Lactoferrin, myeloperoxidase, and ceruloplasmin:complementary gearwheels cranking physiologicaland 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 cerulo-

plasmin (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 intraperi-

toneal injection of Lf to rodents, the ‘‘Cp–Lf’’

complex has been shown to appear in their blood-

stream. Cp prevents the interaction of Lf with

protoplasts of Micrococcus luteus. Upon immunopre-

cipitation 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 thiocy-

anate, as the substrate for Mpo is abrogated when Cp is

added. Hence, Cp can be regarded as an anti-inflam-

matory 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. Struc-

ture 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

Introduction

Ceruloplasmin (Cp, ferro:O2–oxidoreductase) is the

copper-containing protein of vertebrate blood plasma.

Human Cp has M *132 kDa. Along with the soluble

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: biochemsokolov@gmail.com

A. V. Sokolov � V. B. Vasilyev

Saint-Petersburg State University, Mendeleevskaya Line,

Saint Petersburg 199000, Russia

A. V. Sokolov � V. A. Kostevich

Research Institute of Physico-Chemical Medicine,

ul. Malaya Pirogovskaya 1a, Moscow 119435, Russia

V. R. Samygina

Institute of Crystallography, RAS, Leninsky pr 59,

Moscow 117333, Russia

123

Biometals

DOI 10.1007/s10534-014-9755-2

Cp secreted by hepatocytes into plasma, another form

of this protein is known to be anchored by glycosyl-

phosphoinositol 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 anti-

inflammatory 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), glutathi-

one-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 metallo-

proteinase 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

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 sup-

pression 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 (Pana-

senko 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 protein-

ases, 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

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123

properties of Cp were studied, i.e., solely non-prote-

olyzed Cp was capable of efficient protection of low-

density lipoproteins from proatherogenic modification

being the result of the chlorinating activity of Mpo

(Sokolov et al. 2014).

Cp crystal structure obtained at 2.6-A resolution

distinguishes six ß-barrel homologous domains con-

nected 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.

1999). According to a 1.95-A crystal structure (Blair-

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 anion-

radical, 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 Mpo-

inhibiting potential of plasma Cp, and on the

interaction of the latter with the synergistic anti-

microbial system containing Lf and Mpo–H2O2–

chloride/thiocyanate.

Materials and methods

The following reagents were used: arginine, glycerol,

Coomassie R-250, mercaptoethanol, ammonium per-

sulfate, 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 (Labora-

tory MEDIGEN, Russia); heparin (SPOFA, Poland).

All solutions were prepared using apyrogenic deion-

ized water with resistivity 18.2 MX�cm. 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 fol-

lowing 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 ion-

exchange 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-Sephar-

ose, 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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123

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 func-

tioning 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 concentra-

tion 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(3-

ethylbenzotriazoline-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 contain-

ing 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

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 immunoglob-

ulins 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 immu-

noprecipitation 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. Measure-

ments were made using dismountable 0.05-cm cuv-

ettes (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

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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 anti-

microbial agents. Proteins (Cp, Lf bMpo) 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 SDS-

free 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 con-

forms 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 nitrocellu-

lose membrane bound antibodies to human or bovine

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 protop-

lasts. 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 pre-

cipitation 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 introduc-

tion of human Mpo into the bloodstream of rats also

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)

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123

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-con-

taining 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 propor-

tionally 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 oxi-

dation 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

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)

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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 far-

UV, 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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123

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)haloge-

nating cycle of Mpo. These results are in full

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 accom-

panying 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 sup-

pressing its peroxidase activity (Segelmark et al.

1997). Direct interaction of Cp and Lf was docu-

mented 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 com-

plexes 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 intraperi-

toneal 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

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 MIC50a , 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

Biometals

123

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 electropho-

retic band except its complex with either mouse or rat Cp.

The absence of strict species specificity in interaction of

these proteins indicates certain evolutionary conserva-

tism 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). Mean-

while, 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. Indi-

viduals 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).

Fig. 5 Scheme of influence

of Cp (blue arrows), Mpo

(green arrows), Lf (red

arrows) on functions of each

other due to interactions

Biometals

123

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 (Soko-

lov 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 reac-

tion 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 thio-

cyanate (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 com-

plexes 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

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 spe-

cies for the host organism (Chandler and Day 2012).

Antimicrobial synergism of Lf and Mpo function-

ing 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 hypothi-

ocyanate 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., hypoth-

iocyanate (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 pro-

tein from its complex with Lf (Sokolov et al. 2006).

These results are at variance with the data on interaction

Biometals

123

of Lf with two similar peptides, i.e., YYIAAVEVEW-

DYS (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 com-

plex. 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 doc-

umented when both proteins were applied as antiox-

idants 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 erythropoi-

etin (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

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

Biometals

123

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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