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Comparative analysis and characterization of mutated thyroid peroxidases with disturbance expressed on the cell
Kazumi Umeki1, Jun-Ichi Kawano, Ikuo Yamamoto
1Laboratory for Clinical Investigation, Miyazaki Medical College Hospital, Kiyotake, Miyazaki 889-1692, Japan.
Abstract:
Five mutated thyroid peroxidases (TPO) with varying degrees of disturbance in cell surface expression, probably owing to misfolding, were comparatively analyzed. CHO-K1 cells transfected with these mutated mRNAs expressed TPO protein in 65.6-82.1% of cells in antibody staining, and the TPOs were located in intracellular structures like the nuclear envelope and ER as well as cytoplasmically like wild-type TPO. When cell surface expression was examined, three mutated TPOs, G533C-, D574/L575del-, and G771R-TPOs, were expressed to varying degrees. In contrast, R175Q- and R665W-TPOs were thought not to be expressed on the cell surface, although a vague increment in R175Q-TPO was observed with increasing amounts of mRNA. In the kinetic study, three mutated TPOs having insufficient expression on the cell surface showed delays in decrease at 4 and 8 h after chase, although between 8 and 24 h after chase they decreased rapidly, as did the two other mutated TPOs. In immunoprecipitation by anti-TPO antibody, G533C-, D574/L575del-, and G771R-TPOs exhibited increasing interaction with calnexin. The combined evidence suggested that some of the mutated TPOs with disturbance in cell surface expression, probably owing to misfolding, exhibited the delay in kinetics of newly synthesized protein as a result of increasing interaction with calnexin and that such TPOs could be expressed to some extent on the cell surface.
Insights
Misfolded thyroid peroxidases (TPO) show altered cell surface expression. Some mutations cause delays in protein kinetics due to increased calnexin interaction, impacting TPO cell surface presence.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Thyroid peroxidase (TPO) is crucial for thyroid hormone synthesis.
- Mutations in TPO can lead to impaired protein folding and cell surface expression.
- Understanding these mutations is key to understanding thyroid dysfunction.
Purpose of the Study:
- To comparatively analyze five mutated thyroid peroxidases (TPO) with varying cell surface expression disturbances.
- To investigate the relationship between misfolding, calnexin interaction, and TPO cell surface expression.
- To elucidate the kinetic behavior of mutated TPO proteins.
Main Methods:
- Transfection of CHO-K1 cells with mutated TPO mRNAs.
- Antibody staining for TPO protein expression and localization (intracellular and cell surface).
- Kinetic studies analyzing TPO protein decrease over time post-chase.
- Immunoprecipitation assays to assess calnexin interaction.
Main Results:
- Five mutated TPOs showed varying degrees of cell surface expression, with R175Q- and R665W-TPOs exhibiting minimal to no surface presence.
- Mutated TPOs with reduced cell surface expression displayed delayed degradation kinetics.
- Three mutated TPOs (G533C-, D574/L575del-, G771R-) showed increased interaction with calnexin, suggesting misfolding.
- Despite expression issues, some mutated TPOs could still reach the cell surface to some extent.
Conclusions:
- Disturbed cell surface expression of mutated TPOs is likely due to misfolding.
- Increased calnexin interaction in misfolded TPOs leads to delayed protein kinetics.
- These findings provide insights into the molecular mechanisms underlying TPO-related thyroid disorders.
