Cinnamycin (Ro 09-0198) Promotes Cell Binding and Toxicity by
Inducing Transbilayer Lipid Movement*
Asami
Makinoa,
Takeshi
Babab,
Kazushi
Fujimotoc,
Kunihiko
Iwamotoad,
Yoshiaki
Yanoe,
Nobuo
Teradab,
Shinichi
Ohnob,
Satoshi B.
Satoaf,
Akinori
Ohtad,
Masato
Umedag,
Katsumi
Matsuzakie, and
Toshihide
Kobayashiahi
From the a Supra-Biomolecular System Research Group, RIKEN
(Institute of Physical and Chemical Research), Frontier Research
System, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan, the
b Department of Anatomy, Faculty of Medicine, University of
Yamanashi, Yamanashi 409-3898, Japan, the c Section of
Physiological Anatomy, Fukui Prefectural University, 4-1-1 Kenjojima, Matsuoka-cho, Yoshida-gun, Fukui, 910-1195, Japan, the
d Department of Biotechnology, The University of Tokyo,
1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan, the e Graduate
School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan, the f Department of Biophysics, Graduate School of
Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan, the
g Department of Molecular Biodynamics, The Tokyo
Metropolitan Institute of Medical Science, 3-18-22 Honkomagome,
Bunkyo-ku, Tokyo 113-8613, Japan, and h INSERM U352,
Institut National des Sciences Appliquees-Lyon, 20 Ave. A. Einstein,
Villeurbanne 69621, France
Received for publication, October 9, 2002, and in revised form, November 18, 2002
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ABSTRACT |
Cinnamycin is a unique toxin in that its
receptor, phosphatidylethanolamine (PE), resides in the inner layer of
the plasma membrane. Little is known about how the toxin recognizes PE
and causes cytotoxicity. We showed that cinnamycin induced transbilayer phospholipid movement in target cells that leads to the exposure of
inner leaflet PE to the toxin. Model membrane studies revealed that
cinnamycin induced transbilayer lipid movement in a PE
concentration-dependent manner. Re-orientation of
phospholipids was accompanied by an increase in the incidence of
-sheet structure in cinnamycin. When the surface concentration of PE
was high, cinnamycin induced membrane re-organization such as membrane
fusion and the alteration of membrane gross morphology. These results
suggest that cinnamycin promotes its own binding to the cell and causes
toxicity by inducing transbilayer lipid movement.
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INTRODUCTION |
Lipids are asymmetrically distributed in biological membranes. In
the eukaryotic plasma membranes, sphingolipids and phosphatidylcholine are mainly located in the outer leaflet, whereas amino phospholipids such as phosphatidylserine
(PS)1 and
phosphatidylethanolamine (PE) reside in the inner membrane. A number of
protein toxins utilize plasma membrane lipids as their receptors;
e.g. cholera toxin binds ganglioside GM1 (1), shiga toxin
recognizes glycolipid Gb3 (2), and lysenin shows high affinity for
sphingomyelin (3). These receptor lipids are located in the outer
leaflet of the plasma membrane.
Cinnamycin (Ro 09-0198) is a 19-amino acid tetracyclic peptide
produced by Streptomyces species (4). Cinnamycin is a member of the lantibiotics group of toxins. Lantibiotics are
bacteriocins that are characterized by the presence of a high
proportion of unusual amino acids (5). Whereas many lantibiotics are
directed against other Gram-positive species, the action of cinnamycin is not limited to bacterial cells. Lysis of red blood cells has also
been observed upon treatment with this toxin (6). Cinnamycin is unique
in that the toxin specifically binds PE that is located in the inner
layer of the plasma membrane in mammalian cells (7-9). The mechanism
of the recognition of inner leaflet lipid by cinnamycin is not well
understood. In the present study, we showed that cinnamycin induced
transbilayer lipid movement in target cells so that PE was exposed on
the outer leaflet of the plasma membrane. Cinnamycin-induced lipid
flip-flop was reconstituted in model membranes. Model membrane study
revealed that the presence of PE is required to initiate lipid
flip-flop by cinnamycin. Fourier transform infrared-polarized attenuated total reflection (FTIR-PATR) spectroscopy demonstrated that
the order parameter of the hydrocarbon chain of PE was significantly reduced in the presence of cinnamycin. FTIR-PATR also showed that the
re-orientation of PE was accompanied by the
-sheet formation of
cinnamycin. When the surface concentration of PE was high, cinnamycin
induced membrane re-organization such as membrane fusion and the
alteration of gross morphology of the membrane.
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MATERIALS AND METHODS |
Cells and Reagents--
HeLa cells were grown in Dulbecco's
modified Eagle's medium containing 10% fetal calf serum.
1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) was from Nacalai Tesque Inc.,
Kyoto, Japan. Dioleoylphosphatidylcholine (DOPC),
dioleoylphosphatidylethanolamine (DOPE),
palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE),
1-palmitoyl-2-[6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl]-sn-glycero-3-phosphocholine (C6-NBD-PC),
1-myristoyl-2-[6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl]-sn-glycero-3-phosphoethanolamine (C6-NBD-PE), N-(NBD)-dioleoylphosphatidylethanolamine
(N-NBD-PE) and N-(lissamine rhodamine B
sulfonyl)-dioleoylphosphatidylethanolamine (N-LRh-PE) were
from Avanti Polar Lipids (Alabaster, AL). Alexa Fluor 488-conjugated
Annexin V and Alexa Fluor 546-conjugated streptavidin were from
Molecular Probes (Eugene, OR). Calcein was from Sigma (St. Louis, MO).
Cinnamycin (Ro 09-0198) was kindly provided by H. Ishituka (Nippon
Roche Research Center, Japan).
Biotinylation of Cinnamycin--
Biotinylated cinnamycin was
prepared according to Aoki et al. (10) with modification.
0.5 ml of 500 µM cinnamycin in 0.1 M
NaHCO3 was mixed with 0.5 ml of EZ-LinkTM
sulfosuccinimidyl 6-(biotinamido)hezanoate (NHS-LC-biotin, Pierce Biotechnology Inc., Rockford, IL) (8.7 mg/ml sterilized water). After
2-h incubation at room temperature, the reaction was terminated by the
addition of 0.1 ml of 1 M lysine solution. Biotinylated cinnamycin was purified by reverse-phase high-performance liquid chromatography on a column of Cosmosil 5C18-AR-300 (Nacalai Tesque). The peptide was eluted with a linear gradient of increasing
concentration of acetonitrile from 20 to 60% in water in the presence
of trifluoroacetic acid.
ELISA Measurement of Binding of Biotinylated Cinnamycin to
Various Lipids--
The binding of biotinylated cinnamycin to various
lipids was measured by ELISA as described previously (3, 11). In brief, 50 µl of lipid (10 µM) in ethanol was added to a well
of microtiter plate (Immulon 1, Dynatech Laboratories, Alexandria, VA).
After the solvent was evaporated at room temperature, 200 µl of 30 mg/ml bovine serum albumin (BSA) in Tris-buffered saline (TBS, 10 mM Tris-HCl, pH 7.4, 150 mM NaCl) was added to
each well. After washing, the wells were incubated with 50 µl of 100 nM biotinylated cinnamycin in TBS containing 10 mg/ml BSA
(1% BSA-TBS) for 2 h at room temperature. The bound cinnamycin
was detected by incubating with peroxidase-conjugated streptavidin. The
intensity of the color developed with o-phenylenediamine as
a substrate was measured with an ELISA reader (Bio-Rad, model 550 microplate reader), reading the absorption at 490 nm with reference at
630 nm.
Treatment of HeLa Cells with Cinnamycin--
Cells grown on
glass coverslips were washed with the incubation buffer (10 mM Hepes-NaOH, pH 7.4, 140 mM NaCl, and 2.5 mM CaCl2). Cells were then incubated with 1 µM cinnamycin or biotinylated cinnamycin in the
incubation buffer containing Alexa Fluor 488-conjugated Annexin V. At
appropriate intervals, cells were fixed and labeled with Alexa Fluor
546-conjugated streptavidin at room temperature. The stained cells were
examined under a Zeiss LSM 510 confocal microscope equipped with a
C-Apochromat 63XW Korr (1.2 numerical aperture) objective.
Preparation of Lipid Vesicles--
Multilamellar vesicles (MLVs)
were prepared by hydrating a lipid film with 20 mM
Hepes-NaOH (pH 7.4) and vortex mixing. Liposomes were dispersed by a
few seconds of sonication in the bath type sonicator. To prepare large
unilamellar vesicles (LUVs), MLVs were subjected to three freeze-thaw
cycles followed by extrusion through polycarbonate filters with
0.1-µm pore size (Nuclepore, Maidstone, UK) for 25 times using a
two-syringe extruder (12).
Measurement of Transbilayer Lipid Movement in Model
Membranes--
The transbilayer movement of short chain fluorescent
lipids C6-NBD-PE and C6-NBD-PC was measured by mixing labeled LUVs with BSA (13). C6-NBD lipids were incorporated into DOPC or POPC vesicles
containing various amount of DOPE or POPE. Vesicles were mixed with 400 µg/ml BSA before addition of 2.5 µM cinnamycin at
30 °C. Selective extraction of the labeled lipid by BSA was detected
via the decrease in fluorescence due to quenching by BSA (14). The time
course of NBD fluorescence was monitored with a fluorometer (JASCO,
FP-6500) (
ex = 475 nm,
em = 530 nm).
CD Spectra--
CD spectra of 100 µM cinnamycin in
10 mM phosphate buffer (pH 7.4) were measured on a Jasco
J-720 apparatus using 0.5-mm path-length quarts cell. Results from five
scans were averaged.
Transmission IR--
Transmission Fourier transform infrared
spectroscopy (FTIR) spectra were measured on a Bio-Rad FTS-3000MX
spectrometer, using a liquid nitrogen-cooled mercury cadmium telluride
(Hg-Cd-Te) detector and Win-IR software. 1 mM cinnamycin in
D2O containing 40 mM Hepes-NaCl (50 µl) was
applied between the two CaF2 windows. A total of 256 scans
was collected. Difference spectra were obtained by digitally
subtracting solvent spectra.
Fourier Transform Infrared-polarized Attenuated Total Reflection
Spectroscopy--
Dry-cast films of POPE/cinnamycin were prepared by
uniformly spreading an HFIP solution of POPE (5 µmol)/cinnamycin (0.4 µmol) on the surface of a germanium ATR plate (80 × 10 × 4 mm) followed by the gradual evaporation of the solvent. The last
trace of solvent was removed by overnight incubation under vacuum. The
film thickness estimated from the applied amount of the lipid was 5-6
µm. The lipid film was hydrated with a piece of D2O-wet
filter paper put over the plate for 3 h. FTIR-PATR measurements
were carried out on a Bio-Rad FTS-3000MX spectrometer equipped with a
Hg-Cd-Te detector and a PIKE horizontal ATR attachment (PIKE
Technologies Inc., Madison, WI) and an AgBr polarizer (15). The total
reflection number was 10 on the film side. The spectra were measured at
a resolution of 2 cm
1 and an angle of incidence of 45°
and derived from 256 co-added interferograms with the Happ-Genzel
apodization function. The dichroic ratio, R, defined by
A
/
A
, was
calculated from the polarized spectra. The absorbance (
A)
was obtained either as a peak height of the CH2 symmetric
stretching vibration band or as an area of the amino bands. The
subscripts
and
refer to polarized light with its electric
vector parallel and perpendicular to the plane of incidence,
respectively. For ATR correction, refractive indexes of 4.003 and 1.440 were used for germanium and the sample film, respectively.
Measurement of Liposome Fusion--
Liposome fusion was
monitored by measuring the reduction of resonance energy transfer
between NBD- and LRh-labeled lipids in lipid vesicles (16, 17). Donor
MLVs (100 µM total phospholipids) containing 1 mol % N-NBD-PE and N-LRh-PE were mixed with 1 mM acceptor MLVs that do not contain fluorescent lipids. 1 µM cinnamycin was added to the mixture, and the time
course of liposome fusion was measured by monitoring the increase of
NBD fluorescence at 475 nm at 30 °C.
Electron Microscopy--
For negative staining, multilamellar
vesicles containing a total of 50 nmol of lipids were incubated with 2 µg of cinnamycin for 30 min at 37 °C. The mixture were fixed with
2.5% glutaraldehyde for 1 h at 37 °C and washed with PBS by
centrifugation. The suspension was adsorbed on
poly-L-lysine-treated, Formvar/carbon-coated copper grids
and negatively stained with 4% aqueous uranyl acetate. The specimens
were observed in a transmission electron microscope (Hitachi H-7500,
Tokyo, Japan) at an acceleration voltage of 80 kV. For immunogold
labeling, 50 nmol of liposomes was incubated with 2 µg of
biotinylated cinnamycin. Liposomes were then fixed as above, washed in
PBS, and blocked with 2% BSA/PBS for 60 min at 4 °C. The fixed
specimens were adsorbed on
poly-L-lysine/Formvar/carbon-coated nickel grids. The grids
were incubated with 5 nm of gold-conjugated anti-biotin IgG for 60 min
at room temperature, washed with distilled water, and fixed with 1%
glutaraldehyde for 5 min. The fixed grids were negatively stained with
uranyl acetate as above. For freeze-fracture electron microscopy,
multilamellar liposomes treated with cinnamycin as described above were
frozen in a high-pressure freezer (HPM 010, BAL-TEC Inc., Balzers,
Liechtenstein). Immediately after freezing, the frozen samples were
placed into liquid nitrogen for storage. The samples were then
fractured in a freeze-etching machine (BAF 400T, BAL-TEC Inc., Balzers)
at
110 °C, replicated by platinum/carbon, collected on
Formvar-coated grids, and examined with an electron microscope (Tecnai
10, Philips, Eindhoven, The Netherlands).
Other Methods--
The cytotoxicity of cinnamycin was monitored
by the release of lactate dehydrogenase (LDH) as described previously
(18). Liposome lysis assay was performed as described previously (3) using calcein as a fluorescent marker.
 |
RESULTS |
Cinnamycin Induces Transbilayer Lipid Movement Both in Target Cells
and in Model Membranes--
Cinnamycin is cytotoxic to both mammalian
cells (19) and yeast Saccharomyces cerevisiae (20). The fact
that cinnamycin-resistant Chinese hamster ovary cells have a defect in
PE synthesis (19, 21) suggests that the binding of cinnamycin to PE is
essential to induce cell lysis. The specific binding of cinnamycin to
PE has been studied relative to the ability of cinnamycin to bind other
phospholipids (7, 8) or proteins (22). However, the binding of
cinnamycin to other plasma membrane lipid components such as
glycolipids has not been examined. Therefore, we first measured the
binding of cinnamycin to various phospholipids and glycolipids (Fig.
1). Cinnamycin has high affinity for PE
as described previously. Phosphatidylserine (PS) gave a slightly
positive signal, whereas other phospholipids and glycolipids tested did
not bind cinnamycin. The binding of cinnamycin to PE was not dependent on incubation temperature as described previously (10). Incubations of
cinnamycin and PE at 4 °C and 37 °C gave similar degrees of specific binding.

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Fig. 1.
Cinnamycin specifically recognizes PE.
Binding of cinnamycin to various lipids was determined by ELISA as
described under "Materials and Methods." Data are the mean of
duplicate experiments ± difference. When not indicated,
difference values are within the bar. PE,
phosphatidylethanolamine; PS, phosphatidylserine;
PI, phosphatidylinositol; PC,
phosphatidylcholine; SM, sphingomyelin;
GlcCer, glucosylceramide; LacCer,
lactosylceramide; GalCer, galactosylceramide;
GM1, ganglioside GM1; GM3, ganglioside GM3; ,
no lipid added.
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Unlike lipid binding, the cytotoxicity of cinnamycin to HeLa cells
showed a clear temperature dependence (Fig.
2). The toxicity, monitored by the
release of LDH activity, increased in a time-dependent manner at 37 °C, whereas at 4 °C LDH was not released. Both
naturally occurring and biotinylated cinnamycin showed a similar extent of cytotoxicity as reported earlier (10). Cells treated with cinnamycin
at 4 °C, washed and then incubated at 37 °C, did not release LDH.
This result indicates that cinnamycin must be present during high
temperature incubation. Because cinnamycin binds PE at 4 °C, our
result suggests that the amount of PE on the cell surface at steady
state was not enough for cinnamycin to induce cytotoxicity. Previously
it was shown that the number of cinnamycin bound to the cells increased
in a time-dependent manner at 37 °C (10). In Fig.
3, we visualized the binding of
biotinylated cinnamycin to HeLa cells at 37 °C. Within 1 min of
treatment, cinnamycin gave small positive signal on the cell surface
(Fig. 3A). The biotin-cinnamycin-positive area increased
during incubation. During incubation, Annexin V also became bound to
the cell surface. Many of the Annexin V-positive regions were also
stained with cinnamycin. Annexin V is known to recognize negatively
charged phospholipids such as PS in a calcium-dependent
manner (23-26). These results indicate that cinnamycin induces
exposure of both PE and PS in HeLa cells at 37 °C. At 4 °C, the
binding of cinnamycin to the cells was not detected under fluorescence
microscope (Fig. 3B), indicating the presence of very few PE
molecules on the surface of steady-state cells. Like cinnamycin,
Annexin V did not bind cells at 4 °C. In the absence of cinnamycin,
Annexin V did not bind cells even at 37 °C (Fig. 3B).
Although binding of cinnamycin occurs after 1 min of treatment,
prolonged incubation was required to induce cell lysis. This result
indicates that the transbilayer lipid movement precedes cell lysis.

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Fig. 2.
High temperature is required for
cinnamycin-induced LDH release in HeLa cells. 37 °C,
HeLa cells were incubated with cinnamycin at 37 °C in the incubation
buffer as described under "Materials and Methods."
b-cinnamycin, cells were incubated with biotinylated
cinnamycin for indicated time intervals at 37 °C.
4 °C, cells were incubated with cinnamycin for indicated
time intervals at 4 °C. 4 °C-37 °C, cells were
preincubated with cinnamycin for 30 min at 4 °C. Cells were then
washed and incubated for indicated time intervals at 37 °C in the
absence of cinnamycin. LDH activity in the medium was measured as
described under "Materials and Methods." Data are the mean of
duplicate experiments ± difference. When not indicated,
difference values are within the bar.
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Fig. 3.
Exposure of PE and PS to the cell surface
during treatment of HeLa cells with biotinylated cinnamycin.
A, cells were treated with 1 µM
biotinylated cinnamycin in the presence of Alexa Fluor 488-conjugated
Annexin V at 37 °C as described under "Materials and Methods."
At appropriate intervals, cells were fixed and labeled with Alexa Fluor
546-conjugated streptavidin. The distribution of Alexa Fluor 546 fluorescence (cinnamycin) and Alexa Fluor 488 fluorescence (Annexin V)
were monitored using a Zeiss LSM 510 confocal microscope as described
under "Materials and Methods." B: left, HeLa
cells were treated with 1 µM cinnamycin in the presence
of Annexin V for 30 min at 4 °C; right, cells were
incubated in the absence of cinnamycin but in the presence of Annexin V
for 30 min at 37 °C. Cells were then fixed and labeled with Alexa
Fluor 546-conjugated streptavidin. Merged fluorescence of Alexa 488 and
546 is shown. Very little fluorescence was observed under these
conditions. Bar: 10 µm.
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We then asked whether cinnamycin itself has the ability to induce
transbilayer lipid movement in model membranes. In Fig. 4, we measured the transmembrane movement
of fluorescent lipids, C6-NBD-PC and C6-NBD-PE, by the method of
extraction of short chain lipids by BSA (13, 14). Addition of BSA
decreased the fluorescence to 60-70% of initial intensity. This is
very likely because of the extraction of the outer leaflet
C6-NBD-lipids by BSA (13, 14). The effect of BSA was not changed
significantly by the incorporation of POPE into the liposomes. If PE is
included in the membrane, the subsequent addition of cinnamycin further decreased the fluorescence. The decrease of fluorescence depends upon
the amount of PE in the membrane. Inclusion of as little as 1% PE in
the liposomes was effective. Under these conditions liposome lysis
monitored by the release of calcein was less than 2%. These results
indicate that cinnamycin promotes the transbilayer movement of the
fluorescent phospholipids in model membranes. However, without PE
cinnamycin did not induce flip-flop of fluorescent phospholipids.
Because cinnamycin induced transbilayer movement of both fluorescent PC
and PE, it is suggested that there is no lipid selectivity in the
outward translocation of phospholipids.

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Fig. 4.
Cinnamycin induces transbilayer lipid
movement in model membranes. POPC/POPE LUVs were prepared with the
inclusion of 0.25% of short chain fluorescent lipids, C6-NBD-PC
(A) or C6-NBD-PE (B), and the NBD fluorescence
was monitored by measuring fluorescence at 535 nm with excitation 475 nm. BSA and cinnamycin were added where indicated. BSA caused a
substantial decrease of the fluorescence due to the quenching of
extracted lipid. Subsequent addition of cinnamycin induced a further
decrease of the fluorescence in PE-dependent manner.
Results are representative of three independent experiments.
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Re-orientation of Membrane Lipids Was Accompanied by a Structural
Change in Cinnamycin--
Peptide-induced perturbation of bilayer
structures was estimated by examining the CH2 symmetric
stretching band near 2850 cm
1 using Fourier transform
infrared-polarized attenuated total reflection (FTIR-PATR) (27, 28).
The dichroic ratio, R, of an absorption band obtained by
PATR spectra is a measure of the orientation of its transition moment
or molecular axis. For membranes much thicker (~6 µm) than the
penetration depth (under our experimental conditions, 0.2-0.8 µm in
the range of 3000-800 cm
1), an R value
smaller than 2 indicates that the moment lies essentially parallel to
the membrane surface (28-30). From the dichroic ratio R,
the frequency and order parameter, S, is calculated by use of Equation 1 (28-30). This parameter is connected to the
following,
|
(Eq. 1)
|
where the mean orientation angle,
, is between the hydrocarbon
chain and the membrane normal through Equation 2, assuming the uniaxial
orientation of the chain around the normal,
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(Eq. 2)
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The results of the calculations are summarized in Table
I. The presence of cinnamycin in
D2O-hydrated POPE films significantly reduced the order
parameter, indicating that the orientation axis of the lipid was
perturbed by the peptide.
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Table I
Conformations and orientations of lipid hydrocarbon chains
Results are the mean of duplicate experiments ± difference.
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We then asked whether the conformation of cinnamycin was altered in the
presence of PE. The CD spectrum of cinnamycin gave a minimum at 197 nm,
characteristic of a random coil conformation (Fig.
5A) (28). The conformation of
cinnamycin in solution was also examined using transmission IR (Fig.
5B). The major band at 1641 cm
1 originated
from a random coil structure and was in good agreement with the CD
spectrum. Fig. 5C shows ATR spectra of
D2O-hydrated DOPE/cinnamycin (12.5/1) films in the region
of 1600-1700 cm
1. The band at 1633 cm
1 was
assigned to
-sheet conformation whereas the band at 1660 cm
1 corresponds to the formation of turn structure. The
dichroic ratio of the amide I region was close to 2, suggesting a
random orientation of the peptide or an orientational angle close to the magic angle (54.7°) with respect to the membrane normal.

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Fig. 5.
The conformation of cinnamycin alters in the
presence of PE. A, CD spectrum of cinnamycin in 20 mM Hepes-NaOH (pH 7.4); B, FTIR spectrum of
cinnamycin in D2O containing 20 mM Hepes-NaOH
(pH 7.4); C, FTIR-ATR spectrum of POPE/cinnamycin. The
blue line denotes A , whereas
the red line indicates A spectrum.
Data shown are representative of two independent experiments.
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Cinnamycin Induces Re-organization of
Phosphatidylethanolamine-containing Membranes--
When the
surface concentration of PE was high, cinnamycin induced dynamic
membrane re-organization. In Fig. 6, we
measured the activity of cinnamycin to induce liposome
fusion. The increase of NBD fluorescence indicates that fusion occurs
between donor and acceptor membranes. Cinnamycin-induced membrane
fusion in DOPC/DOPE (1:1) and to a lesser degree in POPC/POPE (1:1)
liposomes. However PC liposomes did not fuse in the presence of
cinnamycin. Fusion requires a high concentration of PE in the membrane,
because the addition of 10% POPE to POPC did not cause
cinnamycin-induced membrane fusion (data not shown).

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Fig. 6.
Cinnamycin induces fusion of PE-containing
membranes. In donor liposomes, NBD fluorescence was quenched by
LRh. Liposome fusion caused the release of resonance energy transfer,
which was monitored by the increase of NBD fluorescence. Cinnamycin was
added where indicated. Data shown are representative of three
independent experiments. Cinnamycin selectively induced fusion of
PE-containing membranes.
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The ultrastructure of liposomes in the presence of cinnamycin was
examined by negative staining and freeze-fracture electron microscopy
(Fig. 7). Cinnamycin did not alter the
morphology of DOPC membranes (Fig. 7, A and C).
Addition of 50% DOPE dramatically altered the ultrastructure of the
membranes after the treatment with cinnamycin (Fig. 7, B and
D). Characteristic restiform aggregates were accumulated on
liposomes. Immunogold labeling showed that cinnamycin was present in
these aggregates (Fig. 7B). Freeze-fracture images
visualized that these structures consisted of moniliform aggregates
(Fig. 7D).

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Fig. 7.
Electron micrographs of liposomes treated
with cinnamycin. A and B, negative staining
images; C and D, frozen replica;
A and C, DOPC in the presence of cinnamycin;
B and D, DOPC/DOPE (1:1) in the presence of
cinnamycin. Images are representative of two independent
experiments with similar results. Bars, 100 nm.
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 |
DISCUSSION |
Our results indicate that cinnamycin induces transbilayer lipid
movement in target cells. Flip-flop of plasma membrane phospholipids caused the exposure of PE, which is a specific receptor for cinnamycin. The binding of increased number of cinnamycin to the cell surface induced a dramatic membrane reorganization that eventually leads to
cell death. We cannot exclude the possibility that the observed binding
of cinnamycin was a result of spontaneous flip-flop of cell PE during
incubation. However, unlike inward translocation (t1/2 = minutes), reported outward translocation of
phospholipids is rather slow (t1/2
1.5 h)
(31-34). In addition, the exposure of PS in treated cells indicates
that cinnamycin has the ability to induce transbilayer phospholipid
movement. Although cinnamycin weakly bound PS in ELISA, previously it
was shown that the binding of cinnamycin to the cells was abolished in
the presence of PE-containing liposomes, indicating that cinnamycin
specifically recognized PE in the cell membrane (35). Model membrane
experiments indicate that PE is necessary for cinnamycin to induce
transbilayer lipid movement. Using amino-reactive probe,
trinitrobenzene sulfonic acid, it has been shown that in steady-state
fibroblasts, 2-2.5% of total PE is exposed to the cell surface (36,
37). We could not detect positive signals with biotinylated cinnamycin
at 4 °C incubation under fluorescence microscope. The binding of
cinnamycin to PE monitored by ELISA was not
temperature-dependent. These results suggest that the
steady-state amount of PE on the cell surface was too low to be
detected by biotinylated cinnamycin under fluorescence microscope.
However, cinnamycin bound this small amount of PE and did induce
transbilayer phospholipid movement. Because preincubation of cells with
cinnamycin at 4 °C did not affect the plasma membrane permeability
to LDH even after incubation at 37 °C, the binding of cinnamycin to
the steady-state amount of cell surface PE was not sufficient to induce
membrane damage. Time course experiments indicate that transbilayer
phospholipid movement precedes a membrane permeability change. Like
cinnamycin, the type I lantibiotic nisin, which is a cationic peptide,
induces transbilayer lipid movement without causing membrane leakage
(38). Recently Zhang et al. (39) showed that natural and
synthetic cationic antimicrobial peptides induce lipid flip-flop at
peptide concentrations that were 3- to 5-fold lower than those causing
leakage of membrane. Induction of transbilayer lipid movement has also
been reported with the antimicrobial peptide magainin 2 (40) as well as
synthetic peptides (41). Unlike nisin and flip-flop-inducing synthetic peptides, cinnamycin is electrically neutral. Cinnamycin is unique in
that flip-flop occurs in PE concentration-dependent manner, and the binding of the toxin to cells is dependent on the transbilayer lipid movement. That is, the toxin self-promotes its own binding to
target cells by causing the exposure of the binding PE molecules that
reside in the inner leaflet of the lipid bilayer.
The mechanism of the induction of lipid flip-flop by cinnamycin is not
clear. Proton NMR analysis has revealed that one cinnamycin molecule
binds one PE (42). Our FTIR study indicates that, in the presence of
PE, cinnamycin undergoes conformational change, resulting in the
increase of
-sheet structure. Enrichment of
-sheet structure in
the presence of phospholipid has also been shown for nisin (43). The
observation that the orientation of cinnamycin was random and that the
order parameter of acyl chains of PE was significantly reduced suggests
that the toxin may locally induce a non-bilayer structure, inducing
flip-flop of phospholipids in the cell membrane. This idea is
consistent with the fact that, when the membrane concentration of PE is
high, cinnamycin could induce dramatic membrane alteration. Recently,
using 31P and 2H NMR, Machaidze et
al. (8) demonstrated a perturbation of the bilayer structure of
POPC/POPE (4:1) vesicles in the presence of cinnamycin. Membrane fusion
is often accompanied by non-bilayer structure. Our electron
microscopy image suggests the formation of non-bilayer structure
in cinnamycin-treated PC/PE liposomes. Our results together with the
NMR observation by Machaidze et al. suggest that the dynamic
reorganization of the membrane is associated with cinnamycin-induced
cell death.
 |
ACKNOWLEDGEMENTS |
We thank T. Takakuwa of Jasco for CD
measurement. We are grateful to E. Kiyokawa, R. Ishituka, M. Takahashi,
E. Hayakawa, A. Yamaji, and Y. Shimizu for critically reading the manuscript.
 |
FOOTNOTES |
*
This work was supported by Grants-in-Aid for Scientific
Research 12672143 and 14370753 (to T. K.) and 13670007 (to
T. B.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
i
To whom correspondence should be addressed. Tel.:
81-48-467-9612; Fax: 81-48-467-8693; E-mail:
kobayasi@postman.riken.go.jp.
Published, JBC Papers in Press, November 22, 2002, DOI 10.1074/jbc.M210347200
 |
ABBREVIATIONS |
The abbreviations used are:
PS, phosphatidylserine;
NBD, 7-nitrobenz-2-oxa-1,3-diazole-4-yl;
C6-NBD-PC, 1-palmitoyl-2-[6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl]-sn-glycero-3-phosphocholine;
C6-NBD-PE, 1-myristoyl-2-[6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl]-sn-glycero-3-phosphoethanolamine;
DOPC, dioleoylphosphatidylcholine;
DOPE, dioleoylphosphatidylethanolamine;
ELISA, enzyme-linked
immunosorbent assay;
FTIR-PATR, Fourier transform infrared-polarized
attenuated total reflection;
HFIP, 1,1,1,3,3,3-hexafluoro-2-propanol;
LDH, lactate dehydrogenase;
LUV, large unilamellar vesicle;
MLV, multilamellar vesicle;
N-LRh-PE, N-(lissamine
rhodamine B sulfonyl)-dioleoylphosphatidylethanolamine;
N-NBD-PE, N-(NBD)-dioleoylphosphatidylethanolamine;
PC, phosphatidylcholine;
PE, phosphatidylethanolamine;
POPC, palmitoyloleoylphosphatidylcholine;
POPE, palmitoyloleoylphosphatidylethanolamine;
BSA, bovine serum albumin;
TBS, Tris-buffered saline;
ATR, attenuated total reflection;
PBS, phosphate-buffered saline;
GM1, Gal
1,3GalNAc
1,
4(NeuAc
2,3)Gal
1,4GlcCer;
GM3, NeuAc
2,3Gal
1,4GlcCer.
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