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.

We have demonstrated in prior studies that in mammals, Cu is delivered directly to the cells of various organs by interaction with the plasma proteins that carry it. Since Cu(II) is the main form bound to these proteins, and Cu uptake transporters transfer Cu(I), cell surface reduction would appear to be required and might be rate‐limiting. To begin to explore these concepts, we measured the kinetics of reduction and expression of potential Cu(II)/Fe(III) reductases in HepG2 cells and mouse embryonic fibroblasts that do and do not express Ctr1. Cu(II) reduction rates were measured with whole cells by following formation of Cu(I)‐BCS. Vmax was 3x higher in the hepatocytes vs. fibroblasts (~1 vs. 0.36 nmol/min/mg cell protein); Kms were also higher (~8 vs. 12 uM). Non‐competitive inhibition by Fe(III) was observed. At physiological (5 uM) Cu concentrations, reduction rates exceeded those for uptake. Kinetic plots suggested single reductases were involved. Quantitative Real Time PCR (relative to 18S rRNA) showed HepG2 cells to primarily express Steap 3 (and traces of Steap 2 and dCytb). The fibroblasts expressed Steap 4 almost exclusively. Although we will have to confirm and localize the reductases, our findings suggest that at least in the cell types examined, single but different reductases mediate uptake of Cu from its plasma carrier proteins, and reduction may not be rate‐limiting for uptake.

Ceruloplasmin (Cp) is an abundant multifunctional copper‐binding protein in the blood plasma best known for its ferroxidase activity. Using purified 64Cu‐labeled Cp, we have now established that it also delivers copper directly to cultured cells. Here, we further examined whether this process occurs at the cell surface, involving a reductase, and attempted to identify a new copper transporter and overexpress human Cp. Uptake of copper from 64Cu‐Cp (purified from mouse plasma) by mouse embryonic fibroblasts that do and do not express Ctr1, was not prevented by endocytosis inhibitors; and holoCp appeared to convert to apoCp during uptake. The only known copper reductase expressed was identified as Steap2 by qPCR. Successive siRNA treatments did not reduce cell surface reductase activity, and other approaches are being applied. Identification of copper uptake transporter in the Ctr1‐/‐ fibroblasts was sought using biotinylation‐targeting with mouse Cp, and cross‐linking with formaldehyde. No clear candidates have as yet emerged. To provide a reliable source of 64Cu‐Cp for future studies, attempts were made to clone and express human Cp in Sf9 insect cells using baculovirus (which did not succeed); overexpression in human cell lines using either a conventional or lentivirus construct is now in the process. We conclude that uptake of copper from Cp occurs at the cell surface and may require a reductase that delivers Cu1+ not just to Ctr1 but also to another transporter.