with a mosaic structure made of nuclear and mitochondrial gene modules. EMBO J. 1987 Dec 20;6(13):4193–4196. doi: 10.1002/j.1460-2075.1987.tb02766.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ludgate M., Mariotti S., Libert F., Dinsart C., Piccolo P., Santini F., Ruf J., Pinchera A., Vassart G. Antibodies to human thyroid peroxidase in autoimmune thyroid disease: studies with a cloned recombinant complementary deoxyribonucleic acid epitope. J Clin Endocrinol Metab. 1989 Jun;68(6):1091–1096. doi: 10.1210/jcem-68-6-1091. [DOI] [PubMed] [Google Scholar]
  26. Magnusson R. P., Chazenbalk G. D., Gestautas J., Seto P., Filetti S., DeGroot L. J., Rapoport B. Molecular cloning of the complementary deoxyribonucleic acid for human thyroid peroxidase. Mol Endocrinol. 1987 Nov;1(11):856–861. doi: 10.1210/mend-1-11-856. [DOI] [PubMed] [Google Scholar]
  27. Magnusson R. P., Gestautas J., Taurog A., Rapoport B. Molecular cloning of the structural gene for porcine thyroid peroxidase. J Biol Chem. 1987 Oct 15;262(29):13885–13888. [PubMed] [Google Scholar]
  28. Magnusson R. P., Rapoport B. Modulation of differentiated function in cultured thyroid cells: thyrotropin control of thyroid peroxidase activity. Endocrinology. 1985 Apr;116(4):1493–1500. doi: 10.1210/endo-116-4-1493. [DOI] [PubMed] [Google Scholar]
  29. Magnusson R. P., Taurog A., Dorris M. L. Mechanisms of thyroid peroxidase- and lactoperoxidase-catalyzed reactions involving iodide. J Biol Chem. 1984 Nov 25;259(22):13783–13790. [PubMed] [Google Scholar]
  30. Mariotti S., Anelli S., Ruf J., Bechi R., Czarnocka B., Lombardi A., Carayon P., Pinchera A. Comparison of serum thyroid microsomal and thyroid peroxidase autoantibodies in thyroid diseases. J Clin Endocrinol Metab. 1987 Nov;65(5):987–993. doi: 10.1210/jcem-65-5-987. [DOI] [PubMed] [Google Scholar]
  31. Nagayama Y., Yamashita S., Hirayu H., Izumi M., Uga T., Ishikawa N., Ito K., Nagataki S. Regulation of thyroid peroxidase and thyroglobulin gene expression by thyrotropin in cultured human thyroid cells. J Clin Endocrinol Metab. 1989 Jun;68(6):1155–1159. doi: 10.1210/jcem-68-6-1155. [DOI] [PubMed] [Google Scholar]
  32. Nakajima Y., Howells R. D., Pegg C., Jones E. D., Smith B. R. Structure-activity analysis of microsomal antigen/thyroid peroxidase. Mol Cell Endocrinol. 1987 Sep;53(1-2):15–23. doi: 10.1016/0303-7207(87)90187-0. [DOI] [PubMed] [Google Scholar]
  33. Nilsson M., Mölne J., Karlsson F. A., Ericson L. E. Immunoelectron microscopic studies on the cell surface location of the thyroid microsomal antigen. Mol Cell Endocrinol. 1987 Oct;53(3):177–186. doi: 10.1016/0303-7207(87)90172-9. [DOI] [PubMed] [Google Scholar]
  34. Ohtaki S., Kotani T., Nakamura Y. Characterization of human thyroid peroxidase purified by monoclonal antibody-assisted chromatography. J Clin Endocrinol Metab. 1986 Sep;63(3):570–576. doi: 10.1210/jcem-63-3-570. [DOI] [PubMed] [Google Scholar]
  35. Portmann L., Fitch F. W., Havran W., Hamada N., Franklin W. A., DeGroot L. J. Characterization of the thyroid microsomal antigen, and its relationship to thyroid peroxidase, using monoclonal antibodies. J Clin Invest. 1988 Apr;81(4):1217–1224. doi: 10.1172/JCI113438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Portmann L., Hamada N., Heinrich G., DeGroot L. J. Anti-thyroid peroxidase antibody in patients with autoimmune thyroid disease: possible identity with anti-microsomal antibody. J Clin Endocrinol Metab. 1985 Nov;61(5):1001–1003. doi: 10.1210/jcem-61-5-1001. [DOI] [PubMed] [Google Scholar]
  37. Rousset B., Bernier-Valentin F., Poncet C., Orgiazzi J., Madec A. M., Monier J. C., Mornex R. Anti-tubulin antibodies in autoimmune thyroid disorders. Clin Exp Immunol. 1983 May;52(2):325–332. [PMC free article] [PubMed] [Google Scholar]
  38. Seto P., Hirayu H., Magnusson R. P., Gestautas J., Portmann L., DeGroot L. J., Rapoport B. Isolation of a complementary DNA clone for thyroid microsomal antigen. Homology with the gene for thyroid peroxidase. J Clin Invest. 1987 Oct;80(4):1205–1208. doi: 10.1172/JCI113181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Southern P. J., Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982;1(4):327–341. [PubMed] [Google Scholar]
  40. Tzartos S. J., Kokla A., Walgrave S. L., Conti-Tronconi B. M. Localization of the main immunogenic region of human muscle acetylcholine receptor to residues 67-76 of the alpha subunit. Proc Natl Acad Sci U S A. 1988 May;85(9):2899–2903. doi: 10.1073/pnas.85.9.2899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Weetman A. P., McGregor A. M. Autoimmune thyroid disease: developments in our understanding. Endocr Rev. 1984 Spring;5(2):309–355. doi: 10.1210/edrv-5-2-309. [DOI] [PubMed] [Google Scholar]
  42. Yokoyama N., Taurog A. Porcine thyroid peroxidase: relationship between the native enzyme and an active, highly purified tryptic fragment. Mol Endocrinol. 1988 Sep;2(9):838–844. doi: 10.1210/mend-2-9-838. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES