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J Biol Chem, Vol. 273, Issue 45, 29847-29856, November 6, 1998
From Proenkephalin-A (PEA) and its
derived peptides (PEAP) have been described in neural, neuroendocrine
tissues and immune cells. The processing of PEA has been extensively
studied in the adrenal medulla chromaffin cell showing that maturation
starts with the removal of the carboxyl-terminal
PEAP209-239. In 1995, our laboratory has shown that
antibacterial activity is present within the intragranular chromaffin
granule matrix and in the extracellular medium following exocytosis.
More recently, we have identified an intragranular peptide, named
enkelytin, corresponding to the bisphosphorylated
PEAP209-237, that inhibits the growth of Micrococcus
luteus (Goumon, Y., Strub, J. M., Moniatte, M., Nullans, G.,
Poteur, L., Hubert, P., Van Dorsselaer, A., Aunis, D., and
Metz-Boutigue, M. H. (1996) Eur. J. Biochem. 235, 516-525). As a continuation of this previous study, in order to
characterize the biological function of antibacterial PEAP, we have
here examined whether this COOH-terminal fragment is released from
stimulated chromaffin cells and whether it could be detected in wound
fluids and in polymorphonuclear secretions following cell stimulation. The antibacterial spectrum shows that enkelytin is active against several Gram-positive bacteria including Staphylococcus
aureus, but it is unable to inhibit the Gram-negative bacteria
growth. In order to relate the antibacterial activity of enkelytin with structural features, various synthetic enkelytin-derived peptides were
tested. We also propose a computer model of synthetic
PEAP209-237 deduced from 1H NMR analysis, in
order to relate the antibacterial activity of enkelytin with the
three-dimensional structure. Finally, we report the high phylogenetic
conservation of the COOH-terminal PEAP, which implies some important
biological function and we discuss the putative importance of enkelytin
in the defensive processes.
Secretory granules from adrenal medullary chromaffin cells contain
a complex mixture of low molecular mass constituents such as
catecholamines, ascorbate, nucleotides, calcium, and several water-soluble peptides and proteins. These components are released into
the circulation in response to splanchnic nerve stimulation. Since
relatively large amounts of proenkephalin-A
(PEA)1 and
chromogranin-derived peptides are found in adrenal medullary chromaffin
granules, these organelles have proven to be an excellent model to
study intragranular processing of these proteins. Recently, we have
characterized the processing of bovine chromogranins A and B in
chromaffin granules and in the extracellular medium following their
release from stimulated cultured chromaffin cells (1, 2).
PEA, the precursor protein of Met- and Leu-enkephalin, as well as
larger enkephalin-containing peptides, is highly conserved from
Xenopus (3) to human (4). Originally, PEA mRNA was described to be present in various brain regions, most notably in the
striatum (5) as well as in neuroendocrine tissues, the pituitary (6),
and adrenal gland (6, 7). In addition to their expression in neural
tissues, PEA and its derived peptides (PEAP) are expressed in a variety
of immune cells, including ConA-stimulated CD4 T lymphocytes (8), CD4
thymocytes (9), B lymphocytes (10), as well as T cell lines,
macrophages, and mast cells (11). In adult thymocytes and T lymphocytes
clones, PEA mRNA is not expressed constitutively, but is detected
following cell activation. After exogenous administration, enkephalins
affect several immunologic functions, including antibody production
(12), NK cell activity against tumors and viral infections (13),
macrophage and polymorphonuclear leukocyte functions (14, 15), graft
rejections (16), and mitogen-stimulated lymphocyte proliferation (17).
Recently, it was shown that very low concentrations of PEA and
Met-enkephalin differentially affect IgM and IgG production by B cells
(18). Thus, enkephalins can enhance or inhibit particular immune
functions (13, 19). Moreover, in several studies, bidirectional effects were reported: low concentrations of enkephalins enhance, whereas higher concentrations inhibit the same immune function. Thus, it is
generally accepted that enkephalins act as modulators of immune
reactions, although their physiological function in the immune system
remains unclear. In addition to its expression in cells of the immune
system, PEA mRNA is expressed in other tissues, such as those
comprising the reproductive system (20, 21), heart (22, 23), and in
many developing tissues during gestation and the early postnatal period
(24, 25). Hence, it has been postulated that PEAP may play a role in
cell or tissue growth and differentiation. Recently, it has been
reported that endogenous enkephalins induced in thymocytes, modulate
their own expression and function to inhibit the proliferation of
activated thymocytes (26).
Natural processing of PEA has been extensively studied. Since 1982, it
has been well established that several opioid peptides including
Met-enkephalin and Leu-enkephalin in the ratio 4:1, two COOH-terminal
extended variants, Met-enkephalin-Arg-Phe7 and the
octapeptide Met-enkephalin-Arg-Gly-Leu8 are liberated by
cleavage of the precursor at pairs of basic residues. In these studies,
high concentrations of COOH- or NH2-terminal extended
variants of these peptides have been found in bovine adrenal medullary
chromaffin cells (27, 28). More recently, the processing of PEA has
been well examined in adrenal medulla chromaffin cells (29), as well as
in stably transfected mouse anterior pituitary tumor (AtT-20) cells
(30), showing that PEA maturation proceeds through an orderly series of
steps. Similarly to other precursors, PEA maturation appears to start
with the removal of the carboxyl-terminal fragment, named peptide B
(30, 31), corresponding to PEAP209-239 (32). Four peptide B variants were isolated from bovine adrenal medulla corresponding to
the unmodified form and to this PEAP209-239 containing 1, 2, or 3 phosphate groups (33, 34). These three phosphorylation sites
are clustered together at positions Ser215,
Ser221, Ser223 and the adjacent acidic residues
have been highly conserved during evolution. Interestingly,
immunoreactive forms of this peptide can be found in various regions of
rat brain and circulating in bovine plasma (35).
Our laboratory has recently shown that antibacterial activity is
present within the intragranular chromaffin granule matrix and the
extracellular medium following exocytosis. The first peptide was
identified as secretolytin (2, 36), a peptide corresponding to the
COOH-terminal sequence of bovine chromogranin/secretogranin I
(CGB614-626). Further studies have revealed the
antibacterial activity of a large natural CGA fragment
(CGA79-431), named prochromacin (37), which is generated
by natural cleavage at the previously described site 78-79 and
released during exocytosis (1). Then, we have identified chromacin-(G,
P, and GP), the O-glycosylated and/or phosphorylated
CGA-derived fragment (CGA173-194), as the shortest
antibacterial CGA-derived fragment included in prochromacin.
Secretolytin and chromacin inhibit the growth of Gram-positive bacteria
(Micrococcus luteus and Bacillus megaterium) in
the micromolar range. In addition, antibacterial assays on soluble
chromaffin granule material recovered from HPLC indicated the presence
of several other endogenous peptides with potent antibacterial
activity. Thus, among the complex mixture of intragranular matrix
components, a peptide corresponding to the bisphosphorylated PEAP209-237 was identified (38). This new natural
antibacterial peptide inhibits the growth of M. luteus in
the 0.2 µM range, but has no effect on a Gram-negative
bacteria, Escherichia coli (strain D22) at the same
concentration and does not lyse bovine erythrocytes. Catecholamines and
glucocorticoids play key roles in stress situations. Since these new
antibacterial chromogranin-derived peptides and PEAP are stored with
catecholamines, they may be released during stress and serve as an
early additional protective barrier against bacterial infection. As a
continuation of our previous work (38), we now examine whether
enkelytin is released from stimulated chromaffin cells and
polymorphonuclear neutrophils (PMNs). Furthermore, a potential class of
agents that can simultaneously reduce infection and influence the
action of growth factors, matrix components, and other cellular
effectors has recently been implicated in wound repair. Thus,
antibacterial peptide PR-39, initially identified in pig intestine
kills bacteria as a non-immune defense mechanism (39) and induces
mammalian cells to express cell surface heparan sulfate proteoglycans
(40) which are involved in the wound repair process (41). Since PEAP
also affect cell and tissue growth (24, 25), we decided to analyze
infectious fluids with respect to the antibacterial potency of these peptides.
In addition, various natural and synthetic enkelytin-derived peptides
were prepared and tested to identify the structural features necessary
for a potent antibacterial activity toward M. luteus. In
1996, according to the Homolog method provided in Pro-Explore, we
reported comparative predictions of secondary structure of enkelytin
(38) and the homologous diazepam-binding inhibitor-derived peptide
(42), suggesting an amphipathic helical structure for
PEAP224-237. Here, we generate a computer three-dimensional structure for the synthetic PEAP209-237 on the basis of our 1H NMR study and discuss these
structural features in relation to the antibacterial activity of
enkelytin. Finally, the phylogenetic features of the highly conserved
enkelytin are reported on the basis of the alignment of
PEA198-239 (according to bovine sequence) from several
species, and discussed in terms of enkelytin biological importance.
Isolation of Peptides and Proteins Released from Stimulated
Cultured Cells--
Chromaffin cells were isolated from fresh bovine
adrenal glands and cultured as described previously (1). Cells were
plated at a density of 107 cells/50-mm in plastic Petri
dishes. After 3 days in culture, the medium was removed and cells were
washed four times with Locke's solution (140 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 2.5 mM CaCl2, 11 mM glucose, 0.5 mM ascorbic acid, 15 mM Hepes, pH 7.5) and subsequently stimulated for 10 min with Locke's solution containing 10 µM nicotine. External medium was carefully collected,
completed with trifluoroacetic acid up to 0.1%. Extracellular medium
was lyophilized and stored at Isolation of Peptides and Proteins Released from
Polymorphonuclear Neutrophils--
Human PMNs were prepared to 98%
homogeneity, as described previously (43), from buffy coats 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 Staphylococcus aureus (44).
The secretion was monitored by flow cytometry as described previously
(45) and, when completed, PMNs were centrifuged (800 × g) for 10 min. The supernatant was recovered for further analysis.
Isolation of Proteins from Periarthritis Abscess
Fluids--
Fluid collected from natural bovine knee periarthritis
abscess was extracted with 1 M acetic acid (v/v). After
centrifugation at 12,000 rpm during 15 min at 4 °C, the supernatant
was collected and the soluble material was successively filtered
through Millex filters 0.45 µm and 0.22 µm and then loaded on a
HPLC column.
Purification of PEAP by Reverse Phase HPLC--
PEAP were
isolated from cell secretion and abscess fluids using the Applied
Biosystems HPLC system 140 B. Reverse phase HPLC were successively
performed on Macherey-Nagel Nucleosil columns. In some experiments, a
final purification was performed on a Brownlee C18 column (0.5 × 150 mm; particle size 5 µM and pore size 300 Å).
Absorbance was monitored at 214 nm and the solvent system consisted of
0.1% (v/v) trifluoroacetic acid in water (solvent A) and 0.1% (v/v)
trifluoroacetic acid in acetonitrile (solvent B). Each HPLC elution was
performed using a flow rate and gradient as indicated or shown on chromatogram.
Western Blot Analysis--
Extracts of biological fluids were
separated by SDS-PAGE gels containing 17% acrylamide (46). In order to
detect immunologically reactive fragments, proteins were electrically
transferred to nitrocellulose sheets (47). Electrophoretic blots were
stained with Ponceau red. 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 anti-PEAP224-237 antiserum diluted in
NaCl/Pi (1/1000). 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 (Boehringer) and
0.38 mM 5-bromo-4-chloro-3-indolyl phosphate (Boehringer Mannheim).
Pyroglutamate Aminopeptidase Digestion--
Peptidic material
was digested for 2 h at 37 °C with pyroglutamate
aminopeptidase, at an enzyme/protein weight ratio of 1/50, in 1 mM EDTA, 0.5 mM dithiothreitol, 100 mM sodium phosphate buffer, pH 8.
Sequence Analysis of PEA-derived Peptides (PEAP)--
The
sequence of purified peptides was determined in our laboratory, by
automatic Edman degradation on an Applied Biosystems 473A
microsequencer. Samples purified by HPLC were loaded on
Polybrene-treated and precycled glass-fiber filters (1).
Phenylthiohydantoin-derivatives were identified by chromatography on a
PTH C18 column (2.1 mm × 200 mm).
Mass Spectra Analysis--
Determination of mass was carried out
on a Brucker BIFLEXTM matrix-assisted laser desorption
ionization time of flight mass spectrometer (MALDI-TOF MS) equipped
with the SCOUTTM High Resolution Optics with X-Y
multisample probe, a gridless reflector and the HIMASTM
linear detector. This instrument has a maximum accelerating potential of 30 kV and may be operated either in the linear or reflector mode.
Ionization was accomplished with a 337-nm beam from a nitrogen laser
with a repetition rate of 3 Hz. The output signal from the detector was
digitized at a sampling rate of 250 MHz in linear mode and 500 MHz in
reflector mode using a 1 GHz digital oscilloscope (Lecroy model). The
instrument control and data processing were accomplished with software
supplied by Brucker using a Sun Sparc workstation. These studies were
realized using as the matrix Antibacterial Activity--
Bacteria were grown aerobically at
37 °C in yeast extract-free Luria-Bertani medium (1% bactotryptone,
and 0.5% NaCl (m/v), pH 7.5). Antimicrobial activity was based on the
inhibition of growth of M. luteus (strain A270, from
Institut Pasteur), B. megaterium (strain MA from Dr.
Millet-Obert), Bacillus subtilis (strain QB935, from Dr.
Klier), S. aureus (from Prof. Monteil), E. coli
(strains D22 and 363 from Dr. Bocquet, D31 from Prof. Boman, and wild
strain T13773 from Prof. Monteil) in Luria-Bertani seeded medium,
according to the method previously described (50). Peptide extract
aliquots (10 µl) from HPLC fractions (200 µl of each fraction,
lyophilized, and redissolved in 50 µl of water) 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. Microbial
growth was assessed by the increase of A620 nm
after 16 h of incubation at 37 °C. The A620
nm value of control cultures growing in the absence of peptide
was taken as 100%.
Peptide
Synthesis--
Bisphosphorylated-PEAP209-237
(Ser221 and Ser223 are phosphorylated) or
non-phosphorylated PEAP209-237,
PEAP224-237, PEAP230-237, and
PEAP209-220, were synthesized in our laboratory on an
Applied Biosystems 432A peptide synthesizer, SYNERGY, using the
stepwise solid-phase synthetic approach (51) with
9-fluorenylmethoxycarbonyl (Fmoc chemistry). Synthesis of bisphosphorylated peptide were performed using
Fmoc-Ser[PO(OBzl)OH]-OH. Peptides were further purified by
reverse-phase HPLC on a preparative Macherey-Nagel column Nucleosil RP
300-7C18 (10 mm × 250 mm), and finally on Macherey-Nagel
Nucleosil RP 100-C18 (3 × 250 mm). After lyophilization, the
synthetic peptides were analyzed by sequencing and MALDI-TOF MS.
Antibody Preparation--
A polyclonal rabbit serum was prepared
in our laboratory against a synthetic peptide corresponding to the
PEAP224-237. The first intradermal injection was performed
with 500 µg of peptide coupled to hemocyanin from keyhole limpets
(Megathura crenulata) and emulsified with complete Freund's
adjuvant; a similar injection of the peptide in incomplete Freund's
adjuvant was performed 3 weeks later. Serum was collected a month later
and anti-PEAP224-237 serum was purified and tested by
enzyme-linked immunosorbent assay.
Three-dimensional Model--
The three-dimensional model of
PEAP209-237 is deduced from a 1H NMR
structural analysis of 2 mg of synthetic peptide dissolved in 25 mM sodium acetate/d5 buffer, pH 5, in the
presence of deuterated trifluoroethanol (50% v/v). The
three-dimensional model was obtained using standard simulated annealing
procedure (52) implemented in the X-PLOR program (53). The set of
distances used as input for the structure calculation was derived from
the analysis of a NOESY spectra recorded on a Brucker DRX 600 spectrometer at 283 K. The distance constraints were classified into
three classes on the basis of cross-peak intensity in a 500 ms NOESY
spectrum. Three types of upper limits on interproton distances, 2.7, 3.7, and 5 Å, were assigned to strong, medium, and weak NOE,
respectively. Backcalculated NOESY maps were used to check the
consistency of the resulting three-dimensional models with the
experimental spectra and resolve the initial ambiguous NOE assignments
through several runs of structure calculations. The program Insight II
(Biosym) was used to visualize the structures.
To further characterize the biological role of enkelytin, we
examined whether enkelytin is co-released with catecholamines from
stimulated chromaffin cells and whether it is present in biological
fluids, particularly those involved in immune reactions. In order to
analyze its biological activity, an antibacterial spectrum was realized
with the natural peptide. Several natural and synthetic
enkelytin-derived peptides were also tested to determine the structural
features necessary for the antibacterial activity. Then, the activity
of these peptides was related with the Characterization of Antibacterial COOH-terminal PEAP in Material
Released from Stimulated Cultured Bovine Chromaffin Cells--
The
complex mixture of chromogranins and PEAP recovered in the secreted
material was subjected to separation by HPLC on a reverse-phase C18
column (Fig. 1A). The
different peaks were directly tested for their antibacterial activity
against M. luteus (see "Experimental Procedures") and
sequenced. Several peaks containing antibacterial peptides were eluted
from the column and active PEAP were detected in areas 1 and
2 (including fractions 2a to 2c),
eluted with acetonitrile at 38 and 42%, respectively. After automatic
Edman degradation of these different fractions, a unique NH2-terminal sequence was located at position 209 of PEA.
This sequence (Fig. 1B) possesses three putative
phosphorylation sites (Ser215, Ser221, and
Ser223) (34) and two oxidable residues (Met229
and Met237). The peptidic material present in these
fractions completely inhibited M. luteus (strain A270)
growth at a concentration of 0.2 µM, but was inactive
against E. coli (strain D22) in a similar range
concentration. To determine the molecular differences between PEAP
present in fractions 1, 2a, 2b, and
2c, the sequencing analysis was completed by a detailed
study using MALDI-TOF MS. The mass spectra analysis of the peptides
present in peak 1 (Fig. 1C) indicated, by
comparison with the calculated molecular mass of peptide B (3658 Da),
the presence of a major fragment with a molecular mass of 3836 Da
corresponding to the monooxidized bisphosphorylated form of
PEAP209-239 (peptide B). Three other peptides were also
identified as different forms of PEAP209-237/239. Thus, the molecular masses of 3438 and 3516 Da are attributed to the mono-
and bis-phosphorylated forms of PEAP209-237 (calculated molecular mass of 3355 Da), while the higher masses 3754 and 3931 Da
correspond to the monooxidized monophosphorylated and to the dioxidized
triphosphorylated form of PEAP209-239 (calculated molecular mass of 3658 Da). The occurrence of oxidation states was
explained by the presence of two methionine residues
(Met229 and Met237) in the peptide B sequence
(Fig. 1B). The experimental mass values obtained for
fractions 2a to 2c indicated the exclusive
presence of non-oxidized mono- and bisphosphorylated forms of
PEAP209-239 (data not shown).
From these studies, we can conclude that natural bisphosphorylated
forms of PEAP209-239 and PEAP209-237, named peptide B and enkelytin, respectively, are co-released with
catecholamines and other neuropeptides following nicotine stimulation
of cultured chromaffin cells. These two peptides possess a potent
antibacterial activity against M. luteus growth. To further
characterize the biological function of these antibacterial PEAP,
several biological fluids from injured animals with infection and
polymorphonuclear neutrophil secretions were examined.
Isolation and Characterization of Antibacterial PEAP from
Infectious Fluids--
Periarthritis abscess fluid was collected from
cow knee, extracted by 1 M acetic acid as reported under
"Experimental Procedures" and submitted to a Western blot analysis
against anti-PEA224-237 (Fig.
2D, lane 3). Two bands were
immunodetected with molecular mass of 20 and 4 kDa, respectively. The
broad strongly immunoreactive band (20 kDa) indicated the presence of
several molecular species of PEAP. Sequencing analysis of this material
confirmed that several forms of PEAP72-237/239 and
PEAP80-237/239 were present within this infectious fluid.
Antibacterial assays indicated that these 20-kDa PEAP possessed
activity against M. luteus, but they were less active than
enkelytin (5 versus 0.2 µM). In conclusion, all these PEAP constitute a pool of precursors which have to be processed, during infection, to provide active enkelytin. The lower
4-kDa immunodetected band is likely to be PEAP209-237.
In order to isolate enkelytin, the acid extract was subjected to a
first HPLC on reverse-phase Macherey-Nagel Nucleosil 100-5C18-HD column (4 × 250 mm) (Fig. 2A). Different fractions
were collected, tested in antibacterial assays against M. luteus, and immunoreactivity with anti-PEAP224-237
antiserum was screened by Western blot analysis. The immunoreactive
fraction (Fig. 2A, a) displayed a potent antibacterial
activity against M. luteus and sequencing indicated the
presence of a complex mixture of several peptides. In order to isolate
the shortest antibacterial COOH-terminal PEAP, two additional HPLC were
performed. Peptidic material contained in this fraction was first
separated (Fig. 2B) on a reverse-phase Macherey-Nagel
Nucleosil 300-5C18 column (2 × 125 mm) and for complete
purification of the antibacterial immunoreactive fraction b,
a third chromatography was performed on a Macherey-Nagel Nucleosil 300-5C18 column (3 × 250 mm) (Fig. 2C). Fractions
c2 and c3 were immunoreactive with anti-PEAP224-237 antiserum and after sequencing fraction c1, we detected the
NH2-terminal sequence of defensin BDO1 (DFASXHTNNI; P46159)
(54) and the dodecapeptide (RLXRIVVIRVXR; P2226) (55). MALDI-TOF
analysis have confirmed the presence of these two antibacterial
peptides (4273 and 1485 Da, respectively).
Sequencing of immunoreactive fraction c2
indicated the NH2-terminal sequences of the defensin BDO2
(VRNHVTXRINRGFXVPIR; P46146) (54), bactenecin-5 (RFRPPIRRPPIR; P19660)
(56), bactenecin-7 (RRIRPRPPRLPR; P19661) (57), and histone H2B2
(PEPAKSAPAP; homologous to H2B2 histones of different species)
(58-60). In addition, MALDI-TOF analysis provided an
experimental molecular mass of 4811 Da, which corresponds to the
triphosphorylated form of PEAP199-237 (or the
monophosphorylated form of PEAP199-238), with a pyroglutamic acid as the NH2-terminal end. In some
experiments, mass spectra analysis have provided an experimental
molecular mass of 4964 Da, corresponding to the triphosphorylated form
of PEAP199-238. As the presence of pyroglutamic acid at
the NH2-terminal end (Gln199) prevents Edman
degradation, the peptidic material contained in fraction
c2 (Fig. 2C) was treated with
pyroglutamate aminopeptidase. The resulting digest was separated on a
reverse-phase Macherey-Nagel Nucleosil 300-5C18 column (3 × 250 mm) and the major fraction was characterized by sequencing and
MALDI-TOF analysis (data not shown). In this manner, we identified the
sequence (KRYGGFLKRFAEPLP) corresponding to the
NH2-terminal end of PEAP200-237/238, the
expected fragment generated after digestion with pyroglutamate aminopeptidase. The experimental mass of 4723 Da (with the addition of
a sodium ion by comparison with the theoretical mass of 4700 Da)
confirmed the presence of triphosphorylated
PEAP200-237.
Finally, automated Edman degradation of the immunoreactive fraction
c3 (Fig. 2D, lane 4) indicated the
presence of a NH2-terminal sequence beginning at residue
209. MALDI-TOF analysis (Fig. 3) confirmed the presence of COOH-terminal PEAP with experimental masses
of 3516 Da (the bisphosphorylated form of PEAP209-237), 3508 Da (PEAP209-238), 3523 Da (the monooxidized form of PEAP209-238), and 3805 Da (the dioxidized
triphosphorylated form of PEAP209-238 with addition of a
sodium ion). We also detected a molecular mass of 7027 Da corresponding
to a dimeric form of enkelytin (3516 Da). In some experiments, a
narrow-bore HPLC was performed on a Brownlee C18 column. Elution was
performed at a flow rate of 5 µl/min using successively 15% B over
15 min and a gradient of 5% B to 80% B over 105 min. This additional chromatography confirms the previous HPLC profile and corroborates the
presence of PEAP199/209-237/238 (data not shown).
In order to confirm the presence of antibacterial peptides derived from
the COOH-terminal end of PEA within wounds, we examined two other
infectious fluids. The first liquid was drained from a post-operative
(post-caesarean) abscess in the subcutaneous lining of a cow. Western
blots analysis with anti-PEAP224-237 antiserum (Fig.
2D, lane 5) indicated similar immunoreactivity to that
obtained with the periarthritis abcess (Fig. 2D, lane 3). In
a second experiment, a rabbit abscess induced by subcutaneous injection
of complete Freund's adjuvant was drained 10 days later. The material
collected was treated as for bovine knee periarthritis abscess fluid
and loaded on a HPLC reverse-phase C18 column. The different fractions
were tested for antibacterial assays against M. luteus and
submitted to Western blot immunodetection with
anti-PEAP224-237 antiserum, sequencing, and MALDI-TOF MS.
In the immunodetected fractions (Fig. 2D, lane 6), we
identified the NH2-terminal sequence of two rabbit
defensins, NP1 (P01376) and NP2 (P01377) (61). The experimental mass
values of 3892 and 3849 Da obtained for these peptides correspond to
the theoretical molecular masses of defensins NP1 (3893 Da) and NP2
(3850 Da). In addition, since PEA sequences in several species are
highly conserved (38), the rabbit PEAP sequences and experimental
molecular masses were compared with rat PEAP (62, 63). The most likely
candidates for these fragments are the bisphosphorylated form of
PEAP202-238 and the monophosphorylated form of
PEAP206-237 with experimental molecular masses of 4453 and
3851 Da, respectively, instead of 4453 and 3853 Da for rat PEAP.
In conclusion, the experiments described here reveal the
presence of several peptides with antibacterial activity in fluids from infected wounds: defensins, bactenecins, dodecapeptide as expected, and natural PEAP, such as several forms of
PEAP72/80-237/239, PEAP199-237,
PEAP209-238, and the bisphosphorylated form of
PEAP209-237 (enkelytin). Quantification of isolated enkelytin present at the inflammatory area could be obtained from sequencing and its concentration in (bovine periarthritis abscess fluid) was estimated to be from 0.5 to 1 µM. In this
concentration range, the peptide is fully potent, indicating that
enkelytin locally exerts genuine antibacterial activity in specific
fluids. In contrast, circulating enkelytin concentration is much less as it was hardly detectable in plasma (data not shown). As a
continuation of this study, we have examined the presence of
antibacterial PEAP in secretions from human PMNs. After reverse phase
HPLC on a Macherey-Nagel Nucleosil 100-5C18-HD column (4 × 250 mm), immunoreactivity was detected with anti-PEAP224-237
antiserum (Fig. 2D, lane 7), indicating that PEAP are
secreted for PMNs with a pattern similar to those described for bovine
periarthritis (Fig. 2D, lane 3) and rabbit abscesses (Fig.
2D, lane 5).
Comparison of the Antibacterial Activities of Natural and Synthetic
Enkelytin- and Peptide B-derived Fragments--
In order to further
extend the bacterial spectrum initially reported for enkelytin (38), we
decided to test the antibacterial activity of this natural peptide
against several Gram-positive and negative bacteria. The data reported
on Table I show that enkelytin entirely
inhibits M. luteus and B. megaterium growth at
0.2 µM; it also inhibits the growth of S. aureus, being fully active at a concentration of 4.5 µM. Enkelytin was inactive toward B. subtilis
under similar experimental conditions. Four different strains of
E. coli (D22, D31, 663, and a wild strain, T13773) were
tested with a peptide concentration of 3 µM but no
antibacterial activity was detectable. These tested concentrations were
in accordance with the amount of enkelytin found within physiological
fluids. To conclude, this analysis spectrum indicates that the
antibacterial activity of natural enkelytin is selective for several
Gram-positive bacteria strains. In addition, it is important to point
out that this new antibacterial peptide is able to inhibit the growth
of S. aureus.
In order to characterize the structural features necessary for the
antibacterial activity of enkelytin, we have tested several natural and
synthetic PEAP against the growth of Gram-positive (M. luteus, strain A270) and Gram-negative (E. coli strain
D22) bacteria. Natural enkelytin, PEAP209-237 (peptide 1, Fig. 4A) and natural
bisphosphorylated PEAP209-239, known as peptide B (peptide
2, Fig. 4A) completely inhibit the growth of M. luteus at a concentration of 0.2 µM (Fig.
4B), but were unable to inhibit that of E. coli
in the concentration range from 0.2 to 3 µM.
After preparation of synthetic enkelytin (bisphosphorylated
PEAP209-237), the peptide was loaded on a reverse phase chromatography. The HPLC profile and the MALDI-TOF MS indicated the
presence of different molecular forms. Therefore, synthetic active
enkelytin was further purified on a Macherey-Nagel Nucleosil 300-5C18
column (125 × 3 mm) and analyzed by sequencing and MALDI-TOF. After purification and sequencing of the active synthetic form, we
evaluated that only 10% of the synthetic peptide adopts a conformation with the effective antibacterial activity (peptide 3, Fig.
4A). At this stage, its activity was closer to that of the
natural peptide (100% of bacteria growth inhibition at 3 µM), in contrast with our previous work where we did not
consider that only a low percentage of synthetic peptide adopts the
active conformation (38). These results suggest important
conformational differences between the different synthetic isoforms. In
parallel experiments, we were able to show from the three-dimensional
1H NMR analysis of PEAP209-237 (89) that
proline residues are responsible for conformational changes (cis-trans
isomerization). In contrast with the natural and synthetic
bisphosphorylated peptide, the low antibacterial activity of the
non-modified synthetic peptide (peptide 4, Fig. 4A) suggests
an important role of the two phosphorylated serine residues in active
structure. Thus, at 3 µM the synthetic non-phosphorylated
PEAP209-237 (peptide 4, Fig. 4A) was inactive
against M. luteus; the concentration has to be raised to 100 µM to induce a 20% inhibition of bacterial growth.
Finally, in order to correlate the antibacterial activity with the
NH2 and COOH domain and the length of the peptidic chain of
enkelytin, we have tested four shorter peptides (Fig. 4A): PEAP209-220 (peptide 5, Fig. 4A),
PEAP224-237 (peptide 6), PEAP230-237 (peptide
7), and PEAP233-237; this later fragment corresponds to
Met-enkephalin (peptide 8). As shown in Fig. 4B, the
antibacterial assay of the NH2- and COOH-terminal domains
(peptides 5 and 6) at a concentration of 500 µM indicates a 25 and 20% inhibition of growth, respectively, whereas short COOH-terminal peptides 7 and 8 were inactive at the concentration range
of 500 µM.
These studies were completed with antibacterial assays against E. coli (strain D22) growth and erythrocyte lysis. In the
concentration range from 0.2 to 500 µM, none of the
peptides listed in Fig. 4A showed neither any detectable
antibacterial activity against this Gram-negative bacterium nor any
hemolytic activity. In conclusion, the antibacterial activity of
enkelytin toward M. luteus is directly related to three
structural parameters: (i) the length of the peptidic chain, (ii) the
natural conformational constraints induced by the three proline
residues Pro212, Pro214,
Pro227, and (iii) the phosphorylation of
Ser221 and Ser223.
Computer Model of PEAP209-237--
An extensive study
using biophysical techniques has been carried out on
PEAP209-237 in our laboratory (89). We refer to some of
these data to draw up the computer model of PEAP209-237 fitting with the biological activity. Circular dichroism (CD) spectra
recorded with increasing percentage of trifluoroethanol showed that
synthetic PEAP209-237 folds progressively into an helical
structure, as the percentage of rifluoroethanol is increased up to
50%, as shown by the appearance of a negative band at 220 nm. The CD
spectra displayed no change with trifluoroethanol concentration above
50%.
The presence of helical structure was confirmed in the 1H
NMR spectra of synthetic PEAP209-237 by the presence of
regular H
In both models (Pro227 cis and Pro227 trans)
presented as a ribbon diagram (Fig. 5,
A and B, respectively), the conformations of Pro212 and Pro214 were set to be trans. The
Pro227 residue induces a bend in the three-dimensional
structure, which adopts a L shape and breaks the helical structure
observed on either side of Pro227. It is striking that both
isomers of Pro227 lead to the same kind of spatial
proximity between a glutamic acid and a serine side chain
(Ser223/Glu230, in the cis conformation and
Ser221/Glu228 in the trans one). In enkelytin,
when the two serine residues (Ser221 and
Ser223) are phosphorylated, the negatively charged
phosphate groups probably induce conformational change by electrostatic
interactions (64). In contrast to the COOH-terminal fragment 227-237
which adopts a helical conformation, the structure of the
NH2-terminal end (fragment 209-214) is poorly defined, due
to the lack of medium range NOE, partially explained by an averaging
over a broad range of conformations resulting in the cis-trans
isomerism of Pro214.
Despite intensive research to counter the development of new
bacterial resistance, no novel classes of antibacterial agents have
been discovered in the past 30 years. Currently, there is a great
interest in antibacterial peptides as an attempt to resolve this
challenge. Thousands of such molecules have been synthesized, but just
a few, such as magainins, are currently being tested in clinical
trials. Thus, the structural and biological characterization of new
natural antibacterial peptides, derived from naturally processed
precursors is a topic of growing interest in relation to their
therapeutic use. The intracellular proteolytic processing of protein
precursors occurs in storage compartments in nervous and endocrine
systems. It has been established that the processing takes place at
dibasic sites (65), at single basic residues, at peptide bonds
involving hydrophobic amino acid (1), and at sites marked by the
consensus RX(K/R)R sequence (66). The tertiary structure, in
part due to post-translational modifications (phosphorylation,
glycosylation ... ) must play an important role in cleavage site
accessibility. As large amounts of enkephalins and PEAP are present in
adrenal medullary chromaffin granules, these vesicles have been
extensively used as a source for studying the natural processing of PEA
(28). This protein, which is widely distributed in many cell types,
shows cell-specific processing patterns.
Recently, we have characterized enkelytin, a new antibacterial peptide
which corresponds to the bisphosphorylated PEAP209-237 (38), derived from peptide B (PEAP209-239) (32). As shown
here, this natural peptide displays a potent antibacterial activity
against Gram-positive bacteria M. luteus, B. megaterium, and S. aureus, but was unable to inhibit
the growth of Gram-negative bacteria such as the tested E. coli strains. This COOH-terminal domain of PEA has been well
conserved during evolution, and proteolytic processing of PEA in the
adrenal medulla starts at this COOH-terminal region (31). Recently, it
has been demonstrated that AtT-20 cells transfected with rat
recombinant PEA gene released peptide B 20 min after PEA synthesis
(30), indicating that this peptide is rapidly generated.
In the present study, two antibacterial PEAP, the bisphosphorylated
peptide B (PEAP209-239) and enkelytin
(PEAP209-237) are shown to be secreted from cultured
chromaffin cells following stimulation. This result suggests that these
two peptides that are co-released with catecholamines in stress
situations may play an important role in defense mechanisms.
Furthermore, we have established the presence in bovine infectious
fluids of several antibacterial fragments including
PEAP209-237, PEAP199-237/238, and
major larger precursor fragments, PEAP72/80-237/239. After
extensive extracellular processing, these 20-kDa fragments generate
enkelytin and its derived peptides. Interestingly, in these fluids the
concentration of enkelytin (0.5-1 µM) is in accordance with the antibacterial activity (Table I).
In a previous paper (38), according to the Homolog method provided in
Pro-Explore, we reported comparative structural predictions of
enkelytin and the homologous antibacterial diazepam-binding inhibitor-derived peptide, showing an amphipathic helical structure for
PEAP224-237. In the two models A and B reported here (Fig.
5), it is important to note that Pro227, which is highly
conserved in PEA sequence from several species (Fig.
6), is breaking a regular helical
conformation with a bend formation. This bending brings the glutamic
acid residues (Glu228 and Glu230) close to the
phosphorylated serine residues (Ser221 and
Ser223). The repulsive electrostatic interactions resulting
from the phosphorylation of Ser221/223 may act as molecular
switch for the antibacterial activity. Thus, the phosphorylation of
Ser221 and Ser223 by addition of negative
charges could open the "boomerang angle" (38) or increase the
ability of this peptide to bind divalent ions and thus induce the
antimicrobial activity of enkelytin as described previously for a
poly(Asp) antibacterial peptide (67, 68). The confirmation of this
model will be provided by 1H NMR studies of the
bisphosphorylated synthetic PEAP209-237 and the
bisubstituted glutamic at sites of phosphorylated serine residues, in
aqueous solution including divalent ions and in membrane environment.
Moreover, it is interesting to point out that the helical structure for
the COOH-terminal Met-enkephalin as we report here differs
significantly from 1H NMR structures previously described
for Met- or Leu-enkephalin (69, 70). This is probably due to the
extension of the NH2-terminal region.
Antibacterial peptides have to be positively charged in order to bind
to bacterial surfaces, which are usually negatively charged. Curiously,
the net charges of the most active peptides numbered 1 to 3 (Fig. 4),
were calculated to be PEAP209-239 is the most highly conserved domain of the
protein precursor with a yield of homology around 90% (Fig. 6). Proline residue located in position 212 in bovine sequence is changed
to Ala, Ser, Glu, or Phe residues in other species. Because of the high
conservation of the COOH-terminal domain of PEA, the antibacterial
activity appears to have occurred early in evolution.
The antibacterial COOH-terminal PEAP may originate from chromaffin
cells, since these cells contain high levels of PEAP, or from immune
cells (e.g. PMNs). PEA has been reported to be significantly expressed in the immune system and may provide a basis for neuroimmune interactions (8-11). The local inflammatory response initiates the
synthesis and the secretion of opioid peptides by immune cells. When
Freund's adjuvant is used to induce unilateral hindpaw inflammation in
rats, PEA mRNA are abundant in cells of the inflammed tissue, but
absent in non-inflammed tissue. Numerous cells infiltrating the
inflammed subcutaneous tissue are stained intensively with Met-enkephalin, suggesting that PEAP are synthesized and processed within various types of immune cells at the site of inflammation (73).
Moreover, exposure of rats to lipopolysaccharide endotoxin leads to PEA
mRNA and protein expression in macrophages within lymph nodes and
in chromaffin cells within adrenal glands (74). One physiological
effect of PEAP is to up-regulate or enhance the immune response at low
concentrations, but this effect is abolished at high concentrations.
Other studies performed in invertebrates suggest a potential dual role
of PEA in defensive processes (75, 76). Thus, enkelytin degradation at
the infection site by two endopeptidases, neuropeptide-degrading
endopeptidase and angiotensin-converting enzyme present in
granulocytes, generate Met-enkephalin and its derived peptides (76).
Met-enkephalin enhances the immune reaction in patients with cancer or
AIDS (77). With regard to this immune modulating property,
Met-enkephalin has been proposed to be classified as a cytokine (78).
Moreover, this pentapeptide can bind opioid receptors present in
peripheral inflammed tissues to mediate an analgesic effect (79). The
involvement of opioids in neuroimmunoregulatory events appears to have
a long evolutionary history. Although the relationship between the
immune and nervous systems was discovered in vertebrates, it also
exists in invertebrates (80) and the co-release of enkelytin and
Met-enkephalin represents an unified neuroimmune protective response to
stress situations that may be accompanied with infectious diseases.
Taken together, these two peptides would provide a highly beneficial
survival strategy at the very beginning of a proinflammatory process.
Our studies provide new data concerning the biological
characterization of the COOH-terminal antibacterial PEAP named
enkelytin, first isolated from chromaffin granules and now recovered as
secretory products from stimulated chromaffin cells and in wound
fluids. In view of the widespread distribution of PEA, these peptides may also be present and secreted from other endocrine, neuroendocrine, and immune cells. Due to their nonspecific activity on membranes, the
antibacterial peptides possess cytotoxic activities and may not only
play a role in antimicrobial defense, but also in inflammatory processes. Since antibacterial PEAP are released with catecholamines and chromogranins, the latter being precursors to other peptides with
antibacterial activities (87), they may play a role in stress
situations and act as one immediate protective barrier against
infection. The identification of different classes of antibacterial
peptides in a diverse range of organisms, including prokaryotes,
insects, frogs, and mammals, suggests that they play a potentially
important role in host defense against microbial infections.
We thank Dr. P. Haas, J. Knobloch (CNRS, UPS
840), Dr. D. Colin (Laboratoire de toxicologie bactérienne,
Faculté de Médecine Strasbourg) for help in collecting
biological fluids; Dr. P. Bulet, M. Schneider (CNRS UPR 9022) and Dr.
B. Jaulhac (Laboratoire de toxicologie bactérienne, Faculté
de Médecine Strasbourg) for the generous gift of bacteria. We are
indebted to Drs. O. Sorokine and J. M. Strub (CNRS, URA 31, Strasbourg France) for mass spectrometry analysis of different peptides
and G. Nullans (INSERM U 338) for synthesis of peptides. We are
grateful to Dr. N. Grant (INSERM U 338) for improving the English
version of the manuscript. Finally, we express our sincere gratitude to
the two anonymous reviewers for their suggestions on the first version
of this manuscript that helped us to further characterize enkelytin.
*
This work was supported by grants from INSERM, the Direction
des Recherches, Etudes et Techniques Contract number 96-099 (to D. A.), the Ligue Contre le Cancer (to M. H. M. B.), CNRS,
the Université Louis-Pasteur de Strasbourg (ULP,
Fédération de Recherche Neurosciences), the Région
Alsace Contract number 96 901 13 619 97 (to Y. G.), and the
Association Recherche et Partage (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.
The abbreviations used are:
PEA, proenkephalin-A; HPLC, high performance liquid chromatography; MALDI-TOF MS, matrix-assisted laser desorption ionization
time-of-flight mass spectrometry; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; PEAP, proenkephalin-A-derived peptides; PMNs, polymorphonuclear neutrophils; PAGE, polyacrylamide gel electrophoresis.
Characterization of Antibacterial COOH-terminal
Proenkephalin-A-derived Peptides (PEAP) in Infectious Fluids
IMPORTANCE OF ENKELYTIN, THE ANTIBACTERIAL
PEAP209-237 SECRETED BY STIMULATED CHROMAFFIN CELLS*
,,, and
INSERM, Unité 338 de Biologie de la
Communication Cellulaire, Strasbourg, France, § CNRS, UPR
9003, Cancérogénèse et Mutagénèse
Moléculaire et Structurale, Illkirch Graffenstaden, France, and
¶ CNRS, URA 31, Laboratoire de Spectrométrie de Masse
Bioorganique, Chimie Organique des Substances Naturelles,
Strasbourg, France
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
20 °C.
-cyano-4-hydroxycinnamic acid (Sigma)
prepared as a saturated solution in acetone. Aliquots (1-2 µl) of
the sample-matrix solution were deposited onto probe tips and air
dried. After quick spreading and fast evaporation of the solvent, a
thin layer of matrix crystals was obtained (48, 49). A micromolar
analyte solution was applied to the matrix and allowed to dry under
moderate vacuum. This preparation was washed by applying 1 µl of a
0.5% trifluoroacetic acid in water solution and then flushed after a
few seconds. This cleaning procedure often allows an increase in
sensitivity and mass accuracy by removing the remaining alkali cations.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-helical structure, obtained
from recent 1H NMR data (89).
View larger version (18K):
[in a new window]
Fig. 1.
Purification of
PEAP209-237/239 secreted from
nicotine-stimulated chromaffin cells. A, HPLC elution
profile of soluble secreted peptides on a Macherey-Nagel reverse-phase
Nucleosil 300-5C18 column (4 × 250 mm). Absorbance was monitored
at 214 nm and elution was performed at a flow rate of 700 µl/min,
with a linear gradient as indicated in the right-hand scale.
Fractions numbered 1 and 2 (a-c)
contained different forms of PEAP209-237/239.
B, sequence of PEAP209-239. C,
analysis by MALDI-TOF MS of the secreted peptides present in
fraction 1. By comparison with the calculated molecular mass
of PEAP209-237 (3355 Da), the experimental masses (3438 Da
and 3516 Da) correspond to the mono- and bisphosphorylated forms of
PEAP209-237. The two other detected masses (3754 and 3931 Da) were characterized by comparing with the calculated molecular mass
of PEAP209-239 (3658 Da) and correspond, respectively, to
the monooxidized monophosphorylated and dioxidized triphosphorylated
forms of PEAP209-239.
View larger version (31K):
[in a new window]
Fig. 2.
Characterization of PEAP from cow
knee periarthritis abscess fluid. A, HPLC elution
profile on a Macherey-Nagel reverse-phase Nucleosil 300-5C18-HD column
(4 × 250 mm) of peptidic material included in an acid extract of
cow periarthritis abscess fluid. Absorbance was monitored at 214 nm and
elution was performed at a flow rate of 700 µl/min, with a linear
gradient as indicated in the right-hand scale. Antibacterial
activity and immunoreactivity with anti-PEAP224-237 were
detected in fraction a. B, HPLC elution profile on a
Macherey-Nagel reverse-phase Nucleosil 300-5C18 column (2 × 125 mm) of peptidic material included in fraction a. Absorbance
was monitored at 214 nm and elution was performed at a flow rate of 400 µl/min with a linear gradient as indicated in the right-hand
scale. Antibacterial activity and immunoreactivity were detected
in fraction b. C, HPLC elution profile on a Macherey-Nagel
reverse-phase Nucleosil 300-5C18 column (3 × 250 mm) of peptidic
material included in fraction b. Absorbance was monitored at
214 nm and elution was performed at a flow rate of 400 µl/min with a
linear gradient as indicated in the right-hand scale.
Antibacterial activity was detected in fraction
c1, c2, and
c3 and immunoreactivity in fractions
c2 and c3. D,
Western blot analysis (17%, SDS-PAGE) with
anti-PEAP224-237: lane 1, molecular mass
standard; lane 2, intragranular chromaffin soluble material;
lane 3, peptidic material included in acid extract of cow
periarthritis abscess fluid; lane 4, HPLC fraction
c3; lane 5, peptidic material
included in acid extract of cow post-caesarean abcess fluid; lane
6, peptidic material from induced rabbit abcesses (see
"Experimental Procedures"); lane 7, secretions released
from human PMNs.
View larger version (17K):
[in a new window]
Fig. 3.
MALDI-TOF MS of the peptidic material
included in fraction c3. The four experimental
molecular mass values 3508, 3516, 3523, and 3805 Da correspond to
PEAP209-238, the bisphosphorylated form of
PEAP209-237, the monooxidized form of
PEAP209-238 and the dioxidized triphosphorylated form of
PEAP209-238 with addition of a sodium ion,
respectively.
Activity spectrum of natural enkelytin (bisphosphorylated
PEAP209-237)
View larger version (30K):
[in a new window]
Fig. 4.
Antibacterial activity of natural and
synthetic enkelytin-derived peptides. A, identification
of the 8 different peptides tested. B, peptides at different
concentrations were incubated 16 h at 37 °C with M. luteus (strain A 270) in yeast extract-free Luria-Bertani medium
as described under "Experimental Procedures." Microbial growth was
assessed by measuring the increase at A620 nm.
Values found with control cultures grown in the absence of peptide were
taken as 0%. Numbers in each column indicate the peptide
concentration inhibiting bacterial growth. Experimental values are
given ± 5%.
(i), HN(i+3) NOE for residues from Ser215 to
Gly219 and from Glu228 to Phe236.
PEAP209-237 sequence contains three proline residues which are able to adopt either the cis or trans conformation of the peptide
bond. The two isomers are characterized by distinctive NOE patterns
between the protons of the proline and those of the preceeding residue.
Thus, each three proline residues showed different behavior: (i)
Pro212 has a trans conformation, (ii) cis and trans NOE
patterns were clearly identified from Pro214 and
Leu213, as indicated by two resonance frequency values, and
(iii) both cis and trans NOE patterns were found for
Pro227, but no different chemical shifts were observed for
the two isomers. Therefore, two models were calculated with
Pro227 either in the cis or trans conformation.
View larger version (37K):
[in a new window]
Fig. 5.
Three-dimensional structure of enkelytin
corresponding to PEAP209-237. Ribbon representation
of the three-dimensional structure of PEAP209-237 in a
50% trifluoroethanol/water solution according to X-PLOR program (53).
Both cis (A) and trans (B) conformations of
Pro227 are deduced from the 1H NMR data (89).
The NH2-terminal parts of the two models
(Phe209 to Pro227) have the same orientation.
E, glutamic acid residue; P, proline residue;
S, serine residue.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
View larger version (33K):
[in a new window]
Fig. 6.
Sequence comparison of bovine
PEA198-239 with corresponding fragments from several
species. PEA sequences were retrieved from the Swiss-Prot or
GenBank data base: bovine (P01211) (7), human (P012100) (3, 81), pig
(JL0067) (82), rat (P04094) (62, 63), mouse (P22005) (83),
Mesocricetus auratus (Syrian golden hamster) (MAU09941)
(84), guinea pig (P47969) (85), Xenopus laevis
(P01012) (86), Mytilus edulis and Theromyzon
tessulatum (leech) (87). Leu- and Met-enkephalins were
underlined. S*, phosphorylated serine residues in
bovine sequence. , deletion. Arrows indicate natural
proteolytic cleavage sites.
7, 6, and 7, respectively. However,
enkelytin and peptide B, although negatively charged, may act by a
pore-forming or carpet-like mechanism, as recently described (71).
However, other mechanisms can also be considered such as peptide
membrane receptors on bacterial membranes, the possibility for the
peptides to act as "oblique-oriental" peptides (72) or the ability
for these anionic peptides to bind divalent ions (67, 68). At this
stage, however, the mechanism by which enkelytin and peptide B inhibits
bacteria growth remains to be determined. The presence in infectious
fluids of antibacterial COOH-terminal PEAP together with other
antibacterial peptides supports their potential role in host defense.
Defensins and bactenecins are thought to be released at infection and
inflammation sites. In the present study, several purification steps
were necessary (3 successive HPLC) to isolate the different forms of
active PEAP from periarthritis abscess fluid, suggesting that
interactions occur between these acidic fragments and the cationic
antibacterial peptides, such as defensins or bactenecins. The formation
of molecular complexes including several peptides may be important to
obtain a synergistic antibacterial efficiency. The computer model
obtained for the synthetic PEAP209-237 (Fig. 5) indicated
a long amphipathic -helical structure. This structure completes our previous predicted model concerning the -helical structure of PEAP224-237 (38).
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Unité 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{at}neurochem.u-strasbg.fr.
REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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