Structure-activity analysis of microsomal antigen/thyroid peroxidase

Nakajima, Yoshiyuki; Howells, Roger David; Pegg, C. A. S.; Jones, E. Davies; Smith, Bernard Rees

doi:10.1016/0303-7207(87)90187-0

Cited by 45 publications

(11 citation statements)

References 17 publications

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“…Kimura duced by subjecting the extracted human thyroid microsomal 107-kD protein major band to gel electrophoresis under nonreducing conditions (40). Also, our finding ofa diminished 1 10-kD signal after reduction of the recombinant antigen is in accordance with other findings using the native microsomal antigen (44,45). These data taken together provide evidence that in its native state human TPO exists either as a multimer or in association with another membrane protein of similar size, and that epitope recognition by autoantibodies may be conformation dependent.…”

Section: Resultssupporting

confidence: 91%

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

Kaufman¹,

Rapoport²,

Seto³

et al. 1989

J. Clin. Invest.

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Section: Resultssupporting

confidence: 91%

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

Kaufman¹,

Rapoport²,

Seto³

et al. 1989

J. Clin. Invest.

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“…4). Evidence for a trypsin cleavage site within a disulfide loop of porcine TPO was previously reported by Nakajima et al (12).…”

Section: Discussionmentioning

confidence: 80%

Porcine Thyroid Peroxidase: Relationship between the Native Enzyme and an Active, Highly Purified Tryptic Fragment

Yokoyama

Taurog

1988

Molecular Endocrinology

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We previously described the preparation of highly purified porcine thyroid peroxidase by a procedure that involved initial solubilization of the enzyme with trypsin plus detergent. Recently, the complete amino acid sequence of porcine thyroid peroxidase (TPO) was determined by cDNA cloning, and it became of interest to compare the structure of the purified trypsin-solubilized enzyme with that of the native enzyme. For this purpose we employed antibodies to the purified enzyme and to two synthetic peptides representing defined regions of the protein. We also obtained N-terminal amino acid sequence data on TPO fragments separated by gel electrophoresis. Trypsin cleavage sites in the purified enzyme were observed after arg residues 109 and 561, and also at two undetermined sites close to the putative membrane spanning region at the carboxyl end. Major fragments of approximately 60, 32, and 29 kilodaltons were observed when the purified enzyme was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. This observation is explained by assuming that the cleavage site after arg residue 561 occurred within a disulfide loop. The Mr of the trypsin-solubilized enzyme is approximately 88,000 compared to approximately 106,000 for the native enzyme. The difference can be accounted for by the loss of approximately 90 residues from the amino terminus and of at least 80 residues from the carboxyl end. Despite the loss of these fragments totaling approximately 18 kilodaltons and cleavage of the peptide bond after arg residue 561, the purified trypsin-solubilized TPO appears to retain full enzyme activity.

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“…This restricted heterogeneity in terms of antigenic determinants recognized is similar to that observed by autoantibodies to thyroglobulin where only two or three determinants on each 320 K subunit of thyroglobulin are recognized by the autoantibodies (119). Preliminary studies suggest a similar level of restriction for thyroid microsomal antibodies (120).…”

Section: Do Graves' Sera or Sera From Patients With Other Autoimmmentioning

confidence: 93%

“…Similarly, the autoantibodies to the three proteins do not cross-react (5,19,(120)(121)(122)(123)(124)(125)(126)(127)(128)(129)(130)(131).…”

Section: Relationship Between the Tsh Receptor And Other Thyroidmentioning

confidence: 95%

“…Furthermore, there is some evidence of another thyroid-specific autoantigen which is referred to as second colloid antigen (125). Thyroid microsomal antigen has recently been shown to be thyroid peroxidase (120,(126)(127)(128). It is an integral membrane protein consisting of a single chain of amino acids with mol wt about 110,000 (120,(126)(127)(128)(129)(130).…”

Section: Relationship Between the Tsh Receptor And Other Thyroidmentioning

confidence: 99%

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Autoantibodies to the Thyrotropin Receptor

1988

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This review considers recent developments in our understanding of the properties of TRAb, particularly measurement of the antibodies and their sites of action and synthesis. Two new assay methods have allowed considerable improvements in the sensitivity, specificity, precision, and ease of measuring TRAb. In particular: 1) receptor assays based on inhibition of receptor-purified labeled TSH binding to detergent-solubilized TSH receptors and 2) bioassays based on stimulation of cAMP release from monolayer cultures of isolated thyroid cells. Detailed studies with the two assays indicate that TSH receptor antibodies nearly always act as TSH agonists in patients with a history of Graves' hyperthyroidism. Studies in areas of dietary iodine sufficiency suggest that measurement of the antibodies at various stages in the course of treating Graves' disease can be of value in predicting the outcome of therapy. However, in areas of iodine deficiency, difficulties in the ability of patients' thyroid tissue to recover from the effects of antithyroid drugs may prevent the receptor antibodies from causing a relapse of thyrotoxicosis. Consequently, the predictive value of receptor antibody measurements would be expected to be lower in these geographical areas. Although patients with a history of Graves' hyperthyroidism nearly always have TRAb which act as TSH agonists, about 20% of patients with frank hypothyroidism due to autoimmune destruction of the thyroid have TRAb which act as TSH antagonists (blocking antibodies). There is some evidence that these blocking antibodies can cause hypothyroidism particularly in the neonate. With regard to the site of synthesis of TRAb, there is now direct evidence that they are synthesized by thyroid lymphocytes, particularly the lymphocytes in close proximity to thyroid follicular cells. This is consistent with the well established effects of antithyroid treatment (drugs, radioiodine, or surgery) on TRAb levels in addition to their effects on thyroid hormone synthesis. Recent studies using affinity labeling with 125I-labeled TSH have enabled elucidation of the structure of the TSH receptor. TSH receptors in human, porcine, and guinea pig thyroid tissue have a two-chain structure in which the TSH binding site is formed on the outside surface of the cell membrane by a water-soluble A subunit (Mr approximately 50 K). The A subunit is linked by a disulfide bridge and weak noncovalent bonds to the amphiphilic B subunit (Mr approximately 30 K). This subunit, which penetrates the lipid bilayer, probably forms the site for interaction of the receptor with the regulatory subunits of adenylate cyclase.(ABSTRACT TRUNCATED AT 400 WORDS)

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Structure-activity analysis of microsomal antigen/thyroid peroxidase

Cited by 45 publications

References 17 publications

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

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

Porcine Thyroid Peroxidase: Relationship between the Native Enzyme and an Active, Highly Purified Tryptic Fragment

Autoantibodies to the Thyrotropin Receptor

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