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.
Ionic copper entering blood plasma binds tightly to albumin and the macroglobulin transcuprein. It then goes primarily to the liver and kidney except in lactation, where a large portion goes directly to the mammary gland. Little is known about how this copper is taken up from these plasma proteins. To examine this, the kinetics of uptake from purified human albumin and alpha(2)-macroglobulin, and the effects of inhibitors, were measured using human hepatic (HepG2) and mammary epithelial (PMC42) cell lines. At physiological concentrations (3-6 muM), both cell types took up copper from these proteins independently and at rates similar to each other and to those for Cu-dihistidine or Cu-nitrilotriacetate (NTA). Uptakes from alpha(2)-macroglobulin indicated a single saturable system in each cell type, but with different kinetics, and 65-80% inhibition by Ag(I) in HepG2 cells but not PMC42 cells. Uptake kinetics for Cu-albumin were more complex and also differed with cell type (as was the case for Cu-histidine and NTA), and there was little or no inhibition by Ag(I). High Fe(II) concentrations (100-500 microM) inhibited copper uptake from albumin by 20-30% in both cell types and that from alpha(2)-macroglobulin by 0-30%, and there was no inhibition of the latter by Mn(II) or Zn(II). We conclude that the proteins mainly responsible for the plasma-exchangeable copper pool deliver the metal to mammalian cells efficiently and by several different mechanisms. alpha(2)-Macroglobulin delivers it primarily to copper transporter 1 in hepatic cells but not mammary epithelial cells, and additional as-yet-unidentified copper transporters or systems for uptake from these proteins remain to be identified.
The blood plasma protein, ceruloplasmin (Cp), is well known for its role in enhancing efflux of iron from certain cells; but it also has other functions, and its copper enters cells all over the mammalian organism, as shown by i.v. infusion of 67Cu‐labeled Cp into rats. In current studies, the ability of Cp to directly donate its Cu to cultured cells was investigated. 64Cu‐labeled human Cp secreted by HepG2 cells was purified and incubated in serum‐free medium with polarized human mammary epithelial cell monolayers with tight junctions (PMC42 cells), applied to the basolateral (“blood” side). Most of the radioactivity entered the cells and 30–50% of what entered was released into the apical (“milk”) fluid. Larger amounts of 64Cu‐Cp were produced in mice and purified for incubation with mouse embryonic fibroblasts (MEF) that did and did not express Ctr1 (kindly provided by Dennis Thiele, Duke University). 64Cu from Cp accumulated in both types of MEFs at a linear rate over 3 h, and was internalized, most being present in the cytosol. Uptake was somewhat less in Ctr1−/− MEFs. Uptake of Cp‐64Cu (which is not exchangeable) was inhibited by an excess of Cu(I) and Cu(II)‐histidine; and the presence of external Cu(II) reductase activity was verified. We conclude that Ctr1 and another (unknown) transporter take up Cu from Cp, and that this is mediated by a Cu reductase. Supported in part by US PHS Grant HD46949.
Alpha‐2‐macroglobulin (a2M) is a member of the macroglobulin family of blood plasma proteins. It has a variety of functions, ranging from trapping proteases and transporting zinc to binding and transporting inflammatory and anti‐inflammatory cytokines and, growth factors and the small peptide regulating iron metabolism (hepcidin)[1]. a2M is the main form found in most mammals, while the alpha1‐inhibitor3 form dominates in the plasma of rodents. Some years ago, we determined in rats that a large plasma protein we named transcuprein was involved in transporting copper to the liver (exchanging copper with albumin), and upon purification found it was alpha1‐inhibitor3[2]. We then showed that human a2M also bound Cu(II) tightly, and that this copper was readily delivered by a2M to cultured human cells[3]. As we have been studying aspects of copper metabolism in pigs, we decided to also investigate the structure and copper binding of pig a2M to compare it to that of human a2M. Pig and human a2M were purified from heparinized Yorkshire pig plasma and from the heparinized plasma of human volunteers (under an approved university IRB protocol), using the established procedure for human a2M that uses a combination of PEG 8000 fractionation and Zn(II)‐immobilized metal affinity chromatography. The resulting samples were separated in large pore size exclusion chromatography on Sephacryl S300. Human a2M eluted had one large peak eluting with an Mr of ~900 kDa, and a much smaller peak of about 400 kDa. By SDS‐PAGE, both peaks showed the expected 180 kDa band, indicating that most of the human a2M was a tetramer, with a very small portion as a dimer. In the case of the pig, There was not tetrameric a2M; it was all in dimer form, and the subunits seen in SDS‐PAGE were two bands that together added up to ~180 kDa (120 + 60 kDa). Non‐denaturing PAGE gave single bands indicating that the smaller subunits of the pig a2M were combining into one structure. The copper content of the human a2M was 4 Cu atoms per tetramer (not taking into account potential non‐specific binding), but was 2 Cu atoms per tetramer when copper was first removed and added back in the presence of human or rat albumin. The Cu content of the pig a2M was 4 Cu atoms per dimer. Preliminary data obtained by EPR gave similar but not identical profiles and indicated there were two binding sites, the main one with Cu(II) atoms most likely bound to three Ns and one O. These findings indicate that in some species, a2M forms a dimer, while in others a tetramer; they bind Cu(II) in similar but not identical fashion; and that the subunits of pig a2M may have a vulnerable peptide bond that cleaves the 180 kDa unit into two pieces.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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