Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.11.1.8 (thyroid peroxidase)
3,116 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Generation of recombinant, enzymatically active human thyroid peroxidase and its recognition by antibodies in the sera of patients with Hashimoto's thyroiditis. 247 68

We have characterized thyroid microsomal antigen (M-Ag) prepared from Graves' and normal thyroid tissues using 100,000 x g thyroid membrane fractions in enzyme-linked immunosorbent assays with pooled polyclonal human sera containing high titers of antibody to M-Ag. A ten-fold parallel increase in dose inhibition potencies occurred with M-Ag preparations from Graves' as compared to normal thyroid tissue. The M-Ag preparations were further evaluated by SDS-polyacrylamide gel electrophoresis and proteins visualized by Western blot using high titer microsomal antibody (M-Ab) sera (n = 2) devoid of thyroglobulin antibody activity. We found discrete 100 kD relative molecular mass bands in Graves' M-Ag preparations (n = 3) under nonreducing conditions which were only poorly resolved in normal thyroid M-Ag (n = 3) using up to 100 micrograms of protein per lane. The cellular localization of M-Ag was then investigated using the avidin-biotin-peroxidase technique on frozen sections of Graves' and normal human thyroid tissue with a murine monoclonal antibody reactive with human M-Ag and thyroid peroxidase. M-Ag reactivity was similar in both Graves' and normal thyroid tissues and localized to the entire follicular cell membrane with more intense staining occurring on the inner follicular cell membrane. This was in contrast to follicular cell staining for HLA-DR antigen which was present in 6 of 10 Graves' tissues examined and absent in normal thyroid tissue. Staining for HLA-DR antigen also occurred on the follicular cell surface membrane with occasional enhancement at the thyrocyte apical cell membrane. We conclude: a) M-Ag is induced approximately 10-fold in Graves' thyroid tissue and can be objectively quantified in ELISA systems, 2) There were no detectable qualitative differences between M-Ag from Graves' and normal thyroid tissue, and 3) HLA-DR antigen was detected on 60% Graves' tissues in a cell surface distribution similar to that observed for M-Ag in both Graves' and normal tissues.
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PMID:Induction of microsomal antigen and comparison with histologic localization of HLA-DR in Graves' thyroid tissue. 249 9

The recent cloning of the thyroid peroxidase (TPO) has shown that it is identical to the thyroid microsomal antigen (TMA), a potent antigen involved in autoimmune thyroid disease (ATD), which shares significant sequence homology with myeloperoxidase. The present study shows that autoantibodies (aAb) to the TMA/TPO antigen cross-react with human leucocyte myeloperoxidase, bovine lactoperoxidase and horseradish peroxidase. Cross-reactivity to myeloperoxidase was only apparent by ELISA using reduced and alkylated antigen preparations or by immunoblotting following denaturation with SDS. Sequential absorption of sera on SDS-denatured thyroid microsomes immobilized on Sepharose-4B followed by absorption on native microsomes removed all aAb specificities to TMA/TPO and the three peroxidase preparations, giving compelling evidence on the genuine cross-reactive nature of these aAbs. Sera from different patients contain different qualitative and quantitative specificities of aAb to the TMA/TPO antigen, confirming the polyclonal nature of this autoimmune response.
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PMID:Thyroid microsomal/thyroid peroxidase autoantibodies show discrete patterns of cross-reactivity to myeloperoxidase, lactoperoxidase and horseradish peroxidase. 254 81

In vitro production of antithyroid microsomal antibody (AMA) and antithyroid peroxidase antibody (APA) by peripheral blood lymphocytes from patients with autoimmune thyroid disease (AITD) has been studied and compared, in view of the evidence for identity of the two differently measured antibodies. Peripheral non-T cells (2 x 10(5)) and autologous CD4 (helper/inducer) cells (2 x 10(5)) from patients with positive serum AMA were cultured for 7 days with pokeweed mitogen (PWM). B cells secreting AMA or APA were detected by the enzyme-linked immunosorbent assay (ELISA) spot assay. AMA or APA in the culture supernatants of these cells was also measured by ELISA. There was a significant correlation between the number of AMA- (IgG class) secreting cells and APA- (IgG class) secreting cells (r = 0.89 p less than 0.001). There was also a significant correlation between AMA- and APA-ELISA indices (r = 0.86, p less than 0.001). Furthermore, the number of AMA- or APA-secreting cells significantly correlated with AMA or APA secreted in the culture supernatants (r = 0.91, r = 0.92), respectively. These data show that peripheral blood lymphocytes from patients with AITD were able to produce antibodies against thyroid peroxidase (TPO) in vitro, as well as antibodies against thyroid microsomal antigen, after PWM stimulation. The significant correlation between in vitro AMA versus APA production, or the number of AMA- versus APA-secreting cells, accords with the evidence that TPO is identical to, or at least the major antigenic protein component of, thyroid microsomal antigen.
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PMID:Comparison of measurements of in vitro production of antithyroid microsomal antibody versus antithyroid peroxidase antibody. 285 41

The ultrastructural appearance of colloid vacuoles, considered to be a typical sign of hyperactivity in the human thyroid gland, was studied in human thyroid tissue transplanted to nude mice and in human thyroid tissue fixed directly after surgical removal in patients with thyrotoxicosis. Transplanted normal thyroid tissue and toxic diffuse goiter (TDG) tissue was fixed by vascular perfusion with glutaraldehyde 5 or 12 weeks after transplantation. Light microscopic quantification showed that daily injections for 2 weeks of a gamma globulin fraction of patient sera containing thyroid-stimulating immunoglobulins (TSI) greatly increased the number of colloid vacuoles in both types of transplants. The vacuoles were mainly located in the periphery of the follicle lumen, giving the colloid a scalloped appearance. Electron microscopy of TSI-exposed tissue revealed, in addition to colloid vacuoles, the presence of large amounts of membrane material in the follicle lumen. Only sparse amounts of intraluminal membrane material were present in controls. The colloid vacuoles were almost invariably associated with such membrane material, which lined the border between the vacuole and the surrounding colloid. The intraluminal material consisted of spherical and elongated formations, each structure limited by a triple-layered membrane and often containing a dense interior. The elongated structures were often of the same dimensions as microvilli. The apical surface of follicle cells in TSI-exposed tissue expressed numerous microvilli, of which many showed a similar dense interior as the intraluminal membrane structures. The intraluminal membranes frequently showed, like the apical plasma membrane of the follicle cells, a positive reaction for peroxidase. Organelles, such as mitochondria, lysosomes or rough endoplasmic reticulum, were not encountered among the intraluminal membrane structures. These observations indicate that the intraluminal membrane material is derived from the apical plasma membrane of the follicle cells, presumably by shedding of microvilli. A similar association between colloid vacuoles and membrane material was also found in thyroid tissue from patients with thyrotoxicosis fixed directly at operation. It is suggested that the presence of membrane material in the follicle lumen precipitates the formation of colloid vacuoles in hyperactive thyroid tissue. The possible involvement of intraluminal membrane material in the development of microsomal autoantibodies in Graves' disease, i.e. exposure and presentation of thyroid microsomal antigen (identical to thyroperoxidase) to the immune system, is discussed.
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PMID:Plasma membrane shedding and colloid vacuoles in hyperactive human thyroid tissue. 290 7

This radioimmunoassay was developed for specific and large-scale routine measurement of autoantibodies to thyroperoxidase (TPO), an enzyme recently identified as the thyroid microsomal antigen. Because of the scarcity of purified thyroperoxidase, we did not base the assay on the antigen-coated method but rather on autoantibody inhibition of the binding of labeled TPO to a solid-phase-bound monoclonal antibody to TPO. This assay design ensured highly specific measurements without interference from irrelevant thyroid antigens and autoantibodies. When we used affinity-purified autoantibodies to TPO as standards, the range of the curve extended over 10(3)-fold differences in the autoantibodies' concentrations, which allowed us to assay most sera without dilution. Within- and between-assay coefficients of variation (CVs) ranged from 6.1% to 11.5% and from 6.6% to 12.0%, respectively. The correlation between anti-TPO and antimicrosomal autoantibodies, as assessed by hemagglutination test, was highly significant (r = 0.90, P less than 0.0001). This assay is sensitive, easy to perform, and requires only trace amounts of purified TPO.
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PMID:Novel routine assay of thyroperoxidase autoantibodies. 318 Apr 14

MAb directed to the thyroid microsomal antigen have been developed. All bound to 101- and 107-kD bands in Western blot analysis using thyroid microsomal fraction as antigen. The MAb also bound to microsomal proteins immunoprecipitated by serum from patients having a high titer of anti-microsomal antibody but no antibodies to thyroglobulin or thyrotropin-stimulating hormone receptor. The pattern of binding was related to the amount of reducing agent. The 101- and 107-kD bands were increased by addition of dithiothreitol whereas, in its absence, numerous bands of higher molecular weight were present, suggesting a multimeric protein structure. Despite the inability to immunoprecipitate thyroid peroxidase (TPO) enzymatic activity, the MAb bound intensively in Western blot to denatured purified hog TPO and to denatured immunopurified human TPO. Trypsin digestion of the 101-107-kD antigen produced a doublet of 84-88 kD that was still immunoreactive with MAb. One of five polyclonal sera tested (with a microsomal antibody titer greater than 1/20,480 measured by the tanned red cell hemagglutination technique) also recognized the 84-88 kD trypsin fragments. Addition of V8 protease led to a disappearance of the 107-kD protein, but not the 101-kD protein, proving that this antigen is formed by two different polypeptides. The MAb bound strongly to thyroid epithelium, whereas binding to papillary carcinoma was absent or low and moderate for follicular and Hurthle cell carcinoma. This study indicates that the thyroid microsomal antigen and TPO are identical and are constituted of two different polypeptides. On SDS-PAGE the antigen appears as two contiguous bands which share common epitopes but are not identical, as proven by their size and difference in susceptibility to proteolytic digestion. The immunoreactivity of the molecule is highly dependent on a trypsin-sensitive site, which appears important in the recognition of the antigen by polyclonal sera and may have biological importance. The expression of microsomal antigenicity is variable among various thyroid carcinomas.
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PMID:Characterization of the thyroid microsomal antigen, and its relationship to thyroid peroxidase, using monoclonal antibodies. 328 Jun 2

Although recently the human thyroid microsomal antigen (M-Ag) has been possibly identified as the thyroid peroxidase, its nature remained unknown over almost three decades. One of the difficulties encountered in the identification of M-Ag derived from the conflicting data obtained in the attempts to solubilize active antigenic material from thyroid subcellular fractions. In particular, following detergent treatment, M-Ag could not be detected by complement fixation, while a full recovery of the antigen has been observed using a radioassay technique. In the present investigation, the antigenic activity of Triton X-100 solubilized thyroid microsomes was assessed in parallel by complement fixation and radioassay methods employing the same anti-microsomal antibody (anti-M Ab) preparation for antigen detection. In untreated microsomes antigenic activity was detected by both methods. In contrast, detergent solubilized M-Ag was detected by radioassay, but could not be detected by complement fixation. These data indicate that detergent solubilization diminishes the complement fixing capacity of M-Ag, while the solubilized antigen can still be fully detected by its binding reaction with the autoantibody, and explain the discrepant results obtained in previous studies.
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PMID:Comparison of complement fixation and radioassay techniques to detect solubilized human thyroid microsomal antigenic activity. 336 Oct 83

A hitherto unrecognized thyroid antibody, which reacts with a thyroid cell surface antigen expressed on passaged thyroid cells, was identified in serum from patients with thyroid-associated ophthalmopathy using antibody-dependent cell-mediated cytotoxicity (ADCC) tests. The antibody was detected in 14 of 23 patients with Graves' hyperthyroidism (Gh) and associated ophthalmopathy, in 3 of 4 patients with Hashimoto's thyroiditis (HT) and ophthalmopathy, but in only 1 of 16 patients with Gh without clinically evident eye disease and 4 of 37 patients with HT without eye disease. The ADCC test also was positive in 2 of 30 patients with thyroid cancer, both of whom had had Gh and ophthalmopathy in the past. There was no correlation, in patients with ophthalmopathy, between the levels of the antibody (expressed as percent specific lysis) and the titers of antithyroid microsomal antibody measured using a hemagglutination assay. Based on the results of blocking experiments using mouse monoclonal antibodies against human thyroid peroxidase, now known to be the thyroid microsomal antigen, the corresponding antigen was not thyroid peroxidase. Moreover, the new antigen was expressed on cultured and passaged thyroid cells which do not express the microsomal antigen. In patients with ophthalmopathy there was a close correlation between the degree of lysis of passaged thyroid cells and that of eye muscle cells, and ADCC activity against passaged thyroid cells was absorbed by preincubation of positive serum samples with eye muscle and thyroid cell, but not other cell, monolayers. The reaction of a newly identified cytotoxic thyroid antibody with a shared epitope on eye muscle cells thus appears to be a possible mechanism for the development of ophthalmopathy in patients with Gh and, less often, HT.
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PMID:A thyroid cytotoxic antibody that cross-reacts with an eye muscle cell surface antigen may be the cause of thyroid-associated ophthalmopathy. 341 Sep 41

The thyroid microsomal antigen (MSA) in autoimmune thyroid disease is a protein of approximately 107 kD. We screened a human thyroid cDNA library constructed in the expression vector lambda gt11 with anti-107-kD monoclonal antibodies. Of five clones obtained, the recombinant beta-galactosidase fusion protein from one clone (PM-5) was confirmed to react with the monoclonal antiserum. The complementary DNA (cDNA) insert from PM-5 (0.8 kb) was used as a probe on Northern blot analysis to estimate the size of the mRNA coding for the MSA. The 2.9-kb messenger RNA (mRNA) species observed was the same size as that coding for human thyroid peroxidase (TPO). The probe did not bind to human liver mRNA, indicating the thyroid-specific nature of the PM-5-related mRNA. The nucleotide sequence of PM-5 (842 bp) was determined and consisted of a single open reading frame. Comparison of the nucleotide sequence of PM-5 with that presently available for pig TPO indicates 84% homology. In conclusion, a cDNA clone representing part of the microsomal antigen has been isolated. Sequence homology with porcine TPO, as well as identity in the size of the mRNA species for both the microsomal antigen and TPO, indicate that the microsomal antigen is, at least in part, TPO.
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PMID:Isolation of a complementary DNA clone for thyroid microsomal antigen. Homology with the gene for thyroid peroxidase. 365 79


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