We present evidence that different subfractions of purified human milk LF possess five different enzyme activities: DNase, RNase, ATPase, phosphatase, and malto-oligosaccharide hydrolysis..
Trang 1Multiple enzymic activities of human milk lactoferrin
Tat’yana G Kanyshkova1, Svetlana E Babina2, Dmitry V Semenov1, Natal’ya Isaeva3,
Alexander V Vlassov1, Kirill N Neustroev4, Anna A Kul’minskaya4, Valentina N Buneva1
and Georgy A Nevinsky1
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 Fe3+-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
growthregulation, 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], eachof whichbinds one Fe3+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
growthregulation [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 growthof 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 withATP leads to changes in its interaction with polysaccharides, DNA and proteins [23] We have further demonstrated that LF possesses two DNA-binding sites, whichinteract withspecific 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)
Trang 2apoptosis-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)
(3000 CiÆmmol)1)
Purification and analysis of LF
LF was purified and analyzed individually from the milk
of eachof 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 mM sodium acetate, and
LF (pH 4.0) was loaded and eluted with50 mMTris/HCl
buffer, pH 7.5, and then with a concentration gradient of
NaCl (0–1M) in the same buffer Fractions were collected,
dialyzed at 4C for 12 hagainst 10 mM 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 mM MgCl2, 1.0 mM EDTA, 20 mM Tris/HCl
buffer, pH 7.5, and 0.1–1.0 lMLF incubated for 1–2 hat
37C 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¢-[32P]ribo-oligonucleotides and
deoxyribo-oligonucleo-tides (1–10 lM) were incubated with0.1–1.0 lM LF in
10–20 lL reaction mixture containing 20 mM Tris/HCl,
pH 7.5, and 1.0 mMEDTA withor without 5.0 mMMgCl2
for 2–6 hat 37C, and the products were analyzed in a
20% polyacrylamide gel containing 8 urea
Phosphatase activity was assayed under the same condi-tions: removal of [32P]Pifrom 5¢-[32P]oligonucleotides was assayed by TLC in dioxane/NH4OH/water (5 : 1 : 4, by vol.) on Kieselgel plates (Merck) After chromatography,
th e plates were dried, th e [32P]products localized by autoradiography, and their radioactivity was measured by Cherenkov counting
The same conditions were used to study cleavage of human tRNAPheprepared as described previously [26,27] and labeled at the 5¢ end [26,27] tRNA (0.1 lgÆmL)1; 105 Cherenkov counts per sample) were incubated at 37C for
30 min withLF (0.1–1.0 lM) or RNase A (5· 10)5 mgÆmL)1), and the products were analyzed by electropho-resis in 15% polyacrylamide/8M urea gels, withpartial 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 mM MgCl2, 0.3 mMEDTA, 50 mMTris/HCl, pH 6.8, 100 mMNaCl), 0.05–0.2 mgÆmL)1 LF, and different concentrations of [c-32P]ATP, and were incubated for 0.5–6 hat 37C For screening column fractions during purification of IgG, 2–3 lL of eachfraction was incubated in 10 lL standard reaction mixture containing 0.1 mM[c-32P]ATP or [a-32P] ATP (107 c.p.m.) The products of nucleotide hydrolysis were analyzed by TLC in 0.25MKH2PO4buffer, pH 7.0,
on polyethyleneimine–cellulose plates (Merck); the plates were dried, and the positions of various32P-labeled products were identified using [32P]nucleotide standards and auto-radiography The radioactivity of the regions corresponding
to Piand different nucleotides was measured by Cherenkov counting
Amylase activity of LF Reaction mixtures containing 30 mM Tris/HCl, pH 7.5,
1 mM NaN3, 1–5 mM oligosaccharide, and 1–10 lM LF were incubated at 37C 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 with5% H2SO4in propan-2-ol and again dried at
110C 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-NH2column [28] One unit of activity was defined as the quantity of LF that released 1 lmolÆL)1 reducing sugar from maltoheptaose per min at 37C, 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)1yeast total RNA or 20 lgÆmL)1 calf thymus DNA [25,29–31] added to the gel solution before polymerization After electrophoresis, the gel was
Trang 3washed with a solution of 4Murea and twice withwater to
remove SDS, and then to allow protein renaturation it was
incubated for 16 hat 37C in 20 mM Tris/HCl buffer,
pH 7.5, containing 1 mM EDTA and 5.0 mM MgCl2 To
reveal the regions of DNA or RNA hydrolysis, the gel was
stained withethidium 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 37C withwater (5 times)
and then with 0.5Msodium acetate, pH 6.8 (3 times) To
allow protein renaturation and to detect Piresulting from
ATP hydrolysis, the gel was incubated for 12 h at 37C in
5 mM sodium acetate (pH 6.8) containing 1.0 mMMgCl2,
3 mMPb(NO3)2, and 100 lCi [c-32P]ATP Nonspecifically
adsorbed Pb(NO3)2was 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 [32P]Pi
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 with20 mMTris/HCl, pH 7.5, at 4C for 12 h
The gel slices were removed by centrifugation, and amylase
or phosphatase activity was assayed using 5¢-[32P](pT)8or
EPS as described above
In situgel 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
0.1MTris/HCl (pH 8.2)/25 mMCaCl2at 37C for 4 h [33]
Kmand Vmaxfor 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 37C in 0.1 mL
Dulbecco’s modified Eagle’s medium containing 5% fetal
bovine serum to confluence They were then treated with
mitomycin (1 mgÆmL)1) for 5 hand washed withmedium
Freshmedium containing different concentrations
(10–100 nM) of subfractions of LF-1 to LF-5 (see below)
or tumor necrosis factor (10 nM) was then added The cells
were cultivated for a further 12–48 h, and the percentage of
dead cells, counted after staining withtrypan 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 withLF subfractions (10–100 nM) 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 withethidium 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 withdifferent 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 withphage 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) at 37 C for 1–4 h The details of the experiment are given in Materials and methods.
Trang 4additional 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) withimmobilized 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 32P analogs of their specific ligands is the most sensitive method for revealing any contaminating proteins interacting withthe 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), witha stoichiometry of 1.0 mol [a-32P]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-32P]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.
Trang 5possesses two DNA-binding sites withdifferent affinities
for oligonucleotides, which can be labeled after incubation
withaffinity probes for DNA-binding and RNA-binding
sites, the 2¢,3¢-dialdehyde derivatives of different specific
and nonspecific [5¢-32P]oligonucleotides, including [5¢-32
P]-d(pT)9r(pU) and [5¢-32P](pU)10 [24] These modifications
fulfiled the known criteria of affinity modification [23,24]
As judged by SDS/PAGE analysis, boththe LF polypeptide
purified using anti-LF–Sepharose and the combined LF-1–4
subfractions were specifically affinity-labeled by32P analogs
of [5¢-32P]d(pT)9r(pU) and [5¢-32P](pU)10 oligonucleotides,
and by a-[32P]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 32P-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 withethidium bromide after development
of nuclease activity revealed a sharp dark band on a
fluorescent background of DNA or RNA (Fig 3A, lanes
6 and 7)
We also used an in-gel ATPase assay, adapted from
Gomori’s method of Piprecipitation used previously for
histochemical study of ATPase activity [32], for in situ
detection of enzyme-dependent formation of Pi in SDS/
polyacrylamide gels after establishing conditions for
preci-pitation of Pb2(PO4)3in regions of gels containing Piand for
efficient removal of Pb salts nonspecifically adsorbed to
proteins Enzyme-dependent formation of Pb2(PO4)3,
detec-ted by autoradiography, showed a32P-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) Th e data sh ow th e relative 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 data
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 with5¢-[ 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; [32P]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 {[32P]Pb (PO ) }.
Trang 6detection 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 withtrypsin 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 withmaximal activity in eachof 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 mMNaCl whereas DNase I
is 70% inhibited by 50 mM NaCl [25,30] Cleavage of
oligonucleotides and DNA by LF was stimulated 3–5-fold
by Ca2+, Cu2+, and Zn2+ and 8–9-fold by Mn2+ and
Mg2+ions In contrast withknown human DNases, LF
DNase was activated by ATP, dATP and NAD (150 mM)
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
withcomparable Km values (3.7–7.2 lM) but with
different efficiencies (kcat¼ 0.006–0.042 min)1; Table 2)
Interestingly, Kmand kcatfor different homo-d(pN)10and
homo-(pN)10 (Km¼ 3.0–5.0; kcat¼ 0.026–0.029 min)1) were comparable (Table 2) The Km 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 kcat 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 times faster (kcat¼ 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 tRNAPhe 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 Km¼ 0.5 ± 0.2 mM, and th e kcatvalues varied in the range (0.5–4)· 10)3min)1 Th e Km(ATP) values for LF-1 preparations do not differ significantly from those for corresponding nonfractionated LFs (Km¼ 0.2–1.0 mM)
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 Km¼ 2.0 mMand specific activity of 10 ± 2 standard units/mg The Km values (2.0 ± 0.9 mM) 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.
· k cat (min)1) 10)3· k cat /K m (min)1Æ M )1 )
Trang 7LF 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 withtheir 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 hwas fragmented
as the concentration of the LF increased and became significant at 100 nM (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 withdifferent 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 Enzyme activities of LF recovered by gel filtration on a TSK
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 withcleavage by RNase A (lane 1); lane 3, tRNA
incubated alone (C) The cloverleaf structure of tRNAPheshowing the
major cleavage sites for LF and RNase A Symbol size (and intensity)
corresponds to the relative hydrolytic activity.
Trang 8profound 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 suchas
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 withnucleic 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 Branchof 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 with10 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)1LF-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,
wh ereas LF-1 (lanes 3–5) caused a ladder 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 hrevealed by annexin V
staining.
Trang 9Identification 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 withsuppressor
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 experidevelop-mental 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., Pich on, J., Bellot, F & Figarella, C.
(1984) Effect of lactoferrin on the growth of a human colon
adenocarcinoma cell line-comparison withtransferrin 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) Hepatitis C
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) Human milk lactoferrin binds two DNA molecules
withdifferent 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., Beh r, J.-P & Giege, R.
(1995) Cleavage of tRNA withimidazole and spermine imidazole
constructs: a new approachfor 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 withamylolytic 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 withlupus erythematosus Med Sci Monit 6, 460–470.
32 Burstone, M.S (1962) Enzyme Histochemistry and its Application
in the Study of Neoplasms Academic Press, New York and 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 USA 92, 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 withendogenous 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.