Tài liệu Báo cáo khoa học: Multiple enzymic activities of human milk lactoferrin ppt

Multiple enzymic activities of human milk lactoferrin Tat’yana G. Kanyshkova 1 , Svetlana E. Babina 2 , Dmitry V. Semenov 1 , Natal’ya Isaeva 3 , Alexander V. Vlassov 1 , Kirill N. Neustroev 4 , Anna A. Kul’minskaya 4 , Valentina N. Buneva 1 and Georgy A. Nevinsky 1 1 Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, Novosibirsk, Russia; 2 Novosibirsk State University, Novosibirsk, Russia; 3 Institute of Cytology and Genetics, Siberian Division of the Russian Academy of Sciences, Novosibirsk, Russia; 4 Petersburg Nuclear Physics Institute of Russian Academy of Sciences, St Peterburg, Russia Lactoferrin (LF) is a Fe 3+ -binding glycoprotein, first recognized in milk and then in other human epithelial secretions and barrier fluids. Many different functions have been attributed to LF, including protection from iron- induced lipid peroxidation, immunomodulation and cell growth regulation, DNA binding, and transcriptional acti- vation. Its physiological role is still unclear, but it has been suggested to be responsible for primary defense against microbial and viral infection. We present evidence that different subfractions of purified human milk LF possess five different enzyme activities: DNase, RNase, ATPase, phosphatase, and malto-oligosaccharide hydrolysis. LF is the predominant source of these activities in human milk. Some of its catalytically active subfractions are cytotoxic and induce apoptosis. The discovery that LF possesses these activities may help to elucidate its many physiological functions, including its protective role against microbial and viral infection. Keywords: enzymic activities; human milk; lactoferrin; protection. Lactoferrin (LF) is a single polypeptide chain of 76–80 kDa, containing two lobes [1], each of which binds one Fe 3+ ion and contains one glycan chain [2]. It was first recognized in milk and then in other human epithelial secretions and barrier body fluids [3–6]. Many different functions have been attributed to LF, including protection from iron- induced lipid peroxidation, immunomodulation and cell growth regulation [6,7], DNA binding [6], RNA hydrolysis [8,9], and transcriptional activation of specific DNA sequences [10,11]. It is a potent activator of natural killer cells [12] and may have an antitumor role [7,13], an activity that is independent of iron. LF also influences granulo- poiesis [14], antibody-dependent cytotoxicity [15], cytokine production [16], and growth of some cells in vitro [17]. The physiological role of LF and the mechanisms underlying these activities are still unclear, but it has been suggested to be responsible for primary defense against microbial and viral infection [3,5]. LF is a protein of the acute phase; the highest concentration is usually detected in the inflamma- tory nidus. It is detected in the blood of newborn babies several hours after feeding, and can readily penetrate any cell and nuclear membrane [18]. Owing to its antiviral and antimicrobial activities, LF increases the passive immunity of newborns. It was initially suggested that the antimicro- bial properties of LF may be attributed to its iron-binding capacity; removal of iron from the microbial environment is an important defense mechanism as it is needed for the proliferation of microflora [19]. Many micro-organisms express surface receptors for LF and it may show different iron-independent antimicrobial and antiviral properties [20,21], the mechanisms of which are still a matter of debate. We have proposed that, as LF is a relatively small protein, its polyfunctional properties may result from its existence in several oligomeric forms that have different activities, and that its oligomerization and dissociation are under the control of specific ligands such as ATP [22,23]. In support of this idea, we have shown recently that LF possesses an ATP-binding site and that interaction of the protein with ATP leads to changes in its interaction with polysaccharides, DNA and proteins [23]. We have further demonstrated that LF possesses two DNA-binding sites, which interact with specific and nonspecific DNAs in an antico-operative manner and may coincide or overlap with the known polyanion-binding and antimicrobial domains of the protein [24]. Here we show that this extremely polyfunctional protein possesses five enzyme activities (DNase, RNase, ATPase, phosphatase, and malto-oligosaccharide hydrolysis). The RNA-hydrolyzing and DNA-hydrolyzing subfractions of LF may contribute to its protective role through hydrolysis of viral and bacterial nucleic acids. In addition, we show that some catalytic forms of LF are cytotoxic and Correspondence to G. A. Nevinsky, Laboratory of Repair Enzymes, Novosibirsk Institute of Bioorganic Chemistry, 8, Lavrentieva Ave., 630090, Novosibirsk, Russia. Fax: 007 3832 333677, Tel.: 007 3832 396226, E-mail: nevinsky@niboch.nsc.ru Abbreviations: LF, human milk lactoferrin; EPS, 4-nitrophenyl 4,6-O-ethylidene-a- D -maltoheptaoside. (Received 23 January 2003, revised 13 May 2003, accepted 11 June 2003) Eur. J. Biochem. 270, 3353–3361 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03715.x apoptosis-inducing agents. These findings suggest that LF of milk and other human epithelial secretions and body fluids may contribute to cell defense by policing the function of human cells. Materials and methods Materials and chemicals Reagents were obtained mainly from Sigma and Merck. 4-Nitrophenyl 4,6-O-ethylidene-a- D -maltoheptaoside (EPS) was purchased from Boehringer Mannheim (Germany). We also used heparin, antibodies to human LF (Sigma), DEAE-cellulose DE-52 (Whatman), heparin–Sepharose and Cibacron Blue–Sepharose CL-6b (Pharmacia Fine Chemicals), and Toyopearl HW-55 fine (Toyo Soda). Radioisotopes were purchased from Amersham (3000 CiÆmmol )1 ). Purification and analysis of LF LF was purified and analyzed individually from the milk of each of 30 donors. Electrophoretically and immuno- logically homogeneous LF was obtained by sequential chromatography of human milk proteins on DEAE- cellulose, heparin–Sepharose, and anti-LF–Sepharose col- umns [23,24]. It was further chromatographed on a Cibacron Blue–Sepharose column (15 · 5 mm) as des- cribed previously [9] with the following modifications: the column was equilibrated in 20 m M sodium acetate, and LF (pH 4.0) was loaded and eluted with 50 m M Tris/HCl buffer, pH 7.5, and then with a concentration gradient of NaCl (0–1 M ) in the same buffer. Fractions were collected, dialyzed at 4 °C for 12 h against 10 m M Tris/HCl, pH 7.5, and their enzyme activities measured (see below). The N-terminal amino-acid sequences of five subfractions of LF (see Fig. 2) were determined by the phenyl isothiocyanate procedure using a liquid-chromatography system (Hewlett-Packard Co.). SDS/PAGE and immuno- blotting analysis were performed as described previously [23,24]. Gel filtration of LF after pH shock was performed as in [25]. Synthesis of 2¢,3¢-dialdehyde derivatives of substrates and affinity labeling of LF was carried as described previously [23,24]. Nucleic acid-hydrolyzing and phosphatase activity of LF DNA-hydrolyzing activity was assayed in a mixture (20 lL) containing 150 ng supercoiled pBR322 DNA or phage k DNA, 5.0 m M MgCl 2 ,1.0m M EDTA, 20 m M Tris/HCl buffer, pH 7.5, and 0.1–1.0 l M LF incubated for 1–2 h at 37 °C. The cleavage products were analyzed by electro- phoresis on a 1.0% agarose gel and ethidium bromide staining; gels were photographed and the films scanned to calculate relative activities. For evaluation of the nuclease activities, various 5¢-[ 32 P]ribo-oligonucleotides and deoxyribo-oligonucleo- tides (1–10 l M ) were incubated with 0.1–1.0 l M LF in 10–20 lL reaction mixture containing 20 m M Tris/HCl, pH 7.5, and 1.0 m M EDTA with or without 5.0 m M MgCl 2 for 2–6 h at 37 °C, and the products were analyzed in a 20% polyacrylamide gel containing 8 M urea. Phosphatase activity was assayed under the same condi- tions: removal of [ 32 P]P i from 5¢-[ 32 P]oligonucleotides was assayed by TLC in dioxane/NH 4 OH/water (5 : 1 : 4, by vol.) on Kieselgel plates (Merck). After chromatography, theplatesweredried,the[ 32 P]products localized by autoradiography, and their radioactivity was measured by Cherenkov counting. The same conditions were used to study cleavage of human tRNA Phe prepared as described previously [26,27] and labeled at the 5¢ end [26,27]. tRNA (0.1 lgÆmL )1 ;10 5 Cherenkov counts per sample) were incubated at 37 °Cfor 30 min with LF (0.1–1.0 l M )orRNaseA(5· 10 )5 mgÆmL )1 ), and the products were analyzed by electropho- resis in 15% polyacrylamide/8 M urea gels, with partial RNase T1 and imidazole digests of the tRNAs run in parallel to identify the products [27]. Quantification was performed by analysis on a Fujix BioImaging Analyzer BAS 2000 System (Fuji). Nucleotide-hydrolyzing activity Reaction mixtures (10–20 lL) contained optimal concen- trations of the standard components (1.0 m M MgCl 2 , 0.3 m M EDTA, 50 m M Tris/HCl, pH 6.8, 100 m M NaCl), 0.05–0.2 mgÆmL )1 LF, and different concentrations of [c- 32 P]ATP, and were incubated for 0.5–6 h at 37 °C. For screening column fractions during purification of IgG, 2–3 lL of each fraction was incubated in 10 lL standard reaction mixture containing 0.1 m M [c- 32 P]ATP or [a- 32 P] ATP (10 7 c.p.m.). The products of nucleotide hydrolysis were analyzed by TLC in 0.25 M KH 2 PO 4 buffer, pH 7.0, on polyethyleneimine–cellulose plates (Merck); the plates were dried, and the positions of various 32 P-labeled products were identified using [ 32 P]nucleotide standards and auto- radiography. The radioactivity of the regions corresponding to P i and different nucleotides was measured by Cherenkov counting. Amylase activity of LF Reaction mixtures containing 30 m M Tris/HCl, pH 7.5, 1m M NaN 3 ,1–5m M oligosaccharide, and 1–10 l M LF were incubated at 37 °C. The products of hydrolysis of EPS (12 mgÆmL )1 ) and eight other oligosaccharides were iden- tified using TLC on Kieselgel 60 plates (Merck; ethanol/ butanol/water, 2 : 2 : 1, by vol.). The plates were dried, sprayed with 5% H 2 SO 4 in propan-2-ol and again dried at 110 °C to visualize the carbohydrates as described in [28]. Specificity of the hydrolysis of malto-oligosaccharides was determined after separation of products by TLC and HPLC on a Lichrosorb-NH 2 column [28]. One unit of activity was defined as the quantity of LF that released 1 lmolÆL )1 reducing sugar from maltoheptaose per min at 37 °C, similar to known amylases [28]. In situ gel assay of enzymic activities Enzymic activities of LF were determined in situ by SDS/ PAGE (12% gels). To detect RNase and DNase activities, gels contained 200 lgÆmL )1 yeast total RNA or 20 lgÆmL )1 calf thymus DNA [25,29–31] added to the gel solution before polymerization. After electrophoresis, the gel was 3354 T. G. Kanyshkova et al.(Eur. J. Biochem. 270) Ó FEBS 2003 washed with a solution of 4 M urea and twice with water to remove SDS, and then to allow protein renaturation it was incubated for 16 h at 37 °Cin20m M Tris/HCl buffer, pH 7.5, containing 1 m M EDTA and 5.0 m M MgCl 2 .To reveal the regions of DNA or RNA hydrolysis, the gel was stained with ethidium bromide. Proteins were revealed by Coomassie R250 staining. ATPase was detected using our modification of the Gomori method for histochemical determination of ATP- ases [32]. After electrophoresis, SDS was removed by incubating the gel for 30 min at 37 °C with water (5 times) and then with 0.5 M sodium acetate, pH 6.8 (3 times). To allow protein renaturation and to detect P i resulting from ATP hydrolysis, the gel was incubated for 12 h at 37 °Cin 5m M sodium acetate (pH 6.8) containing 1.0 m M MgCl 2 , 3m M Pb(NO 3 ) 2 , and 100 lCi [c- 32 P]ATP. Nonspecifically adsorbed Pb(NO 3 ) 2 was removed by washing the gel 3 times (10 min) with water, then with hot 5% acetic acid and again with water. The gel was autoradiographed to detect [ 32 P]P i . Phosphatase and amylase activity was determined using gels without substrates. After electrophoresis, SDS was removed by incubating the gels as for analysis of DNase activity. The gels were then cut into 2 mm slices which were incubated with 20 m M Tris/HCl, pH 7.5, at 4 °C for 12 h. The gel slices were removed by centrifugation, and amylase or phosphatase activity was assayed using 5¢-[ 32 P](pT) 8 or EPS as described above. In situ gel assays of the enzymic activities of human milk proteins (3–7 lL dialyzed human plasma) and the limited proteolytic cleavage products of LF (2–7 lg) were as described above for purified LF. Partial proteolytic cleavage of LF was performed using 0.1–0.5% trypsin (w/w of LF) in 0.1 M Tris/HCl (pH 8.2)/25 m M CaCl 2 at 37 °Cfor4h[33]. K m and V max for the hydrolysis of different substrates were determined by the method of initial rates using nonlinear regression analysis. Errors in the values were within 10–30%. Cytotoxicity assays Tumor cell lines L929 (mouse fibroblasts) and HL-60 (human promyelocytes) were cultured at 37 °Cin0.1mL Dulbecco’s modified Eagle’s medium containing 5% fetal bovine serum to confluence. They were then treated with mitomycin (1 mgÆmL )1 ) for 5 h and washed with medium. Fresh medium containing different concentrations (10–100 n M ) of subfractions of LF-1 to LF-5 (see below) or tumor necrosis factor (10 n M ) was then added. The cells were cultivated for a further 12–48 h, and the percentage of dead cells, counted after staining with trypan blue every 3–12 h, was compared with that in a control culture. The results are mean ± SD from at least three different experiments using three preparations of one to five fractions of LF (see below) from different milk donors. DNA fragmentation and annexin V staining of apoptotic cells Cells were incubated with LF subfractions (10–100 n M )as described above for 12–24 h, lysed, centrifuged at 20 000 g, and the supernatant was extracted with phenol/chloroform. DNA fragments were electrophoresed in a 1.2% agarose gel and visualized with ethidium bromide [34]. An Annexin-V- Fluorescein kit was used for analysis of apoptosis according to instructions provided by the manufacturer (Boehringer- Mannheim). Results Purification and characterization of LF subfractions We isolated and analyzed separately LF preparations from the milk of 30 different healthy mothers. LF was purified from the fraction of human milk that was not adsorbed by DEAE-cellulose by chromatography on heparin–Sepharose [22–24], and electrophoretically homogeneous LF was purified on anti-LF–Sepharose (Fig. 1). As shown previ- ously [9], human milk LF could be separated into several distinct isoforms by affinity chromatography on Cibacron Blue–Sepharose. We found that chromatographically, electrophoretically, and immunologically homogeneous LF (after anti-LF–Sepharose chromatography) contains subfractions with different affinities for Cibacron Blue– Sepharose (Fig. 2A–C). They all possessed the N-terminal amino-acid sequence reported for LF, Gly-Arg-Arg-Arg- Arg-Ser-Val-Glu [9], and also a product of partial proteo- lytic cleavage [23,24]. Four prominent protein peaks corresponding to LF were eluted from Cibacron Blue–Sepharose (Fig. 2). The main subfraction of LF (peak 4, Fig. 2) had the highest affinity for this sorbent. Three additional subfractions (peaks 1–3, Fig. 2) represented  10–20% of the total LF dependent on the milk donor. The first protein peak showed no enzyme activity, but the three other peaks showed oligonucleotide 5¢-phosphatase, DNase, RNase, ATPase, and malto-oligo- saccharide-hydrolyzing activities, each activity being eluted in several peaks (Fig. 2A–C). The LF subfraction corres- ponding to peak 2 possessed four different activities: phosphatase, DNase, RNase, ATPase. Eluate correspond- ing to protein peak 3 showed three prominent peaks of oligonucleotide 5¢-phosphatase activity (Fig. 2B) and two peaks of RNase activity (Fig. 2C). Interestingly, two Fig. 1. Chromatography of LF on anti-LF–Sepharose. Solid line, A 280 ; symbols, activity as percentage of the fraction with maximal activity. Aliquots (1–3 lL) of column fractions were incubated with phage k DNA (7.5 lgÆmL )1 ), 5¢-[ 32 P](pU) 10 or 5¢-[ 32 P](pT) 8 (5 l M ), [c- 32 P]ATP (0.5 l M ), or EPS (12 mgÆmL )1 )at37°C for 1–4 h. The details of the experiment are given in Materials and methods. Ó FEBS 2003 Enzymic activities of lactoferrin (Eur. J. Biochem. 270) 3355 additional DNase peaks were revealed in fractions 15–30 (position of peak 3), but profiles of these activity peaks did not correlate with that for protein peak 3, with the second and third DNase peaks occurring between protein peaks 2 and 3 and protein peaks 3 and 4, respectively (Fig. 2A). There was good correlation between the positions of two peaks of malto-oligosaccharide-hydrolyzing activity, and protein peaks 3 and 4 (Fig. 2C). Taking all the data together, we divided the LF subfractions possessing differ- ent activities into five subfractions (LF-1 to LF-5) as shown in Fig. 2. The data on the relative activities of LF-1 to LF-5 in the different enzymatic reactions are collected in Table 1. The samples of all 30 donors of LF not fractionated on Cibacron Blue–Sepharose had detectable levels of all five activities, but these activities were remarkably dependent on the donor. LF preparations from seven different donors were analyzed in more detail after fractionation of LF subfractions on Cibacron Blue–Sepharose. Table 1 shows the range of variation in the relative activities of the subfractions depending on the milk donor. Interestingly, the phosphatase activity of LF varied more than other activities when compared with the DNAse activity. Phosphatase activity varied between 20% and 80% of the DNAse activity for different donors. Catalytic activities of LF Five enzyme activities were ascribed specifically to LF, as shown by several different methods developed in our laboratory to study the enzyme activities of catalytic antibodies [29,30,35,36]. Chromatography of purified (but not fractionated on Cibacron Blue–Sepharose) LF on Sepharose bearing immobilized antibodies to LF led to essentially complete binding of LF to the sorbent (Fig. 1). During protein elution from this column with an acidic buffer, pH 2.6, the five activities analyzed coincided exactly with the LF peak, and there were no other peaks of activity. The same result was obtained with the separated protein subfractions LF-1 to LF-5. In addition, incubation all five enzyme peaks corresponding to the subfractions (Fig. 2) with immobilized LF antibodies led to essentially complete binding of LF to the sorbent and disappearance of all five enzyme activities from the solution. All of the enzyme activities were suppressed by addition of polyclonal LF antibodies to the reaction mixtures (data not shown). Usually strong noncovalent protein complexes dissociate under acidic conditions. To ensure that other proteins were not tightly bound to purified LF, the combined fractions from Cibacron Blue–Sepharose (fractions 5–33, Fig. 2) were incubated at pH 2.4, which usually dissociates strong noncovalent complexes. They were then repurified by gel filtration. A single peak corresponding to LF was recovered (see Fig. 4A), which contained 80–95% of all five enzyme activities loaded on the column. There were no other peaks of activity or protein. The same result was obtained for separated subfractions LF-1, LF-3 and LF-5 (Fig. 2), corresponding to LF from the milk of three different donors (data not shown). Affinity labeling of enzymes with 32 P analogs of their specific ligands is the most sensitive method for revealing any contaminating proteins interacting with the same ligands. As we showed previously, LF possesses an ATP- binding site, which became labeled after incubation with an affinity probe for ATP-binding sites, the 2¢,3¢-dialdehyde derivative of ATP (oxATP), with a stoichiometry of 1.0 mol [a- 32 P]oxATP bound per mol LF [23]. In addition, LF Fig. 2. Chromatography of LF on Cibacron Blue–Sepharose. (A) DNAse (d) and ATPase (s); (B) 5¢-oligonucleotide phosphatase (n); (C) RNase (*) and amylase (j). Aliquots (1–3 lL) of column fractions were used to determine DNAse (k DNA), RNase {5¢-[ 32 P](pU) 10 }, phosphatase {5¢-[ 32 P](pT) 8 }, ATPase ([c- 32 P]ATP), and amylase (EPS) activities as in Fig. 1. The examples of determinations of DNase (agarose electrophoresis), ATPase (TLC), phosphatase (TLC), RNase (PAGE) and amylase (TLC) are given on the right (for details, see Materials and methods). Lane numbers correspond to the numbers of eluate fractions; C, substrate alone. 3356 T. G. Kanyshkova et al.(Eur. J. Biochem. 270) Ó FEBS 2003 possesses two DNA-binding sites with different affinities for oligonucleotides, which can be labeled after incubation with affinity probes for DNA-binding and RNA-binding sites, the 2¢,3¢-dialdehyde derivatives of different specific and nonspecific [5¢- 32 P]oligonucleotides, including [5¢- 32 P]- d(pT) 9 r(pU) and [5¢- 32 P](pU) 10 [24]. These modifications fulfiled the known criteria of affinity modification [23,24]. As judged by SDS/PAGE analysis, both the LF polypeptide purified using anti-LF–Sepharose and the combined LF-1–4 subfractions were specifically affinity-labeled by 32 Panalogs of [5¢- 32 P]d(pT) 9 r(pU) and [5¢- 32 P](pU) 10 oligonucleotides, and by a-[ 32 P]oxATP and showed 1.4, 1.2, and 1.0 binding sites per LF molecule, respectively (Fig. 3A). As the sample preparation for SDS/PAGE dissociates any protein complex, and the electrophoretic mobility of hypothetical contaminating DNases, RNases, phosphatases, and ATP- ases could not possibly all coincide with that of LF, the detection of a 32 P-labeled band in the gel region corresponding to LF, together with the absence of any other labeled bands, provides direct evidence that LF does not contain contaminating enzymes. In addition, immobilized LF antibodies bound LF labeled by these affinity reagents almost completely (data not shown). A further approach provided direct evidence that LF possesses five different enzyme activities. DNase and RNase activities of the LF polypeptide were shown by in-gel in situ assays after SDS/PAGE in gels containing DNA or RNA (Fig. 3). Staining with ethidium bromide after development of nuclease activity revealed a sharp dark band on a fluorescent background of DNA or RNA (Fig. 3A, lanes 6and7). We also used an in-gel ATPase assay, adapted from Gomori’s method of P i precipitation used previously for histochemical study of ATPase activity [32], for in situ detection of enzyme-dependent formation of P i in SDS/ polyacrylamide gels after establishing conditions for preci- pitation of Pb 2 (PO 4 ) 3 in regions of gels containing P i and for efficient removal of Pb salts nonspecifically adsorbed to proteins. Enzyme-dependent formation of Pb 2 (PO 4 ) 3 ,detec- ted by autoradiography, showed a 32 P-labeled product only in the band corresponding to LF (Fig. 3A, lane 8). Phosphatase (lane 9) and amylase (lane 10) activities of LF were also shown by in-gel assays (Fig. 3A). These results were obtained using a mixture of separated subfractions LF-1 to LF-4 (Fig. 2) from the milk of three different donors. In addition, we analyzed, using in situ Table 1. Relative activity of different subfractions of LF obtained by chromatography on Cibacron Blue–Sepharose (Fig. 2). Thedatashowtherelative activity of different subfractions of LF from the milk of one donor (Fig. 2) and the range of variation in the relative activities of LF subfractions purified from milk of seven different donors (in the parentheses). In all cases, the activity of one subfraction with maximal activity was taken as 100% and the activity of other subfractions was calculated as a percentage of that with maximal activity. Zero indicates the absence of any activity in the subfraction analyzed, but in some cases there may be detectable activity from closely positioned peaks of activity. Enzyme activity Relative activity of different LF fractions (%) Additional dataLF-1 LF-2 LF-3 LF-4 LF-5 DNase 100 (100) 0 (0–5) 0 (0) 33 (22–41) 0 (0) 16 (7–20), between LF-2 and LF-3 ATPase 100 (100) 0 (0) 0 (0) 0 (0) 53 (39–62) – Phosphatase 63 (41–68) 26 (17–36) 100 (100) 17 (8–25) 0 (0) – RNase 100 (100) 44 (31–49) 63 (45–69) 0 (0) 0 (0) – Amylase 0 (0) 0 (0) 37 (23–40) 0 (0–6) 100 (100) – Fig. 3. In-gel detection of enzyme activities of the LF polypeptide, its tryptic fragments and proteins of human milk in SDS/12% polyacryl- amide gels. (A) Lane 1, silver stained; lane 2, immunoblot (alkaline phosphatase-conjugated anti-LF); lanes 3–5, LF affinity-labeled by periodate-oxidized [a- 32 P]ATP (3), 5¢-[ 32 P](pU) 10 (4), or 5¢-[ 32 P]- d(pT) 9 r(pU) (5) (autoradiographs); lanes 6–7, (the negatives of the films are shown), DNase and RNase in gels containing calf thymus DNA (6) or yeast RNA (7); lane 8, ATPase; lane 9, phosphatase; lane 10, amylolytic activity (RA, relative activity), respectively {2–3 mm gel slices incubated with 5¢-[ 32 P](pT) 8 or EPS}. (B) Lanes 1 and 2, Comassie R250-stained LF (1) and its tryptic fragments (2); lanes 3–6, the negatives of the films corresponding to DNase (3, 4), RNase (5) and ATPase {6; [ 32 P]Pb 3 (PO 4 ) 2 activity of LF (3) and its tryptic fragments (4–6)}. (C) In situ analysis of DNAse (lanes 1, 2), RNase (lanes 3, 4) and ATPase (lanes 5, 6) of human milk proteins (3–7 lL human plasma); Comassie R250-stained proteins (1, 3, 5), DNase (2), RNase (4) (the negatives of the films) and ATPase (6) activity {[ 32 P]Pb 3 (PO 4 ) 2 }. Ó FEBS 2003 Enzymic activities of lactoferrin (Eur. J. Biochem. 270) 3357 detection of enzyme activities of separated LF-1 (DNAse, RNase, ATPase), LF-3 (phosphatase, RNase, amylase), and LF-5 (ATPase, amylase), subfractions corresponding to LF from two donors, and obtained the the same result as for the LF-1–LF-4 mixture (data not shown). Mild treatment of LF with trypsin at pH 8.2 cleaves the molecule between Lys283 and Ser284 into a N-tryptic lobe (molecular mass  30 kDa) and C-tryptic (molecular mass  50 kDa) fragment [33]. The high-affinity DNA-binding site is located in the N-domain of LF [24], and the ATP- binding site in the C-terminal domain [23]. We obtained these fragments by tryptic hydrolysis of LF (not fraction- ated on Cibacron Blue–Sepharose) and analyzed their activities by SDS/PAGE. Figure 3B shows that the N-tryptic fragment catalyses the hydrolysis of DNA and RNA, whereas the C-terminal domain is responsible for the hydrolysis of ATP. In addition, modification of LF with oxATP did not lead to a decrease in its DNase and RNase activities (data not shown). This result is consistent with the localization of nucleic acid-binding and ATP- binding sites in the N and C lobes, respectively [23,24]. Together, these observations show that all five enzyme activities are intrinsic properties of LF. Substrate specificity of LF Fractions of LF with maximal activity in each of the five enzymatic reactions (Fig. 2) were used for more detailed studies. LF DNase had properties that distinguished it clearly from other known DNases. Its pH optimum was 7.0–7.5, a value markedly higher than that (5.0–5.5) [25,30] of human blood DNase II, and the activity was significantly (100–150%) activated by 100 m M NaCl whereas DNase I is 70% inhibited by 50 m M NaCl [25,30]. Cleavage of oligonucleotides and DNA by LF was stimulated 3–5-fold by Ca 2+ ,Cu 2+ ,andZn 2+ and8–9-foldbyMn 2+ and Mg 2+ ions. In contrast with known human DNases, LF DNase was activated by ATP, dATP and NAD (150 m M ) by a factor of 1.5–2.5 (data not shown). Subfraction LF-1 from the milk of different donors cleaved the deoxyribo-oligonucleotides GGCACTTAC, TAGAAGATCAAA, and ACTACACATCTACA, corres- ponding to sequences to which it is known to bind and activate transcription [10], as well as different d(pN) 10 with comparable K m values (3.7–7.2 l M )butwith different efficiencies (k cat ¼ 0.006–0.042 min )1 ; Table 2). Interestingly, K m and k cat for different homo-d(pN) 10 and homo-(pN) 10 (K m ¼ 3.0–5.0; k cat ¼ 0.026–0.029 min )1 ) were comparable (Table 2). The K m values for different homo-d(pN) 10 and homo-(pN) 10 molecules were also comparable (a difference within 40%) for LF-1 subfractions of nonfractionated LFs from milk of seven different donors (data not shown). More significant differences were observed for k cat values for LF-1 and nonfractionated LF in milk from different donors, but these values correlate with the variation in the relative DNase and RNase activities of nonfractionated LF preparations and their LF-1 subfractions and the relative amounts of LF-1 in total LF [the main LF-5 fraction (80–90%) does not possess DNase activity]. The LF-1 fraction from milk of different donors cleaved plasmid DNAs (phage k, pBR-322, Bluescript) 30–200 timesfaster(k cat ¼ 2–9 min )1 ) than the oligonucleotides, a rate comparable to that of some DNA restriction endonuc- leases [25]. A similar result was obtained for the relative activities of nonfractionated LF in the hydrolysis of oligonucleotides and plasmid DNA. RNase activity has been reported previously in human milk LF [8,9], and we found in the present study that its substrate specificity distinguishes it from RNase A and all other human sera and milk RNases, as shown by its pattern of cleavage of tRNA Phe . It showed major cleavage sites in the double-stranded UGUG region between nucleotides 47 and 48, 50 and 51, and especially 52 and 53, which are unique (Fig. 4B). The data on the difference in tRNA hydrolysis by LF and RNase A are summarized in Fig. 4C. Seven LF-1 preparations from different donors hydro- lyzed ATP with K m ¼ 0.5 ± 0.2 m M ,andthek cat values varied in the range (0.5–4) · 10 )3 min )1 .TheK m (ATP) values for LF-1 preparations do not differ significantly from those for corresponding nonfractionated LFs (K m ¼ 0.2–1.0 m M ). Of the nine oligosaccharides studied, only malto-oligo- saccharide was hydrolyzed by different nonfractionated LF preparations. The LF-5 fraction from one milk donor hydrolyzed malto-oligosaccharide with a K m ¼ 2.0 m M and specific activity of 10 ± 2 standard units/mg. The K m values (2.0 ± 0.9 m M ) for malto-oligosaccharide in the case of seven different LF-5 subfractions and the seven corres- ponding nonfractionated LF preparations were compar- able, their specific activities depending slightly on the milk donor and varying in the range 5–17 standard units per mg. These results agree with the fact that subfraction LF-5 (peak 4, Fig. 2) constitutes  80–90% of total LF. Table 2. K m and k cat values for different ribo-oligonucleotide and deoxyribooligonucleotides characterizing their hydrolysis by LF-1 subfractions of LF from seven samples of different human milk. Results are mean ± SD from three measurements for each of seven LF preparations. Substrate K m (l M )10 3 · k cat (min )1 )10 )3 · k cat /K m (min )1 Æ M )1 ) d(pT) 10 5.6 ± 2.0 42.8 7.6 d(pA) 10 4.2 ± 1.5 6.4 ± 2.0 1.5 d(pN ˜ ) 10 4.5 ± 1.3 28.1 ± 7.0 6.3 (pU) 10 3.4 ± 1.0 29.6 ± 6.3 8.8 (pA) 10 3.0 ± 1.0 26.0 ± 7.9 8.6 (pC) 10 5.0 ± 2.1 29.6 ± 9.1 5.9 d(pGpGpCpApCpTpTpApC) 7.2 ± 1.1 27.2 ± 5.7 3.8 d(pTpApGpApApGpApTpCpApApA) 3.7 ± 1.0 8.2 ± 2.5 2.2 d(pApCpTpApCpApGpTpCpTpApCpA) 5.3 ± 1.8 42.2 ± 4.1 8.0 3358 T. G. Kanyshkova et al.(Eur. J. Biochem. 270) Ó FEBS 2003 LF as the major DNAse, RNase, and ATPase of human milk The hydrolytic function of an enzyme can have a protective role in prokaryotic and eukaryotic cells, and we therefore compared the activities of LF in the hydrolysis of DNA, RNA, and ATP with those of other DNases, RNases, and ATPases of human milk. Published data on these activities in human milk are limited. The14-kDa RNase so far reported [8], like five human blood 14–25-kDa RNases with different affinity for phosphocellulose [37], is relatively small and has substrate specificity similar to that of pancreatic 14-kDa RNase A [38]. Only one 42-kDa DNase with catalytic properties similar to that of DNase II has been described in human blood [39]. In addition, we have recently presented evidence that the milk of healthy human mothers contains subfractions of 150-kDa IgG and 360-kDa sIgA antibodies, which hydrolyze DNA, RNA, and ATP [25,35]. After separation of human milk proteins by SDS/PAGE in a gel containing DNA, an in-gel assay showed DNase activity mostly in the protein band corresponding to LF (Fig. 3C, lane 4). In contrast, DNase activity corresponding to human milk 41-kDa DNAse II (Fig. 3C, lane 4) was detected only in half of 14 analyzed milk samples. A similar situation was observed after separation of human milk proteins in a gel containing RNA; again LF was signifi- cantly more active in hydrolysing RNA than 14-kDa RNase or antibodies (Fig. 3C, lane 2). LF was also significantly more active in hydrolysing ATP than IgG and sIgA antibodies. Furthermore, under the conditions used, we did not detect any other ATPases or phosphatases of low molecular mass (Fig. 3C, lane 6). Thus, LF is the predomi- nant DNase, RNase, and ATPase in human milk, and it is likely that LF may have a protective function during breast feeding of the newborn as well as in human epithelial secretions and barrier fluids during viral and bacterial infections. Cytotoxicity and apoptosis-inducing activities of LF For breast-fed infants, human milk is more than a source of nutrients; it furnishes a wide array of antimicrobial and antiviral molecules, and may also contain substances bioactive toward host cells; for example, it is cytotoxic to human cancer cells [40] because of induction of apoptosis by multimeric a-lactalbumin. We studied the effects of various subfractions of LF with catalytic activities on the growth of L929 (mouse fibroblasts) and HL-60 (human promyelocytes) cells. No effect of the main LF-5 fraction, which possesses only ATPase and amylase activities, was detected [5 mgÆmL )1 ; corresponding to peak 4 (LF-5); Fig. 2], but fraction LF-1 [peak 2 (LF-1); Fig. 2], which possesses at least four enzymic activities (DNase, RNase, phosphatase, and ATPase), showed high cytotoxicity, only 1.5–1.7 times lower than that of tumor necrosis factor (Fig. 5A). All other catalytically active subfractions of LF-2–4 (peaks 3–4) were also cytotoxic, but their effects were estimated as  10–50% of that of LF-1. As LF-5 has ATPase and malto-oligosaccharide- hydrolyzing activities but is not cytotoxic, the cytotoxicity of LF-1 to LF-4 is probably associated with their DNase and/ or RNase activities. DNA of tumor cell lines L929 and HL-60 exposed to fraction LF-1 (peak 2, Fig. 2) for 12–24 h was fragmented as the concentration of the LF increased and became significant at 100 n M (Fig. 5B), and oligonucleosome-size DNAs fragments typical of apoptosis [41,42] were formed. Cells exposed to subfraction LF-1 also display annexin V-staining and morphological changes typical of apoptosis (Fig. 5C). Discussion Here we show for the first time that subfractions obtained by chromatography of homogeneous human milk LF on Cibacron Blue–Sepharose possess different catalytic acti- vities and that DNase, RNase, phosphatase, ATPase, and amylase activities are intrinsic properties of human milk LF. Chromatographic separation (Fig. 2) indicates that these five activities are associated with different isoforms of LF and that, in addition to the three previously reported isoforms [9], one of which is LF RNase, further isoforms of this protein exist that possess other enzyme activities. The nature of the structural variations that give rise to these Fig.4.EnzymeactivitiesofLFrecoveredbygelfiltrationonaTSK HW-55 column. (A) Enzymatic activities of LF recovered by gel fil- tration on a TSK HW-55 column in Tris/glycine (pH 2.4)/0.3 M NaCl after incubation for 30 min at 25 °C in the same buffer to dissociate noncovalently bound proteins; solid line, A 280 ; symbols, relative activity assayed as in Fig. 1. (B) Partial cleavage of human 3¢-[ 32 P]tRNA Phe by LF (lane 2) compared with cleavage by RNase A (lane 1); lane 3, tRNA incubated alone. (C) The cloverleaf structure of tRNA Phe showing the major cleavage sites for LF and RNase A. Symbol size (and intensity) corresponds to the relative hydrolytic activity. Ó FEBS 2003 Enzymic activities of lactoferrin (Eur. J. Biochem. 270) 3359 profound functional differences is not known. The LF molecule contains two potential glycosylation sites [2], the degree of glycosylation of different molecules varies, and they can contain hexose, mannose, hexosamines, or other saccharides [43] and may also differ in the level of phosphorylation. According to X-ray crystallographic analysis, LF consists of two lobes joined by a very flexible amino-acid spacer [1] and is therefore extremely conformationally flexible; its functional state can be influenced not only by iron ions [44] but also by other metal ions and different ligands such as DNA, RNA, and polyanions [45]. Specific conformations of monomeric LF induced by different ligands may modulate its enzymatic functions; binding of DNA is not a rapid process but requires preincubation, and ligands such as ATP and NAD significantly influence this process [45]. The nature of the intersubunit interactions in LF oligomers remains unknown and most investigators do not take into consideration its oligomeric forms. Considering the relat- ively small size of the monomer and its polyfunctionality, we suggested that it may have various oligomeric forms (monomer, dimer, trimer, and tetramer) [23], the intercon- version of which may be controlled by ATP and/or other low-molecular-mass ligands. ATP binding modifies oligomerization which is accompanied by changes in interaction with nucleic acids, polysaccharides, and proteins. Furthermore, as shown above, ATP and NAD stimulate LF-dependent hydrolysis of DNA. Thus, ATP-dependent changes in the oligomeric structure may increase the number of functional states and biological functions of LF. Further study of the structure of its catalytic isoforms and its relation to their various functions is required. Our data indicate that the subfractions of LF constitute the major DNase, RNase, and ATPase in human milk (Fig. 3C) and therefore may contribute to its protective functions; injection of nucleases into the circulatory system or treatment of human respiratory mucosal surfaces with DNases and RNases leads to protection against viral and bacterial diseases [46]. Recently, an inverse correlation between mammary tumor incidence and the amount of RNase activity in human milk was revealed [8]. Several catalytically active LF subfractions are cytotoxic (Fig. 5A), and the most active, the effect of which is comparable to that of tumor necrosis factor, is LF-1, which possesses the highest DNAse, RNase, and ATPase activities. This frac- tion was capable of inducing apoptosis, raising the pos- sibility that LF contributes to mucosal immunity not only by its antimicrobial and antiviral properties but also by policing the function of human cells. The discovery of five enzymatically distinct forms of LF, their cytotoxicity, and the ability of some of them to induce tumor cell apoptosis should contribute to the understanding of its remarkable polyfunctional activities. Acknowledgements This research was made possible in part by a grant from RFBR (98-04- 49719 and 01-04-49759) and a grant for young scientists from the Siberian Branch of RAS. References 1. Anderson, B.F., Baker, H.M., Norris, G.E., Rice, D.W. & Baker, E.N. (1989) Structure of human lactoferrin: crystallographic structure analysis and refinement at 2.8 A ˚ resolution. J. Mol. Biol. 209, 711–734. 2. Van Berkel, P.H., van Veen, H.A., Geerts, M.E., de Boer, H.A. & Nuijens, J.H. (1996) Heterogeneity in utilization of N-glycosyla- tion sites Asn624 and Asn138 in human lactoferrin: a study with glycosylation-site mutants. Biochem. J. 319, 117–122. 3. Masson, P.L., Heremans, J.F. & Schonne, E. (1969) Lactoferrin, an iron-binding protein in neutrophilic leukocytes. J. Exp. Med. 130, 643–453. 4. Brock, J.H. (1985) Transferrins. In Topics in Molecular and Structural Biology: Metalloproteins (Harrison, P.M., ed.) Chemie Verlag, Weinheim. 5. De Sousa, M. & Brock, J.H. (1989) Iron in Immunity Cancer and Inflammation. John Wiley and Sons, New York. 6. Bennett, R.M., Merrit, M.M. & Gabor, G. (1986) Lactoferrin binds to neutrophilic membrane DNA. Br.J.Haematol.63, 105–117. 7. Zimecki, M., Mazurier, J., Spik, G. & Kapp, J.A. (1995) Human lactoferrin induces phenotypic and functional changes in murine splenic B cells. Immunology 86, 122–127. 8. Ramaswamy, H., Swamy, C.V. & Das, M.R. (1993) Purification and characterization of a high molecular weight ribonuclease from human milk. J. Biol. Chem. 268, 4181–4187. 9. Furmanski, P., Li, Z.P., Fortuna, M.B., Swamy, C.V. & Das, M.R. (1989) Multiple molecular forms of human lactoferrin. Fig. 5. Cytoxicity of LF. (A) Loss of viability (trypan blue staining) of L929 cells during incubation with 10 n M tumor necrosis factor (curve 1), 100 n M LF- 1 (curve 2) or LF-4 fraction (curve 3); curve 4, control. (B) Chromatin cleavage induced by LF in L929 cells. The cells were either untreated (lane 1) or treated with 5 mgÆmL )1 LF-5 (lane 2) or 100 n M LF-1 from three different milk donors (lanes 3–5) for 18 h. DNA was electrophoresed in 1.2% agarose gel and stained with ethidium bromide. Lanes 1 and 2 show no detectable fragmentation, whereasLF-1(lanes3–5)causedaÔladderÕ of DNA fragments. Posi- tions of size standards are shown on the right. (C) Apoptosis of HL-60 human cells treated with 100 n M LF-1 for 16 h revealed by annexin V staining. 3360 T. G. Kanyshkova et al.(Eur. J. Biochem. 270) Ó FEBS 2003 Identification of a class of lactoferrins that possess ribonuclease activity and lack iron-binding capacity. J. Exp. Med. 170, 415–429. 10. He, J. & Furmanski, P. (1995) Sequence specificity and tran- scriptional activation in the binding of lactoferrin to DNA. Nature (London) 373, 721–724. 11. Fleet, J.C. (1995) A new role for lactoferrin: DNA binding and transcription activation. Nutr. Rev. 53, 226–227. 12. Mantel, C., Miyazawa, K. & Broxmeyer, H.E. (1994) Physical characteristics and polymerization during iron saturation of lactoferrin, a myelopoietic regulatory molecule with suppressor activity. Adv. Exp. Med. Biol. 357, 121–132. 13. Bezault, J., Bhimani, R., Wiprovnick, J. & Furmanski, P. (1994) Human lactoferrin inhibits growth of solid tumors and develop- ment of experimental metastases in mice. Cancer Res. 54, 2310– 2312. 14. Sawatzki, G. & Rich, I.N. (1989) Lactoferrin stimulates colony stimulating factor production in vitro and in vivo. Blood Cells 15, 371–385. 15. Gahr, M., Speer, C.P., Damerau, B. & Sawatzki, G. (1991) Influence of lactoferrin on the function of human polymorpho- nuclear leukocytes and monocytes. J. Leukocyte Biol. 49, 427–433. 16. De Sousa, M., Breedvelt, F., Dynesius-Trentham, R., Trentham, D. & Lum, J. (1988) Iron, iron-binding proteins and immune system cells. Ann. NY Acad. Sci. 526, 310–322. 17. Amouric,M.,Marvaldi,J.,Pichon,J.,Bellot,F.&Figarella,C. (1984) Effect of lactoferrin on the growth of a human colon adenocarcinoma cell line-comparison with transferrin. In Vitro 20, 543–548. 18. Garre, C., Bianchi-Scarra, G., Sirito, M., Musso, M. & Ravazzolo, R. (1992) Lactoferrin binding sites and nuclear loca- lization in K562 (S) cells. J. Cell Physiol. 153, 477–482. 19. Arnold, R.R., Cole, M.F. & McGhee, F.M. (1977) A bactericidal effect for human lactoferrin. Science 197, 263–265. 20. Yi,M.,Kaneko,S.,Yu,D.V.&Marakami,S.(1977)HepatitisC virus envelope proteins bind lactoferrin. J. Virol. 71, 5997–6002. 21. Ellison, R.T., Giehl, T.J. & Laforse, F.M. (1988) Damage of the outer membrane of enteric gram-negative bacteria by lactoferrin and transferring. Infect. Immun. 56, 2774–2780. 22. Semenov, D.V., Kanyshkova, T.G., Buneva, V.N. & Nevinsky, G.A. (1998) Interaction of human milk lactoferrin with ATP. Biochemistry (Mosc) 63, 67–75. 23. Semenov, D.V., Kanyshkova, T.G., Buneva, V.N. & Nevinsky, G.A. (1999) Human milk lactoferrin binds ATP and dissociates into monomers. Biochem. Mol. Biol. Int. 47, 177–184. 24. Kanyshkova, T.G., Semenov, D.V., Buneva, V.N. & Nevinsky, G.A.(1999)HumanmilklactoferrinbindstwoDNAmolecules with different affinities. FEBS Lett. 451, 235–237. 25. Nevinsky, G.A., Kanyshkova, T.G., Semenov, D.V., Vlassov, A.V., Gal’vita, A.V. & Buneva, V.N. (2000) Secretory immuno- globulin A from healthy human mothers’ milk catalyzes nucleic acid hydrolysis. Appl. Biochem. Biotechnol. 83, 115–130. 26. Perret, V., Garcia, A., Puglisi, J.D., Grosjean, H. & Ebel, J P. (1990) Conformation in solution of yeast tRNA (Asp) transcripts deprived of modified nucleotides. Biochimie 72, 735–743. 27.Vlassov,V.V.,Zuber,G.,Felden,B.,Behr,J P.&Giege,R. (1995) Cleavage of tRNA with imidazole and spermine imidazole constructs: a new approach for probing RNA structure. Nucleic Acids Res. 23, 3161–3167. 28. Savel’ev, E.V., Eneyskaya, K.A., Shabalin, M.V., Filatov, M.V. & Neustroev, K.N. (1999) Antibodies with amylolytic activity. Protein Peptide Lett. 6, 179–184. 29. Kanyshkova, T.G., Semenov, D.V., Khlimankov, Yu, D., Buneva, V.N. & Nevinsky, G.A. (1997) DNA-hydrolyzing activity of the light chain of IgG antibodies from milk of healthy human mothers. FEBS Lett. 416, 23–26. 30. Buneva, V.N., Kanyshkova, T.G., Vlassov, A.V., Semenov, D.V., Khlimankov, D.Y., Breusova, L.R. & Nevinsky, G.A. (1998) Catalytic DNA- and RNA-hydrolyzing antibodies from milk of healthy human mothers. Appl. Biochem. Biotechnol. 75, 63–76. 31. Andrievskaia, O.A., Buneva, V.N., Naumov, V.A. & Nevinsky, G.A. (2000) Catalytic heterogenity of polyclonal RNA-hydrolyz- ing IgM from sera of patients with lupus erythematosus. Med. Sci. Monit. 6, 460–470. 32. Burstone, M.S. (1962) Enzyme Histochemistry and its Application in the Study of Neoplasms.AcademicPress,NewYorkand London. 33. Legrand, D., Mazurier, J., Metz-Boutigue, M., Jolles, J., Jolles, P., Montreuil, J. & Spik, J. (1984) Characterization and localization of an iron-binding 18-kDa glycopeptide isolated from the N-terminal half of human lactotransferrin. Biochim. Biophys. Acta 787, 90–96. 40. Hakasson, A., Zhivotovsky, B., Orrenius, A., Sabharwal, H. & Svanborg, C. (1995) Apoptosis induced by a human milk protein. Proc.Nat.Acad.Sci.USA92, 8064–8068. 34. Zhivotovsky, B., Wade, D., Gahm, A., Orrenius, S. & Nicotera, P. (1994) Formation of 50 kbp chromatin fragments in isolated liver nuclei is mediated by protease and endonuclease activation. FEBS Lett. 351, 150–154. 35. Vlassov, A., Florentz, C., Helm, M., Naumov, V., Buneva, V., Nevinsky, G. & Giege, R. (1998) Characterization and selectivity of catalytic antibodies from human serum with RNase activity. Nucleic Acids Res. 26, 5243–5250. 36. Nevinsky,G.A.,Kit,Yu,Ya.,Semenov,D.V.&Buneva,V.N. (1998) Secretory immunoglobulin A from human milk catalyzes milk protein phosphorylation. Appl. Biochem. Biotechnol. 75, 77–91. 37. Akagi, K., Murai, K., Hirao, N. & Yamanaka, M. (1976) Puri- fication and properties of alkaline ribonuclease from human serum. Biochim. Biophys. Acta 442, 368–378. 38. De, I., Cardayre, S.B. & Raines, R.T. (1995) A residue to residue hydrogen bond mediates the nucleotide specificity of ribo- nuclease A. J. Mol. Biol. 252, 328–336. 39. Gudding, R. (1979) DNases in milk and blood sera from different species. Acta Vet. Scand. 20, 404–416. 41. Schwartz, L.M. & Osborne, B.A. (1993) Programmed cell death, apoptosis and killer genes. Immunol. Today 14, 582–590. 42. Wyllie, A.H. (1980) Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature (London) 284, 555–556. 43. Legrand, D., Salmon, V., Coddeville, B., Benaissa, M., Plancke, Y. & Spik, G. (1995) Structural determination of two N-linked glycans isolated from recombinant human lactoferrin expressed in BHK cells. FEBS Lett. 365, 57–60. 44. Anderson, B.F., Baker, H.M., Norris, G.E., Rumball, S.V. & Baker, E.N. (1990) Apolactoferrin structure demonstrates ligand- induced conformational change in transferrins. Nature (London) 344, 784–787. 45. Kanyshkova, T.G., Buneva, V.N. & Nevinsky, G.A. (2001) Lactoferrin and its biological functions. Biochemistry (Mosc) 66, 1–7. 46. Nevinsky, G.A., Favorova, O.O. & Buneva, V.N. (2002) Natural catalytic antibodies: new characters in the protein repertoire. In Protein–Protein Interactions: A Molecular Cloning Manual (Golemis, E., ed.), pp. 523–534. Cold Spring Harbor Laboratory Press, New York. Ó FEBS 2003 Enzymic activities of lactoferrin (Eur. J. Biochem. 270) 3361 . enzymic activities; human milk; lactoferrin; protection. Lactoferrin (LF) is a single polypeptide chain of 76–80 kDa, containing two lobes [1], each of which. Multiple enzymic activities of human milk lactoferrin Tat’yana G. Kanyshkova 1 , Svetlana E. Babina 2 ,