http://www.elsevier.com/locate/bba

Biochimica et Biophysica Act

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

generating multiple antimicrobial peptide motifs in the newborn stomach

Hisham R. Ibrahima,*, Daisuke Inazaki a, Adham Abdoub, Takayoshi Aoki a, Mujo Kimb

aDepartment of Biochemistry and Biotechnology, Faculty of Agriculture, 1-21-24 Korimoto, Kagoshima University, Kagoshima 890-0065, JapanbPharma 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 wasalso 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 tions and is secreted by polymorphonuclear leukocytes [1].

Lysozyme is an antimicrobial protein widely distributed in

various biological fluids and tissues, including avian egg and

animal secretions, human milk, tears, saliva, airway secre-

0304-4165/$ - see front matter D 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.bbagen.2005.07.008

Abbreviations: cLZ, c-type lysozyme from hen egg white; NLz, native

cLZ; Ppn-Lz(t), pepsin-processed lysozyme (time in hours); cFDA, 5,6-

carboxyfluorescein diacetate; DCFH-DA, 2V,7V-dichlorodihydrofluoresceindiacetate; NPN, N-phenyl-1-naphthylamine; OM, outer membrane; CM,

cytoplasmic membrane; SDS-PAGE, sodium dodecylsulfate-polyacryla-

mide gel electrophoresis; MALDI-TOF-MS, Matrix-assisted laser desorp-

tion ionization-time-of-flight mass spectrometry

* Corresponding author. Tel.: +81 99 285 8656; fax: +81 99 2858525.

E-mail address: hishamri@chem.agri.kagoshima-u.ac.jp

(H.R. Ibrahim).

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 modula-

tory [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

a 1726 (2005) 102 – 114

H.R. Ibrahim et al. / Biochimica et Biophysica Acta 1726 (2005) 102–114 103

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 structure-

related 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 environ-

mental 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 anti-

microbial 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 gran-

ules, including cathepsin G and D, have been reported to

potentiate the antimicrobial activity of cLZ against Gram-

negative 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 res-

piratory and gastrointestinal infections in infants [26,27].

Most of the exposure to bacteria occurs in the gastro-

intestinal tract of neonates. Since most mucosal surfaces in

neonates do not normally contain phagocytes or immuno-

logically 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

[30,31]. Generally, protease-mediated processing events are

vital in the control of essential biological processes, such as

immunological reactions, angiogenesis, apoptosis and acti-

vation 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 pepsin-

treatment [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 pepsin-

released potent bactericidal peptides from cLZ with lethal

action operate via membrane permeabilization and dissipa-

tion 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 (Micro-

coccus lysodeikticus), porcine pepsin A (crystallized and

H.R. Ibrahim et al. / Biochimica et Biophysica Acta 1726 (2005) 102–114104

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 anti-

microbial 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, Pseu-

domodas 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,

Zurich (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).

2.2. In vitro digestion of lysozyme

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 enzyme-

to-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 15000�g 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 lyso-

deikticus 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 (A450/min). The assay was

performed in triplicate with two parallel reactions per sample.

2.4. Isolation of antimicrobial peptides

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

(Bio-Rad). The resultant fractions, designated fractions S1–

S3, were collected and freeze-dried. Peptides in each fraction

were quantified by UVabsorbance at 215 and 220 nm, using

the formula: mg/ml=(A215�A225)�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-TOF-

MS 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 reversed-

phase HPLC, using a TSK gel ODS-120Tcolumn 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 vacuum-

dried and resuspended in milli-Q water to screen for

antibacterial activity, direct microsequencing, MALDI-

TOF-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.

2.5. SDS-PAGE and blot N-terminal microsequencing

Chromatographic fractions were analyzed on SDS-PAGE

(4–15% acrylamide) in the presence and absence of h-mercaptoethanol (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 electro-

phoretic 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 immobi-

lized on PVDF membranes were subjected to automated N-

terminal sequence analysis using an Applied Biosystems

Procise sequencer (Model 610A), equipped with a Fblotcartridge_. 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.

2.6. Mass spectrometry (MS)

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) 102–114 105

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; PE-

Applied Biosystems, Foster City, CA, USA) operated in

positive ion mode. The instrument control and data

processing were accomplished with a Voyager Biospectr-

ometry Workstation ver. 5.1 (PE-Applied Biosystems).

2.7. Antibacterial assay

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) contain-

ing 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 -Cand 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 of10 mM HEPES buffer, NPN (10 mM) and different con-

centrations 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

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.

2.9. Cytoplasmic membrane (CM) permeability

Cytoplasmic membrane permeabilization of the Gram-

negative 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 5000�g for 10 min. Control samples were

treated with 1% TSB without test compound. Fluorescence of

the clear supernatantswasmeasured 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 super-

natant, with the test compound subtracted, with the corre-

sponding 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 redox-

sensitive fluorescent probe (DCFH-DA) using a real-time

kinetics fluorescence spectrophotometer, as described pre-

viously [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

H.R. Ibrahim et al. / Biochimica et Biophysica Acta 1726 (2005) 102–114106

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

Fig. 1. Processing of cLZ by pepsin at 37 -C and pH 4.0. (A) SDS-PAGE of proc

different E/S ratios for 2 h (lower). (B) Residual muramidase activity of processed

analysis was performed under non-reducing (C) and reducing (D) condition. Arro

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

essed cLZ for different lengths of time at an E/S ratio of 1:50 (upper) or at

cLZ for different lengths of time at an E/S ratio of 1:50. MALDI-TOF-MS

w indicates the peak of native lysozyme.

H.R. Ibrahim et al. / Biochimica et Biophysica Acta 1726 (2005) 102–114 107

dissociated into fragments withm/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 dose-

dependent 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.

Fig. 2. Bactericidal activity of pepsin-processed lysozyme (Ppn-Lz). Activity was

eneteritidis (C), and H. pylori and Gram-positive S. aureus (D), S. epidermidis (

doses of NLz, control cLZ-treated without pepsin for 2 or 4 h, and Ppn-Lz 2 or

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 Ppn-

Lz 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

assessed against Gram-negative E. coli K-12 (A), B. bronchiseptica (B), S.

E), B. subtilis (F) and M. luteus (H). The assay was performed at different

4 h. The assays were performed in triplicate.

Fig. 3. Separation of Ppn-Lz-derived fragments by size-exclusion chroma-

tography. (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 No /N, where No

and N are the CFU of control and protein-treated, respectively. Assays were

performed in triplicate and are given TS.E.

H.R. Ibrahim et al. / Biochimica et Biophysica Acta 1726 (2005) 102–114108

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.

3.4. Isolation of the bactericidal peptide(s)

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 cleav-

age 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 N-

terminal 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 h-sheet (Fig. 5E, D18–W62/R114–T118) with calculated pI

10.86, 8.01 and 8.79, respectively.

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 Awas 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.

H.R. Ibrahim et al. / Biochimica et Biophysica Acta 1726 (2005) 102–114 109

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 spectro-

photometrically (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 antimicro-

bial 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-

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 shownwith their observedmolecular 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.

H.R. Ibrahim et al. / Biochimica et Biophysica Acta 1726 (2005) 102–114110

nosa) strains by LZprmp was monitored using the hydro-

phobic 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 demon-

strate 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 metabo-

lism 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

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.

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.

H.R. Ibrahim et al. / Biochimica et Biophysica Acta 1726 (2005) 102–114 111

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 Glc-

induced 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

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 NLz-

induced 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 antimi-

crobial peptide motifs against several Gram-negative and

Gram-positive bacterial strains. The multiple bactericidal

peptides (LZprmp) were able to rapidly interact with and

H.R. Ibrahim et al. / Biochimica et Biophysica Acta 1726 (2005) 102–114112

disrupt the OM of E. coli and P. aeruginosa in a dose-

dependent manner comparable to a known membrane-

active 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 oligomeriza-

tion. 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 con-

tain 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 N-

terminal region of the molecule. Amino acid alignments

revealed that cLZ from different species contained con-

served 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 N-

terminal 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 discov-

ered 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). Cath-

elicidins are proteins with antimicrobial peptides at the C-

terminus 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 protease-

dependent 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 struc-

tural 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 per-

meabilization 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) 102–114 113

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 neutro-

phils. 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 bacter-

icidal 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.

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