A. V. Sokolov

Ceruloplasmin: Macromolecular Assemblies with Iron-Containing Acute Phase Proteins

Copper-containing ferroxidase ceruloplasmin (Cp) forms binary and ternary complexes with cationic proteins lactoferrin (Lf) and myeloperoxidase (Mpo) during inflammation. We present an X-ray crystal structure of a 2Cp-Mpo complex at 4.7 Å resolution. This structure allows one to identify major protein–protein interaction areas and provides an explanation for a competitive inhibition of Mpo by Cp and for the activation of p-phenylenediamine oxidation by Mpo. Small angle X-ray scattering was employed to construct low-resolution models of the Cp-Lf complex and, for the first time, of the ternary 2Cp-2Lf-Mpo complex in solution. The SAXS-based model of Cp-Lf supports the predicted 1∶1 stoichiometry of the complex and demonstrates that both lobes of Lf contact domains 1 and 6 of Cp. The 2Cp-2Lf-Mpo SAXS model reveals the absence of interaction between Mpo and Lf in the ternary complex, so Cp can serve as a mediator of protein interactions in complex architecture. Mpo protects antioxidant properties of Cp by isolating its sensitive loop from proteases. The latter is important for incorporation of Fe3+ into Lf, which activates ferroxidase activity of Cp and precludes oxidation of Cp substrates. Our models provide the structural basis for possible regulatory role of these complexes in preventing iron-induced oxidative damage.

Hypochlorous acid as a precursor of free radicals in living systems.

Hypochlorous acid (HOCl) is produced in the human body by the family of mammalian heme peroxidases, mainly by myeloperoxidase, which is secreted by neutrophils and monocytes at sites of inflammation. This review discusses the reactions that occur between HOCl and the major classes of biologically important molecules (amino acids, proteins, nucleotides, nucleic acids, carbohydrates, lipids, and inorganic substances) to form free radicals. The generation of such free radical intermediates by HOCl and other reactive halogen species is accompanied by the development of halogenative stress, which causes a number of socially important diseases, such as cardiovascular, neurodegenerative, infectious, and other diseases usually associated with inflammatory response and characterized by the appearance of biomarkers of myeloperoxidase and halogenative stress. Investigations aimed at elucidating the mechanisms regulating the activity of enzyme systems that are responsible for the production of reactive halogen species are a crucial step in opening possibilities for control of the development of the body’s inflammatory response.

[Myeloperoxidase-induced biodegradation of single-walled carbon nanotubes is mediated by hypochlorite].

Broadening prospects of using single-walled carbon nanotubes (SWNTs) in medicine and biotechnology raise the concerns about both their toxicity and the mechanisms of biodegradation and elimination from the body. SWNTs biodegradation as a result of catalytic activity of myeloperoxidase (MPO) was shown in the isolated MPO system as well as in the suspension of neutrophils [Kagan V.E. et al., 2010]. In the present study we analyzed the ability of different MPO-produced oxidants to oxidize and to degrade SWNTs. The comparison of the ability of various peroxidases to degrade SWNTs in vitro revealed that myeloperoxidase, due to its ability to produce hypochlorite, and lactoperoxidase, due to its ability to produce hypobromite, are extremely efficient in the degrading of carbon nanotubes. The biodegradation of SWNTs in the model system can also be induced by free radicals generated as a result of heme degradation and, to a lesser extent, by active oxoferryl intermediates of peroxidases. Our experiments showed that in the presence of blood plasma, peroxidase intermediates or free radical products of heme degradation were unable to initiate biodegradation of carbon nanotubes, only the generation of hypochlorite by MPO can cause the biodegradation of carbon nanotubes in vivo. At high concentrations, hypochlorite caused decrease in optical absorbance of plasma-containing SWNTs suspension, which is indicative of the nanotube degradation. Our results unambiguously suggest that hypochlorite can serve as a main oxidizing agent to modify and degrade nanotubes at the sites of inflammation and in phagosomes.

Interaction of Ceruloplasmin, Lactoferrin, and Myeloperoxidase

When lactoferrin (LF) and myeloperoxidase (MPO) are added to ceruloplasmin (CP), a CP-LF-MPO triple complex forms. The complex is formed under physiological conditions, but also in the course of SDS-free PAGE. Polyclonal antibodies to both LF and MPO displace the respective proteins from the CP-LF-MPO complex. Similar replacement is performed by a PACAP38 fragment (amino acids 29–38) and protamine that bind to CP. Interaction of LF and MPO with CP-Sepharose is blocked at ionic strength above 0.3 M NaCl and at pH below 4.1 (LF) and 3.9 (MPO). Two peptides (amino acids 50–109 and 929–1012) were isolated by affinity chromatography from a preparation of CP after its spontaneous proteolytic cleavage. These peptides are able to displace CP from its complexes with LF and MPO. Both human and canine MPO could form a complex when mixed with CP from seven mammalian species. Upon intravenous injection of human MPO into rats, the rat CP-human MPO complex could be detected in plasma. Patients with inflammation were examined and CP-LF, CP-MPO, and CP-LF-MPO complexes were revealed in 80 samples of blood serum and in nine exudates from purulent foci. These complexes were also found in 45 samples of serum and pleural fluid obtained from patients with pleurisies of various etiology.

Identification and isolation from breast milk of ceruloplasmin-lactoferrin complex

The presence of a complex of the copper-containing protein ceruloplasmin (Cp) with lactoferrin (Lf) in breast milk (BM) is shown for the first time. In SDS-free polyacrylamide gel electrophoresis (PAGE), electrophoretic mobility of Cp in BM is lower than that of plasma Cp, coinciding with the mobility of the complex obtained upon mixing purified Cp and Lf. Affinity chromatography of delipidated BM on Cp-Sepharose resulted in retention of Lf. SDS-PAGE of the 0.3 M NaCl eluate revealed a single band with Mr ∼ 78,000 that has the N-terminal amino acid sequence of Lf and reacts with antibodies to that protein. Synthetic peptides R-R-R-R (the N-terminal amino acid stretch 2–5 in Lf) and K-R-Y-K-Q-R-V-K-N-K (the C-terminal stretch 29–38 in PACAP 38) caused efficient elution of Lf from Cp-Sepharose. Cp-Lf complex from delipidated BM is not retained on the resins used for isolation of Cp (AE-agarose) and of Lf (CM-Sephadex). Anionic peptides from Cp-(586–597), (721–734), and (905–914)—provide an efficient elution of Cp from AE-agarose, but do not cause dissociation of Cp-Lf complex. When anti-Lf is added to BM flowed through CM-Sephadex, Cp co-precipitates with Lf. Cp-Lf complex can be isolated from BM by chromatography on CM-Sephadex, ethanol precipitation, and affinity chromatography on AE-agarose, yielding 98% pure complex. The resulting complex Cp-Lf(1: 1) was separated into components by chromatography on heparin-Sepharose. Limited tryptic hydrolysis of Cp obtained from BM and from blood plasma revealed identical proteolytic fragments.

Lactoferrin, myeloperoxidase, and ceruloplasmin: complementary gearwheels cranking physiological and pathological processes

Copper-containing plasma protein ceruloplasmin (Cp) forms a complex with lactoferrin (Lf), an iron-binding protein, and with the heme-containing myeloperoxidase (Mpo). In case of inflammation, Lf and Mpo are secreted from neutrophil granules. Among the plasma proteins, Cp seems to be the preferential partner of Lf and Mpo. After an intraperitoneal injection of Lf to rodents, the “Cp–Lf” complex has been shown to appear in their bloodstream. Cp prevents the interaction of Lf with protoplasts of Micrococcus luteus. Upon immunoprecipitation of Cp, the blood plasma becomes depleted of Lf and in a dose-dependent manner loses the capacity to inhibit the peroxidase activity of Mpo, but not the Mpo-catalyzed oxidation of thiocyanate in the (pseudo)halogenating cycle. Antimicrobial effect against E. coli displayed by a synergistic system that includes Lf and Mpo–H2O2–chloride, but not thiocyanate, as the substrate for Mpo is abrogated when Cp is added. Hence, Cp can be regarded as an anti-inflammatory factor that restrains the halogenating cycle and redirects the synergistic system Mpo–H2O2–chloride/thiocyanate to production of hypothiocyanate, which is relatively harmless for the human organism. Structure and functions of the “2Cp–2Lf–Mpo” complex and binary complexes Cp–Lf and 2Cp–Mpo in inflammation are discussed.

Two-stage method for purification of ceruloplasmin based on its interaction with neomycin

A two-stage chromatography that yields highly purified ceruloplasmin (CP) from human plasma and from rat and rabbit serum is described. The isolation procedure is based on the interaction of CP with neomycin, and it provides a high yield of CP. Constants of inhibition by gentamycin, kanamycin, and neomycin of oxidase activity of CP in its reaction with p-phenylenediamine were assayed. The lowest Ki for neomycin (11 μM) corresponded to the highest specific adsorption of CP on neomycin-agarose (10 mg CP/ml of resin). Isolation of CP from 1.4 liters of human plasma using ion-exchange chromatography on UNO-Sphere Q and affinity chromatography on neomycin-agarose yields 348 mg of CP with 412-fold purification degree. Human CP preparation obtained with A610/A280 ∼ 0.052 contained neither immunoreactive prothrombin nor active thrombin. Upon storage at 37°C under sterile conditions, the preparation remained stable for two months. Efficient preparation of highly purified CP from rat and rabbit sera treated according to a similar protocol suggests the suitability of our method for isolation of CP from plasma and serum of other animals. The yield of CP in three separate purifications was no less than 78%.

Identification of leukocyte cationic proteins that interact with ceruloplasmin

Proteins from leukocytes were investigated for their ability to interact with ceruloplasmin (Cp), a copper-containing glycoprotein of human plasma. Extract from leukocytes was subjected to affinity chromatography on Cp-Sepharose, after which proteins were eluted from the resin with 0.5 M NaCl in Tris-HCl, pH 7.4. SDS-PAGE of the eluate revealed protein bands with molecular weights 78, 57, 40, 30, 16, and 12 kD. Among these, Western blotting detected myeloperoxidase (57, 40, and 12 kD) and lactoferrin (78 kD). Also, the 30-kD component had a sequence 1I-2I/V-3G-4G-5R/H at the N-terminus that is likely to indicate the presence of neutrophilic elastase, cathepsin G, proteinase 3, and azurocidin (CAP 37) — all from the family of serprocidins. Mass spectrometry of tryptic fragments indicated the presence of the 16-kD eosinophilic cationic protein (seven peptides), 27-kD cathepsin G (eleven peptides), 27-kD azurocidin (eight peptides), 29 kD neutrophilic elastase (seven peptides), and 27-kD proteinase 3 (six peptides). Myeloperoxidase was represented by 57-, 40-, and 12-kD fragments (thirteen, ten, and four peptides, respectively). Thus, interaction with Cp of five cationic proteins, i.e. of eosinophilic cationic protein, cathepsin G, neutrophilic elastase, proteinase 3, and azurocidin is reported for the first time.

Interaction of ceruloplasmin with eosinophil peroxidase as compared to its interplay with myeloperoxidase: Reciprocal effect on enzymatic properties

Abstract Myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are involved in the development of halogenative stress during inflammation. We previously described a complex between MPO and ceruloplasmin (CP). Considering the high structural homology between MPO and EPO, we studied the latters interaction with CP and checked whether EPO becomes inhibited in a complex with CP. Disc-electrophoresis and gel filtration showed that CP and EPO form a complex with the stoichiometry 1:1. Affinity chromatography of EPO on CP-agarose (150 mM NaCl, 10 mM Na-phosphate buffer, of pH 7.4) resulted in retention of EPO. EPO protects ceruloplasmin from limited proteolysis by plasmin. Only intact CP shifted the Soret band typical of EPO from 413 to 408 nm. The contact with CP likely causes changes in the heme pocket of EPO. Peroxidase activity of EPO with substrates such as guaiacol, orcinol, o-dianisidine, 4-chloro-1-naphtol, 3,3’,5,5’-tetramethylbenzidine, and 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonate) is inhibited by CP in a dose-dependent manner. Similar to the interaction with MPO, the larger a substrate molecule, the stronger the inhibitory effect of CP upon EPO. The limited proteolysis of CP abrogates its capacity to inhibit the peroxidase activity of EPO. The peptide RPYLKVFNPR (corresponding to amino acids 883–892 in CP) inhibits the peroxidase and chlorinating activity of EPO. Only the chlorinating activity of EPO is efficiently inhibited by CP, while the capacity of EPO to oxidize bromide and thiocyanate practically does not depend on the presence of CP. EPO enhances the p-phenylenediamine-oxidase activity of CP. The structural homology between the sites in the MPO and EPO molecules enabling them to contact CP is discussed.

Effect of lactoferrin on the ferroxidase activity of ceruloplasmin

The effects of various forms of lactoferrin (Lf) interacting with ceruloplasmin (Cp, ferro-O2-oxidoreductase, EC 1.16.3.1) on oxidase activity of the latter were studied. Comparing the incorporation of Fe3+ oxidized by Cp into Lf and serum transferrin (Tf) showed that at pH 5.5 apo-Lf binds the oxidized iron seven times and at pH 7.4 four times faster than apo-Tf under the same conditions. Apo-Lf increased the oxidation rate of Fe2+ by Cp 1.25 times when Cp/Lf ratio was 1 : 1. Lf saturated with Fe3+ or Cu2+ increased the oxidation rate of iron 1.6 and 2 times when Cp to holo Lf ratios were 1 : 1 and 1 : 2, respectively. Upon adding to Cp the excess amounts of apo-Lf (Cp/apo-Lf < 1 : 1) or of holo Lf (Cp/holo-Lf < 1 : 2) the oxidation rate of iron no longer changed. Complex Cp-Lf demonstrating ferroxidase activity was discovered in breast milk.