Identification of the prooxidant site of human ceruloplasmin: A model for oxidative damage by copper bound to protein surfaces

Abstract

Free transition metal ions oxidize lipids and lipoproteins in vitro; however, recent evidence suggests that free metal ion-independent mechanisms are more likely in vivo. We have shown previously that human ceruloplasmin (Cp), a serum protein containing seven Cu atoms, induces low density lipoprotein oxidation in vitro and that the activity depends on the presence of a single, chelatable Cu atom. We here use biochemical and molecular approaches to determine the site responsible for Cp prooxidant activity. Experiments with the His-specific reagent diethylpyrocarbonate (DEPC) showed that one or more His residues was specifically required. Quantitative Transition metal ions such as Cu and Fe are extremely effective promoters of oxidation reactions and can cause the oxidative modification of low density lipoprotein in vitro (1, 2). The involvement of free metal ions in oxidation processes in vivo has been questioned because most of these ions are linked to prosthetic groups or are tightly sequestered by specific binding proteins, and thus the level of free Cu (and Fe) in plasma and interstitial fluids is extremely low (3). In addition, recent mass spectroscopic analysis suggests that the oxidized lipid ''fingerprint'' exhibited by human atherosclerotic lesions is not consistent with free metal-catalyzed processes (4). In view of these findings, recent attention has focused on the investigation of free metal ion-independent mechanisms, such as those using peroxidases (5, 6), and on metal-binding proteins (7-9), which can promote LDL oxidation.Studies by us (10-12) and others (13)(14)(15) show that the copper protein ceruloplasmin (Cp) exhibits potent oxidant activity toward LDL in vitro. Cp is a plasma glycoprotein containing about seven Cu atoms per molecule and Ϸ95% of the total plasma Cu (see refs. 16 and 17 for review). It is a 132-kDa monomer comprised almost entirely of three major domains that have 35-40% sequence homology to each other (18) and to three homologous domains in factors Va and VIIIa (19).

Involvement of Glycosylphosphatidylinositol-linked Ceruloplasmin in the Copper/Zinc-Nitric Oxide-dependent Degradation of Glypican-1 Heparan Sulfate in Rat C6 Glioma Cells

“…However, within the intracellular compartments or under the cell-free conditions used here, the copper in Cp may be available for other Cu(II)-mediated reactions. This may involve only the copper bound at His-426 or also other copper atoms in Cp (21)(22)(23).…”

Inhibition of Endothelial Nitric-oxide Synthase by Ceruloplasmin

“…CP has been detected in human atherosclerotic lesions (14,15), and it has been shown to oxidize low density lipoproteins in the presence of vascular cells (16). A copper binding site labile to Chelex treatment has been proposed to be responsible for the oxidative damage to low density lipoproteins (17). On the other hand, we have shown that CP, at physiological concentrations, inhibits the endothelium-dependent relaxation of rabbit aorta induced by agonists and that this effect is not due to a trapping of NO by the copper sites (18).…”

Site-directed Mutagenesis of Human Ceruloplasmin

“…It occurs mainly in the plasma and plays an important role in iron homeostasis (1,2). Other roles include its participation in the antioxidant defense (3)(4)(5)(6) or in oxidative damage mechanisms (7,8) and its involvement in a number of metabolic processes related to the metabolism of copper (9), biogenic amines (10), and nitric oxide (11).…”

“…The fragments initially do not dissociate and mimic the intact molecule, but the protein suffers changes of its redox properties that lead to a redistribution of electrons within its copper sites (46). In fact, the antioxidant or the prooxidant activities of Cp are specific to the cleaved and to the intact molecule, respectively (8). Thus, it was of outmost importance to have a stable Cp to work with before attempting mutagenesis targeted to the copper sites.…”