Download Processing of lysozyme at distinct loops by pepsin: A novel action for

Biochimica et Biophysica Acta 1726 (2005) 102 – 114
http://www.elsevier.com/locate/bba
Processing of lysozyme at distinct loops by pepsin: A novel action for
generating multiple antimicrobial peptide motifs in the newborn stomach
Hisham R. Ibrahim a,*, Daisuke Inazaki a, Adham Abdou b, Takayoshi Aoki a, Mujo Kim b
a
Department of Biochemistry and Biotechnology, Faculty of Agriculture, 1-21-24 Korimoto, Kagoshima University, Kagoshima 890-0065, Japan
b
Pharma Foods International Co. Ltd., Kyoto 601-8357, Japan
Received 18 April 2005; received in revised form 12 July 2005; accepted 13 July 2005
Available online 3 August 2005
Abstract
C-type lysozyme (cLZ) is an antimicrobial enzyme that plays a major defense role in many human secretions. Recently, we have identified
a helix – loop – helix antimicrobial peptide fragment of cLZ. This finding suggests that processing by coexisting proteases might be a relevant
physiological process for generating peptides that contribute to the in vivo mucosal defense role of cLZ. In this study, we found that pepsin,
under condition relevant to the newborn stomach (pH 4.0), generated various peptides from cLZ with potent bactericidal activity against
several strains of Gram-negative and Gram-positive bacteria. Microsequencing and mass spectral analysis revealed that pepsin cleavage
occurred at conserved loops within the a-domain of cLZ. We found that the bactericidal domain, which was isolated by gel filtration and
reversed-phase HPLC, contains two cationic a-helical peptides generated from a helix – loop – helix domain (residues 1 – 38 of cLZ) by
nicking at leucine17. A third peptide consisting of an a-helix (residues 18 – 38) and a two-stranded h-sheet (residues 39 – 56) structure was
also identified. These peptides share structural motifs commonly found in different innate immune defenses. Functional cellular studies with
outer membrane-, cytoplasmic membrane vitality- and redox-specific fluorescence dyes revealed that the lethal effect of the isolated
antimicrobial peptides is due to membrane permeabilization and inhibition of redox-driven bacterial respiration. The results provide the first
demonstration that pepsin can fine-tune the antimicrobial potency of cLZ by generating multiple antimicrobial peptide motifs, delineating a
new molecular switch of cLZ in the mucosal defense systems. Finally, this finding offers a new strategy for the design of antibiotic peptide
drugs with potential use in the treatment of infectious diseases.
D 2005 Elsevier B.V. All rights reserved.
Keywords: Lysozyme; Muramidase; Proteolysis; Pepsin; Antimicrobial activity; Peptide motif; Membrane damage; Human breast milk; Gastrointestinal
mucosa
1. Introduction
Lysozyme is an antimicrobial protein widely distributed in
various biological fluids and tissues, including avian egg and
animal secretions, human milk, tears, saliva, airway secreAbbreviations: cLZ, c-type lysozyme from hen egg white; NLz, native
cLZ; Ppn-Lz(t), pepsin-processed lysozyme (time in hours); cFDA, 5,6carboxyfluorescein diacetate; DCFH-DA, 2V,7V-dichlorodihydrofluorescein
diacetate; NPN, N-phenyl-1-naphthylamine; OM, outer membrane; CM,
cytoplasmic membrane; SDS-PAGE, sodium dodecylsulfate-polyacrylamide gel electrophoresis; MALDI-TOF-MS, Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry
* Corresponding author. Tel.: +81 99 285 8656; fax: +81 99 2858525.
E-mail address: [email protected]
(H.R. Ibrahim).
0304-4165/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbagen.2005.07.008
tions and is secreted by polymorphonuclear leukocytes [1].
The in vitro antimicrobial activity of lysozyme is directed
against certain Gram-positive bacteria, and to a lesser degree
against Gram-negative bacteria [2 –4]. Lysozyme has many
other functions, including antiviral [5,6], immune modulatory [7], anti-inflammatory [1] and antitumor [8] activities.
The active role played by c-type lysozyme (cLZ) in
defense systems against bacterial infections to the epithelia of
the respiratory and gastrointestinal tract has long been
recognized [3,6,9 –11]. However, the molecular mechanism
for the antimicrobial function of lysozyme remained unclear
until our recent finding that cLZ possesses antimicrobial
activity, which is independent of its catalytic function, and
appears to depend on a structural phase transition in the
molecule [12 – 14]. The independence of antimicrobial action
H.R. Ibrahim et al. / Biochimica et Biophysica Acta 1726 (2005) 102 – 114
on enzyme activity of cLZ was further confirmed by using an
enzymatically inactive mutant of lysozyme (D52S), where its
catalytic residue aspartic acid 52 was substituted with a serine
residue [15]. A further attempt to elucidate the structurerelated antimicrobial action of cLZ was recently made in our
laboratory with a series of synthetic peptides corresponding
to sequences from human and chicken cLZ [4,16]. We found
a specific bactericidal domain within the sequences of both
lysozymes, corresponding to a helix– loop – helix (HLH)
located at the upper lip of the active site cleft of cLZ (residues
87 – 114 in chicken and 87 – 115 in human lysozymes).
Therefore, these findings argue that the generation of lethal
peptide(s) may depend on the location of cLZ and environmental factors, which could regulate its processing.
Examination of physiological fluids in which cLZ is
known to exert its defense role against bacterial infections
suggest a protease-dependent strategy by which its antimicrobial action is modulated in vivo. For instance, cLZ is
proportionally distributed between both azurophil and
specific granules of human polymorphonuclear leukocytes
(PMN), whereas PMN azurophil granules contain several
proteinases beside the abundant cLZ contents [17 – 19].
Upon phagocytosis of bacteria, proteolytic enzymes, such as
cathepsin G and D, elastase and proteinase 3 from PMN, are
discharged with cLZ into the phagocytic vacuole [17 – 21].
Interestingly, the lysosomal proteinases of azurophil granules, including cathepsin G and D, have been reported to
potentiate the antimicrobial activity of cLZ against Gramnegative bacteria [21]. Apart from PMN granules, cLZ is
abundantly secreted in tears, saliva and human milk [1,9].
Tear and saliva components, which play a role in defense
against infections, include cathepsin G and D [17,22 – 25].
Furthermore, the presence of cathepsin D in milk has
recently been reported [22]. Despite the well-recognized
active role of cLZ for breast-feeding, the functional
significance of its distinct presence in human milk and the
molecular mechanism of its action is still undefined.
Breast-feeding has been shown to protect against respiratory and gastrointestinal infections in infants [26,27].
Most of the exposure to bacteria occurs in the gastrointestinal tract of neonates. Since most mucosal surfaces in
neonates do not normally contain phagocytes or immunologically mature cells, a strong antimicrobial defense system
should be pre-existing. Human milk contains a significant
amount of cLZ and seems to play a major role in the local
protection of the infants’ gastrointestinal tract [28,29].
While developing the immune system, the breast-fed
neonate is provided with 0.3– 0.5 g/l of cLZ via the milk
[27]. In parallel, the stomach is well known for its secretion
of pepsin A, a member of aspartic proteases family, secreted
predominantly by chief cells in the gastric mucosa. In the
last two decades, the important role of aspartic proteases in
many pathological processes has become clear [30]. The
functions of these enzymes are manifold, from nonspecific
digestion of proteins to highly specialized processing of
several latent proteins to their biologically active forms
103
[30,31]. Generally, protease-mediated processing events are
vital in the control of essential biological processes, such as
immunological reactions, angiogenesis, apoptosis and activation of defensin-like antimicrobial peptides [30].
In clinical disorders, cathepsin G and D deficiency has
been found to be a significant determinant of the defective
bactericidal activity of human PMN [32], tears and saliva
[24,25], despite their normal level of cLZ and postphagocytic
oxidant production. On the other hand, aspartic proteases,
such like pepsin A, rennin, cathepsin D and E and embryonic
pepsin, are involved in several severe pathologies of the
gastrointestinal mucosa, including bacterial infections and
cancer [30,33,34]. Pepsin was also found with cLZ around
the embryo (embryonic pepsinogen) in chicken [35] and the
amniotic fluid of mammals [36]. At mildly acidic pH (4.0),
pepsin A, rennin, chymosin and cathepsin D specifically
cleaves peptide bonds involving aromatic hydrophobic
amino acids with the most favorable cleavage sites at Phe,
Trp and Leu residues [34]. Consistent with the biological
relevance of the profound colocalization with cLZ of these
proteases and the close similarity of their cleavage specificity,
deficiencies in these enzymes have shown to underlie
important human diseases, such as defective bactericidal
activities of mucosal secretions and PMN [25,30,32,37]. It is
worth noting that retropepsin is an HIV-1 aspartic protease
and the anti-HIV activity of cLZ has recently been reported
[5]. It is likely, therefore, that processing by pepsin A, in the
newborn stomach, might be a relevant biological event to
generate specific antimicrobial peptide(s) from cLZ. The
enhanced antimicrobial action of lactoferrin by pepsintreatment [38] adds further to this evidence.
It is the purpose of this study to examine the operational
complement of pepsin, the major gastric protease, to the
antimicrobial action of cLZ, leading to an understanding of
the in vivo mode of bactericidal action of cLZ, which has
remained a dilemma for decades. For this, conditions
relevant to the newborn stomach, pH 4.0 for 2 and 4 h,
was employed to treat cLZ with pepsin A. This condition
would be expected to produce cLZ with a structure and
antimicrobial function analogous to that in the complicated
milieu of the infant stomach. We explored whether pepsinreleased potent bactericidal peptides from cLZ with lethal
action operate via membrane permeabilization and dissipation of redox-driven membrane potential. The structural
basis of the antimicrobial peptides released by pepsin
processing of cLZ and the relevance of its motif to several
peptides found in innate immunity systems is also discussed.
2. Experimental procedures
2.1. Materials and bacterial strains
Chicken lysozyme was purchased from Wako Chemicals
(Osaka, Japan). The microbial substrate of lysozyme (Micrococcus lysodeikticus), porcine pepsin A (crystallized and
104
H.R. Ibrahim et al. / Biochimica et Biophysica Acta 1726 (2005) 102 – 114
chromatographically purified), 1-N-phenylnaphthylamine
(NPN), 5,6-carboxyfluorescein diacetate (cFDA), and
2V,7V-dichlorodihydrofluorescein diacetate (DCFH-DA)
were from Sigma-Aldrich (Tokyo, Japan). Brain – heart
infusion (BHI) broth and nutrient agar were from Difco
Laboratories (Detroit, MI). Trypticase soy broth (TSB) was
from Becton Dickinson (Tokyo, Japan). All other reagents
were of analytical grade. Test microorganisms for antimicrobial assays, Staphylococcus aureus IFO 14462,
Bacillus subtilis IFO 3007, Salmonella enteritidis IFO
3313 and Escherichia coli K-12 IFO 3301 were obtained
from the Institute of Fermentation, Osaka (Japan). Bacterial
strains of Staphylococcus epidermidis ATCC 12228, Pseudomodas aeruginosa ATCC 27853, Micrococcus luteus
ATCC 4698 were from the American Type Culture
Collection (Rockville, MD, USA). The wild strain of
Bordetella bronchiseptica was gifted by Dr. A. Pellegrini,
the Institute of Bacteriology of the Veterinary Hospital,
Zürich (Switzerland). Helicobacter pylori, from a gasteric
ulcer patient who underwent gastroscopy, was a generous
gift of Dr. N. Fukuda (Central Hospital of Cancer Research
Center, Tokyo, Japan).
(Bio-Rad). The resultant fractions, designated fractions S1–
S3, were collected and freeze-dried. Peptides in each fraction
were quantified by UV absorbance at 215 and 220 nm, using
the formula: mg/ml = (A 215 A 225) 0.144. Chromatography
steps were repeated to collect a greater amount of protein
peaks. Portions of the peaks were analyzed on standard SDS –
PAGE, blot N-terminal microsequencing and MALDI-TOFMS spectrometry. A part of the resultant fractions were
vacuum-dried, resuspended in distilled water then subjected
to antibacterial screening against S. aureus and E. coli K-12.
The most bactericidal fraction S3 was subjected to reversedphase HPLC, using a TSK gel ODS-120T column and a linear
gradient elution was employed using 1 –50% acetonitrile
over 100 min. Peptide elution was monitored at 215 nm.
Peaks designated A – H were collected automatically by the
on-line fraction collector. Collected peptides were vacuumdried and resuspended in milli-Q water to screen for
antibacterial activity, direct microsequencing, MALDITOF-MS analysis and bioassays. The molar concentration
of peptides in bactericidal peak A, referred to as LZprmp (LZ
pepsin-released microbicidal peptides), was estimated from
the average intensity and molecular masses of the constituent
peptides.
2.2. In vitro digestion of lysozyme
2.5. SDS-PAGE and blot N-terminal microsequencing
To mimic conditions in the infant stomach, lysozyme
(0.5 –1.0 mg/ml) in milli-Q sterile water was adjusted to
pH 4.0 with 1 N HCl. Pepsin A in 0.001 N HCl was
added to the samples, which were then placed in a
shaking incubator for 2, 4 or 24 h at 37 -C. The enzymeto-substrate (E/S) ratio was 1:20, 1:50 or 1:100 (w/w).
Then, the pH was increased gradually to 7.0, with 0.5 M
NaHCO3, to simulate conditions in the infant intestine
and irreversibly inactivate pepsin. Controls (Ctrl-Lz) were
treated without the addition of pepsin. Insoluble solids
were removed by centrifugation at 15000g for 15 min
and the resulting supernatants were lyophilized, and
referred to as pepsin-processed cLZ (Ppn-Lz).
2.3. Muramidase activity assay
Lytic activity was determined using Micrococcus lysodeikticus cells, as substrate, according to a previously
described turbidometric method [15]. The activity is
expressed as the rate of decrease in absorbance per min of
the initial velocity of reaction (A 450/min). The assay was
performed in triplicate with two parallel reactions per sample.
Chromatographic fractions were analyzed on SDS-PAGE
(4 – 15% acrylamide) in the presence and absence of hmercaptoethanol (h-ME). The protein bands were either
visualized directly by Coomassie Brilliant Blue R-250
(CBB), or electroblotted onto a polyvinylidene difluoride
(PVDF) membrane by a semidry unit for microsequencing.
The digital image of the gel was analyzed by an electrophoretic documentation and analysis system 120 (EDAS
120) equipped with DC120 camera and Kodak ID 2.02
image analysis software (Eastman Kodak, City, State, USA).
The amounts and molecular size species were estimated
using band intensity of control protein (treated without
pepsin) and standard molecular weight markers. Percent
proteolysis was calculated by dividing the optical density of
the librated peptide bands in a lane containing Ppn-Lz by the
optical density of the total protein bands. Peptides immobilized on PVDF membranes were subjected to automated Nterminal sequence analysis using an Applied Biosystems
Procise sequencer (Model 610A), equipped with a Fblot
cartridge_. The blots were visualized by 0.5% Ponceau S
staining, excised, rinsed in water and 50% methanol solution
and then subjected directly to N-terminal microsequencing.
2.4. Isolation of antimicrobial peptides
2.6. Mass spectrometry (MS)
Ppn-Lz samples were injected into a fast-protein liquid
chromatography system (BioLogic LP; Bio-Rad, Tokyo,
Japan), with a prepacked Sephacryl S-100 column (2.2 90
cm), equilibrated and eluted with pyridine-acetate buffer (pH
5.5). Protein elution was monitored at 280 nm and peaks were
automatically collected using a BioFrac fraction collector
Ppn-Lz samples or chromatographic fractions were
mixed with sinapinic acid (1:1, v/v), as matrix, before
crystallization (2 Al) on a gold-coated 100-position probe.
To identify disulfide-crosslinked fragments, dithiothreitol
(final 2 mM) was added from a concentrated stock to protein
H.R. Ibrahim et al. / Biochimica et Biophysica Acta 1726 (2005) 102 – 114
samples and incubated for 30 min at 37 -C before mixing
with the matrix. MALDI-TOF-MS spectra were acquired by
averaging 100 laser shots, using a MALDI-TOF-MS linear
time-of-flight mass spectrometer (Voyager DE-PRO; PEApplied Biosystems, Foster City, CA, USA) operated in
positive ion mode. The instrument control and data
processing were accomplished with a Voyager Biospectrometry Workstation ver. 5.1 (PE-Applied Biosystems).
105
wells per sample at excitation and emission of 355 and 405
nm, respectively. For each test compound, it was ascertained
that, alone or with 10 mM NPN, there was no fluorescence
increase compared to mere NPN in buffer. The results are
expressed as NPN uptake factors calculated as a ratio of
background-corrected (with value in the absence of NPN
subtracted) fluorescence values of the bacterial suspension
and of the buffer, respectively. Results are representative of
three independent experiments in triplicate.
2.7. Antibacterial assay
2.9. Cytoplasmic membrane (CM) permeability
The bactericidal assay was performed as previously
described [16]. Briefly, mid-logarithmic phase cells, grown
in BHI broth, were washed and resuspended (2 107 cells/
ml) in TSB (pH 7.4). Aliquots (100 Al) of each bacterial
suspension were mixed with 100 Al of TSB containing the
test protein in 2-fold serial dilutions. Controls were
incubated in the absence of protein. The mixture was
incubated at 37 -C for 2 h, serially diluted in physiological
saline solution and plated on nutrient agar. The colony
forming units (CFU) were obtained after incubating the agar
plates at 37 -C for 18 h.
For anti-H. pylori assay, bacteria were grown on Brucella
blood agar plates (BBA; Oxoid, Basingstoke, UK) containing 7% lysed horse blood under micro-aerobic conditions at
37 -C, using CampyPAK plus pouches (Becton Dickinson,
Tokyo, Japan), for 3 days. Cells were harvested and
resuspended (2 105 cells/ml) into TSB containing 7%
horse blood. A 200-Al aliquot of H. pylori suspension was
incubated with 200 Al of the serially diluted test protein
solution. After incubation for 2 h under micro-aerobic
conditions, the mixture was plated on BBA. The plates were
incubated under micro-aerobic conditions for 3 days at 37 -C
and the colonies counted. The antibacterial activity of
different treatments was quantified as log10 reduction in
CFU and was calculated using the following formula: Dlog
killing = log10 nc – log10 np, where nc and np are the CFU
per ml of mock- and protein-treated cells, respectively. All
antimicrobial assays were performed in triplicate and the
results are expressed as log CFU/ml.
2.8. Outer membrane (OM) permeability
Outer membrane permeabilization of E. coli K-12 and P.
aeruginosa was determined by measuring NPN uptake by
bacteria using black fluoroplates and an automated real-time
kinetics fluorometer (Fluoroskan Ascent FL; Labsystems,
Helsinki, Finland), as described recently [39]. Bacteria
grown to log-phase were collected, washed and resuspended
(108 CFU/ml) in 10 mM HEPES buffer (pH 7.2). Aliquots of
bacterial suspension (100 Al) were immediately pipetted onto
preheated fluoroplate wells to 37 -C, containing 100 Al of
10 mM HEPES buffer, NPN (10 mM) and different concentrations of the test cLZ, isolated peptide or polymyxin B
(as positive control). Controls contained buffer instead of test
compounds. Fluorescence was monitored from four parallel
Cytoplasmic membrane permeabilization of the Gramnegative E. coli K-12 and Gram-positive S. aureus was
determined by measuring the leakage of the vitality-specific
fluorescent dye, carboxyfluorescein diacetate (cFDA), from
labeled cells. Bacteria grown to log-phase were suspended
(109 CFU/ml) in PBS buffer (pH 7.4) and stained separately
with cFDA (10 mM final concentration delivered in DMSO)
for 30 min at 37 -C in the dark. Cells were washed and
resuspended in 1% TSB broth. The cFDA-labeled cells were
incubated with different concentrations of NLz, Ppn-Lz or
isolated peptide at 37 -C for 1 h in the dark and then pelleted by
centrifugation at 5000g for 10 min. Control samples were
treated with 1% TSB without test compound. Fluorescence of
the clear supernatants was measured at excitation and emission
of 485 and 538 nm, respectively, in a real-time kinetics
fluorescence spectrofluorometer. Results are expressed as
relative fluorescence units (fluorescence value of cell supernatant, with the test compound subtracted, with the corresponding value of that without the test compound). Assay was
performed in triplicate with four parallel wells per sample.
2.10. Dissipation of membrane potential (DW)
The ability of the isolated peptide to uncouple membrane
potential (DC) of the Gram-negative E. coli K-12 and
Gram-positive S. epidermidis was determined by measuring
the change in intracellular fluorescence of the redoxsensitive fluorescent probe (DCFH-DA) using a real-time
kinetics fluorescence spectrophotometer, as described previously [40]. Bacteria grown in BHI to log-phase were
suspended (109 CFU/ml) in 10 mM HEPES buffer (pH 7.2),
and loaded separately with DCFH-DA (final: 20 AM) for 40
min at 37 -C in the dark, washed and resuspended in
HEPES buffer. A 100-Al aliquot of dye-loaded bacteria was
pipetted onto the fluoroplate wells at 37 -C, containing 100
Al of HEPES, 0.4% glucose and 500 Ag/ml of the test
compound. A steady membrane potential was generated by
the addition of glucose (Glc). Controls included cells treated
with buffer without test compound (Glc) or without both
glucose and test compound (mock cells). Kinetics of
fluorescence increase was monitored from four parallel
wells per sample, at excitation and emission of 385 and 538
nm, respectively. In parallel set of wells, the uncouplers,
valinomycin (1 AM) or nigericin (0.1 AM), were added 15
106
H.R. Ibrahim et al. / Biochimica et Biophysica Acta 1726 (2005) 102 – 114
min before the test compound to collapse membrane
potential (DC) or pH gradient (DpH), respectively, and
serving as indicators that dye oxidation was proportional to
DC. The results are presented as relative fluorescence units
(RFU), after subtracting the values of respective controls.
2.11. Generation of 3D structures
Three-dimensional structures were generated by the
Swiss-PDB Viewer ver. 3.7 (Geneva Glaxo Welcome
Experimental Research) using Brookhaven PDB file of
cLZ (1HEW). Sequence homology analysis was performed
by MPsrch ver. 3.0, via the on-line BLITZ machine
(European Molecular Biology Laboratory). Computation
of the theoretical pI (isoelectric point) and Mw (molecular
weight) of the librated peptides was performed on an
ExPASy server, using Swiss-Prot sequence entries.
3. Results
3.1. Proteolytic processing of lysozyme by pepsin
The production of pepsin A, the principal class of pepsin in
vertebrate stomachs, including infant stomach, is known to
begin 5 days postnatally [34]. Pepsin A is also the major
protease in adult stomach, but the difference between infant
and adult is the acidity of stomach, at pH 4.0 and 2.0,
respectively [34]. However, cLZ is abundantly present in
human milk, while the amount in bovine milk is negligible,
and it has been recognized to play an important defense role
in breast-fed infants [26 – 28]. In parallel, it has been reported
that cLZ is resistant to pepsin hydrolysis at pH 2.0—adult
stomach conditions [41]. Therefore, we tested the ability of
pepsin to process cLZ under conditions similar to the infant
stomach (pH 4.0, for 2, 4 or 24 h). Under this condition (E/S,
1:50, w/w), pepsin was able to proteolyze cLZ, leaving about
60% of the original cLZ intact after 2 h of proteolysis (Fig. 1A
and B). Extending proteolysis time to 4 or 24 h did not lead to
either an increased degree of cLZ hydrolysis or further
degradation of the resulting fragments into smaller peptides
(Fig. 1A upper). The incomplete proteolysis, even with
extended incubation time, suggests that the release of certain
fragments from cLZ may have an inhibitory activity toward
pepsin, or perhaps pepsin was inactivated with extended
incubation time. However, increasing the enzyme/substrate
ratio did not appreciably improve the degree of proteolysis,
but led to a slight increase in the concentration of released
peptides (Fig. 1A bottom), indicating the specificity of cLZ
processing by pepsin under conditions similar to infant
stomach. MALDI-TOF-MS analysis of Ppn-Lz 2h (Fig. 1C)
identified major fragments with molecular masses of 14.3,
7.3 and 5.4 kDa, corresponding to the protein bands identified
by non-reducing SDS-PAGE (Fig. 1C, inset). A signal with
molecular mass of 14.3 (Fig. 1C arrow) corresponds to the
signal obtained with NLz or Ctrl-Lz (data not shown). When
MALDI-TOF-MS analysis was performed on DTT-reduced
Ppn-Lz 2h, the fragment of m/z 7357.9 was further
Fig. 1. Processing of cLZ by pepsin at 37 -C and pH 4.0. (A) SDS-PAGE of processed cLZ for different lengths of time at an E/S ratio of 1:50 (upper) or at
different E/S ratios for 2 h (lower). (B) Residual muramidase activity of processed cLZ for different lengths of time at an E/S ratio of 1:50. MALDI-TOF-MS
analysis was performed under non-reducing (C) and reducing (D) condition. Arrow indicates the peak of native lysozyme.
H.R. Ibrahim et al. / Biochimica et Biophysica Acta 1726 (2005) 102 – 114
dissociated into fragments with m/z signals of 4316.9, 3839.7
and 3241.7 kDa. The fragment of m/z 5437.6 remained
undissociated but gained a mass of 4 Da in MALDI-MS (Fig.
1D), corresponding to the reduction of four half cystines. The
results indicate that the 7.3-kDa fragment is nicked at two
sites but crosslinked through inter-chain disulfide bridges,
while the 5.4-kDa fragment is an intact peptide containing
two intra-chain disulfide bonds. These results demonstrate
the ability of pepsin A to cleave cLZ predominantly at three
sites under conditions relevant to the stomach of the newborn.
3.2. Pepsin processing at pH 4.0 greatly enhance
bactericidal activity of cLZ
After confirming the susceptibility of cLZ to pepsin at
pH 4.0, Ppn-Lz was tested for antibacterial activity against
different strains of Gram-positive and Gram-negative
bacteria (Fig. 2). Both of Ppn-Lz (2 h) and Ppn-Lz (4 h)
showed greatly enhanced bactericidal activity against the
four Gram-negative (E. coli K-12, B. bronchiseptica, S.
enteritidis and H. pylori) and Gram-positive (S. aureus, S.
epidermidis, B. subtilis and M. luteus) bacteria in a dosedependent fashion. NLz and Ctrl-Lz, though much less than
Ppn-Lz derivatives, were only active against S. aureus, B.
subtilis and M. luteus (Fig. 2E, G and H). Interestingly, a
dose-dependent severe reduction in CFU of the highly
resistant strains (E. coli, B. bronchiseptica, wild-type H.
107
pylori and S. epidermidis) to the action of cLZ was
observed with pepsin processing under conditions employed
in this study (Fig. 2A, B, D and F). In addition, Ppn-Lz (2h)
was effective against S. typhimurium and K. pneumonieae,
whereas it produced a 1.9 and 1.7 log-order of killing,
respectively (data not shown). It is worth noting that pepsin
proteolysis at pH 2.0 did not affect the antimicrobial activity
of cLZ, except a very marginal increase in bacteriostatic
activity against S. aureus (data not shown). The results
clearly demonstrate that pepsin processing at pH 4.0 is
necessary to convert cLZ into a potent bactericidal molecule
with a wider antimicrobial spectrum.
3.3. Identification of the cleavage-site(s) for activation of
lysozyme
In an attempt to delineate the potential cleavage site(s)
required for generating such potent bactericidal activity of
cLZ, size-exclusion chromatography was used to isolate
and concentrate the fragments. Ppn-Lz 2h (as well as PpnLz 4h) could be separated into three fractions (peaks S1 –
S3) on Sephacryl S-100 column (Fig. 3A). Fractions S1,
S2 and S3 showed muramidase activities of 0, 12 and
75% relative to the NLz, respectively. When screened
against S. aureus and E. coli (at concentrations of 100 Ag/
ml) both fraction S2 and S3 showed strong bactericidal
activity, but fraction S3 exhibited greater bacterial killing
Fig. 2. Bactericidal activity of pepsin-processed lysozyme (Ppn-Lz). Activity was assessed against Gram-negative E. coli K-12 (A), B. bronchiseptica (B), S.
eneteritidis (C), and H. pylori and Gram-positive S. aureus (D), S. epidermidis (E), B. subtilis (F) and M. luteus (H). The assay was performed at different
doses of NLz, control cLZ-treated without pepsin for 2 or 4 h, and Ppn-Lz 2 or 4 h. The assays were performed in triplicate.
108
H.R. Ibrahim et al. / Biochimica et Biophysica Acta 1726 (2005) 102 – 114
3.4. Isolation of the bactericidal peptide(s)
Fig. 3. Separation of Ppn-Lz-derived fragments by size-exclusion chromatography. (A) Elution profile of Ppn-Lz from Sephacryl S-100 column.
Protein was monitored at 280 nm. (Inset) SDS-PAGE shows the peptides in
the fraction, labeled S1 – S3, and their residual muramidase activity. (B)
Antimicrobial screening of the fractions, Ppn-Lz and control cLZ against S.
aureus and E. coli K-12 (initial viability of 107 CFU/ml) with 100 Ag/ml
peptide for 1 h at 37 -C. Killing activity represented as log N o /N, where N o
and N are the CFU of control and protein-treated, respectively. Assays were
performed in triplicate and are given TS.E.
than S2 (Fig. 7B). To identify the cleavage site(s), peaks
were subjected to electrophoretic and microsequencing
analysis. In SDS-PAGE, the bactericidal fractions (S2 and
S3) showed two intense peptide bands other than the
intact protein, while the inactive fraction (S1) contained an
intense band with one diffused band (Fig. 3A, inset).
However, the most bactericidal fraction (S3) appears to
contain considerable amounts of intact cLZ together with a
sharp low-molecular weight peptide band. MALDI-TOF
analysis identified major peptides with molecular masses
of 14.3, 13.5, 6.8, 5.0 and 5.6 kDa in the most active S3
fraction, while S1 showed two peaks with m/z of 7.5 and
5.6 kDa (data not shown).
N-terminus residues of the two peptides in S2 (as well as
S3) corresponded to Asn39 (upper) and Asp18 (lower) of
cLZ (Fig. 3A). The lower peptide, however, showed another
equimolar sequence with N-terminus corresponding to Lys1
of cLZ. The peptide with Mw of 7.5 kDa in S1 showed an
N-terminus corresponding to Gln57 of cLZ. By combination
of sequencing data and calculation of molecular masses
(MALDI-TOF) of the peptides, we could reveal that pepsin
cleaved cLZ predominantly at the C-terminus of Leu17,
Phe38, Leu56 and Met105, and partially at the N-terminus of
Trp108 – Trp111 residues.
To isolate the bactericidal peptide, the most potent
fraction (S3) was further purified by reversed-phase HPLC,
C18 column, using a linear gradient of acetonitrile. Fraction
S3 was separated into eight subfractions, designated A –H
(Fig. 4A). When screened for bactericidal activity against S.
aureus and E. coli K-12, only peaks A and H showed
bactericidal activity against both strains (Fig. 4B). However,
peak A exhibited the strongest bactericidal activity (six
log10 orders of killing against S. aureus and over seven
log10 order against E. coli), even greater than Ppn-Lz. Direct
sequencing of peak A, eight-residue, yielded two equimolar
peaks with one blank at cycle 6 (Fig. 4A, inset). Although
minor sequences were also observed in the eight cycles,
they were difficult to assign to a certain sequence of cLZ.
The two major sequences corresponded to the sequence
Asp18 – Leu25 and Lys1 –Leu8 of cLZ. MALDI-TOF-MS of
peak A gave five molecular masses of 2414, 3414, 3620,
4535 and 4823 Da (Fig. 4C). Peak H, on the other hand,
showed three molecular masses of 5619, 6815 and 14322
Da, whereas the latter corresponded to the intact cLZ (data
not shown). By a combination of mass-selected peptide
fragmentation (ESI-MS/MS sequencing), identified cleavage sites, specificity of pepsin cleavage and calculation of
molecular masses, the identity of the purified peptides was
determined. The calculated molecular masses (predicted) for
these peptides were in excellent agreement with the
measured masses (signal) by MALDI-MS (Fig. 4C, inset).
These results demonstrate that the isolated bactericidal
peptides (peak A), termed LZprmp (LZ pepsin-released
microbicidal peptides), are exclusively generated from an Nterminal helix –loop –helix domain (Lys1 –Phe38) with the
first two h-strands (Asn39 –Leu56) of the h-domain of cLZ
(Fig. 5A). As shown in Fig. 5A, nicking at Leu17, Phe38,
Leu56 and Met105 (bold circled) and different scissile sites
(arrows) at the C-terminal region of cLZ, produced various
cationic antimicrobial peptide motifs. The structural features
of these bactericidal peptides are predominantly a-helical
(Fig. 5B and C) and helix-sheet (Fig. 5D and E) motifs. It
should be noted that the helix-sheet peptide motif with m/z
5614 (Fig. 5E), released by cleavage at Trp62, was not
present in most of the bactericidal peak A, but detected in
the other bactericidal peak H of RP-HPLC. Each of the four
bactericidal peptide motifs in LZprmp were shown to
contain one cysteine residue, which is engaged in a disulfide
bridge to a short basic peptide from the C-terminal region of
cLZ. The major peptide (m/z 3414) in LZprmp is a cationic
(calculated pI 9.31) a-helix peptide (H2), Asp18 – Phe38,
with one cysteine engaged in an interchain disulfide bridge
(SS-II) with a small segment, Trp111 – Tyr118 (Fig. 5C). The
other peptide motifs, m/z 2414, 4823 and 5614, are helical
(Fig. 5B, K1 – L17/R125 –L129), helix-two-stranded h-sheet
(Fig. 5D, D18 –L56/R114 – T118) and helix-triple stranded hsheet (Fig. 5E, D18 – W62/R114 – T118) with calculated pI
10.86, 8.01 and 8.79, respectively.
H.R. Ibrahim et al. / Biochimica et Biophysica Acta 1726 (2005) 102 – 114
109
Fig. 4. Isolation, on reversed-phase HPLC, of the bactericidal peptide from the size exclusion-derived fraction S3. (A) Elution was achieved with a 1 – 50%
linear gradient of acetonitrile and absorbance was monitored at 215 nm. (B) Eight fractions, labeled A – H, were tested, in triplicate, for antimicrobial activity
against S. aureus and E. coli K-12 and the results are expressed as described in the legend to Fig. 3. The most bactericidal HPLC-derived peak A was subjected
to N-terminal protein sequencing and the results are shown in A (inset). (C) MALDI-TOF-MS spectra of HPLC-derived peak A. (Inset) ESI-MS sequencing,
where signal and predicted refer to the observed and calculated molecular masses of the peptides. The sequence of peptides is shown with a number depicting
residue within the sequence of cLZ.
3.5. Bactericidal action of LZprmp operates through
membrane damage mechanism
Compared with the NLz, LZprmp, as well as Ppn-Lz,
show strong bactericidal activity against both Gram-positive
and Gram-negative bacteria (Figs. 2 and 4). In addition, they
did not show bacterial agglutination, as detected spectrophotometrically (data not shown) and, thus, allow hypothe-
sizing that the potent antimicrobial action of LZprmp
operates through disruption of the integrity of the bacterial
membrane. We adopted several approaches to delineate
the mechanism for the significantly promoted antimicrobial action of the isolated microbicidal peptides of cLZ
(LZprmp).
Permeabilization of the outer membrane (OM) of
susceptible Gram-negative (E. coli K-12 and P. aerugi-
110
H.R. Ibrahim et al. / Biochimica et Biophysica Acta 1726 (2005) 102 – 114
Fig. 5. (A) Ribbon diagram of cLZ illustrating the nick sites by pepsin, which lies in loop regions that are aligned to split the molecule into two domains (bold
dashed lines), and released different antimicrobial peptide motifs from the a-domain (bold circled residues). Structures of the antimicrobial peptide motifs (B – E)
are also shown with their observed molecular masses (m/z) and calculated pI (in parentheses). Basic residues are represented in bold and disulfide connectivities as
thin lines. (F) Sequence alignment and secondary structures of the N-terminal region of different cLZ species demonstrating the conservation of amino acid
residues (bold, underlined) sensitive to pepsin. Arrows indicate the sites for pepsin cleavage specificity.
nosa) strains by LZprmp was monitored using the hydrophobic fluorescent dye NPN, as previously described [39].
The fluorescence of NPN substantially increases when it is
incorporated into the hydrophobic core of a permeabilized
OM compared with its very weak fluorescence in the
presence of intact OM. The results of OM permeabilization
of E. coli and P. aeruginosa by LZprmp compared with
the well-known OM destabilizing antibiotic, polymyxin B
(Plxn B), is shown in Fig. 6A and B. Like Plxn B, both
LZprmp and Ppn-Lz displayed progressive dose-dependent
permeabilization against E. coli and P. aeruginosa, with
LZprmp being much more potent. However, increasing
LZprmp concentration displayed a progressive increase in
NPN uptake, more pronounced than that of Plxn B, against
both strains.
Cytoplasmic membrane (CM) permeabilization was
assessed by following the leakage of cFDA from labeled
cells of E. coli K-12 and S. aureus at different peptide
concentrations (Fig. 6C and D). The fluorogenic dye cFDA
is cell-permeant and undergoes hydrolysis of the diacetate
(DA) groups into carboxyfluorescein (CF) by intracellular
nonspecific esterases, resulting in a highly fluorescent amine
reactive fluorophore (CF). This CF reacts with cytoplasmic
proteins, forming highly stable dye protein adducts [42].
Permeabilization of CM can be detected by measuring the
increase of green fluorescence in the culture medium, which
reflects release of cytoplasmic proteins—CF adducts and
CF. LZprmp permeabilized the CM of both E. coli and S.
aureus in a dose-dependent manner (Fig. 6C and D). A
linear efflux of the cytoplasmic dye with increasing
concentration of LZprmp from 62 up to 500 Ag/ml, but
the onset of CM permeabilization of both strains by Ppn-Lz
was detected at higher concentrations. The results demonstrate that the enhanced bactericidal activity of LZprmp is
attributed to its ability to disrupt bacterial membranes,
whereas its cellular target appears to be the CM of both
Gram-positive and Gram-negative bacteria, obviously by
forming pores into the membrane.
In bacteria, the maintenance of the electrochemical
membrane potential (DC) is dependent on energy metabolism and respiratory activity (electron transport chain), and
is essentially reported to correlate with redox potential status
[43,44]. Therefore, the ability of LZprmp to dissipate the
DC of E. coli K-12 and S. epidermidis is tracked with a
redox-sensitive fluorescent dye (DCFH-DA). The assay is
based on the fact that viable dye-loaded cells can deacetylate
DCFH-DA to DCFH, which is not fluorescent but reacts
quantitatively with the reactive oxygen species (ROS)
coupled to the generation of electrochemical potential
gradient (DAH), by the addition of glucose (Glc) to produce
the fluorescent DCF. The fluorescent dye remains trapped
within the cell and can be kinetically measured to provide an
H.R. Ibrahim et al. / Biochimica et Biophysica Acta 1726 (2005) 102 – 114
111
fluorescence production in LZprmp-treated cells (Fig. 7B).
A similar trend was observed with S. epidermidis (Fig. 7C
and D), except that Nig treatment had no effect on NLzinduced fluorescence production (Fig. 7C). The obvious
collapse of DC by LZprmp was as significant as the
maximum depolarization obtained by Val in both bacterial
strains. The progressive collapse of DC by LZprmp (Fig. 7)
and permeabilization of CM (Fig. 6C and D) clearly indicate
its ability to form pores into the cytoplasmic membrane.
4. Discussion
This study provides evidence that cLZ processing by an
aspartic protease, pepsin, under conditions mimicking the
infant stomach, is a key event in triggering a very potent
bactericidal conformation and generating multiple antimicrobial peptide motifs against several Gram-negative and
Gram-positive bacterial strains. The multiple bactericidal
peptides (LZprmp) were able to rapidly interact with and
Fig. 6. Dose – response curve of membrane permeabilization by the isolated
antimicrobial peptides (LZprmp) and Ppn-Lz. Outer membrane (OM)
permeabilization of E. coli K-12 (A) and P. aeruginosa (B) was monitored
by a fluorescence increase due to NPN partitioning into the OM.
Cytoplasmic membrane (CM) disruption of E. coli K-12 (C) and S. aureus
(D) was monitored by the efflux of cFDA from intracellularly loaded cells.
Samples were NLz, Ppn-Lz and LZprmp, while the antibiotic, polymyxin
B, served as a control. The results are expressed as NPN uptake factors as
described in Section 2. Values represent the mean of three independent
experiments with four parallel wells per sample.
index of DC. As shown in Fig. 7, addition of Glc induced a
linear time-dependent production of intracellular ROS in
both bacterial strains. NLz had no remarkable effect on the
Glc-induced energization of DC. However, LZprmp
exhibited a lag-time of 20 min followed by an increase in
fluorescence, but much slower and less in magnitude than
that produced by NLz (Fig. 7B and D). To distinguish the
ROS generated by the catalytic action of intracellular
oxidases from that driven by the DC, conditions inducing
dissipation of either the membrane potential (Dc? or the
proton motive force (DpH) were employed. The ionophore,
valinomycin (Val), is known to collapse Dc but not DpH,
while nigericin (Nig) will collapse DpH but not DC [45].
Both Val and Nig produced lower fluorescence units in Glcinduced E. coli (Fig. 7A), to a level similar to that produced
in LZprmp-treated E. coli (Fig. 7B). On the other hand,
treatment of E. coli with either Val or Nig had no effect on
Fig. 7. Collapse of bacterial membrane potential (DCm) by LZprmp.
Inhibition of DCm was based on DCm dependence of radical oxygen
species (ROS) production via respiratory control. Fluorescence is plotted
vs. time for E. coli K-12 (A and B) and S. epidermidis (C and D). NLz (A
and C) or LZprmp (B and D) was added (17.5 AM) to DCFH-DA-loaded
cells in the presence of glucose and the intracellular fluorescence intensity
measured in real time. As positive controls, ionophores valinomycin (+Val)
or nigericin (+Nig) were added to verify that the decrease of intracellular
ROS generation is due to collapse of the DCm. Mock cells were treated
without glucose, protein or uncoupler. Data are typical of four experiments
and are given TS.E.
112
H.R. Ibrahim et al. / Biochimica et Biophysica Acta 1726 (2005) 102 – 114
disrupt the OM of E. coli and P. aeruginosa in a dosedependent manner comparable to a known membraneactive antibiotic, polymyxin B. LZprmp was shown to
permeate the CM of E. coli and S. aureus, and dye efflux
was linear with increasing protein concentration. The
results support the classification of LZprmp as an
antimicrobial possessing pore-forming activity, where pore
formation includes a multistep process encompassing
membrane binding, membrane insertion and oligomerization. This was supported by the ability of LZprmp to
dissipate the membrane potential (+Val) and ion gradient
(+Nig) of Gram-negative bacteria (Fig. 4A and B), while
affecting the membrane potential more prominently in
Gram-positive bacteria (Fig. 4C and D) and, thus, acting
via a mechanism that is difficult for bacteria to resist. It
appears that these peptides disrupt the CM membrane via
carpet-like and pore-formation mechanisms, as they contain multiple structure motifs. The ability to interact with
and disrupt cellular membranes of various structures may,
therefore, be a selective advantage for LZprmp, given the
observed potent antimicrobial activity.
Pepsin (at pH 4.0) processed cLZ at structurally distinct
sites. Strikingly, all of the prominently susceptible residues
(Leu17, Phe38, Leu56 and Trp62) of cLZ were found at the Nterminal region of the molecule. Amino acid alignments
revealed that cLZ from different species contained conserved pepsin cleavage sites, (V, T) F/Y, (F, W, Y, N, H) E,
(F, Y) N and (L, F) Q, which may represent conserved sites
in cLZs (Fig. 5F, arrows) for the generation of multiple
potent bactericidal peptides. These cleavage sites are located
within loop regions that approximately split cLZ into its two
half-molecules, a and h domains (Fig. 5A, dashed lines).
The domain which was less bactericidal (Fig. 5A, excluded
by dashed lines), isolated by size-exclusion column (S1),
consisted predominantly of full or part of the h-domain with
the H3- and H4-helices of cLZ, and contained two internal
disulfide bridges SS-III and SS-IV. On the other hand, the
fragments encompass the a-domain, exhibiting potent
bactericidal activity (Fig. 3B; S2 and S3), consisted of
amphiphilic helices H1, H2, H5 and H6 of cLZ. This finding
is of particular importance, as this domain (Fig. 5A, within
bold dashed lines) has been shown to include the second
helix (H5) of the bactericidal helix –loop –helix (HLH)
peptide, reported in our previous study [16], joined to a
HLH motif (H1 and H2, residues 1– 38) located at the Nterminal region of cLZ. This provides additional evidence
for the role of H5 in the antimicrobial action of cLZ, either
as an independent structural element or as a complementary
element for the N-terminal helical peptides, newly discovered in this study by pepsin processing. The identification of
HLH domain (residues 1– 38), at a unique location that falls
within a region of the greatest degree of conservation of all
c-type lysozymes (Fig. 5F), provides strong support for its
major role in the anti-infection activity of cLZ, which can be
triggered by pepsin in the infant stomach or possibly by
cathepsin D or E in saliva or epithelial mucosa.
The structural motifs of the major antimicrobial peptides
purified from Ppn-Lz are either amphiphilic helical, H1 or
H2 of cLZ (Fig. 5C), or helix-sheet, H2+S1 –S3 (Fig. 5D
and E). This amphipathic helix (H2) has two Phe residues
(Phe34 and Phe38) aligned at one terminus and one Trp
residue (Trp28) at the center of the helix (Fig. 5C). Hence,
one can envision that in the different antimicrobial peptides
containing H2 (Fig. 5C – E), when librated by pepsin, the
helix is positioned at the surface of the bacterial membrane.
The hydrophobic array of aromatic residues (Phe34, Phe38
and Trp28) of the helix are most likely localized at the
membrane interface, thus resulting in extrusion of the
conserved basic residues (Arg21, Lys33, Arg45 and those of
the joined peptide Arg112, Arg114 and Lys116) to mediate
insertion of the domain into the membrane.
The mammalian innate defense system, in which cLZ is
involved, includes the secretion of various antimicrobial
peptides commonly derived from precursor proteins
released from leukocytes and epithelia. These microbicidal
peptides are classified by structure into two main families:
defensins and cathelicidins [46,47]. Defensins share a
common structure, either a triple-stranded (mammals) or
two-stranded h-sheet with flanking a-helix (insect). Cathelicidins are proteins with antimicrobial peptides at the Cterminus immediately following a conserved proregion,
which become active when they are cleaved from the
protein by elastase. In human, the majority of the
cathelicidins-derived peptides exist as a-helical structures,
which, like cLZ, is induced during inflammatory disorders
[47]. It should also be pointed out that the cationic a-helical
antimicrobial peptides, buforin I and parasin I, are directly
derived from the N-terminal domain of histone H2A [47].
Interestingly, processing of cLZ by pepsin released various
antimicrobial peptides analogous by structure, a-helical and
helix-sheet structural motifs (Fig. 5B – E), to those derived
from precursor proteins of the innate immune system. This,
together with the fact that cLZ is also stored in and released
upon activation of leukocytes, clearly suggest a proteasedependent strategy by which the antimicrobial action of cLZ
is modulated in vivo.
In conclusion, our results explore the importance of
processing by colocalized protease(s) on the antimicrobial
action of cLZ, with particular emphasis on its defense
role (mothers milk) in the stomach of newborn. The
unique processing of cLZ by pepsin was attributed to the
generation of a-helical and helix-sheet bactericidal structural motifs strictly confined to a highly basic helix–
loop –helix at the N-terminal region (residues 1– 38).
Intriguingly, the degree of cLZ proteolysis by pepsin did
not exceed 40%, even after extending incubation time to
24 h or increasing the E/S ratio, suggesting the important
biological role of the intact molecule. The susceptibility
of a wide range of microbes to Ppn-Lz and the isolated
peptides, LZprmp, was associated with membrane permeabilization and dissipation of DC. Therefore, bacteria
might not easily develop resistance to an antimicrobial
H.R. Ibrahim et al. / Biochimica et Biophysica Acta 1726 (2005) 102 – 114
that trigger such a destructive mechanism. This finding is
noteworthy when considering that the catalytic apparatus
in all aspartic proteases is virtually the same and that cLZ
is predominantly secreted with the aspartic proteases,
cathepsins D and E, in several biological secretions [32],
including saliva, tears and azurophil granules of neutrophils. Finally, the findings presented in this study provide
new information on the understanding of the molecular
mechanism of cLZ action in innate immunity and offer a
fascinating opportunity for the potential use of bactericidal peptides (LZprmp) in the treatment of infectious
diseases.
Acknowledgements
This work was supported in part by a Scientific Research
Grant from the New Energy and Industrial Technology
Development Organization (NEDO), Japan.
References
[1] P. Jolles, J. Jolles, What’s new in lysozyme research? Mol. Cell.
Biochem. 63 (1984) 165 – 189.
[2] G.F. Brooks, J.S. Butel, L.N. Ornston, Medical Microbiology, 19th
edR, Prentice-Hall International Inc., London, 1991.
[3] J.G. Banks, R.G. Board, N.H. Sparks, Natural antimicrobial systems
and their potential in food preservation of the future, Biotechnol. Appl.
Biochem. 8 (1986) 103 – 147.
[4] H.R. Ibrahim, T. Aoki, A. Pellegrini, Strategies for new antimicrobial
proteins and peptides: lysozyme and aprotinin as model molecules,
Curr. Pharm. Des. 8 (2002) 671 – 693.
[5] S. Lee-Huang, P. Huang, Y. Sun, H.f. Kung, D.L. Blithe, H.C. Chen,
Lysozyme and RNases as anti-HIV components in beta-core preparations of human chorionic gonadotropin, Proc. Natl. Acad. Sci. U S A
96 (1999) 2678 – 2681.
[6] F.X. Hasselberger, Uses of Enzymes and Immobilized Enzymes,
Nelson-Hall Inc., Chicago, 1978.
[7] P.L. Kokoshis, D.L. Williams, J.A. Cook, N.R. Di-Luzio, Increased
resistance to Staphylococcus aureus infection and enhancement in
serum lysozyme activity by glucan, Science 199 (1978) 1340 – 1342.
[8] G. Sava, V. Ceschia, G. Zabucchi, Evidence for host-mediated
antitumour effects of lysozyme in mice bearing the MCa mammary
carcinoma, Eur. J. Cancer Clin. Oncol. 24 (1988) 1737 – 1743.
[9] J. Hankiewicz, E. Swierczek, Lysozyme activity in various human
body fluids, Clin. Chem. Acta 57 (1974) 205 – 209.
[10] E.F. Osserman, M. Klockars, J. Halper, R.E. Fischel, Effects of
lysozyme on normal and transformed mammalian cells, Nature 243
(1973) 331 – 335.
[11] P. Venge, T. Foucard, J. Henriksen, L. Hakansson, A. Kreuger, Serumlevels of lactoferrin, lysozyme and myeloperoxidase in normal,
infection-prone and leukemic children, Clin. Chim. Acta 136 (1984)
121 – 130.
[12] H.R. Ibrahim, S. Higashiguchi, L.R. Juneja, M. Kim, T. Yamamoto, A
structural phase of heat-denatured lysozyme with novel antimicrobial
action, J. Agric. Food Chem. 44 (1996) 1416 – 1423.
[13] H.R. Ibrahim, S. Higashiguchi, M. Koketsu, L.R. Juneja, M. Kim, T.
Yamamoto, Y. Sugimoto, T. Aoki, Partially unfolded lysozyme at
neutral pH agglutinates and kills Gram-negative and Gram-positive
bacteria through membrane damage mechanism, J. Agric. Food Chem.
44 (1996) 3799 – 3806.
113
[14] H.R. Ibrahim, On the novel catalytically-independent antimicrobial
function of hen egg-white Lysozyme: a conformation-dependent
activity, Nahrung 42 (1998) 187 – 193.
[15] H.R. Ibrahim, T. Matsuzaki, T. Aoki, Genetic evidence that antibacterial activity of lysozyme is independent of its catalytic function,
FEBS Lett. 506 (2001) 27 – 32.
[16] H.R. Ibrahim, U. Thomas, A. Pellegrini, A helix – loop – helix peptide
at the upper lip of the active site cleft of lysozyme confers potent
antimicrobial activity with membrane permeabilization action, J. Biol.
Chem. 276 (2001) 43767 – 43774.
[17] C.M. Black, M. Paliescheskey, B.L. Beaman, R.M. Donovan, E.
Goldstein, Modulation of lysosomal protease-esterase and lysozyme in
Kupffer cells and peritoneal macrophages infected with Nocardia
asteroides, Infect. Immun. 54 (1986) 917 – 919.
[18] L. Bjermer, O. Back, G. Roos, M. Thunell, Mast cells and lysozyme
positive macrophages in bronchoalveolar lavage from patients with
sarcoidosis. Valuable prognostic and activity marking parameters of
disease, Acta Med. Scand. 220 (1986) 161 – 166.
[19] W. Pruzanski, N.S. Ranadive, S. Saito, Modulation of phagocytosis
and intracellular bactericidal activity of polymorphonuclear and
mononuclear cells by cationic proteins from human granulocytes:
alternative pathway of phagocytic enhancement, Inflammation 8
(1984) 445 – 457.
[20] E. Flescher, Y. Keisari, J. Lengy, D. Gold, On the possible
schistosomulicidal effect of macrophage-derived lysozyme, Parasitology 103 (1991) 161 – 164.
[21] K.J.I. Throne, R.C. Oliver, A.J. Barrett, Lysis and killing of bacteria
by lysosomal proteinases, Infect. Immun. 14 (1976) 555 – 563.
[22] P. Benes, G. Koelsch, B. Dvorak, M. Fusek, V. Vetvicka, Detection of
procathepsin D in rat milk, Comp. Biochem. Physiol. 133 (2002)
113 – 118.
[23] J.D. Coonrod, The role of extracellular bactericidal factors in
pulmonary host defense, Semin. Respir. Infect. 1 (1986) 118 – 129.
[24] S. Sathe, M. Sakata, A.R. Beaton, R.A. Sack, Identification, origins
and the diurnal role of the principal serine protease inhibitors in human
tear fluid, Curr. Eye Res. 17 (1998) 348 – 362.
[25] K.A. McClellan, Mucosal defense of the outer eye, Surv. Ophthalmol.
42 (1997) 233 – 246.
[26] L.A. Hanson, M. Hahn-Zoric, M. Berndes, R. Ashraf, V. Herias, F.
Jalil, T.I. Bhutta, A. Laeeq, I. Mattsby-Baltzer, Breast feeding:
overview and breast milk immunology, Acta Pediatr. Jpn. 36 (1994)
557 – 561.
[27] H. Kohler, S. Donarski, B. Stocks, A. Parret, C. Edwards, H. Schroten,
Antibacterial characteristics in the feces of breast-fed and formula-fed
infants during the first year of life, J. Pediatr. Gastroenterol. Nutr. 34
(2002) 188 – 193.
[28] A. NascimentodeAraujo, L.G. Giugliano, Human milk fractions
inhibit the adherence of diffusely adherent Escherichia coli (DAEC)
and enteroaggregative E. coli (EAEC) to HeLa cells, FEMS Microbiol. Lett. 184 (2000) 91 – 94.
[29] R.T. Ellison III, T.J. Giehl, Killing of gram-negative bacteria by
lactoferrin and lysozyme, J. Clin. Invest. 88 (1991) 1080 – 1091.
[30] N.M. Hooper, Proteases in Biology and Medicine, Portland Press,
London, 2002.
[31] I.Y. Filippova, E.N. Lysogorskaya, V.V. Anisimova, L.I. Suvorov, E.S.
Oksenoit, V.M. Stepanov, Aspartic proteases distribution, Anal.
Biochem. 234 (1996) 113 – 118.
[32] R.I. Lehrer, T. Ganz, Antimicrobial polypeptides of human neutrophils, Blood 76 (1990) 2169 – 2181.
[33] E. Caputo, G. Manco, L. Mandrich, J. Guardiola, A novel aspartyl
proteinase from apocrine epithelia and breast tumors, J. Biol. Chem.
275 (2000) 7935 – 7941.
[34] T. Kageyama, Pepsinogens, progastricsins, and prochymosins: structure, function, evolution, and development, Cell. Mol. Life Sci. 59
(2002) 288 – 306.
[35] K. Hayashi, S. Yasugi, T. Mizuno, Isolation and structural analysis
of embryonic chicken pepsinogen gene: avian homologue of
114
[36]
[37]
[38]
[39]
[40]
[41]
H.R. Ibrahim et al. / Biochimica et Biophysica Acta 1726 (2005) 102 – 114
prochymosin gene, Biochem. Biophys. Res. Commun. 152 (1988)
776 – 782.
M. Ichinose, K. Miki, C. Furihata, M. Tatematsu, Y. Ichihara, T.
Ishihara, DNA methylation and expression of the rat pepsinogen gene
in embryonic, adult, and neoplastic tissues, Cancer Res. 48 (1988)
1603 – 1609.
M.C. Vissers, C.C. Winterbourn, Myeloperoxidase-dependent oxidative inactivation of neutrophil neutral proteinase and microbicidal
enzymes, Biochem. J. 245 (1987) 277 – 280.
K. Yamauchi, M. Tomita, T.J. Giehl, R.T. Ellison, Antibacterial
activity of lactoferrin and a pepsin-derived lactoferrin peptide fragment, Infect. Immun. 61 (1993) 719 – 728.
I.M. Helander, T.M. Sandholm, Fluorometric assessment of Gramnegative bacterial permeabilization, J. Appl. Microbiol. 88 (2000)
213 – 219.
E. Prosperi, Intracellular turnover of fluorescein diacetate. Influence of
membrane ionic gradients on fluorescein efflux, Histochem. J. 22
(1990) 227 – 233.
P. Polverino de Laureto, E. Frare, R. Gottardo, H. Van Dael, A.
Fontana, Partly folded states of members of the lysozyme/lactalbumin
[42]
[43]
[44]
[45]
[46]
[47]
superfamily: a comparative study by circular dichroism spectroscopy
and limited proteolysis, Protein Sci. 11 (2002) 2932 – 2946.
D. Hoefel, W.L. Grooby, P.T. Monis, S. Andrews, C.P. Saint, A
comparative study of carboxyfluorescein diacetate and carboxyfluorescein diacetate succinimidyl ester as indicators of bacterial activity,
J. Microbiol. Methods 52 (2003) 379 – 388.
H. Rottenberg, The generation of proton electrochemical potential
gradient by cytochrome c oxidase, Biochim. Biophys. Acta 1364
(1998) 1 – 16.
K. Bagrarnyan, A. Trchounkm, Decrease of redox potential in the
anaerobic growing E. coli suspension and proton – potassium
exchange, Bioelectrochem. Bioenerg. 43 (1997) 129 – 134.
B.C. Pressman, Biological applications of ionophores, Annu. Rev.
Biochem. 45 (1976) 501 – 530.
M.G. Scott, R.E. Hancock, Cationic antimicrobial peptides and their
multifunctional role in the immune system, Crit. Rev. Immunol. 20
(2000) 407 – 431.
R.M. Epand, H.J. Vogel, Diversity of antimicrobial peptides and
their mechanisms of action, Biochem. Biophys. Acta 1462 (1999)
11 – 28.