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J. Biol. Chem., Vol. 276, Issue 38, 35875-35882, September 21, 2001
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From
Received for publication, May 22, 2001, and in revised form, July 6, 2001
Vasostatin-I, the natural fragment of
chromogranin A-(1-76), is a neuropeptide able to kill a large variety
of fungi and yeast cells in the micromolar range. We have examined the
antifungal properties of synthetic vasostatin-I-related peptides. The
most active shortest peptide, named chromofungin, corresponds to the sequence Arg47-Leu66. Extensive
1H NMR analysis revealed that it adopts a helical
structure. The biophysical mechanism implicated in the interaction of
chromofungin with fungi and yeast cells was studied, showing the
penetration of this peptide with different lipid monolayers. In order
to examine thoroughly the antifungal activity of chromofungin, confocal
laser microscopy was used to demonstrate the ability of the
rhodamine-labeled peptide to interact with the fungal cell wall, to
cross the plasma membrane, and to accumulate in Aspergillus
fumigatus, Alternaria brassicola, and Candida
albicans. Our present data reveal that chromofungin inhibits
calcineurin activity, extending a previous observation that the
N-terminal region of chromogranin A interacts with calmodulin in the
presence of calcium. Therefore, the destabilization of fungal wall and
plasma membrane, together with the possible intracellular inhibition of
calmodulin-dependent enzymes, is likely to represent the
mechanism by which vasostatin-I and chromofungin exert antifungal activity.
The need for effective antifungal agents increases in
parallel with the expanding number of immunocompromised patients at risk for invasive fungal infections. Currently available antifungal compounds act on targets also found in mammalian cells (1), which may
result in toxicity. Thus, it becomes imperative to find antifungal
compounds with no toxicity toward mammalian cells.
We have recently discovered new natural antimicrobial peptides derived
from the processing of chromogranins A
(CGA)1 and B (CGB) and of
proenkephalin-A, which are secreted with catecholamines upon
stimulation of chromaffin cells in adrenal medulla (2-7). These
peptides have been hypothesized to play a role in stress situations,
acting as an immediate protective shield against pathogens (6). Among
these active neuropeptides, we have characterized the antibacterial and
antifungal activities of vasostatin-I, CGA-(1-76), the highly
conserved N-terminal fragment resulting from the natural processing of
CGA (8). Our previous work has also shown that vasostatin-I is secreted
from human polymorphonuclear neutrophils and that the C-terminal moiety
of vasostatin-I CGA-(41-70) is essential for its antifungal activity
(7).
Antimicrobial agents are classified as inhibitors for microbial cell
metabolism (9), lytic peptides that disrupt the membrane structure
(10), inhibitors for protein biosynthesis, and inhibitors for DNA and
RNA synthesis (11). The present work was carried out to understand the
molecular mechanisms involved in the antifungal activity of the
C-terminal region of vasostatin-I; we sought the most active fragment
and found the CGA-(47-66) peptide to be the shortest potent one. This
peptide was named chromofungin. The three-dimensional structure of
chromofungin has been obtained from 1H NMR analysis,
indicating that it adopts a helical structure.
The interaction of chromofungin with fungal membranes was analyzed
using biophysical measurements of the penetration of these peptides
within lecithin monolayers (12). Confocal laser microscopy on several
fungi and yeast cells using rhodamine-labeled chromofungin showed
the ability of this peptide to cross through cell wall and plasma
membrane and to accumulate in microorganisms. Since previous studies
have shown that the CGA N-terminal region interacts with calmodulin
(CaM) in the presence of calcium (13), the sequence of chromofungin was
compared with those of several CaM-binding enzymes and found to display
common features. This property led us to study the effect of
chromofungin on CaM-dependent enzymes choosing calcineurin
(CaN) phosphatase activity.
Preparation of Synthetic Peptides--
Peptides were synthesized
in our laboratory on an Applied Biosystems 432A peptide synthesizer
SYNERGY, using the stepwise solid-phase synthetic approach (7, 14) with
Fmoc (9-fluoromethoxycarbonyl) chemistry. After lyophilization,
synthetic peptides were analyzed by sequencing (7, 15) and
matrix-assisted laser desorption-ionization time-of-flight mass
spectrometry (5, 16, 17). Chromofungin corresponds to the bovine
CGA-(47-66) sequence RILSILRHQNLLKELQDLAL. For control experiments, a
chromofungin-derived peptide with two proline residues in positions 61 and 64 and a bovine CGB-(602-626) peptide corresponding to the
sequence ELENLAAMDLELQKIAEKFSGTRRG have been prepared.
For the fluorescence study, peptides were dissolved in
diisopropylethylamine and labeled by the addition of
5,6-carboxytetramethylrhodamine and
1-hydroxybenzotriazole/o-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate as coupling agent.
Antifungal Activity--
Filamentous fungi were grown on a
five-cereal medium, and spores were harvested as previously described
(18). The following filamentous fungi strains were used:
Neurospora crassa (CBS 327-54), Aspergillus
fumigatus, Alternaria brassicola (MUCL 20297),
Nectria hematococca (160.2.2), Fusarium culmorum
(MUCL 30162), Fusarium oxyporum (MUCL 909), and
Trichophyton mentagrophytes. Yeast cells were precultured on
a Sabouraud medium, and four strains were tested: Candida
albicans, Candida tropicalis, Candida
glabrata, and Cryptococcus neoformans. In order to test
the antifungal activity of CGA-(47-66), spores (final concentration,
104 spores/ml) were suspended in a growth medium containing
potato dextrose broth (Difco; in half-strength) and yeast cells in
Sabouraud medium, with starting absorbance at 620 nm of 0.001. These
media were supplemented with tetracycline (10 µg/ml) and cefotaxime (0.1 µg/ml). Fungal growth was assessed after an appropriate
incubation period (24 or 40 h at 30 °C). Aliquots of peptide
extract (20 µl) were incubated in microtiter plates with 80 µl of
fungal spores or yeast cultures. Growth of fungi was monitored
microscopically and evaluated by measuring the culture absorbance at
595 nm (A595) using a microplate reader. Yeast
cells growth was assessed by the increase of
A620.
1H NMR Analysis of Synthetic Peptide
CGA-(47-66)--
A lyophilized sample of 4 mg of CGA-(47-66) was
dissolved in 600 µl of aqueous 20 mM sodium
acetate-d6 buffer (pH 5.0), 50 mM
KCl with the addition of deuterated trifluoroethanol
(TFE-d3) to yield 50% (v/v) solution. The final
peptide concentration was 3 mM. The
2,2-dimethyl-2-silapentane-5-sulfonate was used as an internal chemical
shift reference (0.00 ppm).
All 1H NMR measurements were carried out at 288 K at 500 MHz on a Bruker AMX500 spectrometer. Standard two-dimensional proton scalar and dipolar coupling experiments were carried out in
phase-sensitive mode using the STATES-TPPI (time-proportional phase
incrementation) scheme (19), with a spectral width of 6009 Hz in both
dimensions and a relaxation delay of 3 s. Water signal suppression
was achieved using a WATERGATE sequence (20), together with a very low
power presaturation pulse on the water frequency. Amino acid proton spin systems were mostly assigned using a homonuclear Hartman Hahn (21)
experiment with a 60-ms DIPSI2 (decoupling in the presence of scalar
interaction) isotropic mixing pulse sequence. Sequential assignment was
achieved using two NOESY spectra recorded with mixing times of 150 and
250 ms.
Processing was performed on an IBM RS/600 computer using the program
FELIX version 2.10 (Biosym Inc.) and on an SGI INDY R5000 computer
using UXNMR software (Bruker). Data were zero-filled and processed by
apodization with a 90°-shifted sine squared bell window in both
dimensions before Fourier transformation. The final size of matrices
was 2048 × 1024 points for all spectra. The computer model was
obtained using the Molmol program (22).
Monolayer Measurements--
Penetration experiments of peptides
within artificial monolayer membrane (12) were performed at constant
area (19.6 cm2) in a thermostated (T = 20 ± 0.2 °C) glass vessel equipped with a short vertical tube and
containing 100 mM NaCl, 20 mM Tris-HCl, pH 7, as a subphase (volume 12 ml). The vessel was put on a miniature Variomag stirring system, and the whole device was enclosed in a
plexiglass box. The lipids were spread at the air/water interface from
an ethanol solution to give the desired initial surface pressure. Aliquots of a peptide stock solution (concentration in water: 10 Confocal Microscopy--
The poly-L-lysine-coated
chambered coverglass system (× 8, Lab Tek, 0.8 cm2) was
covered with 190 µl of medium (one-half potato dextrose broth or
Sabouraud) containing A. fumigatus or A. brassicola (104 spores/ml) or C. albicans
(A620 = 0.001) at 30 °C during 24 or 40 h. Then rhodamine-labeled synthetic peptides (CGA-(47-66)R and
CGB-(602-626)R) were added to culture medium (30 °C). After the
incubation time as indicated, chambers were washed with fresh culture
medium and subsequently treated for 90 min with 4% paraformaldehyde in
0.12 M sodium/potassium phosphate, pH 7.2. After several
rinses with phosphate-buffered saline, chambers were covered with
Elvanol-Mowiol.
Fluorescence staining was monitored with a Zeiss laser scanning
microscope (LSM 410) equipped with a planapo oil (× 63) immersion lens
(numerical aperture = 1.4). Rhodamine emission was excited using
the helium/neon laser 543-nm line, and the emission signal was filtered
with a Zeiss long pass 595-nm filter. Fungi were subjected to optical
serial sectioning (0.2-0.3 µM) to produce images in the
x-y plane. Each optical section was scanned eight times to obtain an average image. Images were recorded digitally in a
768 × 576-pixel format.
Calcineurin Activity Assay--
To measure the
calcineurin phosphatase activity, we have used the BIOMOL
GREENTM assay kit (TEBU), including a 96-well microtiter
plate format with all reagents supplied by the manufacturer. The RII
phosphopeptide substrate (DVPIPGRFDRRVpSVAAE), is the most efficient
peptide substrate known for calcineurin (23, 24). The detection of free
phosphate released is based on the classic malachite green assay.
Antifungal Activity of CGA-(47-66) and
CGA-(47-70)--
Recently, we have shown that vasostatin-I, the CGA
N-terminal 1-76 peptide, displays antibacterial and antifungal
activities (7). After digestion of recombinant human vasostatin-I with endoproteinase Glu-C, the shortest antifungal peptide corresponding to
CGA-(47-60) was isolated and identified (7). In order to investigate
the molecular mechanisms involved in the antifungal activity of
vasostatin-I, several other synthetic peptides have been prepared with
various CGA-41/47-60/70 sequences, and antifungal assays revealed that
the most active ones were CGA-(47-60) and CGA-(47-70) (7). Then the
shortest active peptides with maximum global hydrophobicity and
amphipathic features were synthesized and tested. As shown here, two
peptides were found to be the most efficient; they correspond to
CGA-(47-70) and CGA-(47-66) (Table I).
These two peptides are similarly active against a variety of
filamentous fungi, including N. crassa, A. fumigatus, A. brassicola, N. hematococca, F. culmorum, F. oxyporum, and
T. mentagrophytes in the concentration range from 5 to 30 µM. Furthermore, when tested against the growth of
several strains of yeasts, CGA-(47-70) inhibited completely C. albicans and C. neoformans at a concentration of 200 and 150 µM, respectively, while it inhibited C. tropicalis by 70% at 200 µM. Interestingly, the
second synthetic fragment CGA-(47-66) was more active, since it
inhibited completely the growth of C. albicans, C. tropicalis, and C. neoformans at a concentration of 50 µM; it was inactive against the growth of C. glabrata at 200 µM.
Since the shortest synthetic peptide CGA-(47-66) displayed the highest
antifungal activity, it was used thereafter to investigate the
molecular mechanisms. This synthetic peptide has been named chromofungin.
1H NMR Spectroscopy of Chromofungin--
The first
step was to determine the three-dimensional structure of this peptide
in water/trifluoroethanol solution (50/50) using 1H NMR
spectroscopy. The folding of peptides in the presence of trifluoroethanol is often encountered for peptides able to interact with membranes and has been widely used to study their
three-dimensional properties. Among antibacterial peptides reported so
far, trifluoroethanol has been used to study the structure of
cecropin A (25), pardaxin P-2 (26), magainin 2 (27), and ranalexin
(28). In addition, comparative study of 1H NMR structures
of magainin 2 and ranalexin in the presence of phospholipid micelles
confirmed the helical structure previously found in
trifluoroethanol/water solution (28, 29).
Two regions of the 150-ms NOESY spectrum are shown in Fig.
1. They correspond to the fingerprint
region, which contains the dipolar correlations between amide and H
All sequential and medium range NOEs, which have been observed for the
backbone protons of the CGA-(47-66) peptide are summarized in Fig.
2. The distribution of
Three-dimensional Model of Chromofungin--
A ribbon diagram of
the average structure of the CGA-(47-66) peptide is shown in Fig.
3. This representation shows the
amphipathic helical character of the C-terminal part of the sequence
(Arg53-Leu66). It is shown that hydrophobic
leucine residues in positions 57, 58, 61, 64, and 66 are located on one
side (blue ribbon), while four polar residues
(Arg53, Lys59, Glu60, and
Asp63) are aligned on the other side (red
ribbon). This feature is less pronounced in the N-terminal
side of the Importance of the Amphipathic Helical Fragment CGA-(53-66) for the
Antifungal Activity of Chromofungin--
Chromofungin, in the
concentration range of 1-10 µM, is highly active against
a variety of filamentous fungi, and it completely inhibits the growth
of N. crassa at 5 µM (Table I). In order to
examine the importance of this amphipathic C-terminal sequence, we have
tested the antifungal activity of the chromofungin-derived peptide with
two proline residues in place of Leu61 and
Leu64. These two proline residues disrupt the helical
structure and the amphipathic character (Fig. 4). This control peptide
inhibits the growth of N. crassa by 81% at 100 µM, whereas it was inactive at the concentration of 5 µM. This result shows that the amphipathic helix is
crucial for the antifungal activity.
Many antimicrobial peptides have Penetration of Chromofungin into Lipid Monolayers--
Penetration
experiments were performed at peptide concentrations ranging from
5 × 10 Confocal Laser Microscopy Analysis of the Interaction of
Chromofungin with A. fumigatus, A. brassicola, and C. albicans--
We
used confocal laser microscopy to analyze the interaction of
chromofungin with fungal membranes using the synthetic
rhodamine-labeled CGA-(47-66)R peptide. The fluorescent rhodamine
chromogranin B-derived peptide CGB-(602-626)R was used as a control,
since it was inactive against fungi and yeasts. Prior the confocal
microscopy analysis, we verified that the rhodamine-labeled peptides
possess similar antifungal activities as those of unlabeled peptides.
In these experiments, a concentration of 10 or 30 µM
peptide was used. Fungal spores of A. fumigatus, A. brassicola, and C. albicans were incubated at 30 °C
first during 24 or 40 h and then for 1, 16, or 21 h with
fluorescent peptides.
Fungal spores of A. fumigatus were incubated during 24 h prior to treatment for 1 h at 30 °C with fluorescent peptides
(Fig. 6). By comparison with a control
experiment (no peptide; Fig. 6A), CGA-(47-66)R 10 µM was visible at the level of the cell wall, where it
seemed to accumulate, but also in the inner part of A. fumigatus (Fig. 6B). In contrast, the labeled inactive
peptide CGB-(602-626)R was undetectable in filamentous fungi (A. fumigatus; Fig. 6C) and yeast (C. albicans;
data not shown) at any treatment time.
In a second series of experiments, we have tested the activity of
chromofungin on fungal spores of A. fumigatus prepared by a
first incubation for 40 h (Fig.
7A), prior to treatment for 1 h at 30 °C with CGA-(47-66)R 30 µM (Fig.
7B). The fluorescent active peptide was partly visible on
the cell wall and the inner part of the distal growing end. This result
showed that chromofungin penetrated through the cell wall at the fungus
weakest point. After incubation for 40 h and then for 21 h at
30 °C with CGA-(47-66)R 10 µM, we observed that the
fluorescent peptide invaded the numerous hyphae of A. fumigatus, inducing distortion of the wall and formation of
numerous vacuoles (Fig. 7C). By phase-contrast microscopy, fungi regions invaded with the labeled peptide were visible as darkened areas.
In addition, we also examined the spores of A. brassicola
(Fig. 8A) and C. albicans (Fig. 8B) prepared by a first incubation for
24 h followed by a second incubation for 1 h at 30 °C with CGA-(47-66)R 30 µM or CGA-(47-66)R 10 µM,
respectively. As compared with control experiments, the fluorescent
peptide was detectable in cytoplasm and cell wall of both
microorganisms. Fluorescence was absent, however, in the septum of
A. brassicola (Fig. 8A, lower
panel).
The treatment of A. fumigatus or C. albicans for
1 h with higher concentrations of unlabeled chromofungin (100 µM) together with the inactive CGB-(602-626)R peptide
(10 µM) revealed an intense fluorescence in A. fumigatus (Fig. 6D) and in C. albicans (data not shown). This result indicated that chromofungin at high
concentrations destabilizes cell wall, allowing inert peptide to
penetrate into microorganisms.
Interaction of Chromofungin with Calmodulin--
Besides
destabilization of microorganism cell wall, chromofungin may also exert
activity on intracellular targets. It has previously been shown that
CGA is able to interact with CaM (13) and that the domain responsible
for this interaction is probably located in the N-terminal region. CaM
binds a variety of peptides, hormones, toxins, and enzymes in a
calcium-dependent manner. The affinity of these peptides
(16-25 residues) for CaM appeared to correlate with their abilities to
form positively charged amphiphilic helices named BAA helices (basic
amphipathic
Recently, it has been established that CaN, the CaM-activated
phosphatase B, plays a crucial role in hyphal growth, morphology, and
maintenance of the apical Ca2+ gradient in the
filamentous fungi N. crassa (36). This observation prompted
us to examine the effect of chromofungin on CaN phosphatase activity.
The experimental data reported in Fig.
10 showed that CaN phosphatase activity
was completely inhibited by chromofungin at a concentration of 250 µM. At a concentration of 5 µM,
corresponding to minimal inhibitory concentration against the
filamentous fungi growth, the inhibition of CaN was evaluated to be
32%. The ability of chromofungin to inhibit CaN phosphatase activity
at the same concentration range as that inhibiting fungal growth
suggests that this molecular mechanism might be implicated. However, in addition to CaN, other CaM-dependent enzymes may be the
target to chromofungin, which implies intracellular effects of this
peptide.
The secretory granules of chromaffin cell from adrenal medulla
synthesize, store, and, upon cell stimulation occurring during stress,
exocytotically release catecholamines into circulation to activate
target organs. Besides catecholamines, chromaffin granules contain a
large number of proteins and peptides, including neuropeptides. The
water-soluble proteins named chromogranin A and B and their derived
peptides represent the major components. Furthermore, these proteins
are widely distributed in endocrine, neuroendocrine, and nerve cells
but also in immune cells (7). Sequences of CGA from a variety of
species have now been reported (37), and from them the position of
splicing sites and the size of the seven introns show a strong
phylogenic conservation (38). It is important to point out that the
N-terminal end of CGA is characterized by the presence of a disulfide
loop (7) (which is also present on the N-terminal end of CGB) and that
the sequences of CGA-(1-78) from several species are highly conserved,
exhibiting 71% homology between frogs and humans (39) and up to 97%
between bovine and humans (7).
Studies on CGA and CGB processing have established that in chromaffin
granules these two proteins are attacked at both N- and C-terminal
sides by proteolytic enzymes generating numerous peptides (2, 8, 40).
In addition, the processing of CGA and CGB has been shown to be cell-
and tissue-specific (41, 42), giving rise to the release of multiple
peptides recovered in blood and lymph circulations but also in
biological fluids such as salivary (43), synovial, milk, and
cerebrospinal fluids.2
Vasostatin-I (CGA-(1-76)) is a natural N-terminal fragment of CGA,
released from adrenal medulla (44) and sympathetic nerve terminals (45) in response to stimulation and first shown to exert
vasoconstriction inhibitory activity on isolated human blood vessels
(46). Smooth muscle cells display saturable binding sites to
vasostatin-I and to the shorter fragment CGA-(1-40) with a unique
affinity constant close to 50 nM (47). The role of vasostatin-I in stress response could represent an important biological function in addition to that of catecholamines on regulating
blood circulation. Vasostatin-I also inhibits parathyroid hormone
secretion (48), is neurotoxic in neuronal/microglial cell cocultures
(49), and induces cell adhesion (50, 51). Our recent work has shown that vasostatin-I possesses antifungal activity (7), killing filamentous fungi and yeasts in the micromolar range. Furthermore, we
reported that this new antibacterial and antifungal peptide is released
from polymorphonuclear neutrophils, suggesting that it may act as a
defensive molecule in infectious sites (7).
The past decade has witnessed a dramatic increase in knowledge of
natural antimicrobial peptides, but most data concern antibacterial molecules. The emergence of fungal pathogens resistant to current therapies requires the discovery of new nontoxic antifungal molecules. Among all synthetic vasostatin-derived peptides, chromofungin actually
displayed the highest antifungal activity on both fungi and yeast. The
1H NMR structural analysis of chromofungin demonstrates
that, in our experimental conditions, chromofungin adopts an
Antifungal peptides are classified according to their mode of action. A
first peptide group acts by lysis which occurs via several mechanisms
(e.g. destabilization of membrane, formation of aqueous
pores, and intracellular mechanisms), while a second group interferes
with cell wall synthesis or with the biosynthesis of glucan or chitin
(1, 57). Membrane penetration studies with chromofungin have
demonstrated that this peptide was able to interact with lipid
monolayers. The increase of the surface pressure in presence of
ergosterol indicated a specificity for the fungal membranes. In
addition, chromofungin induced membrane destabilization with the
possible formation of holes, since the inactive peptide CGB-(602-626)
could be recovered within fungi and yeast after preincubation with
chromofungin. Thus, the specific antifungal activity of chromofungin
should be dependent on a molecular mechanism located at the cell wall
rather than at the sole plasma membrane. In addition, chromofungin
penetrates into the cytoplasm, as evidenced by by confocal laser
microscopy. Thus, within the microorganism it may interact with
intracellular targets.
The cell wall is a physically rigid layer that protects the fungal cell
from its environment, mediates cell-cell interaction, and is
responsible for the shape of the cell. The structural skeleton of the
cell wall is composed of The interaction of chromofungin with CaM and CaM-binding proteins
requires careful future investigation, because it opens the way for a
new mechanism concerning antifungal molecules.
We have to mention here that although chromofungin possesses
antifungal activity at micromolar range, it is a nonhemolytic peptide,
unable to penetrate into astrocytes, oligodendrocytes, and
neurons,3 suggesting that
this peptide is nontoxic at this concentration. Furthermore, it has
recently been reported that the CGA-(47-57) peptide (RILSILRHQNL),
included in chromofungin, contains a cell adhesion site for fibroblasts
and smooth muscle cells (51), pointing out multifunctional roles for
this Vasostatin I appears as a peptide with multifunctional potency. Besides
its property as a vascular modulator (46), it is able to kill bacteria
and fungi (7) in in vitro tests. At this stage, it is not
known whether it exerts this activity in vivo. The
concentration of circulating vasostatin-I is in the 100 nM range, and its half-life in blood remains to be determined. However, as
has been shown for enkelytin, a chromaffin granule peptide with
antibacterial activity (5), antibacterial and antifungal peptides may
concentrate in infectious sites and display synergistic activities.
To conclude, vasostatin-I sequence contains a highly conserved region
of 14 amino acids; the corresponding peptide displays a potent
antifungal activity due to its ability to cross microorganism cell
membranes and perhaps to target and inhibit calmodulin-binding enzymes.
Although some important questions concern the expression of its
activity in physiological and pathological situations, chromofungin
represents a prototypic lead molecule useful for the development of new
therapeutic agents.
We are indebted to Dr. W. Broekaert (F. A. Janssens Laboratory of Genetics, Heverlee, Belgium) for the generous
gift of fungi strains.
*
This work was funded by INSERM and supported by grants from
the Meiji Institute of Health Science (Odawara, Japan), the Direction des Recherches, Etudes et Techniques (Grant DRET 96-099 to D. A.),
University Louis-Pasteur (Contrats PPF 1997-2000 to D. A.), the Ligue
Contre le Cancer (to M.-H. M.-B. and D. A.), the Association Recherche et Partage (Ph.D. grant to K. L.), and the Fondation pour la
Recherche Médicale (Ph.D. grant to K. L.).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.
**
To whom correspondence should be addressed: INSERM U. 338, IFR37, 5 rue Blaise Pascal, 67084 Strasbourg Cedex, France. Tel.: 33-3-88-45-66-09; Fax: 33-3-88-60-08-06; E-mail:
metz@neurochem.u-strasbg.fr.
Published, JBC Papers in Press, July 12, 2001, DOI 10.1074/jbc.M104670200
2
M.-H. Metz-Boutigue and D. Aunis, manuscript in preparation.
3
K. Lugardon, D. Aunis, and M.-H. Metz-Boutigue,
unpublished observation.
The abbreviations used are:
CGA, chromogranin A;
CGB, chromogranin B/secretogranin I;
CaM, calmodulin;
CaN, calcineurin;
NOE, nuclear Overhauser enhancement;
NOESY, NOE spectroscopy;
TFE-d3, deuterated trifluoroethanol;
mN, millinewtons.
Structural and Biological Characterization of
Chromofungin, the Antifungal Chromogranin A-(47-66)-derived
Peptide*
,§,,,
,, and**
INSERM Unité 338, IFR37 "Biologie de la
Communication Cellulaire," 5 rue Blaise Pascal 67084 Strasbourg
Cedex, France, § CNRS UPR 2356 IFR37
"Neurotransmission et Sécrétion Neuroendocrine," 5 rue
Blaise Pascal 67084 Strasbourg Cedex, France, CNRS UPR 9003 "Cancérogénèse et Mutagénèse
Moléculaire et Structurale," 67400 Illkirch Graffenstaden,
France, and ¶ Centre de Biophysique Moléculaire CNRS,
H5000 Orléans, France
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5 Mres, expressed in amino acid
residue/liter) were successively injected in the subphase under the
lipid film through the vertical tube, and the bulk phase was gently
stirred for a few minutes. The surface tension was measured with a
Wilhelmy platinum plate hung to a Q11 balance (Schenk SA). The accuracy
of surface pressure measurements was ±0.2 mN/meter.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Antifungal activity of the two synthetic peptides CGA-(47-70) and
CGA-(47-66) chromofungin
protons (Fig. 1A) and correlations between amide protons of
different residues (Fig. 1B). In addition, to sequential
NOEs between the H of one residue and the amide proton of the
following one, several medium range NOEs could be observed between the
H of a residue and the amide proton 3-4 residues toward the C
terminus n(i + 3),
n(i + 4) for several residues along the
sequence. This feature is a clear indication that the peptide adopts an
-helical structure in solution. The analysis of the amide-amide
proton region of the NOESY map (Fig. 1B) shows many strong
sequential and medium range HN-HN cross-peaks
(nn(i + 1), nn(i + 2),
nn(i + 3)), which is expected in helical
conformations.
View larger version (34K):
[in a new window]
Fig. 1.
Two-dimensional proton NMR spectra of
chromofungin (CGA-(47-66)) recorded at 288 K and in 50%
TFE-d3 solution. A,
amide-H proton fingerprint region of the 150-ms NOESY
spectrum recorded at 600 MHz. Cross-peaks corresponding to
intraresidual HN-H correlations are indicated by black
squares labeled with the corresponding residue numbers
(boldface type). Medium range
(i + 3) NOEs are shown with open
circles. B, correlations between amide protons
observed in a 150-ms NOESY spectrum recorded at 600 MHz. Medium range
nn(i + 2) and nn(i + 3) are
indicated with open circles.
n(i + 3) contacts along the sequence shows
that the helical conformation extends through the entire peptide. The
gap observed for residues located around Leu61 is due to
severe overlap between the H protons resonance frequencies of
residues 59, 61, and 62 (Fig. 1A). However, the observation of NOEs between the H of the aspartate 63 and the amide proton of
the C-terminal Leu66 indicates that the C terminus of the
peptide is folded into an -helical conformation.
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Fig. 2.
Summary of the sequential and medium-range
NOEs observed for chromofungin CGA-(47-66) at 288 K in a 50%
TFE-d3 solution. The NOE
connectivities are indicated by black lines with
thickness proportional to the NOE intensity (weak, medium,
strong).
-helix, where a rather hydrophobic segment
(Ile48, Leu49, Ile51) is followed
by a continuous stretch of hydrophilic residues from arginine 53 to
asparagine 56. In Fig. 4, the
representation of the helical wheel corresponding to the N- and
C-terminal fragments of chromofungin (CGA-(47-56) (A) and
CGA-(53-66) (B), respectively) emphasizes the importance of
the region 53-66 for the amphipathic character.
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Fig. 3.
Three-dimensional representation
of chromofungin (CGA-(47-66), RILSILRHQNLLKELQDLAL) structure.
The helical structure was built by assigning a 
and 
angle value
of 
57 and 47°, respectively for all residues. The residues are
colored according to their hydropathy: red has
been assigned to the arginine residue, which is the most hydrophilic
(4.5), and blue represents isoleucine (+4.5). The computer
model was obtained using the Molmol program (22).
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Fig. 4.
Helical wheel projection of the N- and
C-terminal fragments of chromofungin according to the PLOT.A/HEL
program. A, CGA-(47-56); B, CGA-(53-65).
Hydrophobic residues Ile and Leu are in boldface
type. Underlined Leu61 and
Leu64 were changed in proline residues to test the
importance of the amphipathic character of chromofungin for the
antifungal activity.
-helical structures; the majority
are cationic and amphipathic, but some hydrophobic peptides have also
been described. Most antimicrobial peptides interact with inner and
outer membranes and may be able to reach intracellular targets. Thus,
it was important to study the interaction of chromofungin with fungal membranes.
9 to 2 × 108
Mres so that the adsorption to a clean
air/water interface (i.e. in the absence of lipid film) did
not induce any surface pressure increase. The initial pressure of the
lipid films (20 ± 0.5 mN/meter) was near the value generally
considered as the internal pressure in cellular membranes (30). The
addition of chromofungin (5 × 109
Mres in the subphase) beneath lecithin film
resulted in a slow increase, 
, of the surface pressure, which
reached its maximum value, M = 1 mN/meter, after
3 h (Fig. 5, inset). This
modest surface pressure increase is indicative of the penetration of chromofungin into the lipid monolayers, because agents interacting only
with the lipid head groups without penetration do not modify the
surface pressure (31). When ergosterol, the main sterol in yeast (32)
and fungus (33) plasma membranes was included in the lipid monolayer,
M increased to 3.5 mN/meter. Increasing the subphase
peptide concentration from 5 × 109 to 2 × 108 Mres leads to an increase of
M to 2 mN/meter and 4 mN/meter for lecithin and
lecithin/ergosterol monolayers, respectively. Parallel experiments were
carried out with the inactive peptide CGB-(602-626). At 5 × 109 Mres subphase concentration,
CGB-(602-626) induced a surface pressure increase higher than that
obtained with chromofungin (2.5 and 5.5 mN/meter instead of 1 and 4 mN/meter in lecithin and lecithin/ergosterol monolayers, respectively)
(Fig. 5), indicating that CGB-(602-626) is able to interact with lipid
membranes. However, since CGB-(602-626) is inactive toward fungi, this
peptide is unable to interact with the plasma membrane,
probably because of the presence on the cell wall of complex structures
as 
-1,3-glucan and chitin (linear polymer of
-1,4-N-acetylglucosamine) preventing its targeting. At
this stage, it was important to examine with confocal laser microscopy
the interaction of chromofungin with various fungal microorganisms.
View larger version (44K):
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Fig. 5.
Penetration of chromofungin CGA-(47-66) and
CGB-(602-626) into lipid monolayers. Inset, the
initial pressure of the lipid monolayers was evaluated to
1 = 20.5 ± 0.5 mN/meter. The peptide solution was
injected in the subphase beneath the lipid monolayer formed with pure
egg lecithin (black) or lecithin-ergosterol 4:1 monolayer
(dashed). Peptide concentration in the subphase was as
follows: 5 nMres (foreground), 20 nMres (background). Subphase: 100 mM NaCl, 20 mM Tris-HCl, pH 7. T = 20 ± 0.5 °C.
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Fig. 6.
Phase-contrast (left) and
fluorescence confocal laser (right) micrographs of
A. fumigatus after incubation with rhodamine-labeled
synthetic peptides (CGA-(47-66)R; CGB-(602-626)R). A. fumigatus was examined after 24 h in cultured medium
(30 °C). A, in the absence of rhodamine-labeled synthetic
peptide; B, after incubation with 10 µM
CGA-(47-66)R during 1 h; C, after incubation with 10 µM CGB-(602-626)R for 1 h. D, A. fumigatus (or C. albicans) was examined after 24 h
in cultured medium (30 °C) and preincubated during 1 h at
30 °C with 100 µM unlabeled CGA-(47-66) and 10 µM CGB-(602-626)R. An intense staining was observed,
indicating that chromofungin induced cell wall and membrane
destabilization with formation of holes through which CGB-(602-626)R
peptide is able to pass.
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Fig. 7.
Phase-contrast (left) and
fluorescence confocal laser (right) micrographs of
A. fumigatus in the presence of rhodamine-labeled
synthetic peptide (CGA-(47-66)R). A. fumigatus was
examined after 40 h in cultured medium (30 °C). A,
in the absence of rhodamine-labeled synthetic peptide; B,
after incubation with CGA-(47-66)R 30 µM during 1 h; C, after incubation with CGA-(47-66)R 10 µM during 21 h.
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Fig. 8.
Phase-contrast (left) and
fluorescence confocal laser (right) micrographs of
A. brassicola (A) and C. albicans (B) after incubation with
rhodamine-labeled synthetic peptide CGA-(47-66)R. A. brassicola and C. albicans were examined after 24 h in cultured medium (30 °C) in the absence of rhodamine-labeled
synthetic peptide (upper panel) and after
incubation with 30 µM CGA-(47-66)R for 1 h
(lower panel). C. albicans was
examined after 24 h in cultured medium (30 °C) in the absence
of rhodamine-labeled synthetic peptide (upper
panel) and after incubation with 10 µM
CGA-(47-66)R (lower panel) for 1 h.
-helices) (34). The amino acid sequence of a large
number of CaM-binding proteins has been determined and the location of
the binding domains has been mapped (35). The comparison of the
sequence of chromofungin with the CaM-binding regions from various
target enzymes (Fig. 9) revealed a high
60% analogy between chromofungin (14) and the model peptide (13). A
common feature to the sequences is the large number of positively
charged residues (Arg, His, Lys) and the almost complete lack of acidic
residues Asp or Glu. This alignment suggested to us that chromofungin
could represent the region of CGA able to interact with CaM, thus
affecting the activity of several CaM-dependent enzymes.
This possibility was tested by measuring the effect of chromofungin on
the activity of a calmodulin-dependent enzyme.
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Fig. 9.
Sequences of the CaM-binding domains from
various target enzymes (1-12), a model peptide
(13), and chromofungin (14). The
alignment has been obtained manually so that hydrophobic (Trp, Phe,
Val, Leu, Ile, Met) and polar basic residues (Lys, His, Arg)
occupy invariant positions indicated with boldface
letters. The different sequences were produced by the
GenCore software, version 5.0. 1, MLCK(577-602),
myosin light chain kinase (skeletal muscle; P07313); 2,
*MLCK(1742-1761), myosin light chain kinase (smooth muscle;
P11799); 3, CaMKII(294-314),
calmodulin-dependent protein kinase II ( -chain
mouse; P11798); 4, CaMKIV(322-340),
calmodulin-dependent protein kinase type IV (human;
Q16566); 5, hPaseBKgamma(342-365), phosphorylase
B kinase 
-chain (skeletal muscle, human; Q16816); 6,
mPaseBKgamma(302-326), phosphorylase B kinase -chain
(skeletal muscle mouse; P07934); 7,
6-PFK(377-402), 6-phosphofructokinase muscle (P52784);
8, Ca-ATPase(1102-1126), calcium-transporting
ATPase plasma membrane (P11505); 9,
Specalpha(1187-1210), spectrin -chain (Q13813);
10, Hac(238-254), hemolysin-adenylate cyclase
precursor (Q57506); 11, Cald(608-625),
caldesmon; 12, PPB(393-414), Ser-Thr protein
phosphatase B (catalytic subunit calcineurin, P48452).
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Fig. 10.
Calcineurin phosphatase activity
in the presence of chromofungin (CGA-(47-66)). The calcineurin
phosphatase activity was examined using the BIOMOL GREENTM
calcineurin assay kit (TEBU) including the RII phosphopeptide substrate
(DVPIPGRFDRRVpSVAAE) as calcineurin substrate. The detection of
free phosphate released is based on the classic malachite green assay
at 620 nm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical structure composed of two moieties,
Arg47-Leu52 and
Arg53-Leu66, corresponding to a short
N-terminal hydrophobic region and an amphipathic C-terminal fragment.
This C-terminal region is crucial for the antifungal activity, as
demonstrated here when two leucine residues have been replaced by
proline residues. This helical structure is in accordance with
secondary structure predictions of CGA, as early hypothesized by Yoo
(13). Amphipathic -helices were first described as
structure/function designs involved in lipid interaction (52).
Such regions have been described for polypeptide hormones (endorphins)
(53, 54), venoms (bombolitin) (55), certain complex transmembrane
proteins such as bacteriorhodopsin (56), and antibiotics (magainins)
(27).
-1,3-glucan and chitin (linear polymer of
-1,4-N-acetylglucosamine) (58). Cell wall biosynthesis is
a crucial process in the formation, growth, and morphogenesis of fungal
cells. In addition, it has been indicated that Ca2+ and CaM
may activate chitin synthase and that CaM-dependent
phosphorylation of microsomal proteins may be crucial for the
activation of this enzyme (59). Thus, a possible mechanism for the
destabilization of the cell wall induced by chromofungin may be its
binding to CaM in the presence of Ca2+ and thus the
inactivation to the chitin synthase. Because of the homology of
chromofungin with CaM-binding peptides, various CaM-binding proteins
could be affected by this peptide. This is illustrated by the present
observation that CaN is inhibited by chromofungin in in
vitro experiments.
-helical CGA sequence, which is 100% conserved along evolution
(38).
ACKNOWLEDGEMENT
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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