Human Thyroperoxidase in Its Alternatively Spliced Form (TPO2) Is Enzymatically Inactive and Exhibits Changes in Intracellular Processing and Trafficking

Abstract

Thyroid peroxidase (TPO1) is a membrane-bound heme-containing glycoprotein that catalyzes the synthesis of thyroid hormones. We generated stable cell lines expressing TPO1 and the alternatively spliced isoform TPO2. Pulse-chase studies showed that TPO2 half-life was dramatically decreased as compared with TPO1. The sensitivity of TPO2 to endo-␤-N-acetylglucosaminidase H indicated that the protein is processed through the endoplasmic reticulum and bears high mannosetype structures. Cell surface biotinylation experiments showed that the two isoforms also differ in their intracellular trafficking. TPO2 was totally retained in the cell, whereas 15% of TPO1 reached the cell surface. The inability of TPO2 to come out of the intracellular compartments was related to structural changes in the molecule. Evidence of these changes was obtained through the lack of recognition of TPO2 by half of the 13 TPO monoclonal antibodies tested in immunoprecipitation experiments. Our data suggest that because of an improper folding, TPO2 is trapped in the endoplasmic reticulum and rapidly degraded. The failure of incorporation of [ 14 C]aminolevulinic acid in the cultured cells showed that TPO2 did not bind to heme, whereas TPO1 did. This result was confirmed through a guaiacol assay showing that TPO2 is enzymatically inactive.

Expression of tpo mRNA in thyroid tumors: quantitiative PCR analysis and correlation with alterations of ret, Braf , ras and pax8 genes

“…In addition, TPO2 and TPO3 have a shorter half-life than TPO 1 (3 and 7 h vs 11 h respectively), and remain almost exclusively in the intracellular compartment instead of reaching the cell surface (Niccoli et al 1997, Niccoli-Sire et al 2001. Immunological profiles cannot explain the differences in reactivity between MoAb47 and other monoclonal antibodies since MoAb47 recognized both TPO2 and TPO3 under experimental conditions (Niccoli et al 1997, Niccoli-Sire et al 2001. Rapid turnover of TPO2 and TPO3 might provide a more plausible explanation for the weak reaction obtained with MoAb47 on malignant follicular tumors.…”

“…Since it has been shown that TPO2 is folded incorrectly, unable to bind to heme and enzymatically inactive (Niccoli et al 1997), predominance of TPO2 over TPO1 might reduce iodine concentration activity in tumor tissue. In addition, TPO2 and TPO3 have a shorter half-life than TPO 1 (3 and 7 h vs 11 h respectively), and remain almost exclusively in the intracellular compartment instead of reaching the cell surface (Niccoli et al 1997, Niccoli-Sire et al 2001.…”

“…Since it has been shown that TPO2 is folded incorrectly, unable to bind to heme and enzymatically inactive (Niccoli et al 1997), predominance of TPO2 over TPO1 might reduce iodine concentration activity in tumor tissue. In addition, TPO2 and TPO3 have a shorter half-life than TPO 1 (3 and 7 h vs 11 h respectively), and remain almost exclusively in the intracellular compartment instead of reaching the cell surface (Niccoli et al 1997, Niccoli-Sire et al 2001. Immunological profiles cannot explain the differences in reactivity between MoAb47 and other monoclonal antibodies since MoAb47 recognized both TPO2 and TPO3 under experimental conditions (Niccoli et al 1997, Niccoli-Sire et al 2001.…”

Two novel missense mutations in the thyroid peroxidase gene, R665W and G771R, result in a localization defect and cause congenital hypothyroidism

“…Niccoli and co-workers have reported that TPO-2 produced by alternative splicing is rapidly degraded in the ER owing to its improper folding and that TPO-2 is not transported onto the plasma membrane despite an intact transmembrane domain (29). In the case of thymic epithelial cells of TAP (transporter associated with antigen presentation)-deficient mice, the smooth ER compartment represents the site where misfolded proteins are eventually degraded (30).…”

Molecular Model, Calcium Sensitivity, and Disease Specificity of a Conformational Thyroperoxidase B-cell Epitope

“…Construction of the ⌬TPO-pcDNA3-The full-length human TPO cDNA (19) was cloned into HindIII and XbaI sites of the transfer vector pcDNA3 (20). A 710 nucleotides cDNA fragment corresponding to a peptide sequence around the 192 C-terminal aa of TPO was amplified by polymerase chain reaction from the TPO-pcDNA3 construction.…”