Dataset Information

A Novel Missense Mutation in the Thyroid Peroxidase Gene, R175Q, Resulting in Insufficient Cell Surface Enzyme in Two Siblings.

ABSTRACT: Thyroid peroxidase (TPO) abnormality is one of the causes of congenital hypothyroidism. Two missense mutations were found as a compound heterozygous mutation in two siblings with congenital goitrous hypothyroidism. One of these mutations, G614A (R175Q), was a novel mutation. Characterization of the novel mutation and a cotransfection experiment with two mutated TPO mRNAs were carried out. G614A-mRNA introduced into CHO-K1 cells expressed TPO protein with the same molecular weight as that of wild-type mRNA. The R175Q-TPO was thought to possess enzyme activity. In terms of localization, a very small amount of mutated TPO was expressed on the plasma membrane of CHO-K1 cells. This plasma membrane expression of R175Q-TPO was insufficient to perform thyroid hormone synthesis, but was markedly different from R665W-TPO. When G614A- and C2083T-mRNAs were cotransfected, cell surface TPO-positive cells were only 13.1% in contrast to 54.4% for wild-type mRNA. The low positivity and intensity of cell surface TPO suggested that in the patients' thyroids thyroid hormone synthesis was hardly performed. The congenital hypothyroidism of the patients was thought to be a result of the mutations of the TPO gene (G614A/C2083T).

SUBMITTER: Kotani T

PROVIDER: S-EPMC4004912 | biostudies-literature | 2004

REPOSITORIES: biostudies-literature

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A Novel Missense Mutation in the Thyroid Peroxidase Gene, R175Q, Resulting in Insufficient Cell Surface Enzyme in Two Siblings.

Kotani Tomio T Umeki Kazumi K Kawano Jun-Ichi J Suganuma Tatsuo T Yamamoto Ikuo I Aratake Yatsuki Y Ichiba Yozo Y Furujo Mahoko M

Clinical pediatric endocrinology : case reports and clinical investigations : official journal of the Japanese Society for Pediatric Endocrinology 20040707 1

Thyroid peroxidase (TPO) abnormality is one of the causes of congenital hypothyroidism. Two missense mutations were found as a compound heterozygous mutation in two siblings with congenital goitrous hypothyroidism. One of these mutations, G614A (R175Q), was a novel mutation. Characterization of the novel mutation and a cotransfection experiment with two mutated TPO mRNAs were carried out. G614A-mRNA introduced into CHO-K1 cells expressed TPO protein with the same molecular weight as that of wild ...[more]

PMID: 24790296

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Project description:Thyroid peroxidase (TPO) catalyses the biosynthesis of thyroid hormones and is a major autoantigen in Hashimoto's disease--the most common organ-specific autoimmune disease. Epitope mapping studies have shown that the autoimmune response to TPO is directed mainly at two surface regions on the molecule: immunodominant regions A and B (IDR-A, and IDR-B). TPO has been a major target for structural studies for over 20 years; however, to date, the structure of TPO remains to be determined. We have used a molecular modelling approach to investigate plausible modes of TPO structure and dimer organisation. Sequence features of the C-terminus are consistent with a coiled-coil dimerization motif that most likely anchors the TPO dimer in the apical membrane of thyroid follicular cells. Two contrasting models of TPO were produced, differing in the orientation and exposure of their active sites relative to the membrane. Both models are equally plausible based upon the known enzymatic function of TPO. The "trans" model places IDR-B on the membrane-facing side of the myeloperoxidase (MPO)-like domain, potentially hindering access of autoantibodies, necessitating considerable conformational change, and perhaps even dissociation of the dimer into monomers. IDR-A spans MPO- and CCP-like domains and is relatively fragmented compared to IDR-B, therefore most likely requiring domain rearrangements in order to coalesce into one compact epitope. Less epitope fragmentation and higher solvent accessibility of the "cis" model favours it slightly over the "trans" model. Here, IDR-B clusters towards the surface of the MPO-like domain facing the thyroid follicular lumen preventing steric hindrance of autoantibodies. However, conformational rearrangements may still be necessary to allow full engagement with autoantibodies, with IDR-B on both models being close to the dimer interface. Taken together, the modelling highlights the need to consider the oligomeric state of TPO, its conformational properties, and its proximity to the membrane, when interpreting epitope-mapping data.

Project description:BACKGROUND: Angiotensin-converting enzyme (ACE; Kininase II; CD143) hydrolyzes small peptides such as angiotensin I, bradykinin, substance P, LH-RH and several others and thus plays a key role in blood pressure regulation and vascular remodeling. Complete absence of ACE in humans leads to renal tubular dysgenesis (RTD), a severe disorder of renal tubule development characterized by persistent fetal anuria and perinatal death. METHODOLOGY/PRINCIPAL FINDINGS: Patient with RTD in Lisbon, Portugal, maintained by peritoneal dialysis since birth, was found to have a homozygous substitution of Arg for Glu at position 1069 in the C-terminal domain of ACE (Q1069R) resulting in absence of plasma ACE activity; both parents and a brother who are heterozygous carriers of this mutation had exactly half-normal plasma ACE activity compared to healthy individuals. We hypothesized that the Q1069R substitution impaired ACE trafficking to the cell surface and led to accumulation of catalytically inactive ACE in the cell cytoplasm. CHO cells expressing wild-type (WT) vs. Q1069R-ACE demonstrated the mutant accumulates intracellularly and also that it is significantly degraded by intracellular proteases. Q1069R-ACE retained catalytic and immunological characteristics of WT-ACE N domain whereas it had 10-20% of the nativity of the WT-ACE C domain. A combination of chemical (sodium butyrate) or pharmacological (ACE inhibitor) chaperones with proteasome inhibitors (MG 132 or bortezomib) significantly restored trafficking of Q1069R-ACE to the cell surface and increased ACE activity in the cell culture media 4-fold. CONCLUSIONS/SIGNIFICANCE: Homozygous Q1069R substitution results in an ACE trafficking and processing defect which can be rescued, at least in cell culture, by a combination of chaperones and proteasome inhibitors. Further studies are required to determine whether similar treatment of individuals with this ACE mutation would provide therapeutic benefits such as concentration of primary urine.

Dataset Information

A Novel Missense Mutation in the Thyroid Peroxidase Gene, R175Q, Resulting in Insufficient Cell Surface Enzyme in Two Siblings.

Publications

A Novel Missense Mutation in the Thyroid Peroxidase Gene, R175Q, Resulting in Insufficient Cell Surface Enzyme in Two Siblings.

Similar Datasets

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