Cys-881 is essential for the trafficking and secretion of truncated mutant ceruloplasmin in aceruloplasminemia.
OBJECTIVE
Aceruloplasminemia is an inherited iron overload disorder caused by a mutation in the ceruloplasmin gene and characterized by iron accumulation in both the liver and brain. The aim of this study was to elucidate the molecular pathogenesis of aceruloplasminemia by a functional analysis of mutant ceruloplasmin.


METHODS
The effects of nonsense mutations including Y694ter, W858ter and R882ter were studied by the expression in cultured cells.


RESULTS
A biogenesis study demonstrated that the Y694ter and W858ter mutants showed protein synthesis identical to that of wild type protein, however, the mutants were retained in the endoplasmic reticulum (ER), while R882ter mutant was secreted out. Site-directed mutagenesis analyses suggested that Cys-881 was necessary for the secretion of the truncated ceruloplasmin. The W858ter mutant decreased viability in the transfected cells. The expression and the promoter activity of glucose-regulated protein 78 that is an ER stress sensor protein, were up-regulated in the transfected cells.


CONCLUSIONS
The truncated mutant containing Cys-881 was able to pass through the ER and was secreted, while the truncated mutant protein without Cys-881 appeared to accumulate in the ER thus leading to ER stress and eventually resulting in cell death.
Kono
Satoshi
S
First Department of Medicine, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu 431-3192, Japan. satokono@hama-med.ac.jp
Suzuki
Hitoshi
H
Oda
Toshiaki
T
Shirakawa
Kentaro
K
Takahashi
Yoshitomo
Y
Kitagawa
Masatoshi
M
Miyajima
Hiroaki
H
D000818:Animals; D016466:CHO Cells; D016923:Cell Death; D002570:Ceruloplasmin / Q000235:genetics* / Q000378:metabolism*; D018389:Codon, Nonsense / Q000502:physiology*; D006224:Cricetinae; D003412:Cricetulus; D003545:Cysteine; D004721:Endoplasmic Reticulum; D006801:Humans; D019190:Iron Overload / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D021381:Protein Transport; D014162:Transfection
D018389:Codon, Nonsense; D002570:Ceruloplasmin; D003545:Cysteine