From the Centre for Cellular and Molecular Biology,
Uppal Road, Hyderabad, 500 007, India and the
¶ Indian Institute of Chemical Technology, Uppal Road,
Hyderabad 500 007, India
Received for publication, July 25, 2000, and in revised form, September 29, 2000
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ABSTRACT |
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Four broad-spectrum, 11 and 12 residue, novel
antimicrobial peptides have been isolated from the
adrenaline-stimulated skin secretions of the Indian frog Rana
tigerina. Sequences of these peptides have been determined by
automated Edman degradation, by mass spectral analysis and confirmed by
chemical synthesis. These peptides, which we have named as tigerinins,
are characterized by an intramolecular disulfide bridge between two
cysteine residues forming a nonapeptide ring. This feature is not found
in other amphibian peptides. Conformational analysis indicate that the peptides tend to form Antimicrobial peptides constitute a very important component of
the innate immune system in organisms across the evolutionary scale
(1-8). Amphibians being the first group of organisms forming a
connecting link between land and water are forced to adopt and survive
in a variety of conditions laden with pathogenic microbes. Thereby,
they are endowed with an excellent chemical defense system composed of
pharmacological and antimicrobial peptides (9). Bombinins were the
first antimicrobial peptides characterized from the skin of
Bombina variegata in 1969 (10). The discovery of magainins
from the skin secretions of Xenopus laevis in 1987 (11)
triggered extensive search and characterization of antimicrobial peptides from amphibians (12-14). Antimicrobial peptides from genus Rana share an interesting structural motif composed of a
disulfide-bridged cationic heptapeptide segment at the COOH-terminal
end. Peptides with this motif include brevinins and esculentins which
are composed of 24 and 46 amino acids, respectively (14). The primary
structures of large number of peptides belonging to this family have
been determined. Another group of short peptides composed of 13 residues called temporins, which do not contain this
COOH-terminal ring, have also been characterized from frogs of genus
Rana (15). However, considering the large variety of
amphibian species in nature, antimicrobial peptides from only a small
number of them have been characterized, that too only with respect to
primary structure. Also, studies directed toward determining
structure-function relationships have been confined to magainins (16)
and dermaseptins (17, 18). Hence, characterizing host-defense peptides
from other species would be of interest and could conceivably result in
the identification of new structural motifs which would be useful in
designing peptides for therapeutic applications. Rana tigerina is the predominant species of frogs found in India (19). The skin of these frogs have been used traditionally by some tribal communities to heal both open and burn wounds and the antimicrobial components could possibly contribute to the wound healing process (20).
In this study, we have described the isolation and characterization of
antimicrobial peptides from R. tigerina. These peptides are composed of only ~11-12 residues that do not have primary structural homology to any of the known antimicrobial peptides derived from amphibians. They are characterized by a disulfide-bridged loop composed
of 9 amino acids. We have named these peptides as tigerinins.
Collection of Skin Secretions
The frogs of the species (R. tigerina) were
stimulated to release peptides through adrenergic-mediated granular
gland secretion by injecting 0.5 ml of 1 mM adrenaline
(Loba Chemie) into the dorsal sacs. The secretions were collected from
the dorsal surface in ethanol:water (3:1, v/v) and subsequently dried
under reduced pressure so as to remove ethanol and redispersed in water
acidified with 0.1% trifluoroacetic acid. Solid phase extraction on
reverse phase (C18) was carried out with the clarified
homogenate which involved the pumping of the extract through eight
Sep-Pak C18 cartridges (Waters Associates) connected in
series at a flow rate of 1 ml/min. Bound material was sequentially
eluted with 15, 30, and 60% of acetonitrile in acidified water and
freeze dried. These fractions were evaluated for antimicrobial activity.
Purification of the Peptides
The frog skin secretions obtained after partial purification on
Sep-Pak cartridges were redissolved in acidified water
(HPLC1 pure) and purified
further on an analytical reverse-phase Water's µBondapak
C18 column equilibrated with 0.1% (v/v) trifluoroacetic acid/water at a flow rate of 1 ml/min. The acetonitrile concentration in the eluting solvent was raised from 0 to 40% in 30 min and from
40% to 100% in 5 min. Fractions were collected according to detection
at 210 nm and dried in a vacuum centrifuge and evaluated for
antimicrobial activity.
Peptide Characterization
HPLC purified active peptide fractions were subjected to
amino-terminal sequence analysis using a 473 A Applied Biosystems gas
phase sequencer. Cysteine residues were identified by the alkylation of
the HPLC purified active peptide fractions redispersed in pyridine
buffer (pH 8.3) with iodoacetamide incubated in dark for 1 h at
37 °C. Prior to treatment with iodoacetamide, dithiothreitol was added at a concentration of 10-fold molar excess over expected disulfides and nitrogen was flushed to provide an inert atmosphere throughout the reaction (21). The reaction was terminated by using
Mass Spectrometry
The HPLC purified active peptide fractions as well as
iodoacetamide-treated fractions were acidified with 0.1%
trifluoroacetic acid in water and mixed with
COOH-terminal Analysis
The status of the carboxyl terminus was investigated by
digestion of the natural peptides with carboxypeptidase Y that was pretreated with phenylmethylsulfonyl fluoride to inactivate
endopeptidase and amidase activities (22). Aliquots of the enzyme
digests were taken at 1- and 2-h time points and were subjected to
amino acid analysis (LKB 4151 Alpha Plus Amino acid Analyzer).
Peptide Synthesis
Peptides identified were synthesized manually on amide crowns
(Chiron technologies) by the solid phase method using Fmoc chemistry (23). All amino acids were added as Fmoc hydroxy benzotriazole active
esters. The peptides were cleaved from the resin by treatment with
trifluoroacetic acid/thioanisole/phenol/water/ethanedithiol (16.5:1:1:1:0.5, v/v) overnight at room temperature. The peptides were checked for purity on HPLC using a reverse phase column (Waters µBondapak C18) using a solvent system of 0.1%
aqueous trifluoroacetic acid and acetonitrile. The cysteines
were deprotected with mercury (II) acetate in the ratio of 2 equivalents for each equivalent of cysteine. Mercuric sulfide salts
formed were precipitated with 20 eq of Antimicrobial Activity
Minimal inhibitory concentration of the crude 15, 30, and 60%
fractions was monitored by the decrease in turbidity at 600 nm of
Escherichia coli W160.37 cells grown to logarithmic phase in
minimal A medium (10.5 g of KH2PO4, 4.5 g of K2HPO4, 1 g of [NH4]2SO4, 0.5 g of sodium
citrate, 0.1 mM magnesium sulfate, 0.1 g of
L-arginine, and 1% glucose in 1 liter of water) (26).
For evaluating antimicrobial activity of the purified peptides, liquid
culture assays were carried out wherein varying concentrations of the
peptides were added to 100 µl of suspension of the organisms diluted
from a midlogarithmic phase liquid culture to a concentration of
105 cells/ml in sodium phosphate buffer (27, 28). The
microbicidal activity was determined by counting the number of viable
colony forming units on nutrient agar plates after 2 h of
incubation with the individual peptides. The microorganisms used were
Pseudomonas putida, Micrococcus luteus, Bacillus subtilis,
Staphylococcus aureus, E. coli, and Saccharomyces
cerevisiae. The kinetics of killing was also evaluated for
E. coli and S. aureus by determining the viable
cell counts as a function of time.
Outer Membrane Permeability
Outer membrane permeability was assessed by
N-phenyl-1-N-naphthylamine (NPN, Sigma) uptake
assay (29). E. coli W160-37 cells were grown to late
logarithmic phase in bactonutrient broth (Himedia) and the cells
obtained were washed twice with 5 mM HEPES buffer (pH 7.4).
A 1-ml aliquot of cells so prepared, adjusted to an A600 of 0.5 in the same buffer containing 10 µM NPN was taken for each experiment. The excitation
monochromator was set at 350 nm and the emission at 420 nm was
continuously monitored after the addition of the peptide from an
aqueous stock solution.
Inner Membrane Permeability
Inner membrane permeability was monitored by the
o-nitrophenyl-3-D-galactoside (ONPG) influx as
described by Lehrer et al. (30). In brief, E. coli W160-37 cells were grown to late logarithmic phase in
bactonutrient broth (Himedia) in the presence of 5 × 10 Hemolytic Activity
Hemolytic activities of the peptides were evaluated essentially
as described earlier (31), using rat erythrocytes isolated from
heparinized blood by centrifugation. The cells were washed three times
with 5 mM HEPES buffer containing 150 mM sodium
chloride. Aliquots of 1-ml suspension containing 107 cells
in Eppendorf tubes were incubated with different concentrations of
peptides in duplicates at 37 °C for 30 min with gentle mixing. The
tubes were then centrifuged and absorbance of the supernatants was
measured at 540 nm. The lysis obtained with water was considered as
100%.
Conformational analysis
Circular Dichroism (CD) Studies--
CD spectra of the peptides
were recorded in 5 mM HEPES buffer (pH 7.4) in a JASCO
J-715 spectropolarimeter in 0.1-cm path-length cells at 25 °C.
Calibration was carried out with d10-camphor
sulfonic acid. CD band intensities are represented as mean residue ellipticity.
Theoretical Studies--
The starting structures were generated
in extended conformation with Characterization of Peptides--
The lyophilized material
obtained from the adrenaline-stimulated skin secretions or the skin
extracts of R. tigerina was initially processed on Sep-Pak
cartridges. The fraction that eluted between 15 and 30% acetonitrile
contained all the antimicrobial activity against E. coli.
This fraction was subjected to further fractionation on a µBondapak
C18 column. A chromatogram obtained is shown in Fig.
1. The peaks eluting at 21.57, 23.40, and
24.10 min exhibited antimicrobial activity against E. coli.
Mass spectral analysis yielded mass values of 1342, 1368, and 1409, respectively (Fig. 2, A-C).
Amino acid sequence analysis indicated the sequences as
F_TMIPIPR_Y, RV_FAIPLPI_H, and RV_YAIPLPI_Y. To determine whether the gaps in the sequences could arise due to the presence of cysteine residues, the peptides were alkylated with iodoacetamide, purified on
HPLC, and subjected to mass spectral analysis. The data shown in Fig.
2, D-F, indicate mass increases which correspond to the alkylation of two cysteine residues. When sequencing of the modified peptides were carried out, the alkylated cysteine residues eluted at
the blank positions observed for unmodified cysteines. We have named
these peptides as tigerinins. Treatment of tigerinins with carboxypeptidase Y did not result in the release of free amino acids
under conditions where free amino acids were released from peptides
with free COOH-terminal COOH groups, indicating that the COOH-terminal
ends of tigerinins 1, 2, and 3 were amidated. The peptides on
alkylation with iodoacetamide, without prior reduction with
dithiothreitol, did not yield the alkylated derivatives of cysteine
indicating that the two cysteine residues were linked by a disulfide
bridge. The sequences of the peptides were further confirmed by
chemical synthesis. The synthetic peptides coeluted with their natural
counterparts on HPLC and exhibited identical mass values. Reproducible
HPLC profiles were obtained with different batches of skin secretions.
When the skin extracts were processed using similar protocols, along
with the other three peptides an additional peptide, which coeluted
with tigerinin 3, with a mass of 1247 was obtained. On treatment with
iodoacetamide, this peptide could be separated from tigerinin 3. On the
basis of sequence and mass spectral analysis, the primary structure of
this peptide is RVCYAIPLPIC-amide. The primary
structures of the peptides are summarized in Table
I.
Antimicrobial Activity--
The minimal inhibitory concentrations
of the natural tigerinins are shown in Table
II. All the peptides exhibit activities against Gram-positive and Gram-negative bacteria as well as yeast with
minimal inhibitory concentrations varying between 30 and 100 µg/ml.
The bactericidal activity of tigerinins was investigated by studying
the kinetics of killing of two representative organisms, E. coli and S. aureus. The results are shown in Fig.
3. In the first 5 min of incubation
itself, between 60 and 80% of cells are killed in the case of all the
peptides, even at the lowest concentration of 10 µg/ml. Complete
killing is seen between 30 and 120 min. While tigerinin 1 is the most
active peptide for S. aureus, tigerinin 2 is the most active
on E. coli. All the peptides are bactericidal and killing is
rapid.
The rapid killing of microbial cells by cationic peptides is generally
mediated by membrane permeabilization (1-8) and hence the ability of
tigerinins to permeabilize the bacterial membranes was examined. The
extent to which E. coli OM becomes permeable to NPN in the
presence of tigerinins is shown in Fig.
4. Tigerinin 1 is most effective in
permeabilizing the OM of E. coli and tigerinin 2 and 3 are
marginally less active with 50% permeabilizing concentrations (PC50) of 12, 15, and 22 µg/ml, respectively. Tigerinin 4 also possesses considerable OM permeabilizing ability. However, the linear analog of tigerinin 1 (i.e. S-acetamido
protected derivative) did not exhibit antimicrobial activity and also
did not permeabilize the OM of E. coli. Hence, the S-S
bridge appears to be essential for activity.
To determine whether tigerinins are capable of permeabilizing the inner
(cytoplasmic) membrane (IM) of E. coli, the influx of the
chromogenic substrate of the cytoplasmic enzyme Conformation of Peptides--
The CD spectra of tigerinins 1-4 in
buffer are shown in Fig. 6. All the
peptides show a minimum ~204 nm with crossover at wavelengths <195
nm. The spectra indicate a population of unordered and We have described the purification and characterization of two
novel 12-residue peptides and a 11-residue peptide, with broad-spectrum antimicrobial activity, from the skin secretions of R. tigerina. The susceptible microorganisms include Gram-positive and
Gram-negative bacteria as well as yeast with minimal inhibitory
concentrations in the range of 30 to 100 µg/ml. These three peptides
exhibit high homology among themselves, with COOH-terminal amidation
and a disulfide bridge between two cysteine residues to form a
nonapeptide ring but are not related to any described previously
antimicrobial peptides from amphibians. Extensive homology searches
from the protein data banks did not yield any other peptide homologous to these peptides. These three peptides were also obtained from the
skin extracts of R. tigerina. An additional peptide of 11 residues which lacked the COOH-terminal Tyr in tigerinin 3, but marginally less active than tigerinin 3 on all the organisms tested, was also obtained from the skin extracts. None of the peptides exhibited any hemolytic activity up to a concentration of 200 µg/ml.
Tigerinins with 11 and 12 residues are the smallest antimicrobial peptides characterized from amphibians and are different from temporins
which are linear peptides with 13 residues (15).
A large number of antimicrobial peptides have been characterized from
skin tissue of amphibians (10-14). In the genus Rana itself
peptides belonging to eight families based on their structural similarities, have been described. These are brevenin 1 and 2, esculentin 1 and 2, ranatuerins 1 and 2, ranalexin, and temporins (15,
36-41). Of these, the first seven families of peptides are all
characterized by a highly basic, heptapeptide loop linked by a
disulfide bridge at the COOH-terminal end, but with a highly variable
sequence and length at the NH2-terminal end. However, the
presence of the disulfide bridge does not appear to be critical either
for activity or for structure in the case of Rana peptides identified so far (42-45). The primary structures of tigerinins are
compared with other amphibian peptides in Table
III which evidently indicates no
sequence homology. It is thus unlikely that tigerinins are derived from
similar class of peptides from R. tigerina. All the
amphibian antimicrobial peptides, including the Rana
peptides, known thus far, are known to adopt helical structure (15,
36-41). Conformational analysis by CD and theoretical methods suggest -turn structures. The peptides are cationic and exert their activity by permeabilizing bacterial membranes. Tigerinins represent the smallest, nonhelical, cationic antimicrobial peptides from amphibians.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mercaptoethanol at a concentration of 10-fold excess to that of
the peptide. The alkylated peptides were desalted, run on reverse phase
C18 column and were subsequently characterized by protein
sequencing and mass spectrometry. Alkylation with iodoacetamide without
prior reduction with dithiothreitol was also carried out to ascertain
the presence of disulfide bridges.
-cyano-4-hydroxycinnamic acid matrix and loaded on a stainless steel
target and the molecular weights were determined using matrix-assisted
laser desorption/ionization time of flight mass spectrometry (Kratos
PC-Kompact MALDI 4VI.1.2).
-mercaptoethanol (24). The
peptides were then desalted and the disulfide bridges were formed with
20% dimethyl sulfoxide (25). The synthesized peptides were run on
C18 reverse phase HPLC column independently as well as
mixing with natural peptide fractions. The synthetic peptides were then
used for evaluating antimicrobial activity.
4 M isopropyl thiogalactoside for inducing
the cytoplasmic enzyme
-galactosidase. The culture was then diluted
to an A600 of 0.03 with 10 mM sodium
phosphate buffer (pH 7.4) containing ONPG that serves as a substrate.
Aliquots of this were incubated with peptides at 37 °C and the
influx of ONPG into the cells was monitored by absorbance measurements
at 420 and 550 nm at fixed time intervals. ONPG influx into the cells
recorded as (A420
1.75 × A550) reflects the permeability status of inner
membrane. ONPG influx was monitored also in the presence of divalent
cation, calcium.
,
=
180o for
nonproline residues and
,
=
75o,
180o for prolines using BIOPOLYMER module of MSI of
version 98. The cysteines were bonded to form a disulfide bridge. These
structures were minimized for a short duration to remove bad contacts.
The structures were then optimized using a combination of minimizers like Steepest Descent, Conjugate Gradient, and Newton Raphson's methods for 3000 iterations till a final convergence of 0.001 was
achieved. The Amber force field "amber.frc" provided in the MSI 98 (Biosym technologies, San Diego) was used for all calculations. The
final structures were equilibrated for 10 ps duration before they were
subjected to dynamics of 500 ps at a constant temperature of 298 K
using a NVT ensemble temperature control method. The velocity-verlet
integration method was used in this case. The final structures were
once again minimized for 3000 iterations to achieve a convergence of
0.001. The optimization and Molecular Dynamics (MD) studies were
conducted by using DISCOVER module provided in MSI 98. The dihedral
angles for the final structures were calculated by using STRIDE
software program developed by Patrick Argos of EMBL (32). The Procheck
Program indicated that all the dihedral angles were in the allowed
regions in the Ramachandran map.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Reverse-phase HPLC of skin secretions of
R. tigerina. Analysis of adrenergic-mediated skin
secretions of R. tigerina eluting between 15 and 30%
acetonitrile concentration through Sep-Pak cartridges on a
C18 µBondapak column. Conditions: Solvent A, 0.1%
trifluoroacetic acid in water; B, 0.1% trifluoroacetic acid in
acetonitrile; gradient of 0-40% B in 30 min and 40% to 100% in 5 min at a flow rate of 1 ml/min. Detection was at 210 nm.
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Fig. 2.
Mass spectroscopic analysis of peptides.
A-C are the mass spectra of the peaks obtained by HPLC with
retention times 21.57, 23.40, and 24.10 min, respectively.
D-F, correspond to mass spectra of the peptides in
A-C after treatment with iodoacetamide and purified on
HPLC.
Sequences of antimicrobial peptides from R. tigerina
Antimicrobial activity of Tigerinins
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Fig. 3.
Kinetics of killing of bacteria by
tigerinins. A-C, S. aureus; D-F,
E. coli. Cells in the midlogarithmic phase of growth
(105 colony forming units) were incubated with different
concentrations of tigerinins (A and D, 10 µg/ml; B and E, 20 µg/ml, and C
and F, 30 µg/ml) and aliquots were drawn out at different
intervals after incubation and were plated on nutrient broth. The
number of colonies developed were counted after incubating the plates
for 18 h at 37 °C. Open square, tigerinin 1;
open triangle, tigerinin 2; open circle,
tigerinin 3; cross, tigerinin 4. Cells incubated in the
absence of any peptide served as controls.
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Fig. 4.
Peptide-mediated NPN uptake in E. coli W 160.37. E. coli cells were incubated
with NPN in the presence of various concentrations of tigerinins.
Enhanced uptake was measured by an increase in fluorescence caused by
partition of NPN in to hydrophobic interior of the OM. Open
square, tigerinin 1; open triangle, tigerinin 2;
open circle, tigerinin 3; cross, tigerinin 4;
filled square, linear analog of tigerinin 1, tigerinin
1(cys-Acm).
-galactosidase, ONPG
in the absence and presence of the peptides was monitored. The data is
presented in Fig. 5A. It is
evident that tigerinins permeabilize the IM of E. coli
effectively. As tigerinin 2 is the most effective, among tigerinins, in
permeabilizing the IM of E. coli, the effect of stabilizing
the OM with Ca2+ (33) on its IM permeabilizing ability was
also investigated. The results are shown in Fig. 5B.
Ca2+, at a concentration of 600 µM, almost
completely inhibited IM permeabilization by tigerinin. Tigerinins are
thus unable to permeabilize a stabilized OM, suggesting that OM
permeabilization is an essential and critical step for its
activity.
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Fig. 5.
Effect of tigerinins on the influx of ONPG in
E. coli W 160.37. E. coli cells in the
logarithimic phase of growth were diluted to a
A600 of 0.03 in phosphate buffer containing ONPG
and incubated at 37 °C with the peptides (40 µg/ml). The
absorption at 420 and 550 nm were recorded at various time points. The
value of (A420 1.75 × A550) was taken to denote ONPG influx.
Panel A: open square, tigerinin 1; open triangle,
tigerinin 2; open circle, tigerinin 3; cross,
tigerinin 4; diamond, control without any peptide.
Panel B: open triangle, tigerinin 2; filled
triangle, tigerinin 2 in the presence of 600 µM
Ca2+; diamond, control without peptide.
-turn
conformations (34). The conformations of tigerinins 1, 2, and 3 were
analyzed by theoretical methods involving energy minimization and
Molecular Dynamics Simulations. Comparison of the structures are
shown in Fig. 7. The dihedral angles of
the structures obtained after 500 ps Molecular Dynamics
Simulations for both the peptides with those documented for
standard
-turn types (35) indicate that although
-turn structures
are observed for tigerinin 1, 2, and 3, they are not of type I, II, or
III and can be categorized as type IV.
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Fig. 6.
Circular dichroism spectra of tigerinins 1, 2, 3, and 4 in 5 mM HEPES buffer (pH 7.4). Peptide
concentration = 60 µM.
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Fig. 7.
Structures of tigerinins after molecular
dynamics simulations. Side chains of cationic residues are
indicated in red and hydrophobic residues in
blue. Backbone structure is depicted as green
ribbons. Cysteine side chains are in yellow.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-turn conformations for tigerinins and these represent the first examples of nonhelical amphibian antimicrobial peptides. A majority of
cationic antimicrobial peptides (1, 6, 12, 13), including amphibian
skin peptides like magainin (16) and dermaseptins (17, 18), are known
to exert their antimicrobial activity by permeabilizing the cytoplasmic
membrane. Despite being structurally distinct from other amphibian
antimicrobial peptides, tigerinins also are capable of permeabilizing
bacterial membranes. While it is conceivable that tigerinins also exert
their antimicrobial activity by permeabilizing the microbial membranes,
other mechanisms cannot be ruled out. Indolicidin, a 13-residue
Trp-rich antimicrobial peptide from bovine neutrophils, which is known
to permeabilize the outer and cytoplasmic membranes of E. coli also preferentially inhibits synthesis of DNA leading to
filamentation of cells (46). This later mechanism appears to contribute
to its antimicrobial activity. Recently, many other cationic
antimicrobial peptides have been proposed to kill bacteria by alternate
mechanisms (47). Like all other amphibian antimicrobial peptides
(10-14), tigerinins are also cationic (Table III). All of them carry a
charge of +2 with 1 Arg residue and amidated COOH-terminal end. This
low cationicity appears to be sufficient for its biological activity.
Earlier we have shown that SPF, a 13-residue synthetic peptide
corresponding to the most hydrophobic region of bovine seminal plasmin,
with a charge of +1, has both antimicrobial and hemolytic activity (31). Apart from brevinins, another antimicrobial peptide, with a
single disulfide bond, that is well characterized is thanatin from the
hemipteran insect Podisus maculiventus. Thanatin is a 21-residue peptide with 50% homology to brevinins (48). However, in
contrast to a heptapeptide loop at the COOH-terminal end of brevenins,
thanatin has an eight-membered ring. Despite the homology, the
secondary structure and mechanism of action differs from that of
brevenin. Thanatin appears to assume a
-turn structure stabilized by
a disulfide bond and is suggested to exert its action by a nonpore
forming mechanism.
Representative primary structures of antimicrobial peptides from
amphibian skin
The only other short antibacterial peptide with a similar nonapeptide disulfide linkage is bactenecin which has been isolated from bovine neutrophils (49). However, bactenecin has 4 arginine residues as compared with one in tigerinins and therefore considerably more cationic. Reduction of the single disulfide bond in bactenecin was observed to result in a drastic change in the antibacterial spectrum (50). The reduced bactenecin showed high selectivity for Gram-positive bacteria with little activity against Gram-negative bacteria whereas native bactenecin was more active against Gram-negative bacteria. In tigerinin, absence of disulfide bridge resulted in loss of antibacterial activity indicating the importance of disulfide bridge for activity. It is very unlikely that tigerinins are related to bactenecins.
Thus, this paper describes a family of short and nonhelical
antimicrobial peptides in amphibians. As short peptides have the obvious advantage of easy chemical synthesis, the new structural motif
observed in tigerinins would be easily amenable for the synthesis of
analogs with improved activity and conceivably useful against
multidrug-resistant microbes. We are currently investigating other
pharmacological activities of peptides from the skin secretions of
Rana tigerina.
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ACKNOWLEDGEMENTS |
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We thank V. M. Dhople for amino acid analysis, Dr. S. Harinarayan Rao and staff of the animal house, Centre for Cellular and Molecular Biology for their help.
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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.
The peptide sequences of tigerinins reported in this paper has been submitted to the Swiss-Prot Database under Swiss-Prot accession numbers P82651 (tigerinin 1), P82652 (tigerinin 2), P82653 (tigerinin 3), and P82654 (tigerinin 4).
§ Supported by Department of Biotechnology, Goverment of India for post-doctoral fellowship, and Council for Scientific and Industrial Research New Delhi for Research associateship.
To whom correspondence should be addressed. Tel.:
91-40-7172241; Fax: 91-40-7171195; E-mail: nraj@ccmb.ap.nic.in or
sitaram{at}ccmb.ap.nic.in.
Published, JBC Papers in Press, October 12, 2000, DOI 10.1074/jbc.M006615200
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ABBREVIATIONS |
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The abbreviations used are: HPLC, high performance liquid chromatography; MD, molecular dynamics; Fmoc, N-(9-fluorenyl)methoxycarbonyl; NPN, N-phenyl-1-N-naphthylamine; ONPG, o-nitrophenyl-3-D-galactoside; OM, outer membrane; IM, inner membrane.
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REFERENCES |
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