From the Department of Comparative Physiology,
Evolutionary Biology Centre, Uppsala University, Norbyvägen 18A,
SE-752 36, Sweden and the § College of Pharmacy, Pusan
National University, Jangjeon Dong, Kumjeong Ku, Busan, 609-735, Korea
Received for publication, September 9, 2002, and in revised form, December 1, 2002
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ABSTRACT |
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An antibacterial peptide with 16 amino acid
residues was found in plasma of the freshwater crayfish,
Pacifastacus leniusculus. This peptide, designated
astacidin 1, was purified by cation-exchange column chromatography and
reverse-phase high performance liquid chromatography. Astacidin 1 has a
broad range of antibacterial activity, and it inhibits growth of both
Gram-positive and Gram-negative bacteria. The primary sequence of
astacidin 1 was FKVQNQHGQVVKIFHH-COOH. The molecular mass was 1945.2 Da, and no carbohydrate-linked amino acid residues could be found by
mass spectrometry. A synthetic astacidin 1 resulted in similar activity
as the authentic astacidin 1 against Gram-positive bacteria, whereas it
had less or no activity against Gram-negative bacteria. Three
amino-terminal-truncated synthetic peptides were made; they all showed
low activity, suggesting that the amino-terminal part of astacidin 1 contributes to the antibacterial activity. The structure of astacidin 1 based on the CD results showed that it has a Antimicrobial peptides have become recognized as important
components of the nonspecific host defense or innate immune system in a
variety of organisms ranging from plants and insects to animals, including mollusca, crustaceans, amphibians, birds, fish, mammals, and
humans (1-3). The primary structures of antimicrobial peptides with
positively charged and hydrophobic amino acids are highly diverse, yet
their secondary structures share a common feature of amphipathicity,
and many of these peptides are membrane-active by ion-channel formation
or carpet effect (4, 5). Although they exhibit great structural
diversity, they are often divided into four major groups according to
composition and secondary structural patterns. The first group has an
antiparallel The Toll signaling pathway is involved in regulating dorsal-ventral
polarity in developing embryos and synthesis of antimicrobial peptides
in Drosophila. Antimicrobial peptides synthesized in the fat
body are secreted into the hemolymph. One role of the Toll pathway in
Drosophila immune response is to activate the synthesis of
these peptides after fungal or Gram-positive bacterial infection (17),
whereas the immune deficiency pathway is involved in producing peptides
aimed at Gram-negative bacteria in Drosophila.
Several antimicrobial peptides have been characterized from insects and
chelicerates, and only a few peptides have been reported from
crustaceans such as the shore crab Carcinus maenas (18) and
the shrimp Penaeus vanamei (19, 20). Here we present the isolation, biochemical characterization, and synthesis of a new antimicrobial peptide, astacidin 1, from plasma of the freshwater crayfish, Pacifastacus leniusculus.
Animals--
Freshwater crayfish, P. leniusculus,
were purchased from Berga Kräftodling, Södermanland, Sweden
and were maintained in tanks with aerated water at 10 °C. Only
intermolt crayfish were used in this study.
Purification of Antibacterial Peptides--
Hemolymph was
prepared by collecting blood from 400 crayfish in anticoagulant buffer
(0.14 M NaCl, 0.1 M glucose, 30 mM
trisodium citrate, 26 mM citric acid, 10 mM
EDTA, pH 4.6) (21). After centrifugation at 4 °C and 800 × g for 10 min, the plasma was removed from hemocytes and
stored at Determination of Amino Acid Sequence and Mass Analysis--
The
homogenous purified peptide was identified based on Edman sequence
analysis using an Applied Biosystem 476A automated amino acid
sequencer. For mass analysis and for confirming amino acid sequences,
MALDI-TOF-MS1 was performed
in a Q-tof tandem mass spectrometer (Micromass, Manchester, UK)
equipped with nanospray interphase. Interpretation of mass spectra was
done by using the MassLynx (Micromass) suite of software programs.
Peptide Synthesis--
An amidated 16-residue antibacterial
peptide and three different truncated peptides were synthesized by the
solid method (22). The molecular masses of the synthetic peptides were
determined with MALDI mass spectra.
Acid-urea PAGE--
The purity of the authentic and synthetic
peptides was checked with 20% acetic acid-urea polyacrylamide gel
electrophoresis followed by Coomassie staining for peptides as
described by Selsted and Becker (23). A low molecular mass calibration
kit for electrophoresis (Amersham Biosciences) was used, containing
rabbit muscle phosphorylase b (94 kDa), bovine serum albumin
(67 kDa), egg white ovalbumin (43 kDa), bovine erythrocyte carbonic
anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa), bovine milk
Assay of Antibacterial Activity--
During the purification
procedure, the antimicrobial activities of samples were monitored by a
radial diffusion using Bacillus megaterium BM11 and
Escherichia coli D21 as test organism as described by Lehrer
et al. (24). Briefly, a 10-ml culture of bacterial cells in
mid-logarithmic phase was subjected to centrifugation at 900 × g for 5 min, washed with 10 mM sodium phosphate
buffer, pH 7.4, and then resuspended in 10 ml of the same buffer. One hundred µl of bacterial solution containing 1 × 106
colony-forming units was added to 10 ml of previously autoclaved agar
(10 mM sodium phosphate, pH 7.4, 1% (v/v) LB
medium, 1% (w/v) agarose, 0.02% (v/v) Tween 20), and the
mixture was poured into a petri dish. Peptide samples were added
directly to 3-mm wells made on the solidified underlayer agar. After
incubation for 3 h at 37 °C, the plates were overlaid with 10 ml of sterile agar containing a double-strength (6% v/v) solution of
LB and 1% agarose, and then they were incubated for 12-24 h at
30-37 °C. The minimal inhibitory concentration was determined using
the same method and tested against several species of Gram-negative and
Gram-positive bacteria. The lowest concentration of the antibacterial
peptide that showed visible suppression of growth was defined as the
minimal inhibitory concentration.
A liquid growth inhibition assay was performed as described in Lee
et al. (25). Bacteria grown in LB medium (peptone 10 g,
yeast extract 5 g, NaCl 5 g, glucose 1 g/distilled water 1 liter) was collected in the exponential phase of growth and
resuspended with phosphate-buffered saline, pH 6.0, at a density of
1 × 108 cells/ml. Samples were suspended in 200 µl
of 0.2% (w/v) bovine serum albumin and then incubated in 190 µl of
LB medium with 10 µl of bacterial suspension and shaking for 3 h
at 37 °C. The optical density at 650 nm was measured on each sample.
cDNA Cloning and Nucleotide Sequencing of Astacidin
1--
The cDNA library was screened with
5'-[ Processing of Astacidin 1 from Hemocyanin--
To test the
production of astacidin 1 in crayfish, plasma was separated from
hemocytes, diluted twice with anticoagulant buffer or CAC buffer (10 mM sodium cacodylate, 5 mM CaCl2,
pH 7.0), and treated with trifluoroacetic acid at a final concentration
of 0.1%. Plasma (14 mg/ml) treated with trifluoroacetic acid in
anticoagulant buffer or CAC buffer was incubated for different time
intervals, such as 0.5, 1, 2, 3, 4, and 5 days, at 4 °C. As a
control, plasma was treated with trifluoroacetic acid and then
immediately centrifuged without any incubation. Each sample was
centrifuged at 16,000 × g for 20 min, and the
resulting supernatant was subjected to SEP-PAK chromatography. The
samples were eluted with 80% acetonitrile containing 0.05%
trifluoroacetic acid and then vacuum dried. The dried samples were
dissolved in sample loading buffer, and 50 µg of each sample was
subjected to 20% acid-urea PAGE.
To confirm the involvement of a proteinase in the processing of
hemocyanin, plasma was treated with several different proteinase inhibitors such as pepstatin (1 µM), EDTA (1 mM), E-64 (10 µM, trans-epoxysuccinyl-L-leucylamido (4-guanidino)-Butane),
leupeptin (50 µM), iodoacetamide (100 µM),
2-mecaptoethanol (0.1%), and 1 mM phenylmethylsulfonyl
fluoride. The different proteinase inhibitors were separately incubated
with plasma (14 mg/ml) in CAC buffer for 1 h at room temperature
and then treated with trifluoroacetic acid. After incubation for
12 h at 4 °C, the procedures were followed as described above.
Immunization of Crayfish for Production of Astacidin 1--
Ten
crayfish were injected with 100 µg of LPS (E. coli
serotype 055:B5; Sigma) or laminarin ( Circular Dichroism Measurements--
All CD spectra were
obtained by JASCO-720 spectropolarimeter. Cellular path length was 1 mm. The concentration of stock solution of protein was determined by
bicinchoninic acid assay (27). The stock solution was diluted to
50-100 µg/ml in appropriate buffers. All the experiments were
carried out at 25 °C. Scan speed was set with 10 or 20 nm/min. The
scan was carried out three times and averaged to the mean value. The
contents of secondary structure were calculated using the method of
Yang (28).
Purification of Astacidin 1--
An antibacterial peptide,
astacidin 1, was purified from crayfish plasma by adding the acidified
plasma solution to C-18 reverse-phase column chromatography. The eluted
fractions were assayed for their antibacterial activity against two
bacterial strains, B. megaterium BM11 and E. coli
D21. The fractions containing antibacterial activity were collected,
vacuum dried, and subjected to Mono S cation exchange column
chromatography for further purification of astacidin 1. Most proteins
did not bind to the cation exchange column. Finally, the antibacterial
peptide was purified to homogeneity by reverse-phase HPLC. The purity
of samples was monitored by 20% acid-urea PAGE (Fig.
1A), because astacidin 1 could
not be detected in 20% SDS-PAGE (Fig. 6B).
Determination of Primary and Secondary Structures--
The new
antibacterial peptide from crayfish plasma was analyzed for its primary
structure by Edman degradation and MALDI-TOF-MS. Astacidin 1 consists
of 16 amino acid residues with the sequence FKVQNQHGQVVKIFHH-COOH. The
mass of astacidin 1 determined by MALDI-TOF-MS was 1945.2 Da, and it is
COOH-terminal carboxylated. The peptide does not contain
carbohydrate-linked amino acid residues or cysteine residues.
For determination of the secondary structure of astacidin 1, CD spectra
were determined in citric acid buffer at different pH values as well as
at different temperatures. No significant changes in CD spectra
were observed under various temperature conditions. However, the CD
spectra showed that 40-53.8% of the molecule has a Antibacterial Activity Spectrum of Authentic and Synthetic
Astacidin 1--
To fully characterize the biochemical properties of
astacidin 1, we performed solid-phase synthesis of the 16-amino acid
peptide and three different amino-terminal-truncated peptides,
designated SP-1 to -4 (Table I). The
purity of the synthetic peptides was confirmed by 20% acid-urea PAGE
(Fig. 1C). The synthetic peptide SP-1 had similar
antibacterial activity as the authentic native astacidin 1 against
Gram-positive bacteria such as B. megaterium BM11,
Bacillus subtilis ATCC 6633, and Micrococcus
luteus Ml 11, whereas the synthetic peptide had lower
antibacterial activity toward Gram-negative bacteria. The
amino-terminal-truncated peptides had much lower antibacterial activity
than the complete synthetic 16-amino acid peptide (Table
II). This result indicates that the amino-terminal amino acids contribute to the antibacterial activity. The difference in antibacterial activity between native and synthetic astacidin 1 may be because of the solubility of the synthetic peptide
because the synthetic peptide is less soluble than the native peptide
in water. Therefore, the synthetic peptide was dissolved in water
containing 0.05% trifluoroacetic acid. In this solution with low pH,
the secondary structure of the synthetic peptide is changed from a
random coil to a Cloning and Nucleotide Sequence Analysis of Astacidin 1--
We
obtained a positive clone from a crayfish hepatopancreas cDNA
library. The amino acid sequence of astacidin 1 was used to
design and synthesize degenerate primers. Using the
5'-[
Comparisons of the deduced amino acid sequence of hemocyanin cDNA
with shrimp hemocyanin and crayfish Prophenoloxidase shows 58 and 33% identity, respectively (Fig.
3). It also has high similarity with
other hemocyanins such as hemocyanin Processing of Astacidin 1 from Crayfish Hemocyanin and Induction of
Astacidin 1 Production by Injection of LPS or Glucan in
Crayfish--
To reveal whether astacidin 1 is processed from
hemocyanin, plasma was incubated under acidified condition in a
time-dependent manner using the anticoagulant buffer or CAC
buffer. The processing of astacidin 1 from hemocyanin was detectable
after 12 h of incubation under acidic condition, and it further
increased up to 5 days of incubation (Fig.
4). This processing occurs under neutral
pH such as in the CAC buffer with a final concentration of 2.5 mM CaCl2. However, higher CaCl2
concentration, i.e. more than 2.5 mM, prohibited
this processing (data not shown).
To evaluate whether a proteinase is involved in this processing,
several proteinase inhibitors were added to crayfish plasma. The
generation of astacidin 1 was strongly inhibited by pepstatin or E-64
(Fig. 5). EDTA (5 mM) could
also block the production of astacidin 1 (data not shown). This result
suggests that some cysteine proteinase is likely to be involved
in processing of the antibacterial peptide from hemocyanin.
The effect of LPS or glucans for the generation of astacidin 1 was
examined after injection of these carbohydrates into crayfish. Plasma
was prepared from hemolymph withdrawn from crayfish 6 h post-injection. The concentration of astacidin 1 was shown to be
increased in plasma from animals previously injected with LPS or glucan
compared with control animals (Fig.
6A). If a purified homogeneous
hemocyanin were incubated with LPS or glucan in parallel with
experimental and control animals, no astacidin 1 was produced. SDS-PAGE
was simultaneously performed using the same amount of protein as in
acid-urea PAGE to compare protein patterns (Fig. 6B).
Astacidin 1 could not be detected under SDS-PAGE condition, but new
proteins could be detected after injection of crayfish with LPS or
glucan. Several immune reactions in invertebrate have previously been
known to be initiated by such bacterial or fungal cell wall components
as LPS and glucan, for instance, the prophenoloxidase activation system
(29, 30), the clotting system (32), and the synthesis of antibacterial
peptides (33). The result in this study shows that the
carboxyl-terminal part of crayfish hemocyanin is processed by a
cysteine-like proteinase and this processing is up-regulated by LPS or
glucan treatment to produce a biologically active antimicrobial
peptide, astacidin 1.
The cells of invertebrates and mammals produce various
antimicrobial substances that act as endogenous antibiotics or
disinfectants. Most antimicrobial peptides consist of fewer than 100 amino acids; these peptides are amphipathic, carry a net positive
charge, and manifest a well defined Here we describe the molecular and functional characterization of a
novel peptide with a broad-spectrum antibacterial activity from the
hemolymph of the freshwater crayfish, P. leniusculus, which
we have named astacidin 1. The antibacterial molecule was purified to
homogeneity and is fully characterized at the level of its primary and
secondary structure by a combination of reverse-phase chromatography,
cation exchange chromatography, MALDI-TOF-MS, and CD spectrum.
Astacidin 1 consists of 16 amino acid residues with no cysteine, a
strong cationic property, and a Many antimicrobial peptides are derived from larger precursors, and
processing and generation of antibacterial peptides have been reported
from several species. For example, in amphibians, buforin I from the
stomach gland cells of the Asian toad Bufo bufo is generated
by a pepsin-mediated processing of the cytoplasmic histone H2A (41). In
mice, the precursor Hemocyanin is an interesting molecule that serves as an oxygen carrier
for many chelicerates and crustaceans. In a recent study, hemocyanins
were suggested to have phenoloxidase activity after proteolytic
cleavage at the amino-terminal part of hemocyanins in chelicerates
such as the spider, Eurypelma californicum (45, 46), and
the horseshoe crab, Tachypleus tridentatus (47). Several
physicochemical properties of hemocyanins are very similar to those of
phenoloxidase (EC 1.14.18.1) (48, 49). Phenoloxidase is an efficient
immune molecule for non-self recognition and is the terminal compound
of the so-called prophenoloxidase activating system that is involved in
immune reaction such as melanin production, cell adhesion,
encapsulation, and phagocytosis as well as sclerotization of the
arthropod cuticle (29). It is expressed in hemocytes without a signal
peptide and synthesized as a zymogen that is activated by a proteolytic
cleavage of an amino-terminal peptide. In contrast, hemocyanin is
produced in hepatopancreas and then released to plasma. Evolution seems
to have developed a double function of hemocyanin in the chelicerates
(50). Under normal conditions the hemocyanin has a function as an
oxygen carrier, but it may be converted to phenoloxidase after
infection to prevent microbial invasion. The amino acid sequence of
crayfish hemocyanin reveals high homology with shrimp hemocyanin and
crayfish prophenoloxidase, but there is no homology in the
carboxyl-terminal part (Fig. 3). Therefore, only crayfish hemocyanin,
and not crayfish prophenoloxidase, can produce and release astacidin 1. In this study, we report that crustacean hemocyanin can be processed by
a cysteine-like proteinase, most likely from a lysosomal organelle, to
generate an antimicrobial peptide under acidic condition and that this production can be further enhanced by injecting LPS and glucan into the animal.
-sheet structure in
citric acid buffer at pH 4, 6, and 8. Cloning of astacidin 1 shows that it is the carboxyl-terminal part of crayfish hemocyanin and that astacidin 1 is produced by a proteolytic cleavage from hemocyanin under
acidic conditions. The processing and release of astacidin 1 from hemocyanin is enhanced when crayfish are injected with lipopolysaccharide or glucan.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheet structure containing three disulfide bridges,
and these defensin peptides can be divided into two subgroups according
to their structure. The mammalian defensins have a triple-stranded
-sheet structure (6), whereas insect defensins form two-stranded
-sheets with a flanking
-helix (7). Although all defensins
contain three disulfide bonds, the mammalian and insect defensins show different three-dimensional structures. Cecropin and magainin family
peptides contain linear peptides forming
-helices and are deprived
of cysteine residues. This group generally has a random coil structure
in aqueous solution and can penetrate bacterial membranes and disrupt
the membrane structure by ion channel formation (8-10). A third group
of peptides has a loop structure containing one or more cysteine
residues such as bactenecin, and the fourth group comprises peptides
with an over-representation of specific amino acids, such as proline,
arginine (11-13), and glycine residues (14, 15) or tryptophan-rich
peptide (16). The proline-rich peptides are present in insects,
crustaceans, and mammals. However, until now, no glycine-rich
molecules have been reported in mammals.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C until further analysis. For purification of
antibacterial proteins from plasma, the frozen samples were thawed and
trifluoroacetic acid was added to a final concentration of
0.1%. After incubation at 4 °C for 12 h, the sample was
centrifuged at 16,000 × g for 20 min, and the
supernatant was diluted twice with 0.05% trifluoroacetic acid water
and directly subjected to C-18 reverse-phase chromatography (
2.7 × 9 cm, Waters) equilibrated with 0.05% trifluoroacetic acid
water. The sample was eluted with a step gradient of 20, 50, and 80%
acetonitrile containing 0.05% trifluoroacetic acid, and then 50%
acetonitrile elution fraction was applied to the second C-18 column
using 0-50% acetonitrile/water/0.05% trifluoroacetic acid as a
linear gradient. For the next purification step, Mono S cationic ion
exchange chromatography was performed using a FPLC system (Amersham
Biosciences). The absorbed proteins were eluted with a linear
gradient of 0-1 M NaCl containing 10 mM sodium phosphate
buffer, pH 6.0. The sample was then further purified to homogeneity by
reverse-phase HPLC (Amersham Biosciences smart chromatography system)
on C-18 column using acetonitrile/water/0.05% trifluoroacetic acid
gradients of 0-60% acetonitrile in 60 min at a flow rate of 100 µl/min. Ultraviolet absorption was monitored at 280, 254, and 214 nm. The eluted peak fractions were vacuum-dried and used for assay of
antibacterial activity and determination of amino acid sequences.
-lactalbumin (14.4 kDa), and aprotinin (6.5 kDa). A synthetic
peptide of astacidin 1 (1.9 kDa) was also used.
-32P]ATP-labeled mixed probe
(AT(C/T)TTIACIAC(C/T)TGICC(A/G)TG(C/T)TG(A/G)TT(C/T)TGIAC; I is
inosine), which was designed according to the following amino acid
sequence of astacidin 1, VQNQHGQVVKI. For the initial screening, ~120,000 recombinants of crayfish hepatopancreas
gt 11 cDNA
library were used. The membranes were prehybridized at 65 °C for
1 h in 5× SSC (750 mM NaCl, 75 mM
Na-citrate, pH 7.0), 5× Denhardt's solution (100× Denhardt's
solution is 2% (w/v) bovine serum albumin, 2% (w/v) Ficoll, and 2%
(w/v) polyvinylpyrrolidone), 100 µg/ml salmon sperm DNA, and 0.5%
SDS. The membranes were then hybridized at 65 °C for 12 h in
the same solution of prehybridization. After the second screening,
positive clones were amplified with PCR using a pair of nested
gt
11-specific primers (GGATTGGTGGCGACGACT and GCTTTATGCCCGTCTGTA). PCR
conditions were 94 °C for 45 s, 55 °C for 30 s, and
72 °C for 2 min carried out for 30 cycles. The largest PCR product
was subcloned into TOPO cloning vector (Invitrogen). The plasmids were
released according to the instructions of the manufacturer (Sigma). For
confirming the size of plasmids, the insert was digested out by the
restriction enzyme EcoRI and then run on 1% agarose gel. It
was sequenced with an Applied Biosystems PRISM dye terminator cycle
sequencing ready reaction kit (PerkinElmer Life Sciences). The cDNA
sequence was analyzed with MacVector 6.5.1. software (Kodak). The
nucleotide and the deduced amino acid sequences were compared using the
BLAST program (National Center for Biotechnology Information,
Bethesda, MD).
-1-3-glucan; Sigma) dissolved in 100 µl of distilled water. After incubation of injected crayfish for 6 h in water at 16 °C, hemolymph was collected in a test
tube and plasma was isolated by centrifugation for 5 min at 2,800 × g. Purified hemocyanin was also treated with the same
amount of LPS and glucan and then incubated for 6 h at 16 °C.
Both plasma and hemocyanin were diluted twice with CAC buffer and
treated with trifluoroacetic acid at a final concentration of 0.1% and then further incubated for 12 h at 4 °C. The samples were
centrifuged at 16,000 × g for 20 min, and the
resulting supernatant was subjected to SEP-PAK chromatography. The
samples were eluted with 80% acetonitrile containing 0.05%
trifluoroacetic acid and then vacuum dried. The dried samples were
dissolved in sample loading buffer, and 40 µg of protein was
subjected to 20% acid-urea PAGE or 15% SDS-PAGE under reducing
conditions according to Laemmli (26).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
A, 20% acid-urea PAGE of the
purified astacidin 1. A low molecular size marker was used: rabbit
muscle phosphorylase b, bovine serum albumin, egg white ovalbumin
(43 kDa), bovine erythrocyte carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa), bovine milk
-lactalbumin (14.4 kDa), aprotinin (6.5 kDa),
and synthetic peptide of astacidin 1. B, the predicted
secondary structure of astacidin 1 based on CD spectrum. The
black lines indicate
-sheet structures. C,
20% acid-urea PAGE of the synthetic peptide of astacidin 1. Lane
1, size marker; lane 2, SP-1 (16-amino acid residue
peptide); lane 3, SP-2 (15-amino acid residue peptide);
lane 4, SP-3 (14-amino acid residue peptide); lane
5, SP-4 (12-amino acid residue peptide); lane 6,
purified astacidin 1.
-sheet
structure at pH 4, 6, and 8. Moreover, the addition of up to 80% (v/v)
acetonitrile to astacidin 1 results in ~27%
-sheet structure
content (data not shown). The predicted secondary structure of
astacidin 1 based on CD data is shown Fig. 1B. Two putative
-sheet structures in each terminus of astacidin 1 may help to
disturb the cell wall and membrane of bacteria, as is known from
several other antimicrobial peptides.
-sheet according to our CD data, which might have
affected the antibacterial activity against Gram-negative bacteria.
Contamination with other antibacterial peptides in the native astacidin
1 can be excluded, because the amino acid sequence and homogeneity of
native astacidin 1 used for these experiments were determined by
MALDI-TOF-MS.
Primary structure of synthetic peptide of astacidin 1
Minimal inhibition concentration of astacidin 1 and synthetic peptides
-32P]ATP labeling method, specific DNA fragments
representing astacidin 1 were amplified. The largest clone was shown to
code for the complete amino acid sequence of astacidin 1. The
nucleotide sequence and deduced amino acid sequence are shown in Fig.
2. The cDNA has an open reading frame
of 1,980 nucleotides corresponding to a 660-amino acid residue, and
this sequence turns out to be a hemocyanin. The underlined amino acid
sequences of the cDNA perfectly match the amino acid sequences of
astacidin 1, and a termination codon directly follows the astacidin 1 sequence. This indicates that the isolated antibacterial peptide
corresponds to the carboxyl terminus of hemocyanin. The first 17 amino
acid residues of hemocyanin form a typical signal sequence. Therefore,
hemocyanin of crayfish consists of a 660-amino acid residue with a
calculated molecular mass of the protein portion of 75,316 Da and an
estimated pI of 5.47. Hemocyanin is a blue copper-containing
oxygen-transporting molecule and is the predominant protein in the
plasma of many crustaceans. The six histidine residues with open
circles in Fig. 2 are essential for binding the two oxygen-binding
copper atoms in hemocyanin, and they are conserved in all hemocyanins
as well as in invertebrate prophenoloxidases (29-31).
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Fig. 2.
Nucleotide and deduced amino acid sequences
of crayfish P. leniusculus hemocyanin.
Nucleotides (upper) are numbered on the right.
Amino acids (lower) are also numbered on the
right and counted from the initiating methionine. The first
17 amino acids are the putative signal peptide. The histidine residues
of Cu(A), His-200, His-214, and His-237, and of Cu(B), His-357,
His-361, and His-397 are indicated by a circle. The
underlined amino acid sequences denote the sequences
of astacidin 1 purified from crayfish plasma. A polyadenylation signal
is shown with underline.
-subunit of Homarus americanus (AJ272095) and hemocyanin subunits 1 (AJ344361), 2 (AJ344362), and 3 (AJ344363) of Palinurus vulgaris.
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Fig. 3.
Alignment of crayfish P. leniusculus hemocyanin, crayfish P. leniusculus
prophenoloxidase (GenBankTM
X83494), and shrimp hemocyanin
(GenBankTM X82502). The
shadowed boxes indicate that the residues are identical. The
dots indicate that the amino acids have similar properties.
The underlined amino acid sequences in hemocyanin of
crayfish and shrimp represent astacidin1 and shrimp antifungal peptide
(PvHct), respectively. Crayfish-Hc, crayfish hemocyanin;
shrimp-Hc, shrimp hemocyanin; crayfish-proPO,
crayfish prophenoloxidase.
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Fig. 4.
Processing of astacidin 1 from hemocyanin
under acidic conditions. The plasma (14 mg/ml) was prepared in
anticoagulant buffer and was treated with trifluoroacetic acid in a
time-dependent manner. After SEP-PAK chromatography, 50 µg of protein was subjected to 20% acid-urea PAGE. The
arrow shows produced astacidin 1. Lane 1, size
marker; lane 2, 0 h of incubation after trifluoroacetic
acid treatment; lane 3, 12 h of incubation after
trifluoroacetic acid treatment; lane 4, 1 day of incubation
after trifluoroacetic acid treatment; lane 5, 2 days of
incubation after trifluoroacetic acid treatment; lane 6, 3 days of incubation after trifluoroacetic acid treatment; lane
7, 4 days of incubation after trifluoroacetic acid treatment;
lane 8, 5 days of incubation after trifluoroacetic acid
treatment.
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Fig. 5.
Inhibition of production of astacidin 1 from
hemocyanin by different proteinase inhibitors. Pepstatin (1 µM), EDTA (1 mM), E-64 (10 µM),
leupeptin (50 µM), iodoacetamide (100 µM),
2-mecaptoethanol (0.1%), or phenylmethylsulfonyl fluoride (1 mM) were each incubated with plasma protein for 1 h at
room temperature, and then the samples were treated with
trifluoroacetic acid for 12 h at 4 °C. After SEP-PAK
chromatography, 50 µg of protein was subjected to 20% acid-urea
PAGE. The arrow shows the produced astacidin 1. Lane
1, 0 h of incubation after trifluoroacetic acid treatment
without any proteinase inhibitor as a negative control; lane
2, 12 h of incubation after trifluoroacetic acid treatment
without any proteinase inhibitor as positive control; lane
3, pepstatin; lane 4, EDTA; lane 5, E-64;
lane 6, leupeptin; lane 7, iodoacetamide;
lane 8, iodoacetamide with 2-mercaptoethanol; lane
9, phenylmethylsulfonyl fluoride; lane 10, size
marker.
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Fig. 6.
The generation of astacidin 1 from hemocyanin
in crayfish injected with LPS or glucan. Plasma was collected from
crayfish hemolymph 6 h post-injection, treated with
trifluoroacetic acid, and incubated for 12 h at 4 °C. 40 µg
of protein was subjected to 20% acid-urea PAGE (A) and 15%
SDS-PAGE under reducing conditions (B). The arrow
indicates the produced astacidin 1. Lane 1, size marker;
lane 2, 12 h of incubation after trifluoroacetic acid
treatment as positive control; lane 3, plasma injected by
LPS and treated trifluoroacetic acid, incubated for 12 h at
4 °C; lane 4, plasma injected by glucan and treated
trifluoroacetic acid, incubated for 12 h at 4 °C; lane
5, 0 h incubation after trifluoroacetic acid treatment as
negative control.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical or
-sheet structure
in membrane-like environments. Expression of antimicrobial peptides can
be constitutive, inducible, or both; several reviews of this topic have
appeared in recent years (2, 33-36). Although there are extensive
studies on antimicrobial proteins as important immune molecules in
various animals, a few antibacterial proteins have been characterized from crustaceans (18, 19).
-sheet structure based on CD
spectrum, which is likely to be important for its antibacterial
activity. Another antibacterial peptide, thanatin from the bug
Podisus maculiventris (37), shows a structure similar to
that of astacidin 1. Thanatine is a 21-amino acid residue peptide containing two cysteine residues that form an internal disulfide bridge. This peptide has two
-sheet stranded sheets (five residues each), which are held together by a single disulfide bridge. Such an
antiparallel two-stranded
-sheet structure is also found in brevinins from frog (38), protegrins from porcine leukocytes (39), and
tachyplesins isolated from the horseshoe crab, Tachypleus tridentatus (40). However, there is no sequence homology between astacidin 1 and these peptides including thanatin. Three truncated synthetic peptides were made to elucidate the minimal structure required for antibacterial activity. The amino-terminal-truncated synthetic peptides had less antibacterial activity than authentic astacidin 1, which suggests that the amino-terminal amino acids are
important for antibacterial activity.
-defensin is cleaved by metalloproteinase, a
matrilysin to produce
-defensin (42), and human defensin-5 is also
processed by paneth cell trypsin (43). The organization and processing
of peptides from one large precursor molecule is an efficient way to
synthesize different effector molecules and amplify the antibacterial
response. Interestingly, astacidin 1 is released from the
carboxyl-terminal part of crayfish hemocyanin by a cysteine-like
proteinase and is up-regulated by injection of LPS or glucan. The LPS
injection results in the generation of other proteins than astacidin 1, whereas glucan injection mainly leads to production of astacidin 1. Recently, three kinds of small antimicrobial peptides were reported
from shrimp, which could also be produced from the carboxyl-terminal
part of hemocyanin (44). The production of these peptides can be
enhanced by exposure to LPS. The small antimicrobial peptide with a
molecular mass of 2.7 kDa named PvHCt purified from Penaeus
vannamei has a similar size to astacidin 1 (Fig. 3), whereas two
other peptides, PsHCt 1 and PsHCt 2 purified from Penaeus
stylirostris, are large (7.9 kDa and 8.3 kDa, respectively), but
none of them has any homology to astacidin 1.
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FOOTNOTES |
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* This work has been supported by the Swedish Research Council (NT), STINT (the Swedish Foundation for International Cooperation in Research and Higher Education), and Teknikbrostiftelsen in Uppsala.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 nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number(s) AF522504.
¶ To whom correspondence should be addressed: Dept. of Comparative Physiology, Evolutionary Biology Center, Uppsala University, Norbyvägen 18A, SE-752 36, Sweden. Tel.: 46-18-4172818; Fax: 46-18-4716425; E-mail: Kenneth.Soderhall@ebc.uu.se.
Published, JBC Papers in Press, December 18, 2002, DOI 10.1074/jbc.M209239200
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ABBREVIATIONS |
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The abbreviations used are: MALDI-TOF-MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; CD, circular dichroism; LPS, lipopolysaccharide; HPLC, high performance liquid chromatography; LB, Luria-Bertani.
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