Comparative analysis and characterization of mutated thyroid peroxidases with disturbance expressed on the cell surface.
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.
Umeki
Kazumi
K
Laboratory for Clinical Investigation, Miyazaki Medical College Hospital, Kiyotake, Miyazaki 889-1692, Japan.
Kawano
Jun-Ichi
J
Yamamoto
Ikuo
I
Aratake
Yatsuki
Y
Kotani
Tomio
T
D000818:Animals; D016466:CHO Cells; D037281:Calnexin / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology*; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D007453:Iodide Peroxidase / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D017354:Point Mutation / Q000235:genetics*; D017510:Protein Folding; D012333:RNA, Messenger / Q000235:genetics
D012333:RNA, Messenger; D037281:Calnexin; D007453:Iodide Peroxidase