Glycosylphosphatidylinositol-linked ceruloplasmin is expressed in multiple rodent organs and is lower following dietary copper deficiency

Abstract

Ceruloplasmin (Cp), a multicopper ferroxidase, is expressed as both a secreted (sCp) plasma enzyme from the liver and a membrane-bound glycosylphosphatidylinositol-anchored (GPI-Cp) splice variant protein. Cp is thought to be essential for iron mobilization as selective iron overload occurs in aceruloplasminemia in humans and in Cp null mice. Dietary copper-deficient (CuD) rodents have near total loss of Cp activity, severe loss of Cp protein and develop anemia. Hepatic iron augmentation is often observed, suggesting that loss of Cp function may be correlated with anemia. The impact of CuD treatment on GPI-Cp has not previously been evaluated. Our hypothesis was that CuD rodents would have lower levels of GPI-Cp and this would correlate with higher tissue iron retention. In these studies, GPI-Cp was detected in purified membranes of multiple organs of rats and mice but not Cp -/- mice. Immunoreactive Cp protein was released with phosphatidylinositol phospholipase C treatment and expressed ferroxidase activity. Following perinatal and postnatal copper restriction, GPI-Cp was markedly lower in the spleen and modestly lower in the liver of CuD rats and mice, when compared with copper-adequate (CuA) rodents. However, spleen non-heme iron (NHI) was lower in CuD than CuA rats, and not different in CuD mice. Hepatic iron was higher only in CuD mice. Spleen and liver membranes of CuD rats expressed augmented levels of ferroportin, the iron efflux transporter, which may explain lower NHI content in the spleen of CuD rats despite a greater than 50% lower level of the multicopper ferroxidase GPI-Cp. Spleen and liver levels of GPI-Cp mRNA were not impacted in CuD rats, suggesting that turnover rather than biosynthesis may explain the lower steady-state levels of GPI-Cp following dietary copper restriction. Lower GPI-Cp did not correlate with tissue iron retention and thus the role, if any, of Cp in anemia of copper deficiency is unknown.

Copper deficiency has minimal impact on ferroportin expression or function

“…1b, d). Analysis of spleen NHI in several models confirmed lower levels in CuD rats (Mostad and Prohaska 2011). However, in the same study CuD mice consuming the newer diet with extra iron exhibited spleen NHI concentration equivalent to CuA controls.…”

“…Thus, it would follow that spleen of CuD rats and mice with very low GPICp protein and ferroxidase activity should have lower Fpn; but, in fact CuD spleen Fpn protein levels are either not impacted or augmented (Jenkitkasemwong et al 2010;Mostad and Prohaska 2011). Spleen Fpn mRNA content was not altered in postweanling CuD rats (Fig.…”

“…Spleen GPI-Cp is much greater in abundance than cerebellum GPI-Cp. Furthermore abundance of spleen GPI-Cp was greatly reduced by dietary Cu deficiency in both CuD rats and mice (Mostad and Prohaska 2011).…”

“…Several copper-iron interactions can impact iron efflux but especially the MCO Cp working with Fpn (Collins et al 2010). Recently, we reported that the GPI-Cp splice variant of Cp was widely expressed in rat and mouse tissues (Mostad and Prohaska 2011). Previously, emphasis on this membrane associated MCO was brain astrocytes; the organ and cell type where it was first identified (Patel and David 1997).…”

“…Cu-deficient means were significantly different than Cu-adequate, *P \ 0.01 (Student's t-test) Liver response to copper deficiency Conditional deletions of mouse Fpn that allowed survival but loss of Fpn function resulted in iron retention in both Kupffer cells and heptatocytes, illustrating the key role of Fpn in hepatic iron homeostasis (Donovan et al 2005). CuD mice exhibit greater than twofold hepatic iron augmentation even with a new AIN-93G modified diet that eliminates hepatic iron overload in rats (Bastian et al 2011;Mostad and Prohaska 2011). Mouse liver Fpn level was not reported in those studies.…”