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Processing of an antibacterial peptide from hemocyanin of the

freshwater crayfish Pacifastacus leniusculus

So Young Lee*, Bok Luel Lee#, and Kenneth Söderhäll*¶

Department of Comparative Physiology, Evolutionary Biology Centre,

*Uppsala University, Norbyvägen 18A, SE-752 36, Sweden; #College of Pharmarcy, Pusan National

University, Jangjeon Dong, Kumjeong Ku, Busan, 609-735, Korea

*¶To whom correspondence should be addressed : Department 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.

Running title : Astacidin 1 has antibacterial activity

Key words : antibacterial protein, astacidin 1, hemocyanin, crayfish, Pacifastacus leniusculus, innate

immunity

Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on December 18, 2002 as Manuscript M209239200 by guest on A

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ABSTRACT

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 chromatograph. Astacidin 1 has a

broad range of antibacterial activity and it inhibits growth of both Gram-positive bacteria 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 a similar activity as the authentic astacidin 1 against

Gram-positive bacteria, whereas it had less or no activity against Gram-negative bacteria. Three N-

terminal truncated synthetic peptides were made and they all showed low activity suggesting that the N-

terminal part of astacidin 1 contributes to the antibacterial activity. The structure of astacidin 1 based on

the CD1 results showed that it has a beta-sheet structure in citric acid buffer at pH 4, 6, and 8. Cloning

of astacidin 1 shows that it is the C-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.

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INTRODUCTION

Antimicrobial peptides have become recognized as important components of the non-specific 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 β-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 β-sheet structure (6), whereas insect

defensins form two stranded β-sheets with a flanking α-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 α-helices and are deprived of

cysteine residues. This group generally have 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 have 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), 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.

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 (imd) pathway is involved in producing peptides aimed at gram-

negative bacteria in Drosophila.

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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.

EXPERIMENTAL PROCEDURES

Animals

Freshwater crayfish, Pacifastacus 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 oC and 800 g for 10 min, the plasma was removed from hemocytes and the plasma

were stored at -80 oC until further analysis. For purification of antibacterial proteins from plasma, the

frozen samples were thawed and TFA was added to a final concentration of 0.1 % TFA. After

incubation at 4 oC for 12 hrs, the sample was centrifuged at 16,000 g for 20 min and the supernatant was

diluted twice with 0.05 % TFA water and directly subjected to C-18 reverse-phase chromatography (φ

2.7 x 9 cm, Waters) equilibrated with 0.05 % TFA water. The sample was eluted with a step gradient of

20 %, 50 %, and 80 % acetonitrile containing 0.05 % TFA and then 50 % acetonitrile elution fraction

was applied to the second C-18 column using 0-50 % acetonitrile/ water/ 0.05 % TFA as a linear

gradient. For the next purification step, Mono-S cationic ion exchange chromatography was performed

using a FPLC system (Pharmacia). 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 (Pharmacia Smart chromatography system) on C-18 column using

acetonitrile/ water/ 0.05 % TFA gradients of 0-60 % acetonitrile in 60 min at a flow rate of 100 µl/min.

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Ultraviolet absorption was monitored at 280 nm, 254 nm, and 214 nm. The eluted peak fractions were

vacuum dried and used for assay of antibacterial activity and determination of amino acid sequences.

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-MS was performed in a Q-tof tandem mass spectrometer (Micromass,

Manchester UK) equipped with nanospray interphase. Interpretation of mass spectra was done by using

MassLynx (Micromass, Manchester UK) suite of software programs.

Peptide synthesis

An amidated 16 residues 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

et al.1986 (23). A low molecular mass calibration kit for electrophoresis (Amersham Pharmacia

Biotech) 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 α–lactalbumin (14.4 kDa), and aprotinin (6.5 kDa). A synthetic

peptide of astacidin 1 (1.9 kDa) was also used.

Assay of Antibacterial activity

During the purification procedure, the antimicrobial activities of samples were monitored by a

radial diffusion using Bacillus megaterium BM 11and Escherichia coli D21 as test organism as

described by Lehrer et al. 1991 (24). Briefly, 10 ml culture of bacterial cells in mid-logarithmic phase

was subjected to centrifugation at 900 x g for 5 min, washed with 10 mM sodium phosphate buffer, pH

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7.4 and then resuspended in 10 ml of the same buffer. One hundred µl of bacterial solution containing 1

x 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 hrs at 37 oC, 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 hr at 30-37 oC. 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., 1994 (25). Bacteria

grown in LB medium (peptone 10 g, yeast extract 5 g, NaCl 5 g, glucose 1 g/distilled water 1L) was

collected in the exponential phase of growth and resuspended with phosphate buffered saline, pH 6.0 at

a density of 1 x 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 hrs at

37 oC. 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’-[γ-32P] ATP-labeled mixed probe [AT(C/T) TTI ACI

AC(C/T) TGI CC(A/G) TG(C/T) TG(A/G) TT(C/T) TGI AC; I is inosine], which was designed

according to the following amino acid sequence of astacidin 1, VQNQHGQVVKI. For the initial

screening approximately 120,000 recombinants of crayfish hepatopancreas λgt 11 cDNA library was

used. The membranes were prehybridized at 65 oC for 1 hr in 5 x SSC (750 mM NaCl, 75 mM Na-

citrate, pH 7.0), 5 x Denhardt’s solution (100 x 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 oC for 12 hr in the same solution of prehybridization. After

the second screening, positive clones were amplified with PCR using a pair of nested λgt 11-specific

primers (GGA TTG GTG GCG ACG ACT and GCT TTA TGC CCG TCT GTA). PCR conditions were

94 oC for 45 sec, 55 oC for 30 sec, and 72 oC for 2 min carried out for 30 cycles. The largest PCR

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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. Then it was sequenced

with an Applied Biosystems PRISM dye terminator cycle sequencing ready reaction kit (Perkin-Elmer).

The cDNA sequence was analyzed with the MacVector 6.5.1. Software (Kodak). The nucleotide and the

deduced amino acid sequences were compared with the BLAST program (National Center

Biotechnology International, Bethesda, MD).

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 TFA at a final concentration of 0.1 % TFA. Plasma (14 mg/ml) treated with TFA 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 oC. As a control, plasma was treated with TFA 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% TFA 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 PMSF (1 mM, phenylmethylsulfonylfluorid). The different

proteinase inhibitors were separately incubated with plasma (14 mg/ml) in CAC buffer for 1hr at room

temperature and then treated with TFA. After incubation for 12 hrs at 4 oC, the procedures were

followed as described above.

Immunization of crayfish for production of astacidin 1

Ten crayfish were injected with 100 µg of LPS (Escherichia coli serotype 055;B5 from Sigma) or

laminarin (β-1-3-glucan from Sigma) dissolved in 100 µl of distilled water. After incubation of injected

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crayfish for 6 hrs in water at 16 oC, 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 hrs at 16 oC. Both plasma and hemocyanin were diluted twice with

CAC buffer and treated with TFA at a final concentration of 0.1 % and then further incubated for 12 hrs

at 4 oC. 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% TFA 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).

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 (BCA) assay (27).

The stock solution was diluted to 50-100 µg/ml in appropriate buffers. All the experiments were carried

out at 25 oC. 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 Yang's

method (28).

RESULTS

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 BM 11 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), since astacidin

1 could not be detected in 20 % SDS-PAGE (Fig. 6B).

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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

following sequence FKVQNQHGQVVKIFHH-COOH. The mass of astacidin 1 determined by

MALDI-TOF-MS was 1945.2 Da, and it is COOH-terminally 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. Any significant changes in CD

spectra were not observed under various temperature conditions. However, the CD spectra showed that

40-53.8 % of the molecule has a beta-sheet structure at pH 4, 6, and 8. Moreover, the addition of up to

80 % (v/v) acetonitrile to astacidin 1 results in approximately 27 % beta-sheet structure content (data

not shown). The predicted secondary structure of astacidin 1 based on CD data is shown Figure 1B.

Two putative beta-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.

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 N-terminally truncated peptides, designated SP-1 to 4

(Table 1). 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 Bacillus megaterium B11, Bacillus subtilis ATCC 6633, Micrococcus luteus

Ml 11, whereas the synthetic peptide had lower antibacterial activity towards gram-negative bacteria.

The N-terminal truncated peptides had much lower antibacterial activity than the complete synthetic 16

amino acid peptide (Table 2). This result indicates that the N-terminal amino acids contribute to the

antibacterial activity. The difference in antibacterial activity between native and synthetic astacidin 1

may be due to the solubility of the synthetic peptide since the synthetic peptide is less soluble than the

native peptide in water. Therefore, the synthetic peptide was dissolved in water containing 0.05% TFA.

In this solution with low pH, the secondary structure of the synthetic peptide is changed from a random

coil to a beta-sheet according to our CD data, which might have affected to the antibacterial activity

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against Gram-negaive bacteria. Contamination with other antibacterial peptides in the native astacidin 1

can be excluded, since the amino acid sequence and homogeneity of native astacidin 1 used for these

experiments were determined by MALDI-TOF-MS.

Cloning and nucleotide sequence analysis of astacidin 1

We obtained a positive clone from a crayfish hepatopancreas cDNA library. The amino acid

sequences of astacidin 1 was used to design and synthesize degenerate primers. Using 5’-[γ-32P] ATP-

labelling method, specific DNA fragments representing astacidin 1 was 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 Figure 2. The cDNA has an open reading frame of 1,980

nucleotides corresponding to 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 C-terminus of hemocyanin. The first 17 amino acid

residues of hemocyanin form a typical signal sequence. Therefore, hemocyanin of crayfish consists of

660 amino acid residues with a calculated molecular mass of the protein portion of 75,316 Da and an

estimated pI is 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

figure 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).

Comparisons of the deduced amino acid sequence of hemocyanin cDNA with shrimp hemocyanin

and crayfish proPO shows 58 % and 33 % identity, respectively (Fig. 3). It has also high similarity with

other hemocyanins such as hemocyanin alpha-subunit of Homarus americanus (AJ272095),

hemocyanin subunit 1 (AJ344361), 2 (AJ344362), and 3 (AJ344363) of Palinurus vulgaris.

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

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processing of astacidin 1 from hemocyanin was detectable after 12 hrs 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 proteinases 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 hrs

post-injection. The concentration of astacidin 1 was shown to be increased in plasma from animals

previously injected with LPS or glucan compared to control animals (Fig. 6A). If a purified

homogeneous hemocyanin was 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 crayfish with

LPS or glucan. Several immune reactions in invertebrate have previously been known to be initiated by

bacterial or fungal cell wall components such 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 paper shows that the C-terminal part of crayfish hemocyanin is processed by a cysteine-

like proteinases and this processing is up-regulated by LPS or glucan treatment to produce a

biologically active antimicrobial peptide astacidin 1.

DISCUSSION

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 and these peptides are amphipathic, carry a net positive charge and manifest a well-defined α-

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helical or β-sheet structure in membrane-like environments. Expression of antimicrobial peptides can be

constitutive, inducible or both and several reviews of this topic have appeared during the past 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).

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, Pacifastacus

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 beta-

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 residues peptide containing two cysteine

residues which form an internal disulfide bridge. This peptide has two beta-sheet stranded sheets (five

residues each), which are held together by a single disulfide bridge. Such antiparallel two-stranded beta-

sheet structure is also found in brevinins from frog (38), protegrins from porcine leukocytes (39), and

tachyplesins isolated from the horseshoes 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

N-terminal truncated synthetic peptides had less antibacterial activity than authentic astacidin 1, which

suggests that the N-terminal amino acids are important for antibacterial activity.

Many antimicrobial peptides are derived from larger precursors and the 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 precusor α-defensin is cleaved by metalloproteinase,

a matrilysin to produce α-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 are an efficient

way to synthesize different effector molecules and amplify the antibacterial response. Interestingly,

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astacidin 1 is released from the C-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 C-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 1and 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.

Hemocyanin is an interesting molecule, which 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 N-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 which 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 N-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 hemocyanins has a

function as 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 C-terminal part (Fig. 3).

Therefore, only crayfish hemocyanin can produce and release astacidin 1 and not crayfish

prophenoloxidase. In this paper, 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.

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FOOTNOTES

The nucleotide sequences(s) reported in this paper has been submitted to the GenBank/NCBI Data Bank

with accession number(S) AF522504.

1 The abbrevation used are: CD, circular dichroism; LPS, lipopolysaccharide; TFA, trifluoroacetic acid;

HPLC, high performance liquid chromatography; MALDI-TOF-MS, matrix-assisted laser

desorption/ionization time-of-flight mass spectrometry.

Acknowledgments-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.

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FIGURE LEGENDS

Figure 1. A, 20 % acid-urea PAGE of the purified astacidin 1. A low molecular size marker was used:

rabbit muscle phosphrylase 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 α–lactalbumin (14.4 kDa), aprotinin (6.5 kDa) and synthetic peptide of astacidin 1 (1.9 kDa). B,

The predicted secondary structure of astacidin 1 based on CD spectrum. The black lines indicate beta-

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 residues peptide); lane 3, SP-2 (15 amino acid residues peptide); lane 4, SP-

3 (14 amino acid residues peptide); lane 5, SP-4 (12 amino acid residues peptide); lane 6, purified

astacidin 1.

Figure 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 putative signal peptide is in italics. 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 with bold type amino acid sequences denote the sequences of astacidin 1

purified from crayfish plasma. A polyadenylation signal is shown with underline.

Figure 3. Alignment of crayfish P. leniusculus hemocyanin, crayfish P. leniusculus prophenoloxidase

(GenBank X83494) and shrimp hemocyanin (GenBank X82502). The shadowed boxes indicate that the

residues are identical. The dots indicate that the amino acids have similar properties. The underlined

amino acid sequence 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.

Figure 4. Processing of astacidin 1 from hemocyanin under acidic conditions. The plasma (14 mg/ml)

was prepared in anticoagulant buffer and was treated with TFA in a time-dependent manner. After SEP-

PAK chromatography, 50 µg of protein was subjected to 20 % acid-urea PAGE. The arrow shows

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produced astacidin 1. Lane 1, size marker; lane 2, 0 hr incubation after TFA treatment; lane 3, 12 hrs

incubation after TFA treatment; lane 4, 1 day incubation after TFA treatment; lane 5, 2 days incubation

after TFA treatment; lane 6, 3 days incubation after TFA treatment; lane 7, 4 days incubation after TFA

treatment; lane 8, 5 days incubation after TFA treatment.

Figure 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 PMSF (1 mM) were each incubated with plasma protein for 1 hr at room

temperature and then the samples were treated with TFA for 12 hrs at 4 oC. 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 hr incubation after TFA treatment without any proteinase inhibitor as a

negative control; lane 2, 12 hr incubation after TFA 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, PMSF; lane 10, size marker

Figure 6. The generation of astacidin 1 from hemocyanin in crayfish injected with LPS or glucan.

Plasma were collected from crayfish hemolymph 6 hrs post-injection and treated with TFA and then

incubated for 12 hrs at 4 oC. 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 hr incubation after TFA treatment as positive control; lane 3, plasma injected by LPS

and treated TFA, incubated for 12 hrs at 4 oC; lane 4, plasma injected by glucan and treated TFA,

incubated for 12 hrs at 4 oC; lane 5, 0 hr incubation after TFA treatment as negative control.

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F K V Q N Q H G Q V V K I F H H

kDa43.020.114.4

6.5

30.0

A

B

CkDa43.020.114.4

6.5

30.0

1 2 3 4 5 6

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CAGTATCGGCGGAATTCCGAAATGCAGTGGACAGTGCTTGTAGCAGCGCTCCTGGTGGCCACTGTGTCCGCTGACACAGACGTG 84 M Q W T V L V A A L L V A T V S A D T D V 21GCCCACCAGCAGCAGGCTATCAACCGTCTGCTCTACAAGGTCACCAGCCACATCAAGTCAAGTTTCACTGACCTGAAGGAGGCA 168 A H Q Q Q A I N R L L Y K V T S H I K S S F T D L K E A 49GCAGAAACATGGAACCCAAGAGACCACACTGACAAGAGCAGCGATGGAGGCGCAGCCATAAAGCACCTTATGGATGAGCTGGAC 252 A E T W N P R D H T D K S S D G G A A I K H L M D E L D 77GACCACCGCTTGCTCGAGCAGCACCACTGGTTCTCCCTCTTCAACGACCGTCAGCGTGAAGAAGCCCTATTACTTGTCGATGTC 336 D H R L L E Q H H W F S L F N D R Q R E E A L L L V D V 105CTGCTGCACAGCACAGACCTTGAAGCCTTCAAGAACAATGCCGCTTACTTCCGTGAGCATACGAATGAAGGAGAGTTTGTTTAT 420 L L H S T D L E A F K N N A A Y F R E H T N E G E F V Y 133GCTCTGTATGTGGCTGTGACCCACTCTGACCTAACACCACATGTGGTCTTGCCACCTCTCTACGAAGTCACCCCTCATTTGTTC 504 A L Y V A V T H S D L T P H V V L P P L Y E V T P H L F 161ACAAACTCAGAGGTCATCGACCAAGCTTATGCTGCTAAGATGACACAAACACCAGGCAACTTTAAGATGGAATTTACTGGTAGC 588 T N S E V I D Q A Y A A K M T Q T P G N F K M E F T G S 189AAGAAAAACCGTGAACAGCGGGGGGCCTACTTCGGTGAGGATGTTGGCCTCAACTCTCACCACGTCCACTGGCATATGGATTTC 672 K K N R E Q R G A Y F G E D V G L N S H H V H W H M D F 217CCCTTCTGGTGGAATGGAGCTAAGATCGACCGCAAGGGAGAACTCTTCTTCTGGGCTCATCATCAGCTAACTGCCAGATACGAC 756 P F W W N G A K I D R K G E L F F W A H H Q L T A R Y D 245GCTGAGCGCCTTTCTAACTTCCTGCCTGCAGTAGACGAACTGTACTGGGACCGCCCCATTAAAGACGGTTTTGCTCCCCACACA 840 A E R L S N F L P A V D E L Y W D R P I K D G F A P H T 273ACTTACAAATATGGTGGAGAGTTCCCCGCTCGTCCCGACAACAAGGAATTTGAAGATGTTGACGGCGTGGCCCGTATTCGTGAC 924 T Y K Y G G E F P A R P D N K E F E D V D G V A R I R D 301TTGAAGGAGCTGGAAAGTCGAATCCGTGATGCCGTTGCTCACGGGTACATCATCAACGCCGATGGCACAAAAACTGACATCAAC 1008 L K E L E S R I R D A V A H G Y I I N A D G T K T D I N 329AACGAACATGGTATTGATATCTTGGGTGACATCATCGAGTCTTCCACCTATAGCACCAATGCTGCTTACTATGGCGCTCTACAT 1092 N E H G I D I L G D I I E S S T Y S T N A A Y Y G A L H 357AACCAGGCTCACCGCGTCTTGGGTGCCCAGTCTGATCCCAAACACAAGTTCAACATGCCTCCAGGAGTCATGGAACACTTCGAG 1172 N Q A H R V L G A Q S D P K H K F N M P P G V M E H F E 385ACTGCGACCCGCGACCCCGCATTCTTCCGACTCCACAAATACATGGATGGTATCTTCAAGGAACACAAGGACAACCTTCCACCT 1260 T A T R D P A F F R L H K Y M D G I F K E H K D N L P P 413TATACCGAGGAAGATTTACTCTACAGCAATGTCAAAATCACTGGTGTTGATGTCACTGAACTTTCTACTTTCTTCGAAGATTTT 1344 Y T E E D L L Y S N V K I T G V D V T E L S T F F E D F 441GAGTTTGACCTGAGTAATGCTCTTGACACAACAGAGAACGTCAATGAAGTTTCCGTCAAAACCCACATATCTCGCCTGAACCAC 1428 E F D L S N A L D T T E N V N E V S V K T H I S R L N H 469AAGCCATTCTCCCTCAACATTCACGCCCACGCCGAGCACGACGACAAAGTCACCGTTCGCGTCTACATAAGCGCCAAGCACGAT 1512 K P F S L N I H A H A E H D D K V T V R V Y I S A K H D 497GAGAACCACATCGCCCTCGACATCGACGAGTCTCGCTGGGGAGCCATCTTGCTCGACACCTTCTGGACTGAAGTACATGCGGGA 1596 E N H I A L D I D E S R W G A I L L D T F W T E V H A G 525GACAACGAAATCAAACGTAAGTCCTCCGAGTCGTCAGTGGCCATCCCTGATCGGGTGTCCTTCCCTCAACTGATCCACGACGCT 1680 D N E I K R K S S E S S V A I P D R V S F P Q L I H D A 553GACGAAGCTGTGGCCAATGGCGCCGAGCTGCCCCACAAGGAGAGTCGCAGCTGTGGTCACCCCCAGAGGCTGCTGCTGCCCAAG 1764 D E A V A N G A E L P H K E S R S C G H P Q R L L L P K 581GGCAAGGAACAAGGCATGGATTTCTGGCTGGACATTATCATTACCAGCGGAGATGATGCTGTCCAGGATGACTTGACTGTTAAC 1848 G K E Q G M D F W L D I I I T S G D D A V Q D D L T V N 609GCCCACGGCAGCACCCATGGTTACTGCGGTATTCACGGAGAGAAGTACCCTGACAAACGTCCTATGGGCTTCCCCTTCGACCGT 1932 A H G S T H G Y C G I H G E K Y P D K R P M G F P F D R 637 CCCATTCCTGACCTCCGAGTCTTCAAGGTGCAAAACCAGCATGGCCAGGTCGTCAAGATTTTCCATCACTAATCAACTTTCTTT 2016 P I P D L R V F K V Q N Q H G Q V V K I F H H * 661GGATTCTCGAATTCTTTATGACAAATAATTCCTGTTGTGATCATTATGTTCCCTGAAACGACACGATATAACAATGCATGTAAA 2100CCAACATTAAAGATGGAGCAATAAAAAAAAAAAA 2134

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kDa

20.114.4

6.5

Syntheticastacidin 1

1 2 3 4 5 6 7 8

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20.114.4

6.5

Syntheticastacidin 1

1 2 3 4 5 6 7 8 9 10

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kDa20.114.4

6.5

Syntheticastacidin 1

1 2 3 4 5kDa

20.114.4

1 2 3 4 5

30.043.0

A B

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Table 1. Primary structure of synthetic peptide of astacidin 1

Peptide Amino acid sequence

SP-1 (1-16) FKVQNQHGQVVKIFHH-COOH

SP-2 (2-16) KVQNQHGQVVKIFHH-COOH

SP-3 (3-16) VQNQHGQVVKIFHH-COOH

SP-4 (5-16) NQHGQVVKIFHH-COOH

Table 2. Minimal inhibition concentration of Astacidin 1 and synthetic peptides

Organism Astacidin 1 SP-1 (16) SP-2 (15) SP-3 (14) SP-4 (12)

Shigella flexneri ATCC 203 15 µM 205 µM 0.8 mM 4.8 mM 6mM

Proteus vulgaris OX19 ATCC 6380 >20 µM >1mM >6mM >6mM >6mM

Escherichia coli D21 15 µM 410 µM 3.2 mM 6mM 3.2 mM

Psedomonas aeruginosa OT 97 >20 µM 617 µM >6mM >6mM >6mM

Bacillus megateriun B11 1.9 µM 1.95 µM 1.6 mM 4.8mM 0.8 mM

Bacillus subtilis ATCC 6633 15 µM 20 µM 0.8 mM 6mM 3.2mM

Staphylococcus aureus Cowan 1 >20 µM >1mM >6mM >6mM >6mM

Staphylococcus aureus JC-1 >20 µM 94 µM 0.8mM 3.2mM 1.6mM

Micrococcus luteus Ml 11 12.8 µM 23 µM N.A N.A N.A

N.A ; not assayed.

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So Young Lee, Bok Luel Lee and Kenneth SöderhällPacifastacus leniusculus

Processing of an antibacterial peptide from hemocyanin of the freshwater crayfish

published online December 18, 2002J. Biol. Chem. 

  10.1074/jbc.M209239200Access the most updated version of this article at doi:

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