From the Departments of Medicine and
¶ Haematology, The Queen's University of Belfast, Belfast BT12
6BJ, United Kingdom, the
Forschungsinstitut für
Molekulare Pharmakologie, Alfred Kowalke Strasse 4, 10315, Berlin,
Germany, ** Novo Nordisk A/S, DK-2880 Bagsvaerd, Denmark, and the
Department of Biotechnology, University
of Ulster, Coleraine BT52 1SA, United Kingdom
Received for publication, October 23, 2000
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ABSTRACT |
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On the basis of histamine release from rat
peritoneal mast cells, an octadecapeptide was isolated from the skin
extract of the Northern Leopard frog (Rana pipiens). This
peptide was purified to homogeneity using reversed-phase high
performance liquid chromatography and found to have the following
primary structure by Edman degradation and pyridylethylation:
LVRGCWTKSYPPKPCFVR, in which Cys5 and Cys15 are
disulfide bridged. The peptide was named peptide leucine-arginine (pLR), reflecting the N- and C-terminal residues. Molecular modeling predicted that pLR possessed a rigid tertiary loop structure with flexible end regions. pLR was synthesized and elicited rapid, noncytolytic histamine release that had a 2-fold greater potency when
compared with one of the most active histamine-liberating peptides,
namely melittin. pLR was able to permeabilize negatively charged
unilamellar lipid vesicles but not neutral vesicles, a finding that was
consistent with its nonhemolytic action. pLR inhibited the early
development of granulocyte macrophage colonies from bone marrow stem
cells but did not induce apoptosis of the end stage granulocytes,
i.e. mature neutrophils. pLR therefore displays biological
activity with both granulopoietic progenitor cells and mast cells and
thus represents a novel bioactive peptide from frog skin.
Studies on the array of biologically active substances present in
amphibian skin have been reported in the scientific literature for
almost 40 years (1). In particular, skin is a rich source of bioactive
peptides, many of which are present in copious amounts (mg/g wet weight
skin) (2). Many peptides occur at concentrations that are several
orders of magnitude higher than the circulating levels in the frog,
indicating that a major source of biosynthesis is in the skin (3).
These peptides are putative components in the defense of the frog
against predation or invading microorganisms (1).
Several peptides have been isolated from the skins of ranid frog
species, which have structural analogs in mammalian neuroendocrine systems. Bradykinin and related family members have been identified in
several Rana species (4-8) and others include the
bombesin-like peptide, ranatensin (9), thyrotropin-releasing hormone
(10, 11), caerulein (12), xenopsin-related peptide, margaratensin (13),
and the tachykinin ranamargarin (14). The hemolytic/chemotactic ranid
frog peptides (7, 12) and the family of temporin peptides (15)
demonstrate structural similarity to the mast cell-activating and
phospholipase A2-facilitating peptide, crabrolin, from the venom of the
wasp, Vespa crabro (16).
Many peptides activate rat peritoneal mast cells; thus, histamine
release can be utilized as a screen for putative bioactive peptides.
There are several frog skin peptides that have been identified in this
manner, namely, peptide XO-4 from Kassina maculata (17),
granuliberin R from Rana rugosa (18), and the pipinins from
Rana pipiens (19). A number of peptides have also been isolated from the venoms of hornets, wasps, and bees on the basis of
histamine release (Refs. 16 and 20-26 and Table
I). Many mammalian peptides can activate
mast cells, including substance P, neurokinin A, calcitonin
gene-related peptide, neuropeptide Y, and neurotensin (27-31). Some of
these mast cell-activating peptides also have modulatory effects on a
variety of immune cells (32-35).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Mast cell activating peptides identified on the basis of histamine
releasing ability
The present study describes the isolation and structural
characterization of peptide leucine arginine
(pLR)1 from the skin of the
frog R. pipiens. We have shown that pLR is one of the most
active, noncytolytic histamine-liberating peptides, which also inhibits
granulocyte macrophage colony formation without induction of neutrophil
apoptosis. This novel amphibian peptide may have an unrecognized
counterpart with a regulatory function in the mammalian immune system.
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EXPERIMENTAL PROCEDURES |
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Tissue Extraction-- The frogs were lightly anesthetized and killed by pithing. Dorsal skin (6.2 g wet weight) was removed from six adult specimens of R. pipiens, chopped into small pieces, and incubated in acidified ethanol (ethanol, 0.7 M HCl (3:1 v/v) and 8 ml/g of tissue) for 12 h at 4 °C. Tissue debris was removed by centrifugation (3000 × g, 4 °C, 30 min), and the ethanol was removed from the decanted supernatant prior to lyophilization.
Size Exclusion Chromatography-- The extract was reconstituted with acetic acid (3 ml, 2 M) and applied to a precalibrated 90 × 1.6 cm chromatographic column packed with Sephadex G50 (fine) gel, equilibrated with acetic acid (2 M). The column was eluted at a flow rate of 10 ml/h, and 2.5-ml fractions were collected.
Reversed-phase HPLC-- Size exclusion chromatographic fractions 55-64 (500 µl of each fraction) were pooled and subjected to reversed-phase HPLC using a 30 × 1-cm Vydac 218TP1010 C18 column and a Waters HPLC system: mobile phase A, trifluoroacetic acid and water (0.1:99.9; v/v); mobile phase B, trifluoroacetic acid, acetonitrile, and water (0.1:49.9:50.0 v/v/v); and linear gradient 0-35% B in 70 min (gradient rate of 0.25% acetonitrile/min). Aliquots (200 µl) of each fraction were removed, lyophilized, and assayed for mast cell histamine-releasing activity. The fraction demonstrating maximum histamine release was purified in two consecutive runs using a 0.46 × 15 cm Hypersil PEP 300 C3 column: mobile phase A and B, as above; linear gradient, 0-25% B in 50 min (gradient rate of 0.25% acetonitrile/min). Aliquots (100 µl) of each fraction were removed, lyophilized, and assessed for mast cell histamine-releasing activity.
Structural Analyses-- The molecular mass of the purified histamine-releasing peptide was determined by 252Cf plasma desorption mass spectroscopy using a BioIon 20 K time-of-flight instrument. Spectra were recorded at 16 kV for 106 primary fission events, and internal mass calibration of the instrument with known standards established the accuracy of mass determination as ± 0.1%. Automated Edman degradation was performed using an Applied Biosystems 470A gas phase sequencer. The limit for detection of phenylthiohydantoin-amino acids was 0.5 pmol. Subsequent to the initial sequencing run, a further sample of the peptide was pyridylethylated, using 4-vinylpyridine under reducing conditions. This was achieved by incubation in 100 µl of guanidine hydrochloride (6 M) containing DL-dithiothreitol (10 mM) and 4-vinylpyridine (1 µl) for 1 h at room temperature in the dark. The reaction mixture was purified by reversed-phase HPLC, and the pyridylethylated peptide was subjected to automated Edman degradation on an Applied Biosystems 491 Procise sequencer with phenylthiohydantoin-pyridylethyl-cysteine incorporated into the phenylthiohydantoin-amino acid standards. The primary structure was compared with those deposited in the SWISSPROTTM data base. The initial quantity of peptide was estimated using automated sequence analysis data (~20-30% accuracy).
Peptide Synthesis-- The peptide pLR was synthesized automatically using the solid phase method (Wang-resin, 0.6 mmol/g, Calbiochem-Novabiochem AG, Läufelfingen) and standard 9 Fmoc chemistry (double couplings with 8 equivalent of Fmoc-amino acid derivatives). Couplings were performed by use of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (Calbiochem-Novabiochem AG, Läufelfingen). The final cleavage/deblocking was performed using trifluoroacetic acid, phenol, water, and triisopropylsian (88:5:5:2 v/v/v/v) for 3 h. The crude peptide (213 mg, 0.01 mmol) was then dissolved in 100 ml of dimethyl sulfoxide with water (2:8 v/v, pH 8). The final purification was performed by preparative HPLC (Nucleosil C-18, 250 × 20 mm): mobile phase A, trifluoroacetic acid and water (0.1:99.9 v/v); mobile phase B, trifluoroacetic acid, acetonitrile, and water (0.1:80.0:19.9 v/v/v); linear gradient 10-70% B in 70 min (gradient rate 0.68% acetonitrile/min). The mass of the purified peptide was verified by electrospray mass spectroscopy.
Conformational Studies-- Stock solutions of pLR were prepared by dissolving the samples in 10 mM Tris buffer (pH 7.4 and 150 mM NaCl). For CD measurements, aliquots of the solution were diluted with buffer or mixed with 2,2,2-trifluoroethanol (50%) to give a final concentration of 50 µM and the desired solvent composition. Measurements were carried out on a J 720 spectrometer (Jasco, Japan).
Molecular Modeling and Data Base Screening--
Protein data
base screening for similar sequences was performed using BLAST and
FASTA programs available in the bioinformatic program GCG Wisconsin
package. The Sybyl program package version 6.4 was used to construct
the cyclic peptide. The initial conformation of pLR was predicted using
fragments of consensus conformations found within the three-dimensional
protein data bank. The conformation was then minimized by AMBER 4.1 force field and was subjected to an annealing simulation protocol,
where, in a cyclic procedure (50 cycles) the peptide was heated to
1000 K during 2000 fs and cooled down during 5000 fs. The phi/psi
torsion angles of the polyproline motif PPKP were weakly constrained
(force constant 2.0 kcal mol1 rad
2) during
the cooling down phase.
Histamine Release and Lactate Dehydrogenase Release Assays-- Male Hooded Lister rats (150-250 g body weight) were lightly anesthetized with CO2 and then killed by cervical dislocation and exsanguination. Mixed peritoneal cells were obtained as previously described (36). These cells were washed twice in Tyrode's buffer (137 mM NaCl, 5.6 mM glucose, 10 mM HEPES, 2.7 mM KCl, 1 mM MgCl2·6H2O, 1 mM CaCl2·2H2O, and 0.4 mM NaH2PO4·2H2O, pH 7.4) and recovered by centrifugation (100 × g, 4 °C, 2 min). Isolated peritoneal cells (100 µl) were aliquoted into conical polystyrene test tubes and prewarmed to 37 °C for 5 min. Lyophilized aliquots of chromatographic fractions were reconstituted in Tyrode's buffer (100 µl) and added to the cell suspensions. Following incubation (10 min, 37 °C), the reaction was quenched by addition of ice-cold Tyrode's (2.8 ml). The cell suspensions were centrifuged as above, and the supernatants removed for histamine assay. The remaining cell pellets were resuspended in buffer (3 ml) and then placed in a boiling water bath (10 min) to release the residual histamine. The histamine content was determined in both the supernatants and the cell pellets using the fluorimetric method based on the method described previously (37). Histamine release was expressed as a percentage of total content, and values were corrected for spontaneous release in the absence of added peptides (not exceeding 5%).
Lactate dehydrogenase (LDH) release assay was performed utilizing a commercially available colorimetric assay kit (Sigma). LDH release was measured in both the supernatants and cell pellets and is expressed as a percentage of total LDH. Values were corrected for the spontaneous release in the absence of added stimuli.
Characterization of pLR-induced Mast Cell Histamine Release
Mechanism--
Stock solution (200 µM) of synthetic pLR
was stored at 20 °C, and dilutions were prepared that gave final
concentrations of between 0.001 and 50 µM. Assays were
performed in duplicate, and the individual experiments were repeated
four to six times. The potency of the synthetic peptide was compared
with serial dilutions of the purified peptide from the skin. The time
course of pLR-induced histamine release was investigated by incubating pLR (final concentration, 500 nM) with mast cells for time
periods ranging from 1 s to 30 min. The temperature dependence of
histamine release was investigated by incubating pLR (final
concentration, 500 nM) at a range of temperatures between 4 and 45 °C. To determine the role of extracellular calcium in
pLR-induced histamine release, cells were preincubated with Tyrode's
buffer containing a range of different calcium concentrations (0-5
mM) prior to stimulation with pLR (final concentration, 500 nM) as above. To assess whether pLR-induced histamine
release was via an energy-dependent mechanism, cells were
preincubated for 20 min in the absence of glucose but in the presence
of antimycin A (Sigma, 1 µM) and 2-deoxyglucose (Sigma, 5 mM). The cells were then stimulated with pLR (final concentration, 500 nM) as described above. To investigate
whether pLR induced mast cell activation was via a cytolytic mechanism, LDH release was measured. In a further series of experiments, the
histamine release induced by a range of pLR concentrations and various
other known histamine-releasing peptides were compared.
Membrane Permeabilizing Actions of pLR-- Membrane perturbation was studied by synthetic peptide induced dye release from negatively charged 1-palmitoyl-2-oleoylphosphatidyl-DL-glycerol (POPG) and neutral 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) large unilamellar vesicles. Large unilamellar vesicles (diameter, 0.1 µm) containing calcein at self-quenching concentrations were prepared by extrusion as described (38). Calcein release was monitored fluorimetrically by measuring the decrease of self-quenching (excitation, 490 nm; emission, 520 nm) on a PerkinElmer LS 50B spectrofluorimeter after mixing peptide solution and vesicle suspension. pLR was tested over the concentration range of 1-40 µM for POPG and 1-110 µM for POPC vesicles; magainin-2 amide was used at concentrations between 0.1 and 0.5 µM for POPG and 1 and 50 µM for POPC vesicles, and melittin was tested at concentrations ranging from 0.01 to 1 µM for POPG and 0.01 to 0.1 µM for POPC vesicles. The fluorescence intensity corresponding to 100% dye release was determined by addition of Triton X-100. The percentage of leakage after 1 min was used to create dose response curves, and the concentration required to evoke 50% dye release (EC50) for each peptide was determined from duplicate experiments. Hydrophobicity of pLR, magainin-2 amide and melittin were determined from methods using the Consensus Scale of Hydrophobicity (39)
Hemolytic Activity-- The hemolytic activity of the synthetic peptide was determined using human red blood cells (40). Suspensions containing 1.8 × 108 cells/ml where incubated (30 min, 37 °C) with varying concentrations of peptides. Concentration ranges used were 1-40 µM, 1 µM to 1 mM, and 0.1-10 µM for pLR, magainin-2 amide, and melittin, respectively. After cooling in ice water (5 min) followed by centrifugation (2000 × g, 4 °C, 5 min), the supernatant (200 µl) was diluted with NH4OH (1800 µl, 0.5%), and the optical density was measured at 540 nm. Peptide concentrations that produced 50% hemolysis (EC50) were determined from dose response curves.
Measurement of Granulopoiesis by Colony Forming Units-Granulocyte Macrophage Formation (CFU-GM)-- Semi-solid agar cultures were performed utilizing normal human bone marrow obtained from thoracotomy rib specimens as previously described (41). In brief, bone marrow cells (1 × 105) were cultured in the upper layer of triplicate cultures (1 ml), with human umbilical cord conditioned medium in the lower layer as the source of colony stimulating activity. Synthetic pLR was incorporated into both layers of the cultures in the range 0-4.7 µM. CFU-GM colonies (>20 cells) were scored using an inverted microscope after incubation (37 °C, 7 days, 5% CO2/air). Colony inhibition in the presence of pLR was expressed as percentage inhibition of granulopoiesis compared with that observed in the absence of any stimuli. These experiments were carried out in triplicate.
Measurement of Neutrophil Apoptosis-- Neutrophils were prepared by layering human peripheral blood on an equal volume of Histopaque 1119 and Histopaque 1077 (Sigma) (42). Cells were washed in phosphate-buffered saline (0.01 M, pH 7.4, Sigma), resuspended in RPMI 1640 (Life Technologies, Inc.) containing 10% (v/v) fetal calf serum (Life Technologies, Inc.), and counted by hemocytometer. A suspension of neutrophils (1 × 106/ml) was cultured in the presence of synthetic pLR (0-4.7 µM) in 5% CO2/95% air at 37 °C in 6-well multi-dishes (Nunc). After 24 h the neutrophils were removed, and the cell viability was estimated by trypan blue exclusion. Three cytocentrifuge slide preparations were made per peptide concentration. One slide was stained with Giemsa and examined for morphological signs of apoptosis. At least 500 cells were counted per slide, and the percentage of apoptotic cells was calculated. Two slides were labeled using the terminal deoxyuridine triphosphate nick end labeling technique (43) for further evaluation of apoptotic status. All experiments were repeated three times.
Data Presentation and Statistical Analysis--
Unless otherwise
stated all values are given as the means ± S.E. Statistical
analyses were performed using Student's t test. For
multiple comparisons, an analysis of variance was first performed followed by the protected t test.
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RESULTS |
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Isolation of Peptide Using Histamine-releasing Activity--
The
maximal histamine release was detected in the pool of fractions 33-36
from the reversed-phase HPLC (Fig.
1A). Other histamine-releasing fractions were detected that were identified as related to the previously documented pipinins (44). The individual fraction 35 possessed all of the activity (data not shown). Fraction 35 was further
resolved into two major peptides (Fig. 1B). The second, more
hydrophobic peptide evoked all histamine-releasing activity, and 2.23 nmol of peptide demonstrated a highly effective histamine-releasing ability of 80.6%. There was negligible LDH release evoked with this
peptide (data not shown; LDH release 0.2% ± 0.01%). Furthermore, significant histamine release could still be detected at a 1:100 dilution (223 pmol) and had a comparable dose response curve to the
synthetic pLR (data not shown). Therefore, synthetic pLR was used for
all subsequent functional studies.
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Structural Analyses-- 252Cf plasma desorption mass spectroscopy of the isolated peptide indicated a molecular mass of 2137 Da [MH]+ or 2136 Da in nonprotonated native form. Automated Edman degradation established the identity of residues through 18 cycles with two blanks in positions 5 and 15. The most likely reason for this was the presence of an intramolecular disulfide bridge between two cysteinyl residues. The calculated molecular mass of the peptide, taking this into account, was 2135.6 Da. However, the identity of the cysteinyl residues at these sites, following reduction and pyridylethylation of the natural peptide, was unequivocally confirmed (Table II). From automated sequence analysis, the initial yield of purified peptide was determined as 2.23 nmol, which enabled the quantity of pLR in R. pipiens to be estimated as 45 µg/g wet weight of skin. This is an estimation of peptide quantity, which was achieved on the basis of peptide sequence analysis data and not the preferred method of amino acid analysis. Amino acid analysis has a reported accuracy of 5% compared with 20% by peptide sequence analysis, and therefore the reported quantification may be suboptimal but still is satisfactorily accurate to supply an estimated quantity.
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Conformational Studies-- The CD spectra of pLR in buffer and structure inducing buffer with nd 2,2,2-trifluoroethanol mixture (1:1 v/v) are characterized by a broad negative band with a minimum in the region of 200 nm (data not shown). Compared with the spectrum in buffer, the spectrum in buffer with 2,2,2-trifluoroethanol shows a weak shoulder in the 220-nm range, and there is a slight decrease in the intensity of the negative band. The minor differences in the CD characteristics of pLR in the different solvent systems point to pronounced conformational constraints in the peptide.
Molecular
Modeling--
pLR2 displayed
no identity to sequences from any of the contemporary data bases nor
with peptides that were isolated on the basis of histamine releasing
ability or which demonstrate this activity (Table I). A number of
proteins were found that contained cysteinyl and prolyl residues with
identical positioning to those found in pLR. Proteins from the
three-dimensional structure protein data base also demonstrated the
motifs TKSXPPK and YPPKP, which are found within the cyclic
portion of pLR and this polyproline consensus sequence PPKP resulted in
near identical backbone conformations in the three-dimensional
structure of all these proteins. A relatively rigid cyclic structure is
therefore predominantly determined by the consensus polyproline motif
Pro11-Pro12-Lys13-Pro14
and the disulfide bridge between Cys5 and
Cys15. The resultant molecular model, from simulations with
the PPKP conformation weakly constrained during simulated annealing
procedures, also displays a -strand-like conformation formed by
Pro12-Lys13-Pro14-Cys15,
a sharp turn between Ser9 and Pro12, and N- and
C-terminal tails that appear to remain flexible (Fig. 2).
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Characterization of Histamine Release by Synthetic
pLR--
Stimulation of rat peritoneal mast cells with synthetic pLR
caused a significant and dose-dependent histamine release
after correction for spontaneous release, which was less than 5% of total (Fig. 3). Maximal histamine release
occurred at a concentration of 2.5 µM, and the
concentration required to elicit release of 25% of the total cellular
histamine content (EC25) was 330 ± 8 nM.
The molar potency of the synthetic analog was comparable with that of
natural pLR, except that the maximal release obtained with the natural
pLR was 80% compared with 67.9 ± 2.4% for the synthetic peptide
(data not shown).
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On comparing the histamine-releasing activity of pLR to other mast cell-activating peptides including substance P, neuropeptide Y, bradykinin, magainin-2 amide, and melittin, the cytolytic bee venom peptide (a very potent histamine liberator), pLR was the most potent of all of these peptides (Fig. 3).
pLR induced a rapid release of histamine, and within 15 s,
maximal release had occurred (Fig.
4A). Histamine release was
still maximal after 30 min of stimulation with pLR. pLR-induced
histamine release was temperature-dependent with an optimum
at 37 °C. Both increasing and decreasing temperatures significantly
reduced the histamine-releasing potential of pLR, and at 4 °C there
was negligible release (Fig. 4B).
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Histamine release induced by pLR was also affected by extracellular
calcium concentrations (Fig.
5A). The optimal extracellular calcium concentration was 0.1 mM. pLR-induced histamine
release was significantly reduced by calcium concentrations higher than 1 mM (p < 0.05). In nominally free calcium
medium, there was no significant difference in the histamine release by
pLR. Preincubation of the mast cells with the metabolic uncouplers,
2-deoxyglucose and antimycin A, significantly inhibited pLR-induced
histamine release, indicative that pLR induced histamine release is
energy-dependent (Fig. 5B, p < 0.05). pLR was devoid of LDH releasing ability at all concentrations
tested (0-5 µM), thus supporting a noncytolytic mode of
activation (data not shown).
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Membrane Permeabilization and Hemolysis by pLR-- The EC50 of pLR from negatively charged POPG vesicles was determined as 15 µM (Table III). Complete lysis of POPG liposomes occurred at 40 µM. In contrast, pLR was completely inactive against neutral POPC liposomes up to a concentration of 110 µM. The low lytic activity of pLR against electrically neutral membranes is supported by low hemolytic activity. Although melittin (100 32 µM) and magainin-2 amide (100 µM) produced complete and 25% hemolysis, respectively, pLR was practically inactive (Table III).
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Effect of pLR on CFU-GM Formation and Neutrophil
Apoptosis--
pLR exerted a dose-dependent
inhibition of normal CFU-GM formation (Fig.
6). At a concentration of 46.8 nM, pLR evoked inhibition of granulopoiesis (34.3 ± 5.4%). Near maximal inhibition was achieved by a pLR concentration of
4.68 µM (92.8 ± 0.7%). pLR had no effect on either
viability or neutrophil apoptosis up to concentrations of 4.7 µM (data not shown).
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DISCUSSION |
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This study describes the isolation and characterization of pLR from the skin of R. pipiens. The peptide has potent immunomodulatory effects on mast cells and granulopoietic progenitor cells. This is the first documentation of a peptide that has dual effects on histamine release and on granulocyte/macrophage colony formation. Concentrations of pLR, which elicited the observed effects, are comparable with the estimated concentration of pLR within the skin of R. pipiens, therefore reflecting that the in vitro responses were generated using physiological concentrations.
pLR is one of the most potent naturally occurring, noncytolytic
histamine-releasing peptides discovered to date. This peptide shows no
known sequence similarity with other peptides, which have also been
isolated on the basis of mast cell activation or with other
histamine-releasing peptides, such as melittin and magainin-2-amide
(Table I). The EC25 of pLR (330 ± 8 nM;
Fig. 3) is 2-fold greater than that of melittin (600 ± 2 nM; Fig. 3) whose action is purely cytolytic. The rapid,
energy/temperature-dependent characteristics of pLR-induced
histamine release were similar to those of other classical mast
cell-activating peptides (46); however, compared with other
noncytolytic mast cell activating peptides, pLR was 10-20 times more
active on a molar basis, e.g. substance P (4.15 ± 1.65 µM; Fig. 3). This high potency can be related to its
primary structure as pLR is highly cationic (pI = 10.2) with a net
positive charge of +4. It has been previously documented that an
increasing net positive charge increases the histamine-releasing
ability of peptides (47). Mast cell activation involves initial
electrostatic interaction between the polycationic peptides and the
anionic sialic acid residues on the mast cell membrane (48). From the
primary structure, because of the presence of the helical breaking
residues, proline and glycine, and the disulfide-bridge at
Cys5 and Cys15, pLR is predicted to be devoid
of -helical regions. This is consistent with previous reports that
histamine-releasing ability is independent of helicity (36, 49). The
very high potency of pLR has yet to be explained and may relate to the
mechanisms by which pLR activates intracellular signaling mechanisms,
which are currently being investigated.
Peptide membrane interaction can also be related to total hydrophobicity of the peptides and the distribution of hydrophobic and charged residues in the sequence (50). High cationic peptide charge favors the accumulation of peptides to negatively charged POPG bilayers by electrostatic interactions. The high permeabilizing effect of melittin and magainin-2 amide is supported by insertion of the extensive hydrophobic N-terminal helix domain (H = + 0.248) of melittin and the hydrophobic face of the amphipathic magainin helix in the hydrophobic acyl chain region of the lipid bilayer. Strong hydrophobic interactions are also responsible for the high lytic effect of melittin on neutral POPC and red blood cell membranes, whereas the reduced activity of magainin-2 amide correlates with the lower peptide hydrophobicity. The inactivity of the cationic pLR against POPC bilayers and red blood cells is consistent with minor electrostatic interactions of the cationic peptide with the neutral membrane and weak hydrophobic interactions caused by the very low peptide hydrophobicity. It is clear from these data that permeabilizing mechanisms utilized by other peptides, such as melittin and magainin-2 amide, are only in part responsible for the effects instigated by pLR.
The conformational constraints in pLR indicated by CD studies are
comparable with properties that have been described for a cyclic,
18-residue peptide corresponding to the -hairpin region of the
neutrophil defensin peptide-2 (51). A hairpin turn and
-strand
region between Pro12 and Cys15 were also
predicted by the molecular model of pLR, in addition to a rigid cyclic
structure with N- and C-terminal tails that appear to remain flexible.
This predicted structure has further been supported by NMR
experiments.3
The granulopoietic actions of pLR were cell stage-specific. Whereas pLR
exerted a strong inhibitory effect on myeloid progenitor cells, there
was no effect on apoptosis of mature neutrophils. In contrast,
transforming growth factor and macrophage inflammatory protein-1
, two well characterized inhibitors of granulopoiesis, have
previously been shown to act via an apoptotic mechanism (52).
It has recently been reported that pLR possessed potent anti-proliferative effects shown to be mediated via highly specific binding sites expressed on ovarian and breast cancer cell lines (53). Therefore, the immunomodulatory effects of pLR described here may be mediated via similar binding sites. The presence of specific binding sites would be consistent with the presence of a natural mammalian ligand, which may be involved in the regulation of the immune system and control of cellular proliferation.
pLR represents a prototype of a novel class of amphibian skin
secretory peptides. The high biological potency and
nonhemolytic/cytolytic actions of pLR, inhibition of progenitor cell
development, anti-proliferative effects, and the reported presence of
high affinity binding sites merit further investigations for future
development as a potential chemotherapeutic agent.
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ACKNOWLEDGEMENT |
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Many thanks are given to H. Hans for technical assistance.
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FOOTNOTES |
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* 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.
§ Recipient of a studentship from the Department of Education for Northern Ireland. To whom correspondence should be addressed: Dept. of Medicine, The Queen's University of Belfast, Grosvenor Road, Belfast BT12 6BJ, UK. Tel.: 44-2890-240503; Fax: 44-2890-329899.
Published, JBC Papers in Press, November 29, 2000, DOI 10.1074/jbc.M009680200
2 The amino acid sequence of this peptide has been deposited in the SWISSPROTTM data base under the accession number P82110.
3 S. Rothemund, G. Krause, J. Klose, M. Beyermann, L. J. M. Cross, A. L. Salmon, M. Bienert, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: pLR, peptide leucine arginine; HPLC, high performance liquid chromatography; Fmoc, 9 fluorenylmethoxycarbonyl; LDH, lactate dehydrogenase; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine; POPG, 1-palmitoyl-2-oleoylphosphatidyl-DL-glycerol; CFU-GM, colony forming units-granulocyte macro- phage formation; EC50, concentration required to elicit 50% release of calcein or produced 50% hemolysis; EC25, concentration required to elicit release of 25% of the total cellular histamine content; Clipid, concentration of POPG or POPC in the large unilamellar vesicles.
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REFERENCES |
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