Thyroid peroxidase forms thionamide-sensitive homodimers: relevance for immunomodulation of thyroid autoimmunity.
Thyroid peroxidase (TPO) is the key enzyme in thyroid hormone production and a universal autoantigen in Graves' and other autoimmune thyroid diseases. We wished to explore the expression of TPO and whether it was affected by thionamide antithyroid drugs. We studied recombinant TPO, stably expressed by a Chinese hamster ovary cell line (CHO-TPO) and transiently expressed TPO-enhanced green fluorescent protein (eGFP) and -FLAG fusion proteins. Immunoblotting of CHO-TPO cell extracts showed high-molecular weight (HMW) TPO isoforms that were resistant to reduction, as well as 110 kDa monomeric TPO. Co-immunoprecipitation and enzyme-linked-immunosorbent assay (ELISA) binding studies of FLAG- and eGFP-tagged TPO demonstrated TPO dimerisation. CHO-TPO cells cultured in methimazole (MMI) for 10 days showed a significant reduction in HMW-TPO isoforms at MMI concentrations of 1 microM and above (p < 0.01), whereas monomeric TPO expression was unchanged. We observed a similar reduction in HMW-TPO in CHO-TPO cells cultured in propylthiouracil (10 microM and above). Binding of Graves' disease patient sera and TPO-Fabs to enzymatically active TPO that was captured onto solid phase was not abrogated by MMI. The cellular localisation of TPO in CHO-TPO cells was unchanged by MMI treatment. Our demonstration of homodimeric TPO and the reduction in HMW-TPO isoforms during thionamide treatment of CHO-TPO cells shows, for the first time, an effect of thionamides on TPO structure. This suggests a structural correlate to the effect of thionamides on TPO enzymatic activity and opens up a novel potential mechanism for thionamide immunomodulation of autoimmune thyroid disease.
McDonald
David O
DO
Institute of Human Genetics, Newcastle University, International Centre for Life, Central Parkway, Newcastle upon Tyne, UK.
Pearce
Simon H S
SH
D000818:Animals; D013956:Antithyroid Agents / Q000378:metabolism; D015551:Autoimmunity / Q000276:immunology*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D056747:Immunomodulation / Q000276:immunology*; D007453:Iodide Peroxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008713:Methimazole / Q000378:metabolism; D008970:Molecular Weight; D011441:Propylthiouracil / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism; D013961:Thyroid Gland / Q000276:immunology*
D013956:Antithyroid Agents; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D013844:Thiazoles; D008713:Methimazole; D011441:Propylthiouracil; D007453:Iodide Peroxidase