Biometals DOI 10.1007/s10534-014-9755-2
Lactoferrin, myeloperoxidase, and ceruloplasmin: complementary gearwheels cranking physiological and pathological processes Alexey V. Sokolov • Elena T. Zakahrova • Valeria A. Kostevich • Valeria R. Samygina Vadim B. Vasilyev
•
Received: 24 February 2014 / Accepted: 30 May 2014 Springer Science+Business Media New York 2014
Abstract 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 A. V. Sokolov (&) E. T. Zakahrova V. A. Kostevich V. B. Vasilyev N-W Branch of the Russian Academy of Medical Sciences, Institute for Experimental Medicine, Pavlov Street 12, Saint Petersburg 197376, Russia e-mail:
[email protected] A. V. Sokolov V. B. Vasilyev Saint-Petersburg State University, Mendeleevskaya Line, Saint Petersburg 199000, Russia
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. Keywords Ceruloplasmin Lactoferrin Myeloperoxidase Protein–protein interactions Synergism of antimicrobial proteins Inflammation Thiocyanate Halogenative stress Abbreviations Cp Ceruloplasmin Lf Lactoferrin Mpo Myeloperoxidase
A. V. Sokolov V. A. Kostevich Research Institute of Physico-Chemical Medicine, ul. Malaya Pirogovskaya 1a, Moscow 119435, Russia
Introduction
V. R. Samygina Institute of Crystallography, RAS, Leninsky pr 59, Moscow 117333, Russia
Ceruloplasmin (Cp, ferro:O2–oxidoreductase) is the copper-containing protein of vertebrate blood plasma. Human Cp has M *132 kDa. Along with the soluble
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Cp secreted by hepatocytes into plasma, another form of this protein is known to be anchored by glycosylphosphoinositol to membranes of some cells in the nervous, immune, and other systems (Salzer et al. 1998; Vassiliev et al. 2005; Marques et al. 2012). Cp synthesis is increased in response to hypoxia, iron deficiency (Mukhopadhyay et al. 2000), and copper excess (Martin et al. 2005). A similar result is envoked by the rise of insulin (Seshadri et al. 2002), thrombin (Yang et al. 2006), estradiol (Voronina and Monakhov 1980), and proinflammatory cytokine (Mazumder et al. 1997) level. A number of enzymatic and antiinflammatory activities are characteristic of Cp as an acute phase reactant (Gitlin 1988). The distinctive feature of Cp is its capacity to oxidize Fe2? to Fe3? (Osaki 1966). However, the physiological roles of this protein are not likely to be reduced to Fe2? oxidation, even though deficiencies of the Cp gene in humans (aceruloplasminemia) are known to provoke the oxidative stress resulting from accumulation of ferrous iron in tissues (Vassiliev et al. 2005). Cp as the enzyme actively precludes the formation and persistence of free radicals, having the activities of ferroxidase (Osaki 1966), cuproxidase (Stoj and Kosman 2003), superoxide dismutase (Vasilyev et al. 1988), glutathione-linked peroxidase (Kim and Park 1998), and NO– oxidase (Shiva et al. 2006). Plasma concentration of Cp in inflammation can grow from 0.3 to 0.9 mg/ml, which allows suggesting its role in the regulation of inflammatory reactions (Glezer et al. 2007). In the last 10 years we were the first to characterize complexes of Cp with cationic proteins of neutrophils, such as lactoferrin (Lf), myeloperoxidase (Mpo) (Sokolov et al. 2007a), the members of the serprocidin family (elastase, cathepsin G, proteinase 3 and azurocidin) (Sokolov et al. 2007b), matrix metalloproteinase 2 and 12 (Sokolov et al. 2009a), and 5-lipoxygenase (Sokolov et al. 2010a). Anionic Cp (pI 4.7) interacts with cationic proteins in a somewhat similar manner, yet the complexes formed display certain diversity. We succeeded in showing the high affinity of components within the complexes. For example, the affinity of Cp to Lf and azurocidin is characterized by Kd *13 nM (Sokolov et al. 2009b, 2010b). Both in vitro and in vivo Cp is able to form multimeric complexes that include Lf and Mpo (Sokolov et al. 2007a; Samygina et al. 2013). Lf increases the ferroxidase activity of Cp upon forming a complex
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with the latter (Sokolov et al. 2005a). Considering that one of the mechanisms of antimicrobial activity of Lf is its high-affinity binding of Fe3? that is essential for bacterial growth (Tenovuo 2002), it cannot be excluded that formation of complexes with Cp favors sequestration by Lf of iron from the milieu. Interaction between Cp and Mpo results in suppression of the prooxidative activity of the leukocytic enzyme (Segelmark et al. 1997). Participation of Mpo in protection of the host organism against pathogenic bacteria is unquestionable (Panasenko et al. 2013), and yet the enzyme plays an important role in the development of the halogenative stress associated with inflammation. Antimicrobial effect of Mpo is provided by a cycle of reactions in which Mpo reacts with hydrogen peroxide and is transformed into Compound I possessing a high two-electron redox potential (1.16 V). Being highly reactive, Compound I oxidizes halogenides (Cl-, Br-) and thiocyanate to respective (pseudo) hypohalide acids, i.e., HOCl, HOBr, and HOSCN as the enzyme returns to its native state. Thiocyanate is the most specific substrate for Mpo (van Dalen et al. 1997). Along with the (pseudo)halogenating cycle described above, Mpo is capable of oxidizing a number of substrates using single-electron transfer (Panasenko et al. 2013) in course of its peroxidase cycle (e.g., the chromogenic substrate ABTS (Sokolov et al. 2008). Our latest data show that the inhibiting effect of Cp on Mpo depends on the integrity of the copper protein, i.e., partially proteolyzed Cp is inefficient as an inhibitor of the chlorinating activity of Mpo (Panasenko et al. 2008; Sokolov et al. 2008). Likewise, it loses the capacity to inhibit synthesis of leukotrienes catalyzed by 5-lipoxygenase (Sokolov et al. 2010a). When bound to an intact Cp molecule, Mpo makes the vulnerable interdomain loop inaccessible to proteinases, which protects Cp against the attack of trypsin, elastase, and plasmin, preventing the cleavage of the Cp molecule between domains 5 and 6. Proteolytic cleavage of peptide bonds in Cp beyond the region of protein–protein interaction was also inhibited when it formed a complex with Mpo. This may be explained by a trigger effect: the proteinases are known to hydrolyze peptide bonds in Cp in a certain order. Therefore, unless the first one is cleaved, splitting of other bonds does not occur (Sokolov et al. 2008). This observation was confirmed when antiatherogenic
Biometals
properties of Cp were studied, i.e., solely non-proteolyzed Cp was capable of efficient protection of lowdensity lipoproteins from proatherogenic modification being the result of the chlorinating activity of Mpo (Sokolov et al. 2014). ˚ resolution Cp crystal structure obtained at 2.6-A distinguishes six ß-barrel homologous domains connected by flexible loops (Samygina et al. 2008). Six tightly bound copper ions, which can be divided into three types according to their spectral characteristics, are distributed irregularly among these six domains. Domains 2, 4, and 6 contain one type I copper each. Three copper ions (two type III and one type II) form a trinuclear cluster with ligands provided by domains 1 and 6. Lf (78 kDa) is composed of two highly homological sequences known as N- and C-lobes. Each lobe contains one specific metal-binding site in a deep cleft between two dissimilar domains (Sun et al. ˚ crystal structure (Blair1999). According to a 1.95-A Johnson et al. 2001), Mpo is a homodimer of 140 kDa, each monomer consisting of two polypeptides of 108 a.a. (light chain) and 466 a.a. (heavy chain) and containing a heme. Revealing the sophisticated molecular assembly including three metal-containing proteins (Cp, Lf, and Mpo) is a prerequisite for a detailed study of their interaction with reactive oxygen and halogen species that are formed in inflammation (Samygina et al. 2013). The damage of Cp in reaction with hydrogen peroxide (Sokolov et al. 2012a), superoxide anionradical, and HOCl (Sharonov et al. 1988, 1989) has been documented. Here we present data concerning the specificity of interaction of Cp with Lf and Mpo. In particular, this communication is focused on the selectivity of interaction occurring in the bloodstream between Cp and exo- and endogenous Lf, on the Mpoinhibiting potential of plasma Cp, and on the interaction of the latter with the synergistic antimicrobial system containing Lf and Mpo–H2O2– chloride/thiocyanate.
Materials and methods The following reagents were used: arginine, glycerol, Coomassie R-250, mercaptoethanol, ammonium persulfate, Tris (Serva, Germany); SDS, NaSCN, KSCN, neomycin trisulfate, resazurin, phenylmethylsulfonyl
fluoride (PMSF), 4-chloro-1-naphtol (Sigma, USA); acrylamide, N,N’-methylene-bis-acrylamide, N,N,N’,N’-tetramethyleneethylenediamine (Laboratory MEDIGEN, Russia); heparin (SPOFA, Poland). All solutions were prepared using apyrogenic deionized water with resistivity 18.2 MXcm. Cyanogen bromide was obtained by bromination of KCN in biphasic system ‘‘water-dichloroethane’’. The obtained solution of BrCN in dichloroethane was used to activate Sepharose for immobilization of neomycin and heparin (Sokolov et al. 2005b). Molecular mass of proteins was evaluated in PAAG SDS electrophoresis (Laemmli 1970). Optical spectra and the changing absorption rates were registered on a SF-2000-02 spectrophotometer (OKB-Spectr, Russia). Concentrations of substances were measured by spectrophotometry using the following extinction coefficients: dimeric Mpo— e430 = 178,000 M-1 cm-1 (Bakkenist et al. 1978), Cp—e610 = 9,780 mM-1 cm-1 (Noyer et al. 1980), apo-Lf—e280 = 87,360 M-1 cm-1 (Zakharova et al. 2000), H2O2—e240 = 43.6 M-1 cm-1 (Beers and Sizer 1952). Protein purification To obtain a stable preparation of monomeric Cp containing 95 % of non-fragmented protein with M 132 kDa and A610/A280 [0.049 human plasma to which PMSF and EDTA were added, respectively, to 1 mM and to 0.1 mM, was subjected to chromatography on UNOSphere Q and neomycin-agarose (Sokolov et al. 2012b). Lf was purified from breast milk using ionexchange chromatography on CM-Sepharose and gel filtration on Sephadex G-75 Superfine (Zakharova et al. 2000). Lf from cow milk was isolated similarly. Using chromatography on heparin-Sepharose, phenyl-Sepharose, and gel filtration on Sephacryl S-200 HR, Mpo preparation was purified from human leukocytes to the ratio A430/A280 (RZ) = 0.85, which characterizes the homogeneous protein (Sokolov et al. 2010c). Revealing heterologous complexes of Lf and Cp after intraperitoneal injection of Lf Wistar rats and mice C57Black were narcotized with ether and injected intraperitoneally, respectively, 20 mg and 1 mg of Lf from cow or breast milk. Blood
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was sampled from the tail vein 15, 0, 60, and 120 min past the injection. Animal serum (5 ll) was analyzed by SDS-free disc-electrophoresis (Davis 1964) and then by Western blotting (Anderson et al. 1982) with antibodies against CP (rat or mouse) and Lf (bovine or human). Evaluation of the effect of Cp on Mpo activity Electrochemical measurements of the rate of H2O2 concentration decrease were done with planar sensors constructed on the basis of Prussian blue nanofilms (Borisova et al. 2009). Our device included the planar electrode-containing unit placed on a permanently functioning magnetic stirrer, and the potentiostat P-8 Elins (Chernogolovka, Russia) that registered the electric current proportional to H2O2 concentration. The initial current strength was registered upon introducing H2O2 to 50 lM into the medium containing 100 mM KCl, 10 mM potassium phosphate buffer, pH 6.2, 10 mM Tau. The reaction was launched by adding an aliquot of Mpo (to the final concentration 1–5 nM) and the time-dependent current strength was tracked. The rate of H2O2 concentration decrease and the turnover number (1/s) for the catalytic center of Mpo were determined using the linear part of the plot. Also, the effect on H2O2 utilization resulting from adding to this system of 0.2 mM KSCN and of 0.2, 0.4, 0.8 b 1.6 lM Cp was quantified. Graphs reflecting the effect of Cp on Mpo-catalyzed H2O2 utilization in presence of various substrates were plotted. Peroxidase activity of Mpo was assayed by oxidation of the chromogenic substrate sodium 2,20 -diasinobis(3ethylbenzotriazoline-6-sulphonate) (ABTS) (Sokolov et al. 2008). Oxidation of the substrate gives origin to the stable radical ABTS•?, the amount of which is measured by light absorption. The reaction mixture for this assay contained 3 nM Mpo, 100 lM H2O2, 1 mM ABTS in 100 mM sodium acetate buffer, pH 5.5. Upon adding H2O2 to the mixture, the activity of Mpo was assayed at room temperature as DA414/min having set the ‘‘Kinetics’’ mode in SF 2000–2002. Peroxidase activity of Mpo was measured upon adding samples of blood plasma containing various amounts of Cp, after its immunoprecipitation. Sample dilution providing IC50 was determined. Immunoprecipitation of Cp from plasma To achieve immunoprecipitation, we incubated blood plasma with increasing concentrations of high-affinity
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rabbit antibodies against human Cp (Sokolov et al. 2010c), which was followed by precipitation of an immune complex by goat antibodies against rabbit immunoglobulins. In 30 min of plasma incubation with rabbit affinity antibodies against Cp (0.1–2 mg/ml), the immunoprecipitation of Cp was accomplished. Then goat antibodies (2 mg/ml) against rabbit immunoglobulins were added with subsequent incubation for 30 min. Samples were centrifuged for 10 min at 15,000 9 g (4 C). Cp content in plasma after immunoprecipitation was assayed by ELISA (Sokolov et al. 2010c), Lf content was assayed by commercial ELISA (Vector-Best, Russia). Agglutination of Micrococcus luteus protoplasts Agglutination of Micrococcus luteus protoplasts was followed by changes in A450 using a model system containing 0.5 % suspension of freeze-dried M. luteus, 5 lg/ml lysozyme, 0.5 mg/ml Lf in 0.1 M NaCl, 66 mM sodium acetate buffer, pH 5.4 (Perraudin and Prieels 1982). Cp was added to molar ratio with Lf 2:1, 1:1, and 1:2, and its effect was studied. Circular dichroism spectroscopy Circular dichroism (CD) spectra were registered on a CD6 dichrograph (Jobin–Yvon, France), calibrated with (?)-10-D-camphora-sulfonic acid. Measurements were made using dismountable 0.05-cm cuvettes (near-UV and visible regions) and 0.001-cm cuvettes (far-UV). To carry out these measurements, 5 lM Mpo, 10 lM Cp, 10 lM Lf, and mixtures of proteins corresponding to complexes 2Cp:1Mpo and 2Lf:2Cp:1Mpo were used. Antimicrobial activity (MIC50) The slowing growth of Escherichia coli, strain ML-35p, was evaluated by absorption spectrum of the metabolic indicator resazurin, which allowed estimating the antimicrobial activity of Lf and of the system Mpo– H2O2–chloride/thiocyanate (Cooper 2013). Cells were cultured overnight at ?37 C in 3 % soybean tryptic hydrolysate. Thus, grown suspension of E. coli culture was centrifuged at 6,000 9 g for 5 min, then washed with PBS cooled to ?4 C, and again centrifuged under the same conditions. Cell precipitate was resuspended in PBS, after which the concentration of cells was
Biometals
determined by measuring A620, on account that 2.5 9 108 CFU/ml corresponds to one optical density unit. A 96-well flat-bottom plate was filled with studied substances in PBS, i.e., the bacterial suspension (final concentration in a well was 4 9 104 CFU/ml), soybean tryptic hydrolysate (final concentration 0.18 %), 30 lM resazurin. Lf and Mpo in the presence of H2O2 (10 lM) and sodium thiocyanate (10 lM) were tested as antimicrobial agents. Proteins (Cp, Lf b Mpo) were added in amounts providing the ratio 2Lf:2Cp:1Mpo. The plate was put on a shaker at ?37 C. The metabolic activity of bacteria was evaluated by the growth of A530–A630. This index was registered every 30 min in a multichannel spectrophotometer Stat Fax (USA). The antimicrobial activity was expressed as the protein concentration that caused a two-fold drop of A530–A630 as compared to the control cell culture (MIC50). Interaction between protein regions analyzed using a 3D model of complex To reveal the sites of interaction in Cp and Lf previous 3D models obtained in a SAXS study of the 2Lf–2Cp– Mpo complex (Samygina et al. 2013) was used.
Results The specificity of interaction between Cp and Lf To explore the selectivity of interaction between Lf and Cp we used a heterologous system, when either bovine or human Lf was injected intraperitoneally to rats and mice, after which electrophoretic mobility of Cp and Lf was revealed by Western blotting of SDSfree PAAG in which samples of serum had been subjected to disc-electrophoresis (Fig. 1). It is seen that 15 min after injection of Lf into animals the immunoreactive band corresponding to rat or mouse Cp had an altered mobility as compared to the serum sampled before injection. The novel mobility conforms to that of the Cp–Lf complex which is formed when Lf is added to either rat or mouse serum (Fig. 1a). Lf (either bovine or human) detected in sera of rats and mice also migrated with the speed of the Cp–Lf complex (Fig. 1b). The intensity of the band corresponding to the heterologous Cp–Lf complex did not change for 2 hours. No other part of a nitrocellulose membrane bound antibodies to human or bovine
Fig. 1 Detection of mouse and rat Cp (a), of human and bovine Lf (b) before and after the intraperitoneal injection into mice and rats of 1 and 20 mg Lf, respectively. Western blotting of human serum samples (5 ll) after SDS-free electrophoresis. 1—serum before injection of Lf, 2–5 to 15, 30, 60, and 120 min after injecting Lf, 6—blood serum after adding Lf (0.5 lg)
Lf hence, Cp is likely to be the preferable partner of Lf. We have shown previously that interaction of Cp with Lf is prevented by polyanionic substances bound to the N-terminal polycationic cluster in Lf, such as LPS, DNA, and heparin (Pulina et al. 2002). A study of Lf-mediated agglutination of M. luteus protoplasts showed that Cp blocks this process (Fig. 2). In the presence of Lf, instead of monotonous decrease of turbidity, a temporary increase of A450 is observed, which results from agglutination of protoplasts. In the presence of Cp that peak goes down in a dose-dependent manner, so that the curve practically coincides with that observed when protoplasts are lyzed essentially by pure lysozyme. We studied Lf content in plasma samples subjected to immunoprecipitation of Cp. It appeared that plasma Lf concentration goes down concomitantly with precipitation of Cp. Hence, upon precipitation, 98 % of Cp the plasma became depleted of Lf for about 92 % (Fig. 3a). Control experiments showed that antibodies against Cp alone cause no precipitation of Lf. The specificity of Mpo inhibition by Cp Our previous study provided evidence that introduction of human Mpo into the bloodstream of rats also
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Fig. 2 Effect of Cp on Lf-dependent agglutination of 0.5 % cell suspension of M. luteus in the presence of lysozyme, evaluated by an increase of the solution transparency (A450). The system contained 5 lg/ml lysozyme, 0.5 mg/ml Lf in 0.1 M NaCl, 66 mM sodium phosphate buffer, pH 5.4, and portions of
Cp: 4, 2, and 1 mol per mole of Lf. 1 lysis caused by lysozyme, 2 in the presence of lysozyme and Lf, 3 in the presence of lysozyme, and Lf with Cp (2:1), 4 in the presence of lysozyme, and Lf with Cp (1:1), 5 in the presence of lysozyme, and Lf with Cp (1:2)
results in forming a heterologous complex (Sokolov et al. 2007a). Besides, upon adding excessive amounts of Mpo to human plasma, it got bound to Cp, including the incorporation into multimeric LDL/VLDL-containing complexes (Sokolov et al. 2010c). Cp is likely to be the physiological inhibitor of Mpo. We studied the effect of immunoprecipitation of Cp from plasma (adding varying amounts of affinity antibodies against Cp) on inhibition of Mpo activity by such plasma. This approach allowed obtaining plasma preparations varying in Cp content (from 80 nM to 3.4 lM). In every such case, a dilution corresponding to IC50 for peroxidase activity of Mpo with ABTS was determined. The direct approximating dependence of plasma dilution on Cp concentration appeared to get interpolated to zero (Fig. 3b). This is evidence of Cp being the major inhibitor of Mpo peroxidase activity in blood plasma. However, an electrochemical sensor used to study the effect of added plasma on the rate of hydrogen peroxide utilization allowed showing that the rate of H2O2 utilization is not decreased proportionally to the degree of the Mpo peroxidase activity inhibition provided by that same portion of plasma (Fig. 3c). When under the same conditions, plasma preparations containing varying amounts of Cp were
used, no effect on the rate of H2O2 utilization was observed (data not shown). Therefore, adding plasma capable of inhibiting Mpo peroxidase activity (by virtue of interacting with Cp) does not prevent to the same extent hydrogen peroxide utilization. We suggested that Cp cannot counteract the oxidation of some plasma-contained substrate in the halogenating cycle of Mpo, e.g., of thiocyanate. Indeed, when H2O2 utilization by chlorinating Mpo in the presence of Cp and SCN- was measured, it appeared that Cp does not preclude the utilization of hydrogen peroxide in course of Mpo-catalyzed oxidation of SCN- to HOSCN (Fig. 3d). CD spectra of the 2Cp–Mpo and of the 2Lf–2Cp– Mpo complexes showed noticeable changes in the ellipticity of the heme in Mpo, occurring upon forming of a complex, once the experimental spectra and their arithmetic sum were compared (Fig. 4). For instance, a significant decrease of the ellipticity was registered in the far-UV region with a shift of the minimum from 284 nm in Mpo to 281 nm in the 2Cp–Mpo and the 2Lf–2Cp–Mpo complexes, while no shift of that was observed in the arithmetic sum of the spectra (Fig. 4a). In the spectra of Mpo, of its complex with Cp and of the triple complex the ellipticity in the region of the
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Fig. 3 Dose-dependent interactions of Cp with Lf and Mpo. a Changes of Lf content in course of Cp immunoprecipitation from plasma. b Dependence of plasma dilution to IC50 of Mpo peroxidase activity with ABTS on Cp concentration in samples of Cp-depleted plasma. c Dependence of the rate of H2O2 utilization on the proportion of plasma (3 lM Cp) in the system containing 3 nM Mpo, 100 mM KCl, 20 mM potassium
phosphate buffer, pH 7.4, 0.4 mM taurine, 50 lM H2O2. d Effect of Cp on catalytic turnover of Mpo in course of substrates’ oxidation in the systems containing 100 mM KCl (200 lM KSCN), 10 mM potassium phosphate buffer, pH 6.2, 10 mM Tau, 50 lM H2O2, 1–5 nM Mpo, 0–1.6 lM Cp (reaction rate registered using electrochemical sensor for H2O2)
Fig. 4 CD-spectra (a farUV, b near-UV, and visible regions) of 5 lM Mpo (green), 10 lM Cp (blue), 10 lM Lf (red), 2Cp: 1Mpo (black) and 2Lf:2Cp: 1Mpo (orange). Arithmetical sums of spectra for Cp?Mpo and Lf?Cp?Mpo are shown by dotted black and orange lines, respectively
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Biometals Table 1 Effect of Cp on antimicrobial activity of Lf and of the system Mpo–H2O2–chloride/thiocyanate with respect to E. coli System under study
MICa50, nM
Cp
Cannot be determined
apo-Lf
980 ± 40
apo-Lf: Cp
990 ± 30
Mpo (H2O2–chloride)
45 ± 12
2Cp: Mpo (H2O2–chloride)
Cannot be determined
2apo-Lf: 1Mpo (H2O2–chloride)
4±1
2: Cp: 2apo-Lf: 1Mpo (H2O2–chloride)
860 ± 30
Mpo (H2O2–thiocyanate)
38 ± 11
2Cp: Mpo (H2O2–thiocyanate)
42 ± 9
2apo-Lf: 1Mpo (H2O2–thiocyanate)
7±2
2: Cp: 2apo-Lf: 1Mpo (H2O2–thiocyanate)
8±2
a
Expressed as concentration of the protein that was present in the system at a lower concentration as compared to other components, which resulted in a two-fold decrease of A530– A630 from the control level
Soret band (maximum at 412 nm) is virtually the same between 405 and 425 nm (Fig. 4b). However, when the experimental spectra of complexes are compared with the arithmetic sum of the proteins’ spectra, the heme ellipticity in complexes of Mpo with Cp and Lf appears to be greater than in Mpo alone. Effect of Cp on antimicrobial activity of Lf and Mpo The results of assaying the antimicrobial activity of Lf, of the system Mpo–H2O2–chloride/thiocyanate, of their combined effect, and of the influence of Cp, are summarized in Table 1. It is seen that Cp blocks the antimicrobial effect of Mpo once chloride becomes the substrate of the latter. Besides, in the presence of Cp, the synergizing effect of such a system with Lf goes down to the activity of Lf acting separately. These results are in good agreement with the data on inhibited chlorinating activity of Mpo obtained by measuring the luminol-dependent chemiluminescence when the enzyme forms a complex with other proteins (Panasenko et al. 2008). On the other hand, Cp had no effect on the system Mpo–H2O2–thiocyanate and did not alter the synergism of Mpo and Lf in the presence of thiocyanate as the substrate of the (pseudo)halogenating cycle of Mpo. These results are in full
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agreement with the incapacity of Cp to inhibit the Mpo-dependent production of hypothiocyanate.
Discussion Our results allow concluding that Cp, Lf, and Mpo function in concord like tooth-wheels in a chain gear, and the functions of each metal-containing protein composing the complex are interactively aimed at decreasing the oxidative/halogenative stress accompanying inflammation (Fig. 5). The interaction of Cp with Mpo and Lf was discovered only at the turn of the XX and XXI centuries. However, of all the antioxidant proteins of blood plasma, Cp was shown to possess the highest potential as the scavenger of superoxide anion radical and hypochlorous acid which are known, respectively, as the substrate and the product of reaction catalyzed by Mpo (Sharonov et al. 1988, 1989). For instance, Cp was identified as the protein adsorbed on immobilized Mpo and capable of suppressing its peroxidase activity (Segelmark et al. 1997). Direct interaction of Cp and Lf was documented in our laboratory when studying the properties of Cp in breast milk. It turned out that Lf is retained on immobilized Cp during the chromatography of breast milk and once these two proteins are mixed, they form a complex with the same electrophoretic mobility as the abnormal mobility of Cp in breast milk (Zakharova et al. 2000; Sokolov et al. 2006). We were the first to show that after injection of Lf and Mpo into the rat bloodstream, these proteins form heterologous complexes with the host Cp (Zakharova et al. 2000; Sokolov et al. 2007a). The Cp–Lf complex was isolated from breast milk and lacrimal fluid of healthy donors (Sokolov et al. 2006, 2013). Complexes composed of Cp, Lf, and Mpo at a time can be formed in vitro upon mixing the purified proteins, but also are found in biological fluids obtained from patients with inflammatory diseases (Sokolov et al. 2007a). Both Lf and Mpo were shown to interact with a number of other plasma proteins, such as albumin (Lampreave et al. 1990; Tiruppathi et al. 2004). Considering these data, we analyzed the selectivity of interaction of Cp with Lf after intraperitoneal injections of human and bovine LF to mice and rats (Fig. 1). We have observed previously the peculiar changes in electrophoretic mobility of Cp upon adding Lf to a
Biometals Fig. 5 Scheme of influence of Cp (blue arrows), Mpo (green arrows), Lf (red arrows) on functions of each other due to interactions
sample of blood plasma, however, such observations provided no evidence of the extent to which Lf is engaged in complexes with Cp and other plasma components. In this study we revealed no other Lf-positive electrophoretic band except its complex with either mouse or rat Cp. The absence of strict species specificity in interaction of these proteins indicates certain evolutionary conservatism of such complex. Similar results were obtained in our study of interaction of human and canine Mpo with Cp of a number of mammalian species (Sokolov et al. 2007a). Our observation that practically all the Lf of plasma co-immunoprecipitates with Cp (concentration of the former drops from 6.3 to 0.55 nM, see Fig. 3a) indicates that interaction of these two proteins can take place in plasma under normal conditions. This means that Kd 13 nM determined in a model system with Sepharose-immobilized Cp (Sokolov et al. 2009b) in reality can be even lower. Structural studies of complexes formed by Cp with Lf and Mpo indicate the stoichiometry 2Cp–2Lf–Mpo (Sokolov et al. 2009c; Samygina et al. 2013). Meanwhile, such methods as SAXS, laser correlation spectroscopy, and fluorescence studies showed the presence of a complex with 1:1 stoichiometry in solution (Sabatucci et al. 2007; Sokolov et al. 2009c; Ha-Doong et al. 2010). Lf participates in forming the complex 2Lf–2Cp–Mpo and, using a number of
mechanisms, in this way restricts the production of reactive oxygen species. Firstly, it enhances the oxidation of Fe2? by Cp and thus decreases the oxidative potential of the iron pool (Sokolov et al. 2005a, 2009b). Secondly, it binds Cu2?, which precludes the production of hydroxyl radicals in course of H2O2-induced degradation of Cp (Sokolov et al. 2012a). Lastly, Lf does not hamper the inhibition by Cp of the chlorinating activity of Mpo (Panasenko et al. 2008) and binds Fe3?; on the whole this decreases the production of hydroxyl radicals in reaction of HOCl with Fe2?. The physiological role of Cp as the inhibitor of Mpo activity is beyond doubt (Segelmark et al. 1997; Sokolov et al. 2008; Chapman et al. 2013). Cp inhibits Mpo even in the presence of C-reactive protein, which also interacts with Mpo (Xu et al. 2013). The activity of Mpo provoking the halogenative stress in inflammation (Panasenko et al. 2008) is important for antimicrobial protection of an organism. However, the role of this enzyme does not seem to be limited exclusively to the antibacterial defense. Individuals with autoantibodies against Mpo (ANCA) causing dissociation of its complex with Cp suffer from systemic vasculitis (Griffin et al. 1999; Xu et al. 2012). On the other hand, hereditary deficiency of Mpo results in the development of candidosis (Lehrer and Cline 1969).
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Mpo has no effect on the activity of Cp (Park et al. 2000), except for the capacity to enhance its oxidase activity in reaction with p-phenylene diamine (Sokolov et al. 2008). As shown by an X-ray study of the 2Cp–Mpo complex, in this case the effect of Mpo results from its direct contact with the binding site of p-phenylene diamine in Cp (Samygina et al. 2013). Each of the two protomers of Mpo interacts with one Cp molecule in the 2Cp–Mpo complex. This study has shown that upon elimination of Cp by immunoprecipitation blood plasma loses much of its capacity to inhibit the peroxidase activity of Mpo (Fig. 3b), but retains the ability to utilize hydrogen peroxide in Mpo-catalyzed oxidation of substrates that can be regarded as more physiological (Fig. 3c). Several substrates of Mpo-catalyzed peroxidase reaction are known to be present in blood plasma, e.g., tyrosine, urate, ascorbate, and nitrite (Vlasova et al. 2012). Along with those, however, the most specific substrate of the halogenating cycle of Mpo can be found, which is thiocyanate (van Dalen et al. 1997). Yet, in our experiments, the presence of Cp had no inhibitory effect on Mpo-catalyzed oxidation of thiocyanate (Fig. 3d) performed with the minimum Km and the highest specificity for the enzyme (van Dalen et al. 1997). It has been suggested that inhibition of Mpo by Cp is realized via direct contact of the peptide loop of the latter (amino acids 883–892) with the heme pocket of Mpo (Samygina et al. 2013). This means a competition between Cp and various substrates for the access to the active center of Mpo. Taking into account the results described above, SCN- seems to be a more successful competitor than Cp. We have shown previously that the efficiency of inhibition by Cp of Mpo peroxidase activity depends on the dimensions of a peroxidized substrate, but also on the integrity of the peptide loop in Cp (a.a. 883–892) connecting domains 5 and 6 (Sokolov et al. 2008). Properly this loop, as judged by the X-ray studies, directly contacts with the entrance into the heme pocket of Mpo, and the synthetic peptide RPYLKVFNPR mimicking its amino acid sequence efficiently inhibits the Mpo peroxidase activity (Samygina et al. 2013). CD spectra showed that when Mpo forms complexes with Cp and Lf, the ellipticity of its heme changes (Fig. 4). This observation supports the notion of interaction of Cp with the entrance into the heme pocket in Mpo, while Lf does not prevent Cp from inhibiting the chlorinating activity of Mpo (Panasenko
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et al. 2008). In line with this observation are the data on the Mpo heme ellipticity changes occurring upon forming a complex with low-density lipoproteins, which increases the chlorinating activity of Mpo (Delporte et al. 2014). A competitive mechanism of Mpo inhibition seems to be inefficient when such a specific substrate as thiocyanate is oxidized. It should be noted that Km for hydrogen peroxide does not change in the presence of Cp (Sokolov et al. 2008). Another important notion is that the catalytic turnover of Mpo (ca. 90/s) is two orders higher than that of Cp (0.5/s) when it oxidizes its most specific substrate, i.e., Fe2? (Stoj and Kosman 2003). In view of all of this, Cp has poor chances to reduce the highly reactive Compound I, contrary to the suggestion of Chapman et al. (2013). It seems important that by redirecting the activity of Mpo at oxidizing thiocyanate, but not chloride, Cp favors the production of hypothiocyanate, an efficient antimicrobial agent, though relatively harmless species for the host organism (Chandler and Day 2012). Antimicrobial synergism of Lf and Mpo functioning in the (pseudo)halogenating cycle is well known (Kokryakov 1999; Tenovuo 2002). Cp interfered with the antimicrobial effect of Mpo only when chloride was the substrate of the halogenating cycle. However, it did not suppress the antimicrobial activity of the system when, in PBS buffer, production of hypothiocyanate was possible in the (pseudo)halogenating cycle of Mpo (Table 1). The fact that Cp does not interfere with the antimicrobial synergism of Lf and Mpo and redirects the system towards synthesis of the relatively harmless antimicrobial agent, i.e., hypothiocyanate (Table 1), favors the notion of participation of the complex 2Cp–2Lf–Mpo in the protection of an organism against the halogenative stress developed in inflammation. Considering the results of Lf displacement from its complex with Cp by polyanionic structures (DNA, LPS, heparin) and by peptides mimicking the N-terminal cationic cluster of Lf (RRRR), it can be concluded that the site of interaction with Cp is located at the N-terminus of Lf (Pulina et al. 2002; Sokolov et al. 2006). However, anionic peptides homologous to amino acid stretches in Cp, such as DQVDKEDEDFQE (586–597), EVEWDYSPQREWE (721–734), and DENESWYLDD (905–914) did not displace this protein from its complex with Lf (Sokolov et al. 2006). These results are at variance with the data on interaction
Biometals
Fig. 6 Details of the interaction sites in Cp and Lf. a Labile Fe(II) binding sites (LS1 and LS2, red), anionic peptides 715–727 and 904–916 (blue) in Cp and Fe(III) binding sites in Lf (violet). b N-terminal cationic peptides 2–5 and 28–32 (magenta) in Lf and amino acid 50–109 and 929–1,012 stretches (blue) containing ligands for copper ions of trinuclear cluster (yellow triangle) in Cp
of Lf with two similar peptides, i.e., YYIAAVEVEWDYS (715–727) and FDENESWYLDDNI (904–916), obtained in experiments with absorption of labeled Lf on peptide library of Cp (White et al. 2012). In our recent model of the Cp–Lf complex (Samygina et al. 2013), these two stretches are not included in the contact area
with Lf (Fig. 6a). Cp contacts Lf using the amino acid stretches from its domains 1 and 6, which contain ligands for copper ions of trinuclear cluster in Cp (Fig. 6b). As shown by SAXS studies, there is no direct contact between Lf and Mpo within the ternary complex. On the one hand, this is in line with the observation that Lf does not affect the activity of Mpo (Panasenko et al. 2008), and on the other hand, it shows that the enzyme’s activity is likely to be inhibited by the N-terminal peptide of Lf (1–11) only if the latter is proteolyzed, but not intact Lf (van der Does et al. 2012). Protective effect of the Cp–Lf complex was documented when both proteins were applied as antioxidants in treatment of patients with malignancies (Edeleva et al. 2001). Experimental data on involution of mammary gland in mice provided evidence that genes encoding Cp and Lf become activated at that period (Nakamura et al. 2006). The Cp–Lf complex is found in breast milk, which is more evidence of the protective effect of these two proteins (Sokolov et al. 2006). Our recent study (Zakharova et al. 2012) showed that apo-Lf has a pronounced anti-hypoxic effect and possesses the properties of a physiological mimetic of hypoxia as it stimulates the synthesis of Cp and erythropoietin by stabilizing the hypoxia-inducible factor 1-alpha (Zakharova et al. 2012). Iron-saturated Lf has no such features, which allows suggesting a negative feedback in regulation of the system that includes Cp and apo-Lf. Firstly, apo-Lf increases the ferroxidase activity of Cp and becomes saturated with Fe3? (Sokolov et al. 2005a, 2009b). Secondly, apo-Lf triggers the synthesis of Cp and erythropoietin, which favors an increase of the plasma ferroxidase activity (egress of iron from tissue storages) and stimulates erythropoiesis. These two mechanisms provide a good explanation of the anti-anemic properties of apo-Lf (Pulina et al. 2010; Zakharova et al. 2012). Moreover, once saturated with iron, Fe2–Lf is unable to activate the hypoxia-inducible synthesis of Cp and erythropoietin (Zakharova et al. 2012). Under conditions of focal inflammation and poor oxygenation, Cp can become a factor that favors iron binding by apo-Lf with further realization of the antimicrobial function of the latter. The selectivity of interaction of the three metal-containing proteins, i.e., Cp, Lf, and Mpo, which participate in inflammatory reactions and antimicrobial defense of an organism, does not seem accidental, since Cp is the preferred
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Biometals
partner of Lf among other plasma proteins and is capable of specific inhibition of the peroxidase and chlorinating activities of Mpo, leaving unaffected the production of hypothiocyanate, an antimicrobial agent. Acknowledgments This study was supported by RFBR grants § 12-04-00301; 13-04-01191, MK-6062.2014.4 and by the Program ‘‘Human Proteome’’. The authors are grateful to Professor V. N. Kokryakov for generously providing leukocytes of healthy donors, to Dr. M. N. Berlov for kind assistance in mastering the evaluation of antimicrobial activity of proteins, to Dr. M. O. Pulina and Dr. A. N. Skvortsov for CD-spectra measurement.
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