Mechanism of Copper Uptake from Blood Plasma Ceruloplasmin by Mammalian Cells

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 transpor...

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Published in:PloS one 2016-03, Vol.11 (3), p.e0149516-e0149516
Main Authors: Ramos, Danny, Mar, David, Ishida, Michael, Vargas, Rebecca, Gaite, Michaella, Montgomery, Aaron, Linder, Maria C
Format: Article
Language:English
Subjects:
Amino acids
Animals
Apoproteins - metabolism
Biochemistry
Biological transport
Biological Transport - physiology
Biology and Life Sciences
Blood
Blood plasma
Bone marrow
Cation Transport Proteins - metabolism
Cell culture
Cell Line
Cell Line, Tumor
Cell surface
Cells (Biology)
Ceruloplasmin
Ceruloplasmin - metabolism
Copper
Copper (Nutrient)
Copper - metabolism
Cytochrome
Cytosol
Efflux
Embryo fibroblasts
Embryos
Endocytosis
Epithelial cells
Epithelial Cells - metabolism
Female
Fibroblasts
Fibroblasts - metabolism
Genetic aspects
Hep G2 Cells
Humans
Iron
Iron - metabolism
Male
Mammalian cells
Mammals
Mammary gland
Mammary Glands, Human - metabolism
Medicine and Health Sciences
Metabolism
Metabolites
Mice
Milk
Milk - metabolism
Monolayers
Oxidation-Reduction
Oxidoreductases - metabolism
Physical Sciences
Physiological aspects
Plasma - metabolism
Protein transport
Proteins
Reductase
Reductases
siRNA
Spectral analysis
Substrates
Tight junctions
Transport
Trypsin
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Summary: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 (64)Cu-labeled ceruloplasmin secreted by HepG2 cells. Monolayers took up virtually all the (64)Cu over 16h and secreted half into the apical (milk) fluid. This was partly inhibited by Ag(I). The (64)Cu in ceruloplasmin purified from plasma of (64)Cu-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 (64)Cu 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 (64)Cu 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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0149516