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J Biol Chem, Vol. 275, Issue 15, 10745-10753, April 14, 2000
From Vasostatin-1, the natural N-terminal 1-76
chromogranin A (CGA)-derived fragment in bovine sequence, has been
purified from chromaffin secretory granules and identified by
sequencing and matrix-assisted laser desorption time-of-flight mass
spectrometry. This peptide, which displays antibacterial activity
against Gram-positive bacteria at micromolar concentrations, is also
able to kill a large variety of filamentous fungi and yeast cells in
the 1-10 µM range. We have found that the
C-terminal moiety of vasostatin-1 is essential for the antifungal
activity, and shorter active peptides have been synthesized. In
addition, from the comparison with the activity displayed by related
peptides (human recombinant and rat synthetic fragments), we could
determine that antibacterial and antifungal activities have different
structural requirements. To assess for such activities in
vivo, CGA and CGA-derived fragments were identified in secretory
material released from human polymorphonuclear neutrophils upon
stimulation. Vasostatin-1, which is stored in a large variety of cells
(endocrine, neuroendocrine, and neurons) and which is liberated from
stimulated chromaffin and immune cells upon stress, may represent a new
component active in innate immunity.
A rapid and effective response to challenge pathogens is essential
for the survival of all living organisms. The need for efficient agents
increases with the expanding number of immunodeficient patients
(chemotherapy, grafts, human immunodeficiency virus type-1 syndrome, prolonged antibiotherapy, etc.) and with the emergence of
bacterial and fungal pathogens resistant to current therapies. Among
the several different mechanisms that have evolved to act efficiently,
the production of a large variety of natural antimicrobial peptides
from both animals and plants is attracting increasing attention. The
importance of these molecules is clearly established in the immune
defense of invertebrates (1, 2), whereas in vertebrates they act as a
first line of innate defense against pathogens and in the control of
the natural flora (1, 3). These antimicrobial peptides are located at
sites exposed to microbial invasion such as the epithelia of
amphibian (3), mammals (4-6), and insects (2, 7). They are present in
the hemolymph of insects and stored in the secretory granules of immune
cells of mammals and birds (8-11). In addition, We have recently discovered new natural antimicrobial peptides derived
from the processing of chromogranins
(CGs)1 and proenkephalin-A,
which are secreted with catecholamines upon stimulation of chromaffin
cells from adrenal gland (20-24). These peptides may play a role in
stress situations and act as one immediate protective barrier against infection.
In the present paper we report that bovine vasostatin-1, an abundant
natural secreted peptide (24-25), well conserved along evolution and
corresponding to the N-terminal domain of bovine chromogranin A
(CGA1-76), displays both antibacterial and antifungal
activities. By using recombinant and synthetic peptides corresponding
to vasostatin-related fragments from various species, structural
features necessary for the expression of antibacterial and antifungal
activities have been pointed out. We have shown that the C-terminal
moiety of vasostatin-1 displays a potent antifungal activity, and we
have prepared several synthetic antifungal vasostatin-1-derived fragments. Furthermore, in order to study the biological function of
CGA-derived peptides in innate immunity, we have investigated their
presence in material secreted from human polymorphonuclear neutrophils.
Purification of Natural Bovine Vasostatin-1
(CGA1-76), Production of Human Recombinant VS-1
(Ser-Thr-Ala CGA1-78), and Rat CGA7-57
Synthetic Peptides--
Secretory granules were isolated from bovine
adrenal medulla (26), and soluble proteins were separated from
membranes after lysis and centrifugation (27). CGA1-76 was
purified by HPLC on a Macherey Nagel Nucleosil 300-5C18 column (4 × 250 mm; particle size 5 µm and pore size 100 nm) with the Applied
Biosystems HPLC system 140 B. Absorbance was monitored at 214 nm, and
the solvent system consisted of 0.1% trifluoroacetic acid in water (solvent A) and 0.1% trifluoroacetic acid in acetonitrile (solvent B).
Material was eluted at a flow rate of 0.7 ml/min using successively a
gradient of 0-30% B in A over 15 min followed by a gradient of
30-50% over 40 min and achieved by a gradient of 50-100% over 10 min. Each fraction was manually collected and concentrated by
evaporation. Human recombinant Ser-Thr-Ala-CGA1-78 peptide (VS-1) was cloned, purified, and characterized as described previously (28). Rat CGA7-57 peptide was synthesized on an ABI 431 A
(Applied Biosystems) peptide synthesizer using the standard procedures
of Fmoc (9-fluorenylmethoxycarbonyl) chemistry (29). Formation of the
disulfide bridge between Cys17 and Cys38 was
obtained in the presence of N-ethyldiisopropylamine at pH 8.5, leaving the mixture to stand under open atmosphere for 24 h
until complete reaction, according to Ellman test (30). After reduction
of the disulfide bridge in the presence of 8 M guanidine HCl in 1 M Tris-HCl, pH 8.5, containing 4 mM
EDTA, S-pyridylation of cysteine residues was obtained by
treatment with 4-vinylpyridine (31). The reduced and alkylated peptide
was then isolated by HPLC on a Macherey Nagel Nucleosil 300-5C18
column. In order to oxidize natural bovine vasostatin-1 and recombinant
human VS-1, the oxidizing agent was prepared by addition of formic acid
to 30% hydrogen peroxide (v/v, 9:1) and stirring at 20 °C for 45 min. This mixture was then chilled at 0 °C, and the oxidative agent
(100 µl) was added to the protein. The reaction was performed at
0 °C for 30 min, stopped by dilution with water (500 µl). Samples were then concentrated by evaporation but not to dryness. This washing
step was repeated three times to eliminate completely performic acid.
Purification of VS-1-derived Peptides--
Recombinant VS-1 was
digested for 18 h at 30 °C by endoproteinase Glu-C at a protein
to enzyme weight ratio of 50:1 in 50 mM Tris-HCl, pH 8.3. Released fragments were separated by HPLC using a Macherey Nagel
Lichrospher 100-5-RP-18 column (3 × 250 mm; particle size 5 µm
and pore size 100 nm). Absorbance was monitored at 214 nm, and the
solvent system consisted of 0.1% trifluoroacetic acid in water
(solvent A) and 0.1% trifluoroacetic acid in acetonitrile (solvent B).
Material was eluted at a flow rate of 0.7 ml/min using successively a
gradient 0-10% solvent B in 5 min, followed by a gradient 10-40%
over 55 min.
Sequence Analysis--
The sequence of purified peptides was
determined in our laboratory by automatic Edman degradation onto an
Applied Biosystems 473A microsequencer. Samples purified by HPLC were
loaded on Polybrene-treated and precycled glass fiber filters (25).
Phenylthiohydantoin (PTH)-derivatives were identified by chromatography
on a PTH C18 column (2.1 × 200 mm).
Mass Spectra Analysis--
Determination of molecular mass was
carried out on a Brucker BIFLEXTM matrix-assisted laser
desorption time-of-flight mass spectrometer (MALDI-TOF) according to
the procedure previously reported (23). A micromolar analyte solution
(0.5 µl) was applied to the matrix and dried under moderate vacuum.
After fast spreading and evaporation of the solvent, we obtained a thin
layer of matrix crystals (32, 33). This preparation was washed by
applying 1 µl of a 0.5% trifluoroacetic acid in water and then
flushed after a few seconds.
Antibacterial Activity--
Bacteria were precultured
aerobically at 37 °C in a Mueller-Hinton Broth medium, pH 7.3. The
following bacteria strains were tested: Micrococcus luteus
(A270), Bacillus megaterium (MA), Bacillus cereus
(ATCC11778), Bacillus subtilis (QB935), Streptococcus
pyogenes, Mycobacterium fortuitum (K23430),
Staphylococcus aureus, Listeria monocytogenes
(S47014), Escherichia coli (D22 and D31), Enterobacter cloacae, Salmonella typhimurium, Klebsiella
pneumonia, and Pseudomonas aeruginosa (ATCC82118).
Bacteria were suspended in the Mueller-Hinton Broth medium, and
antibacterial activity was tested by measuring the inhibition of
bacterial growth (34). Aqueous peptidic solutions (10 µl) were
incubated in microtiter plates with 100 µl of a midlogarithmic phase
culture of bacteria with a starting absorbance of 0.001 at 620 nm (35).
Microbial growth was assessed by the increase of absorbance after
16 h incubation at 30 °C. The A620 nm value of control cultures growing in the absence of peptide was taken
as 100%.
Antifungal Activity--
Filamentous fungi were grown on medium
including a five-cereal medium, and the spores were harvested as
described previously (36). The following filamentous fungi strains were
used: Alternaria brassicola (MUCL 20297), Trichophyton
mentagrophytes, Nectria hematococca (160.2.2),
Fusarium culmorum (MUCL 30162), Fusarium oxyporum (MUCL 909), Neurospora crassa (CBS 327-54),
and Aspergillus fumigatus. Yeast cells were precultured on a
Sabouraud medium, and two yeast strains were tested,
Saccharomyces cerevisiae (TGY481 pJM600) and Candida
albicans. In order to test the antifungal activity of CGA-derived
peptides, 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 appropriate incubation period, specific to each
fungi (24 or 48 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
also evaluated by measuring the culture absorbance at 595 nm using a
microplate reader. Yeast cells growth was assessed by the increase of
absorbance at 620 nm.
Isolation of Peptides and Proteins Released from
Polymorphonuclear Neutrophils (PMNs)--
Human PMNs were prepared to
98% homogeneity, as described previously (37), from buffy coat of
healthy donors of either sex, kindly provided by the Center de
Transfusion Sanguine de Strasbourg (France). PMNs were suspended in a
buffer solution containing 140 mM NaCl, 5 mM
KCl, 1.1 mM CaCl2, 0.1 mM EGTA, and
10 mM Hepes, pH 7.3, at 5 × 106 cells per
ml. Exocytosis of the content of the specific and primary granules of
PMNs was initiated at room temperature by application of 2.3 nM LukS-PV and 0.6 nM LukF-PV, the two
components of leukocidin from S. aureus (38). The secretion
was monitored by flow cytometry as described previously (39), and when
completed, PMNs were centrifuged (800 × g) for 10 min.
The supernatant was recovered for further analysis.
Western Blot Analysis--
Samples from PMNs secretions were
separated by SDS-PAGE gels containing 15% acrylamide (40). In order to
detect immunologically reactive fragments, proteins were
electrotransferred to nitrocellulose sheets (41). They were first
soaked in 3% bovine serum albumin in 25 mM sodium
phosphate containing 0.9% NaCl at pH 7.5 (NaCl/Pi). Nitrocellulose sheets were quickly washed with NaCl/Pi and
incubated 2 h at room temperature with monoclonal anti-CGA5A8
corresponding to anti-CGA47-68 antiserum (28) diluted in
NaCl/Pi (2 µg/ml). The second antibody was an
anti-rabbit IgG conjugated with alkaline phosphatase (Bio-Rad). The
nitrocellulose sheets were stained for enzyme activity in 100 mM NaCl, 50 mM MgCl2, 100 mM Tris-HCl, pH 8.5, containing 0.4 mM nitro
blue tetrazolium and 0.38 mM 5-bromo-4-chloro-3-indolyl phosphate.
In addition to catecholamines, material present in bovine
chromaffin intragranular matrix contains a large number of peptides derived from chromogranins and proenkephalin-A. After separation by
HPLC, antibacterial activity against M. luteus
(Gram-positive bacteria) was detected in several fractions, and we
focused on a major active fraction eluted at 47% B (Fig.
1A). Automatic Edman degradation revealed a unique sequence (LPVNSPMNKGDT) corresponding to
the N-terminal end of bovine CGA (42). To determine further the full
primary structure of this N-terminal CGA-derived fragment, mass
spectrometry analysis was obtained using the MALDI-TOF technique (Fig.
1B). The experimental molecular mass values of 8583.9, 4292.9, and 2863.6 correspond to the ratio m/z (z = 1, 2 and 3) and are in agreement with the theoretical molecular mass of
the peptide CGA1-76 (8584.9 Da), named vasostatin-1,
including the disulfide bridge Cys17-Cys38
(43). The alignment of the primary structure of this natural fragment
with the corresponding fragments of several species (43-48) shows the
high conservation of this sequence along the evolution (Fig.
2).
Structural and Biological Characterization of the Antimicrobial
Activity of Vasostatin-1-derived Fragments
Natural Bovine Vasostatin-1 (CGA1-76)--
When
tested against several Gram-positive and -negative bacteria, natural
bovine vasostatin-1 displays antibacterial activity against M. luteus and B. megaterium after 16 h
incubation at 30 °C, inhibiting completely bacterial growth at 2 and
0.2 µM, respectively (Table
I). In contrast, no activity is
detectable at the concentration of 10 µM against other
Gram-positive bacteria such as B. cereus, B. subtilis, S. pyogenes, M. fortuitum, S. aureus, L. monocytogenes, and several Gram-negative
bacteria such as E. coli D31 and D22, E. cloacae,
S. typhimurium, K. pneumoniae, and P. aeruginosa
(data not shown).
To complete the spectrum of activity of vasostatin-1, we also tested
its ability to affect the growth of filamentous fungi and yeast cells
(Table I). Interestingly, this peptide in the concentration range of
1-10 µM is highly active against a variety of
filamentous fungi including N. crassa, A. fumigatus, A. brassicola, N. hematococca,
F. culmorum, and F. oxyporum, but it is inactive against T. mentagrophytes. In addition, it is active against
the yeast forms of S. cerevisiae and C. albicans
at the concentration of 10 µM (Table I). Furthermore,
after removal of medium containing vasostatin-1 and substitution with
fresh vasostatin-1-free growth medium, fungi are unable to restart its
growth, strongly suggesting that vasostatin-1 possesses fungicidal
activity. In parallel experiments, we have found that vasostatin-1 is
not able to induce the lysis of bovine erythrocytes (data not shown).
In addition, the antimicrobial properties of vasostatin-1 against
M. luteus, B. megaterium, and N. crassa disappear after treatment with a mixture of proteolytic enzymes such as trypsin, chymotrypsin, and the protease V8 of S. aureus, indicating that the activity is due to peptidic material.
The importance of the disulfide bridge for the antibacterial and
antifungal activities of natural vasostatin-1 was then examined. After
reduction, alkylation using 4-vinylpyridine and desalting by HPLC, the
peptide was modified on the two cysteine residues (Cys17
and Cys38), which were S-pyridylethylated as
verified by MALDI-TOF analysis (data not shown). The modified fragment
(pS-vasostatin-1) is still active against the growth of M. luteus and B. megaterium but at the concentration of 10 and 5 µM, respectively
(Table II). In addition, modification of
disulfide bridge does not alter antifungal activity since the
pS-vasostatin-1 inhibits the growth of N. crassa at a
concentration of 3 µM similarly to the natural form
(Table II). These data indicate that the intactness of the disulfide bridge is not crucial for antifungal property but seems to be important
for full antibacterial activity.
After performic oxidation of vasostatin-1, all peptide molecules were
modified, as shown from MALDI-TOF analysis that revealed the presence
of five oxidations corresponding to the modifications of cystine
(cysteic acids Cys17 and Cys38) and methionine
(methionine sulfoxide/sulfone Met7, Met15, and
Met32) residues (data not shown). This oxidized form
[O]-vasostatin-1 is inactive against the growth of
M. luteus and B. megaterium at a
concentration up to 30 µM, but it inhibits the growth of N. crassa at a concentration of 30 µM (Table
II). These data indicate that performic oxidation induces the complete
loss of the antibacterial activity and strongly decreases the
antifungal activity. The conformational changes induced by the
oxidative treatment inhibit the antimicrobial activity of
vasostatin-1.
Human Recombinant Ser-Thr-Ala-CGA1-78
(VS-1)--
Recombinant VS-1 corresponds to the sequence of human
CGA1-78 bearing the tripeptide STA at the N-terminal end.
This peptide was expressed in E. coli, purified (28), and
tested after sequencing and mass spectrometry analysis. The
experimental mass values of 9069.7, 4536.1, and 3024.1 correspond to
the ratio m/z (z = 1, 2 and 3) and fully
agree with the theoretical mass of 9069.5 Da corresponding to the
recombinant fragment including the disulfide bridge (data not shown).
This fragment is active against the growth of M. luteus and
B. megaterium at a concentration of 30 µM
(Table I). In addition, it inhibits the growth of several fungi such as
N. crassa, A. brassicola, N. hematococca, and
F. culmorum at concentration range of 1-10 µM
(Table I). However, VS-1 is inactive against the growth of yeast cells
at a concentration up to 50 µM (Table I).
After reduction and alkylation, VS-1 was modified by
S-pyridylethylation of the two cysteine residues
(Cys20 and Cys41) as indicated by MALDI-TOF
analysis (data not shown). This modified fragment (pS-VS-1) is inactive
against the growth of M. luteus at a concentration up to 30 µM, but it is active against B. megaterium at
a concentration of 20 µM (Table II). pS-VS-1 inhibits the
growth of N. crassa at a concentration of 5 µM, similarly to unmodified VS-1 (Table II). These data
indicate that the alkylation of VS-1 leads to the specific loss of its
antibacterial activity against M. luteus but does not alter
the antibacterial activity against B. megaterium nor its
antifungal activity.
The oxidized form of recombinant VS-1 is inactive against the growth of
M. luteus and B. megaterium at a concentration up to 30 µM, but it kills N. crassa at a
concentration of 20 µM (Table II).
Synthetic Rat CGA7-57--
The synthetic rat
CGA7-57 peptide was purified by HPLC and analyzed by
sequencing and MALDI-TOF mass spectrometry. The experimental molecular
mass values of 5654.4 and 2827.1 correspond to the ratio m/z
(z = 1 and 2) and well agree with the expected mass of
5655.7 Da for the rat CGA7-57 fragment with two cysteine
residues (Cys17 and Cys38) (data not shown).
This peptide is inactive against the growth of Gram-positive bacteria,
M. luteus and B. megaterium at a concentration of 30 µM (Tables I), revealing that it does not possess
the structural features necessary for antibacterial activity. In
contrast, we found that this peptide is active against N. crassa, A. brassicola, N. hematococca, and
F. culmorum at the concentration range of 10-20
µM (Table I); it is inactive against the growth of yeast cells at a concentration of 50 µM.
The importance of the disulfide bridge was examined after preparation
of the synthetic peptide including the disulfide loop Cys17-Cys38. The formation of the disulfide
bridge was controlled by the reaction with Ellman's reagent and
MALDI-TOF mass spectrometry (data not shown). After chemical
modification, the synthetic peptide was purified by HPLC. The
antibacterial test revealed that the synthetic rat CGA7-57
with disulfide bridge is inactive against M. luteus and
B. megaterium at a concentration of 30 µM (Table I). In contrast, this peptide kills N. crassa,
A. brassicola, N. hematococca, and F. culmorum at a concentration of 10-20 µM (Table I).
These data indicate that synthetic peptides corresponding to rat
CGA7-57 with or without the disulfide bridge possess similar antifungal activity.
In conclusion, natural vasostatin-1 is the most active peptide, since
the fragments rat CGA7-57 seem too short to display the
antibacterial activity. Furthermore, in natural vasostatin-1 the
presence of the disulfide bridge is crucial for this antibacterial activity, but in contrast it seems not to be important for the antifungal activity. We then attempted to characterize the shorter antifungal peptide after proteolytic digestion of rhVS-1 and
purification of the generated fragments.
Identification of Antifungal rhVS-1-derived Peptides
After digestion of recombinant VS-1 by endoproteinase Glu-C, short
fragments were separated by HPLC (Fig.
3A). The analysis by
sequencing and MALDI-TOF mass spectrometry of the different peaks
indicate the presence of 5 major cleavage sites located, in the
vasostatin-1 sequence at positions Glu13-Val14,
Glu40-Thr41,
Glu46-Arg47,
Glu60-Leu61, and
Glu71-Arg72. Then, the antifungal properties of
the peptides included in the different peaks were tested. An active
peptide corresponding to CGA47-60 with an experimental
molecular mass of 1732.9 Da (Fig. 3B; expected molecular
mass, 1733.1 Da) is active against N. crassa showing a
minimal inhibitory concentration giving 100% inhibition of 7 µM. Incubation of fungi with synthetic peptide corresponding to CGA47-60 at increasing concentrations
from 3 to 10 µM (Table III)
induces a decrease of fungal proliferation and the inhibition in the
growth of filaments. For instance, as illustrated in Fig.
4, incubation of A. brassicola
with increasing concentrations of synthetic peptide
CGA47-60 (3 µM-10 µM) shows
that a complete stop was reached at 10 µM.
Antibacterial and Antifungal Activities of Vasostatin-1, the
N-terminal Fragment of Chromogranin A*
,,,
,, and**
INSERM Unité 338, "Biologie de la
Communication Cellulaire," 5 Rue Blaise Pascal 67084 Strasbourg
Cedex, France, § Department of Biological and Technological
Research, San Raffaele Scientific Institute, 20132 Milan, Italy,
¶ CNRS UPR 4301, Centre de Biophysique Moléculaire, Rue
Charles Sadron, 45071 Orléans, France, and CNRS,
UPR 9022, Institut de Biologie Moléculaire et Cellulaire, 15 Rue
René Descartes, 67084 Strasbourg Cedex, France
ABSTRACT
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MATERIALS AND METHODS
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REFERENCES
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
REFERENCES
-defensins are
expressed by human intestinal Paneth cells that are secretory
epithelial cells located at the bottom of crypts in the small intestine
(12). Furthermore, 
-defensins that are produced in various epithelia such as psoriatic skin (6) and trachea (13, 14) are not stored in
cytoplasmic granules, and their local concentration is directly related
to their synthesis and secretion rates. Surveys of antimicrobial
peptides have revealed links between the antimicrobial peptides found
in vertebrates and those found in invertebrates (15-18). Recently, new data have highlighted similarities
between pathogen recognition, signaling pathways and
effector mechanisms of innate immunity in Drosophila and
mammals (19). Thus, it became apparent that innate immunity is an
evolutionary ancient defense mechanism.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (20K):
[in a new window]
Fig. 1.
Isolation on HPLC (A) and
mass spectrometry analysis (B) of natural
CGA1-76 (vasostatin-1). Soluble bovine chromaffin
granule peptides were separated on a Macherey Nagel reverse-phase C18
column (4 × 250 mm; particle size 5 µM and pore
size 100 nm). The solvent system consisted of 0.1% trifluoroacetic
acid in water (solvent A) and 0.1% trifluoroacetic acid in
acetonitrile (solvent B). Absorbance was monitored at 214 nm, and
elution was performed with a linear gradient as indicated on the
right-hand scale. Arrow indicates the fraction
corresponding to bovine vasostatin-1.
View larger version (16K):
[in a new window]
Fig. 2.
Sequence alignment of bovine
CGA1-78 (42) with hrVS-1 (28) and corresponding fragments
from several species. pCGA1-78 (porcine
(p) (43)), rCGA1-78 (rat (r) (44)),
mCGA1-78 (mouse (m) (45)),
oCGA1-78 (ostrich (o) (46)),
eCGA1-78 (equine (e)),3
fCGA1-78 (frog (f) (48)).
Antimicrobial activity of vasostatin-1, human recombinant VS-1, and
synthetic rat CGA7-57
Antimicrobial activity of vasostatin-1, human recombinant VS-1, and
related peptides
View larger version (18K):
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Fig. 3.
Separation by HPLC (A) of
rhVS-derived peptides generated after proteolysis by endoproteinase
Glu-C and mass spectrometry analysis (B) of an active
C-terminal derived fragment. rhVS-1 digest was separated on a
Macherey Nagel Lichrospher 100-5-RP-18 column (3 × 250 mm;
particle size 5 µm and pore size 100 nm). The solvent system
consisted of 0.1% trifluoroacetic acid in water (solvent A) and 0.1%
trifluoroacetic acid in acetonitrile (solvent B). Absorbance was
monitored at 214 nm, and elution was performed with a linear gradient
as indicated on the right-hand scale. Arrow
indicates the fraction corresponding to the antifungal peptide
CGA47-60.
Antifungal activity of hrVS-1-related peptides
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Fig. 4.
Inhibition of fungal growth by synthetic
peptide bCGA47-60. Phase-contrast photomicrographs
were taken after 48 h incubation of A. brassicola
spores suspension in potato dextrose broth (Difco, in half-strength) in
the absence (a) and in the presence of 3 (b), 5 (c), 7 (d), and 10 µM
(e) of bCGA47-60. Magnification, × 40.
In order to investigate the structural features crucial for the antifungal activity of this highly conservative C-terminal moiety of VS-1 (Fig. 2), we decided to examine the role of the tripeptide Arg43-Gly44-Asp45 and the length of the peptidic chain, testing the activities of the synthetic peptides CGA41-60, CGA41-70, and CGA47-70, respectively (Table III). The comparison of their activity with the activity of CGA47-60 highlights several important points as follows: (i) CGA41-60 displays a weak activity, suggesting that the tripeptide Arg43-Gly44-Asp45 may confer a hindrance for the antifungal activity; (ii) CGA41-70 is able to kill several fungi suggesting the importance of the C-terminal end of this fragment that counteracts the inhibitory effect of the tripeptide Arg43-Gly44-Asp45; (iii) fragments CGA47-60 and CGA47-70 are active against filamentous fungi, but the increase in their activities seems related with the lengthening of the peptidic chain; (iv) in contrast with natural vasostatin-1 (CGA1-76), these C-terminal synthetic peptides at a concentration of 100 µM are inactive against yeast cells.
In separate experiments we have found that chromogranin-derived peptides are present in inflammatory fluids,2 and to establish further a correlation between the antifungal activity of vasostatin-1 and its potential biological function in innate immunity, we looked for anti-CGA immunoreactivity in secretions of immune cells.
Characterization of CGA-derived Fragments in Secretions of PMNs
The secretions released from human PMNs were submitted to Western
blot immunodetection (Fig. 5) with
monoclonal anti-CGA 5A8, directed against the epitope
CGA47-68 (28). We immunodetected several N-terminal
CGA-derived fragments corresponding to the processing of CGA in
secretions of PMNs. The difference between the apparent and theoretical
molecular mass of CGA and CGA-derived fragments possibly results from
post-translational modifications (O-glycosylation,
phosphorylation) and the abundance of acidic amino acids (25%) causing
a reduced migration in SDS-PAGE gels (25, 54). By taking into account
the primary structure of human CGA (42), the apparent molecular mass of
the different released fragments, the reactivity observed with the
monoclonal antisera, and the cleavage points previously reported (25), the immunodetected bands are likely to correspond, respectively, to the
following: the native protein (band 1),
CGA1-394 (band 2), CGA1-272
(band 3), CGA1-209 (band 4),
CGA1-115 (band 5), and CGA1-78
(band 6). The presence of diffuse bands results from the
presence of post-translational modifications (O-glycosylation and phosphorylation) (49) and from an
endogenous cleavage point (Val3-Asn4) located
at the N-terminal end of the protein as it has been previously reported
for bovine CGA (25). These data reveal the presence of CGA-derived
fragments in secretions of PMNs with a pattern similar to that
described for secretions of stimulated chromaffin cells (50). In
conclusion, CGA-derived fragments are secreted by PMNs and thus may
locally be recovered in specific infectious fluids where they exert
their antimicrobial activity.
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Chromogranins/secretogranins (CGs) constitute a family of acidic secretory glycophosphoproteins widely expressed in a large number of endocrine and neuroendocrine cells and in neurons (50-53). At the subcellular level, chromogranins are exclusively found in the soluble core of hormone and neurotransmitter storage vesicles and are released during exocytosis. CGs are considered as precursor proteins that are processed into peptides within the secretory granules (25, 50, 54, 55). Chromogranin A (CGA), the major member (40% of total soluble bovine chromaffin granule proteins) of this family, has been extensively studied. CGA is a ubiquitous 48-kDa secretory protein stored and released from most endocrine and neuroendocrine cells and neurons. Different biological activities, in relation to the autocrine or paracrine role in hormone secretion, have been attributed to peptides located along the sequence of CGA (for review see Ref. 56). Among these peptides, the collective term of vasostatins refers to bovine N-terminal fragments CGA1-76 (vasostatin-1) and CGA1-113 (vasostatin-2) to specify their vascular inhibitory effects as has been originally reported (57). Our previous study on the natural processing of CGA has indicated that vasostatins are predominantly generated in the matrix of chromaffin granules and co-released with catecholamines in the extracellular medium upon chromaffin cell stimulation (25). A study on the characterization of CGA-derived fragments, generated by the isolated retrogradely perfused bovine adrenal gland under basal conditions and during stimulation with acetylcholine, indicates that vasostatins are secreted from bovine adrenal medulla (58). In addition, vasostatins have been immunodetected in the large dense core vesicles traveling down sympathetic axons and are released from the nerve terminal in response to stimulation (59). The vasoinhibitory effect of vasostatins, which does not involve interference with endothelin-1 (a potent vasoconstrictor peptide) binding to its vascular receptor (60), has recently been associated with recombinant and synthetic human vasostatin-derived fragments (61). Synthetic peptides corresponding to the sequence CGA1-76 mimic natural vasostatin-1 in its inhibition of contractions induced by endothelin-1 (62). Furthermore, vasostatin-1 exhibits other biological activities such as the inhibition of parathyroid hormone secretion (63), the regulation of cell adhesion (64), and the neurotoxicity in neuronal/microglial cell co-cultures (65). The predominant presence of vasostatin-1 in bovine chromaffin granules, but also in numerous neuroendocrine tissues, and its release from sympathetic nerves suggest an important function for this fragment. A role in the regulation of vascular smooth muscle has been reported (57), although the specific receptor has not been identified yet. It has also been demonstrated that the disulfide-bonded loop included in vasostatin-1 plays a crucial role for oligomerization of CGA (66). This N-terminal natural bovine CGA-derived fragment corresponds to a very conserved domain showing high yield of identity with human (97.3% (67)), pig (97.3% (43)), rat (85.5% (44)), mouse (86.8% (45)), ostrich CGA (80.2% (46)), equine CGA (92.1%),3 and frog CGA (75.7% (48)), respectively. In all species examined so far the disulfide bridge Cys17-Cys38 is conserved, and the sequence SILRHQNLLKELQ (CGA50-62) is strictly unchanged. Furthermore, it has been shown that vasostatin-1 interacts with the secretory vesicle membrane at pH 5.5 and dissociates from it at pH 7.5 (68). The biological role of these interactions is still not understood, but they may reflect some function of this domain in intragranular lattice organization.
Data reported in the present paper establish that bovine vasostatin-1, human recombinant VS-1, and rat synthetic CGA7-57 possess antimicrobial activities at micromolar concentration. Bovine vasostatin-1 results from the cleavage of CGA at the dibasic site Lys77-Lys78 and the subsequent action of carboxypeptidase B (25). This peptide which is composed of 76 residues with a disulfide bridge (Cys17-Cys38) forming a loop (Fig. 2) possesses structural features specific for antimicrobial peptides as follows: (i) a global positive charge (+3), (ii) an equilibrated number of polar (21:76) and hydrophobic residues (23:76), and (iii) the presence of a helical region corresponding to sequence CGA40-65, as previously indicated (69).
In addition to its antibacterial activity, vasostatin-1 was found to display unexpected antifungal activity, provoking the death of filamentous fungi and yeast cells at micromolar concentrations. After removal of vasostatin-1 no re-growth of fungi or yeast cells was observed after a 24-h incubation demonstrating the fungicidal property of the peptide. In order to understand the molecular mechanisms implicated in the antibacterial and antifungal properties of vasostatin-1, we have attempted to relate these activities with some structural parameters.
Antimicrobial Activity in Relation with the Length of the CGA-derived Fragments-- The loss of the antibacterial activity of synthetic rat CGA7-57, with and without the disulfide bridge, (Table I) indicates that the regions comprising 6 and 19 residues on the N- or C-terminal ends, respectively, are essential for the activity (Fig. 2). The 19 residues on C-terminal end (residues 58-76) are important for the secondary structure of CGA1-76, probably with regard to the formation of helical structure. A previous study has shown the importance of the N-terminal sequence CGA1-15 in the inhibition of parathyroid hormone secretion evoked by CGA1-40 (62), whereas residues 1-5 on the N-terminal end are not necessary for such an activity (70). Concerning recombinant VS-1 peptide, the addition of STA and KK residues at the N- and C-terminal ends, respectively (Fig. 2), may be responsible of the antibacterial activity decrease.
Synthetic rat CGA7-57 peptides with and without the disulfide bridge possess antifungal activity lower than that of bovine vasostatin-1, with MIC100 against N. crassa (3×), A. brassicola (7×), N. hematococca (10×), and F. culmorum (20×). They are inactive A. fumigatus, F. oxyporum, T. mentagrophytes, and yeast cells (Table I). Addition of STA and KK on N- and C-terminal ends of VS-1, respectively, may induce the loss of the antifungal activity against A. fumigatus, F. oxyporum, T. mentagrophytes, and yeast cells and some decrease of antifungal activity against N. crassa (3×), N. hematococca (3×), and F. culmorum (5×) (Table I).
Antimicrobial Activity in Relation with the Disulfide Bridge-- The opening of the disulfide bridge and the S-pyridylethylation of CGA-derived peptides induce a slight decrease in the antibacterial activity of vasostatin-1 and the loss of the inhibition against M. luteus for VS-1. In contrast, the activity of VS-1 against B. megaterium is fully preserved. In addition, we found that the opening of the disulfide bridge and the S-pyridylethylation of vasostatin-1 and VS-1 induce no alteration in the antifungal activity. A similar result is obtained with the two synthetic rat CGA7-57 peptides indicating that the disulfide bridge is not crucial for the antifungal activity. Thus, it appears that the antibacterial and antifungal activities require different structural parameters.
Antimicrobial Activity in Relation with Oxidation-- Performic oxidation of vasostatin-1 and of VS-1 induces the complete loss of the antibacterial activity against M. luteus and B. megaterium and partially modifies the antifungal activity at the concentration range of 20-30 µM. These data suggest the importance of methionine residues Met7, Met15, and Met32 and cysteine residues Cys17 and Cys38 for the antimicrobial activity of vasostatin-1.
Antimicrobial Activity in Relation with Residue Modifications-- Comparison of bovine vasostatin-1 and human VS-1 sequences shows two changes on Lys36/Gln36 and Thr73/Ala73 (Fig. 2). As Thr73/Ala73 change corresponds to a conservative modification, the decrease of antibacterial activity is likely to result from the modification of Lys36 which may induce electrostatic interactions with Glu37 and Glu40 to stabilize the active conformation.
In addition, comparison of vasostatin-1 sequence with that of rat synthetic CGA7-57 indicates two changes on hydrophilic residues Glu13/Lys13 and Lys36/Pro36 (Fig. 2) that may be responsible for conformational changes. The chemical synthesis of peptides including prolyl residues in the sequence is well known to provide several molecules corresponding to different prolyl isomers and, among these structures, only one corresponds to the natural conformation (23). Thus, the presence of four prolyl residues in rat CGA7-57 (Pro29, Pro31, Pro33, and Pro36) may explain the attenuated antifungal activity (Fig. 2).
Characterization of a Short Antifungal CGA-derived Fragment-- After proteolytic digestion of rhVS-1, we have isolated an active peptide corresponding to CGA47-60. Then several synthetic peptides have been prepared and then antifungal assays show that the most active CGA-derived fragments correspond to sequences 47-60 and 47-70 (Table III). This observation indicated that helical structure might be crucial for the activity (69). Our studies are currently in progress to characterize the molecular mechanisms responsible for this activity.
Biological Function-- Vasostatin-1 is a 76-residue peptide displaying antibacterial and antifungal activities at micromolar concentrations in biological assays, as standardized here. The secretory granules present in adrenal chromaffin cells and other neural and neuroendocrine tissues represent a compartment storing several antibacterial peptides (secretolytin, chromacin, and enkelytin) for which a role in innate immunity during stress situations has been suggested (24). The new antifungal activity of vasostatin-1 described here looks remarkable because it supplements the antibacterial activity in innate immunity. Taken together these two antibacterial and antifungal activities would provide a highly beneficial survival strategy. To further characterize the biological function of vasostatin-1, secretion of PMNs was examined by Western blot. Indeed, the local inflammatory response may initiate the synthesis and the secretion of these peptides by immune cells at the concentration necessary for the antimicrobial activity, as we have recently shown for enkelytin (23). Our data indicate for the first time that stimulated immune cells (PMNs) release CGA and CGA-derived peptides, including vasostatin-1, resulting from the natural processing of the precursor (Fig. 5). The potency of antimicrobial activities associated with peptides may be modulated by different agents present in biological fluids (47), and the activity of antibacterial and antifungal peptides in biological fluids is still a matter of debate. In infectious fluids and in blood, there is a large variety of antimicrobial peptides that can act on same target with cooperative mode (23).
VS-1 expresses antifungal activity indicating that this recombinant
fragment can be used for future structural investigation including NMR
analysis and the understanding of the mode of action. Comparison of the
human sequence with the corresponding bovine CGA fragment (Fig. 2)
indicates 97.3% homology. Because of the high conservation of the
N-terminal domain of CGA, the antibacterial and antifungal activities
appear to have occurred early in evolution. Peptides with antifungal
activity have been described in plants, insects, and invertebrates, and
they fully protect these organisms against pathogenic invasion. In
mammals vasostatin-1, which is the first natural antifungal CGA-derived
peptide predominantly isolated so far from adrenal chromaffin cells,
may represent a weapon in innate immunity during stress situations, a
role that is presently under investigation in our laboratory.
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ACKNOWLEDGEMENTS |
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We thank Dr. W. Broekaert (F. A. Janssens Laboratory of Genetics, Heverlee, Belgium) for the generous gift of fungi strains and Dr. D. Colin (Laboratoire de Toxicologie Bactérienne, Faculté de Médecine, Strasbourg) for help in collecting secretions from human polymorphonuclear neutrophils. We are indebted to Drs. A. Van Dorsselaer and J. M. Strub for mass spectrometry determinations (CNRS URA 31 Strasbourg). We are grateful to M. Schneider (CNRS, UPR 9022), P. Gadroy, and G. Nullans (INSERM U. 338) for expert technical assistance.
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FOOTNOTES |
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* This work was funded by INSERM and supported by grants from Meiji Institute of Health Science (Odawara, Japan), the Direction des Recherches, Etudes et Techniques Grant DRET 96-099 (to D. A.), Université Louis-Pasteur Contrats Pluriformation 93-96 and 97-2000 (to D. A.), the Ligue Contre le Cancer (to M. H. M. B.), the Association Recherche et Partage (to K. L.), the Fondation pour la Recherche Médicale (to K. L.), and the Région Alsace (to Y. G.).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, 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.
2 M. H. Metz-Boutigue, K. Lugardon, R. Raffner, P. Gadroy, and D. Aunis, manuscript in preparation.
3 F. Sato, N. Ishida, T. Hasegawa, and H. Mukoyama, accession number Q9W7AO, DDBJ/EMBL/GenBankTM databases.
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ABBREVIATIONS |
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The abbreviations used are: CG, chromogranin; CGA, chromogranin A; MALDI-TOF, matrix-assisted laser desorption time-of-flight; PAGE, polyacrylamide gel electrophoresis; PEA, proenkephalin-A; PMNs, polymorphonuclear neutrophils; PTH, phenylthiohydantoin; VS-1, human recombinant Ser-Thr-Ala-CGA1-78; HPLC, high pressure liquid chromatography.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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 L. Taupenot, K. L. Harper, and D. T. O'Connor The Chromogranin-Secretogranin Family N. Engl. J. Med., March 20, 2003; 348(12): 1134 - 1149. [Full Text] [PDF]
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 B. Colombo, R. Longhi, C. Marinzi, F. Magni, A. Cattaneo, S. H. Yoo, F. Curnis, and A. Corti Cleavage of Chromogranin A N-terminal Domain by Plasmin Provides a New Mechanism for Regulating Cell Adhesion J. Biol. Chem., November 22, 2002; 277(48): 45911 - 45919. [Abstract] [Full Text] [PDF]
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 G. M. Portela-Gomes and M. Stridsberg Chromogranin A in the Human Gastrointestinal Tract: An Immunocytochemical Study with Region-specific Antibodies J. Histochem. Cytochem., November 1, 2002; 50(11): 1487 - 1492. [Abstract] [Full Text] [PDF]
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 A. Tasiemski, H. Hammad, F. Vandenbulcke, C. Breton, T. J. Bilfinger, J. Pestel, and M. Salzet Presence of chromogranin-derived antimicrobial peptides in plasma during coronary artery bypass surgery and evidence of an immune origin of these peptides Blood, June 28, 2002; 100(2): 553 - 559. [Abstract] [Full Text] [PDF]
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 K.C. Wollert and H. Drexler Chromogranin A in heart failure Eur. Heart J., June 2, 2002; 23(12): 926 - 927. [Full Text] [PDF]
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G. M. Portela–Gomes and M. Stridsberg Selective Processing of Chromogranin A in the Different Islet Cells in Human Pancreas J. Histochem. Cytochem., April 1, 2001; 49(4): 483 - 490. [Abstract] [Full Text]
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S. Ratti, F. Curnis, R. Longhi, B. Colombo, A. Gasparri, F. Magni, E. Manera, M.-H. Metz-Boutigue, and A. Corti Structure-Activity Relationships of Chromogranin A in Cell Adhesion. IDENTIFICATION OF AN ADHESION SITE FOR FIBROBLASTS AND SMOOTH MUSCLE CELLS J. Biol. Chem., September 15, 2000; 275(38): 29257 - 29263. [Abstract] [Full Text] [PDF]
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Y. Goumon, K. Lugardon, P. Gadroy, J.-M. Strub, I. D. Welters, G. B. Stefano, D. Aunis, and M.-H. Metz-Boutigue Processing of Proenkephalin-A in Bovine Chromaffin Cells. IDENTIFICATION OF NATURAL DERIVED FRAGMENTS BY N-TERMINAL SEQUENCING AND MATRIX-ASSISTED LASER DESORPTION IONIZATION-TIME OF FLIGHT MASS SPECTROMETRY J. Biol. Chem., December 1, 2000; 275(49): 38355 - 38362. [Abstract] [Full Text] [PDF]
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K. Lugardon, S. Chasserot-Golaz, A.-E. Kieffer, R. Maget-Dana, G. Nullans, B. Kieffer, D. Aunis, and M.-H. Metz-Boutigue Structural and Biological Characterization of Chromofungin, the Antifungal Chromogranin A-(47-66)-derived Peptide J. Biol. Chem., September 14, 2001; 276(38): 35875 - 35882. [Abstract] [Full Text] [PDF]
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