Tomio Kotani

Regional localization of the gene for thyroid peroxidase to human chromosome 2p25 and mouse chromosome 12C

Thyroid peroxidase (TPO) plays a central role in thyroid gland function. The enzyme catalyzes two important reactions of thyroid hormone synthesis, i.e., the iodination of tyrosine residues in thyroglobulin and phenoxy-ester formation between pairs of iodinated tyrosines to generate the thyroid hormones, thyroxine and triiodothyronine. Previously, we isolated the cDNAs encoding human and mouse TPOs and assigned the human TPO gene to the short arm of chromosome 2 by somatic cell hybrid mapping. By a similar analysis of DNA from somatic cell hybrids, the human TPO gene was mapped to 2pter-p12. The mouse TPO gene was localized to chromosome 12 using a rat TPO cDNA as a probe to hybridize with mouse-hamster somatic cell hybrids. In this study, we used fluorescence in situ hybridization (FISH) to confirm the localization of human and mouse TPO genes to human chromosome 2 and mouse chromosome 12 and to assign them regionally to 2p25 and 12C, respectively. 7 refs., 1 fig.

Partial iodide organification defect caused by a novel mutation of the thyroid peroxidase gene in three siblings

background Three siblings with goitre and latent to mild hypothyroidism were suspected of having thyroid peroxidase (TPO) abnormality. Direct sequencing of their genomic DNAs showed two novel mutations of the TPO gene, one of which was G1687T (Gly533Cys; exon 9) and the other 1808–13del (Asp574/Leu575del; exon 10). The two mutations were compound heterozygous, as the former was found in their fathers DNA as heterozygous, and the latter was found in DNA from their mother, also as heterozygous. As Gly533 and Asp574/Leu575 were well‐conserved amino acids in the peroxidase superfamily, Gly533Cys‐ and Asp574/Leu575del‐TPOs were thought to be affected structurally or functionally. In expression studies using CHO‐Kl cells and mRNAs introduced with individual mutations, both mutated TPO proteins were expressed at the same molecular size as wild‐type TPO and had enzyme activity, although Gly533Cys‐TPO was slightly lower in efficiency of expression and more degenerative than wild‐type TPO.

Characterization of rat and human steroid sulfatases

Rat and human steroid sulfatases were purified from liver and placenta, respectively, by the same procedure. The rat and human enzymes were solubilized with Triton X-100, and purified by immunoaffinity chromatography with a monoclonal antibody showing high binding activities to both the enzymes. They were further purified by high-pressure anion-exchange chromatography to compare their structural and catalytic properties. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that both enzymes had a molecular weight of 62,000. The enzymes had similar amino acid compositions and amino-terminal amino acid sequences. Significant differences of the optimum pH, Michaelis constant and maximum velocity were observed between these enzymes. The optimum pH of each enzyme varied from 6.0 to 8.0, depending on substrates and with or without Triton X-100. In detergent-free media, steroid sulfates competitively inhibited the ability of these enzymes to hydrolyze 4-nitrophenyl sulfate. In media containing Triton X-100, on the other hand, the inhibition types of the steroid sulfates on the hydrolyzing activities of the rat and human enzymes were noncompetitive- and mixed-types, respectively.

Coexistent anaplastic and differentiated thyroid carcinoma : An immunohistochemical study

The aim of the present study was to clarify the underlying molecules that might contribute to the highly aggressive behavior of anaplastic thyroid carcinoma. We selected 5 cases of anaplastic thyroid carcinoma that had a differentiated area to determine differences in the molecules of undifferentiated and differentiated cancer cells. We immunohistochemically examined the localization of nuclear antigen (Ki-67), proliferating cell nuclear antigen (PCNA), p53, apoptotic protease-activating factor-1 (Apaf-1), CD26, galectin-3, E-cadherin, and CD147. We found increased Ki-67, PCNA, and p53 labeling indices; decreased levels of Apaf-1, CD26, galectin-3, and E-cadherin; and overexpression of CD147 in the undifferentiated area compared with the differentiated area. These findings indicate high proliferative properties, suppression of apoptosis, disruption of cell-cell interaction, and induction of matrix metalloproteinases in the undifferentiated areas. Thus the molecules examined might be useful for evaluating the aggressive nature of this tumor and the prognosis.

Purification of thyroid peroxidase by monoclonal antibody-assisted immunoaffinity chromatography

A rapid method was developed for purification of hog thyroid peroxidase by immunoaffinity chromatography on a column of Sepharose 4B coupled to a monoclonal antibody to the peroxidase. The purified enzyme had a specific activity of 194 units/mg and showed the same absorption spectrum in the Soret and visible regions as that of the enzyme purified after trypsin treatment. The ratio of A413 nm to A280 nm was 0.24, being much less than that for the trypsinized enzymes. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it gave a broad protein band in the 100,000-dalton region. It is concluded that the preparation purified in this study represents a native form of thyroid peroxidase.

Dipeptidyl peptidase IV (DPP IV/CD26) staining predicts distant metastasis of ‘benign’ thyroid tumor

Because follicular thyroid carcinoma is extremely difficult to diagnose, several cases were encountered which have been rediagnosed as carcinoma due to distant metastasis. In the search for a method of correctly diagnosing ‘benign’ thyroid tumor, dipeptidyl peptidase (DPP) IV immunostaining was applied to 10 cases whose diagnoses had been corrected to follicular thyroid carcinoma because of distant metastases. The positive rate of immunostaining using paraffin sections in the rediagnosed follicular thyroid carcinoma group (7/10) was much higher than that of the control group (1/29), which consisted of 15 cases of follicular thyroid adenoma and 14 cases of nodular hyperplasia. These results suggested that pre‐ or postoperative DPP IV staining is useful for predicting distant metastasis of ‘benign’ thyroid tumor.

CALNUC (nucleobindin) is localized in the Golgi apparatus in insect cells

A mouse monoclonal antibody 12B1 was raised against Golgi fractions from Sf21 insect cells and selected as Golgi-specific by immunostaining of the cells. The antigen was purified from the cells by immunoaffinity chromatography with the monoclonal antibody, and its N-terminal and internal amino acid sequences were determined. Based on the partial amino acid sequences, cDNA encoding the antigen protein was cloned and sequenced. The amino acid sequence deduced from the cDNA nucleotide sequence showed a homology to those of CALNUC family proteins, CALNUC (or nucleobindin, a calcium-binding Golgi protein with DNA-binding activity) and protein NEFA (a cell surface protein with DNA-binding, EF-hand, and acidic domains). The insect protein had two EF-hand loops at the same sites as the mammalian CALNUC family proteins, but had no leucine zipper which the mammalian homologues commonly have. An electron microscopic immunoperoxidase study demonstrated that the insect protein was localized in the cis-Golgi cisternae and cis-Golgi networks. Since this localization is identical to that of mammalian CALNUC, the insect protein was considered to be a homologue of CALNUC rather than that of NEFA. Assays involving proteinase K digestion, sodium carbonate extraction and Triton X-114 extraction revealed that the insect CALNUC-like protein was a soluble protein tightly associated with the luminal surface of Golgi membranes as reported for mammalian CALNUC. The insect protein was also shown to have calcium-binding activity as does mammalian CALNUC. These data verify that the insect protein is CALNUC. The existence of CALNUC in insect cells suggests that CALNUC is an essential calcium-binding Golgi protein in a wide range of the animal kingdom. A phylogenetic tree analysis, however, suggested that NEFA was derived from CALNUC long after the segregation of a mammalian ancestor from an insect ancestor.

Stable high level expression of human thyroid peroxidase in cultured Chinese hamster ovary cells

An expression plasmid containing both human thyroid peroxidase and mouse dihydrofolate reductase cDNAs was transfected into chinese hamster ovary cells. The stably transformed cells constitutively expressed immunoreactive thyroid peroxidase on the cell surface. These cells were further used to establish a subline producing a large amount of thyroid peroxidase by selecting clones resistant to methotrexate. The molecular weight of the expressed thyroid peroxidase was the same as purified human thyroid peroxidase. This expressed protein had peroxidase activity when determined by guaiacol oxidation. Furthermore, the expressed thyroid peroxidase was immunoreactive to sera of patients with autoimmune thyroid disease in which autoantibodies to thyroid peroxidase appeared.

Iodide organification defects resulting from cosegregation of mutated and null thyroid peroxidase alleles.

This report describes an intriguing combination of the thyroid peroxidase (TPO) alleles resulting in an iodide organification defect. Sequence analysis of the patients TPO gene showed the presence of T-deletion in exon 14 of the TPO gene (T2512del). From the sequencing pattern, this new mutation of the TPO gene was thought to be homozygous. mRNA transfection studies in which mutated mRNA was transfected to CHO-K(1) cells by electroporation showed that the cells transfected with mutated mRNA expressed smaller TPO molecules than those of cells transfected with wild-type mRNA and that they had TPO activity. However, the smaller TPO molecules could not translocate onto the cell surface. To investigate T2512del in the parents, their genomic DNAs were sequenced. Results showed that the mother had T2512del but the father did not. However, when seven polymorphic positions reported earlier were analyzed, the mother showed two kinds of nucleotides at four positions but the patient and father showed only one nucleotide at all seven positions. We suspected a deletion of the TPO gene (2p25) in one of two second chromosomes, and analyzed the patients chromosomes by FISH using TPO cDNA and N-myc genomic DNA as probes. N-myc genomic DNA exhibited two signals and TPO cDNA only one signal, although the G-band showed no morphological abnormalities. T2512-deleted and 2p25-deleted null alleles cosegregated from her parents, resulting in iodide organification defect in the patient.

cDNA-directed expression of human thyroid peroxidase

A human thyroid peroxidase cDNA, hTPO‐1 [(1987) Proc. Natl. Acad. Sci. USA 84, 5555–5559], was expressed in human Hep G2 cells using a vaccinia virus cDNA‐expression system. When examined by immunoblot analysis, the level of hTPO‐1 protein expression reached a maximum approx. 24 h after infection and remained at a similar level up to 72 h post‐infection. The expressed protein was enzymatically active as measured by guaiacol oxidation. Monoclonal antibody‐assisted immunoaffinity column chromatography was used for partial purification of vaccinia‐expressed hTPO‐1, resulting in more than 300‐fold higher specific activity and a measurable difference spectrum of the hTPO‐1 (Fe3+)‐CN complex.