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Mechanism of Ceruloplasmin‐Copper Uptake by, and Overexpression in, Mammalian Cells
Author(s) -
Vargas Rebecca,
Islas Leonel,
Gaite Michaella,
Berger Devon,
Ramos Danny,
Mar David,
Kidane Theodros,
Cuajungco Math,
Linder Maria
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.1011.4
Subject(s) - ceruloplasmin , copper , transporter , endocytosis , biotinylation , chemistry , clone (java method) , cell , cell culture , embryonic stem cell , biochemistry , microbiology and biotechnology , biology , gene , genetics , organic chemistry
Ceruloplasmin (Cp) is an abundant multifunctional copper‐binding protein in the blood plasma best known for its ferroxidase activity. Using purified 64 Cu‐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 64 Cu‐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 64 Cu‐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.