Abstract
Ceruloplasmin is a multicopper oxidase required for correct iron homeostasis.Previously, we have identified a ceruloplasmin mutant associated with the iron overload disease aceruloplasminemia, which was unable to acquire copper from the mammalian pump ATP7B but could be produced in an enzymatically active form in yeast. Here, we report the expression of recombinant ceruloplasmin in the yeast Pichia pastoris and the study of the role of five surface-exposed loops in copper incorporation by comparing the efficiencies of mammalian ATP7B and yeast Ccc2p. The possibility to "mix and match" mammalian and yeast multicopper oxidases and copper ATPases can provide clues on the molecular features underlying the process of copper loading in multicopper oxidases.
Highlights
Five C-terminal amino acids of the secreted protein by 30 alternative residues that lead to the addition of the GPI anchor [4]
The ferroxidase activity of Cp is required for proper iron homeostasis; lack of oxidase-active Cp leads to internalization and degradation of ferroportin (Fpn), the only known mammalian iron exporter [5]
Missense Cp mutants associated with aceruloplasminemia are beginning to be characterized and can be broadly classified in different groups according to their ability to stabilize Fpn on the plasma membrane of cells silenced for endogenous Cp-GPI
Summary
Ceruloplasmin; BPS, bathophenanthroline disulfonic acid; Fpn, ferroportin; GFP, green fluorescent protein; GPI, glycosylphosphatidylinositol; MD, minimal dextrose. We have previously reported that Cp R701W is unable to be loaded with copper by ATP7B, but it can acquire the prosthetic metal from the yeast copper ATPase Ccc2p This mutant is dominant over wild type Cp and induces fragmentation of the Golgi complex with re-localization of ATP7B [8]. All three mutants (R701W, K340W, and R883W) are enzymatically active when produced in yeast [8] These findings suggest that Cp loops could play a critical role in copper incorporation and that the process of copper loading in yeast versus mammalian cells is less structurally demanding. The possibility of “mixing and matching” yeast and mammalian ferroxidases and copper ATPases can provide clues on the molecular mechanism of copper incorporation into complex proteins such as the multicopper oxidases
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