Abstract
Ceruloplasmin, the main copper binding protein in blood plasma, has been of particular interest for its role in efflux of iron from cells, but has additional functions. Here we tested the hypothesis that it releases its copper for cell uptake by interacting with a cell surface reductase and transporters, producing apoceruloplasmin. Uptake and transepithelial transport of copper from ceruloplasmin was demonstrated with mammary epithelial cell monolayers (PMC42) with tight junctions grown in bicameral chambers, and purified human 64Cu-labeled ceruloplasmin secreted by HepG2 cells. Monolayers took up virtually all the 64Cu over 16h and secreted half into the apical (milk) fluid. This was partly inhibited by Ag(I). The 64Cu in ceruloplasmin purified from plasma of 64Cu-injected mice accumulated linearly in mouse embryonic fibroblasts (MEFs) over 3-6h. Rates were somewhat higher in Ctr1+/+ versus Ctr1-/- cells, and 3-fold lower at 2°C. The ceruloplasmin-derived 64Cu could not be removed by extensive washing or trypsin treatment, and most was recovered in the cytosol. Actual cell copper (determined by furnace atomic absorption) increased markedly upon 24h exposure to holoceruloplasmin. This was accompanied by a conversion of holo to apoceruloplasmin in the culture medium and did not occur during incubation in the absence of cells. Four different endocytosis inhibitors failed to prevent 64Cu uptake from ceruloplasmin. High concentrations of non-radioactive Cu(II)- or Fe(III)-NTA (substrates for cell surface reductases), or Cu(I)-NTA (to compete for transporter uptake) almost eliminated uptake of 64Cu from ceruloplasmin. MEFs had cell surface reductase activity and expressed Steap 2 (but not Steaps 3 and 4 or dCytB). However, six-day siRNA treatment was insufficient to reduce activity or uptake. We conclude that ceruloplasmin is a circulating copper transport protein that may interact with Steap2 on the cell surface, forming apoceruloplasmin, and Cu(I) that enters cells through CTR1 and an unknown copper uptake transporter.
Highlights
Copper is a trace element required for a wide variety of enzymatic reactions critical to most living cells and for the functions of an ever-growing number of other proteins, especially in mammals, whose function is less understood [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15]
As described in the Introduction, previous studies from our laboratory had provided evidence that ceruloplasmin (Cp), the main copper-binding protein in the blood plasma, is a circulating source of copper for many tissues in the mammalian organism, and for heart, placenta, and the fetus (1,53,54), the main evidence being that radioactive copper in Cp accumulated in most organs over time
This could have occurred through direct interaction of ceruloplasmin with cells in the various organs leading to docking with copper uptake transporters and/or endocytosis, and/or effects of other factors on the cell surface or in extracellular fluid, mediating release of Cu from Cp or otherwise mediating its cell uptake
Summary
Copper is a trace element required for a wide variety of enzymatic reactions critical to most living cells and for the functions of an ever-growing number of other proteins, especially in mammals, whose function is less understood [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15]. Ceruloplasmin (Cp) the main Cucontaining blood plasma protein has other functions These include the ability to oxidize Fe(II) (ferroxidase activity)–implicated in the mediation of iron efflux from certain cells [18], and the oxidative inactivation of NO [12] and some biogenic amines (like catecholamines and serotonin) [1,13,19,20,21,22]. Lack of Cp expression (as in genetic aceruloplasminemia) or activity (as in severe copper deficiency) does result in iron overload in certain tissues (like brain, liver, and pancreas) [23] This accumulation takes a long time to develop (by age 45–55 in humans), and we would not expect that to be the case if Cp ferroxidation were essential for iron efflux. Other circulating ferroxidases detected in the blood plasma are taking up the slack and substituting for Cp (in the absence of active Cp) [30,31], but that would detract from viewing Cp as an essential ferroxidase
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