Abstract
The multicopper oxidase ceruloplasmin plays a key role in iron homeostasis, and its ferroxidase activity is required to stabilize cell surface ferroportin, the only known mammalian iron exporter. Missense mutations causing the rare autosomal neurodegenerative disease aceruloplasminemia were investigated by testing their ability to prevent ferroportin degradation in rat glioma C6 cells silenced for endogenous ceruloplasmin. Most of the mutants did not complement (i.e. did not stabilize ferroportin) because of the irreversible loss of copper binding ability. Mutant R701W, which was found in a heterozygous very young patient with severe neurological problems, was unable to complement per se but did so in the presence of copper-glutathione or when the yeast copper ATPase Ccc2p was co-expressed, indicating that the protein was structurally able to bind copper but that metal loading involving the mammalian copper ATPase ATP7B was impaired. Notably, R701W exerted a dominant negative effect on wild type, and it induced the subcellular relocalization of ATP7B. Our results constitute the first evidence of "functional silencing" of ATP7B as a novel molecular defect in aceruloplasminemia. The possibility to reverse the deleterious effects of some aceruloplasminemia mutations may disclose new possible therapeutic strategies.
Highlights
Individuals heterozygous for the disease mutations have a partial Cp deficiency and usually display normal iron metabolism and no clinical symptoms, a few cases of affected heterozygous patients have been reported
This finding provides a straightforward explanation for brain iron overload in patients with aceruloplasminemia, where the lack of a functional Cp-GPI would lead to defective export of iron from cells because of degradation of Fpn
Because brain iron overload is a hallmark of aceruloplasminemia, we chose the rat C6 glioma cell line as our model system
Summary
Constructs—The cDNA for human Cp-GPI was cloned by RT-PCR on total RNA from U373MG human glioma cells with the cMaster RT-PCR System (Eppendorf). For Western blot analysis of secreted Cp, culture supernatants of transfected cells grown in serum-free medium were supplemented with protease inhibitors (phenylmethylsulfonyl fluoride 1 mM, leupeptin 2 g/ml, pepstatin 2 g/ml), concentrated with 30 units of Microcon (Millipore), and fractionated by SDS-PAGE under denaturing (samples heated in the presence of reducing agents) or nondenaturing (samples loaded as such) conditions. This technique was used to discriminate apo- and holo-Cp, as reported [16]. Deglycosylation with endoH (PerkinElmer Life Sciences) was performed in denaturing conditions, according to the manufacturer’s instructions
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