Purification and characterization of three isoforms of chrysophsin,
a novel antimicrobial peptide in the gills of the red sea bream,
Chrysophrys major
Noriaki Iijima
1
, Norio Tanimoto
1
, Yohko Emoto
1
, Yohko Morita
1
, Kazumasa Uematsu
2
,
Tomoya Murakami
3
and Toshihiro Nakai
4
1
Laboratory of Molecular Cell Biology,
2
Laboratory of Fish Physiology and
4
Laboratory of Fish Pathology, Graduate School of
Biosphere Science, Hiroshima University, Japan;
3
Hiroshima Fisheries Experimental Station, Ondo, Aki, Japan
We report here the isolation of three isoforms of a novel
C-terminally amidated peptide from the gills of red sea
bream, Chrysophrys (Pagrus) major. Peptide sequences
were determined by a combination of Edman degrada-

antibacterial substances are thought to exist at these sites to
prevent penetration of bacteria into the circulatory system.
In fact, skin mucus, eggs and serum of fish contain a variety
of nonspecific defense substances, such as lysozyme, com-
plement, C-reactive protein, transferrin, lectin, and antimi-
crobial proteins [5–10]. Furthermore, antimicrobial peptides
have been purified from fish skin mucus: pardaxin from the
moses sole fish Pardachirus marmoratus [11], pleurocidin
from the winter flounder Pleuronectes americanus [12] and
parasin I from the catfish Parasilurus asotus [13], and the
gene expression of pleurocidin-like antimicrobial peptides
found in the skin and intestine of the winter flounder [14].
The antimicrobial peptide, misgurin, has also been purified
from the whole body of the loach Misgurnus anguillicau-
datus [15]. These antibacterial peptides show potent
antimicrobial activity against Gram-negative and Gram-
positive bacteria and act as nonspecific defense substances in
fish skin.
Fish gills are constantly being flushed with water that
may contain fish pathogens, but are covered with only a
thin layer of protective mucus and are constructed of only
a single layer of fragile cells that separate the vascular
system from the external environment. Thus, they are a
very important site of pathogen penetration. Therefore,
potent antimicrobial peptides can be expected to be found
in fish gills to prevent such penetration. However, there is
a paucity of information on nonspecific defense systems in
the gills. An antimicrobial peptide has been identified in
the gills of only one fish species, hybrid striped bass
(Morone saxatilis · M. chrysops) [16–18]. In this study, we

accepted 9 December 2002)
Eur. J. Biochem. 270, 675–686 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03419.x
Materials and methods
Chemicals
Fmoc-
L
-amino acids, Fmoc-
L
-amino acid resins and
TentaGelÒ S (TGS)-RAM were purchased from Shimadzu
(Kyoto, Japan). Chemicals for peptide synthesis, trifluoro-
acetic acid, ethylmethylsulfide, ethanedithiol, thiophenol,
2-methylindole, thioanisole, phenol, and anisole were
obtained from commercial sources and were of the highest
purity available. Lysyl endopeptidase, a silver staining II kit
and 2,2,2-trifluoroethanol were purchased from Wako Pure
Chemicals (Tokyo, Japan). A TSKgel G2000SW column
was obtained from Tosoh (Tokyo, Japan), an Inertsil C8-3
column from GL Science (Tokyo, Japan), and a Capcellpak
C18 column from Shiseido (Tokyo, Japan). SP-Sephadex
C-25 was purchased from Pharmacia Biotech (Uppsala,
Sweden), and PolySulfoethyl Aspartamide column was
from Poly LC Inc. (Columbia, MD, USA). Melittin and
BSA were obtained from ICN Biomedicals Inc. (Aurora,
OH, USA), Imject maleimide activated mariculture keyhole
limpet hemocyanin (mcKLH) and Imject Alum were from
Pierce (Rockford, IL, USA), Simple Stain MAX-PO
(Multi) and Simple Stain DAB solution were from Nichirei
(Tokyo, Japan), peroxidase-labeled affinity-purified anti-
body to rabbit IgG (H + L) was from KPL (Gaithersburg,

(219 mL) was used as the acid extract and was applied to
a Sep-Pak C18 cartridge (Waters, Milford, MA, USA).
After a wash with 0.1% trifluoroacetic acid, the peptide
was eluted with 80% acetonitrile/0.1% trifluoroacetic
acid. The dried eluate was dissolved in 1
M
acetic acid
and then adsorbed on SP-Sephadex C-25 resin. Successive
elution with 1
M
acetic acid, 2
M
pyridine and 2
M
pyridine/acetic acid (pH 5.0) afforded three respective
fractions of SP-1, SP-2 and SP-3. The SP-3 fraction was
further lyophilized and dissolved in 40% acetonitrile
containing 0.1% trifluoroacetic acid. An aliquot of the
solution was loaded on a TSKgel G2000SW column
(7.6 · 600 mm) equipped with a Tosoh HPLC pump
(CCPM) and a UV-VIS detector (UV-8010) and was
eluted with 40% acetonitrile containing 0.1% trifluoro-
acetic acid. The SP-3 fraction was repeatedly injected, and
fraction A, estimated to be less than 5 kDa, was pooled
and subjected to RP-HPLC.
The on-line HPLC separation was performed on a
Hewlett-Packard HP1100 series HPLC system equipped
with an auto sampler, thermostatically controlled column
compartment, UV-VIS detector, and degasser. Solvent A
was 5% acetonitrile containing 0.1% trifluoroacetic acid,

/H
3
PO
4
(pH 3.0)
containing 25% acetonitrile, and further loaded on a
PolySulfoethyl Aspartamide column (4.6 · 200 mm)
equipped with a Tosoh HPLC pump. The column was
eluted with a linear gradient of KCl. The gradient was
0–3 min 0–0.1
M
KCl, 3–43 min 0.1–0.4
M
KCl, and
43–48 min 0.4–1
M
KCl.
Mass spectrometry
MS analysis of peptides was performed with a Finnigan
LCQ ion trap mass spectrometer (ThermoQuest, San Jose,
CA, USA) equipped with an electrospray ionization source
(ESI-ITMS) as described previously [19]. The mass scale
was calibrated using Ultra-mark provided by the manufac-
turer. Ions were detected and analyzed in the positive mode
on the basis of their m/z ratio.
Solid-phase peptide synthesis
The protected peptide chain was assembled with a
Shimadzu peptide synthesizer (PSSM8) according to
standard Fmoc chemistry [20]. Non-amidated peptides
were prepared with Fmoc-

Aliquots of peptides purified by RP-HPLC were charac-
terized with ESI-ITMS as described below.
Lysyl endopeptidase digestion
The three purified peptides, P-1 (2.9 lg), P-2 (0.6 lg) and
P-3 (0.46 lg), dissolved in 20 lL20m
M
Tris/HCl (pH 8.0)
were mixed with lysyl endopeptidase and incubated for
2–4 h at 37 °C. The ratio of enzyme to peptide was 1 : 100
for P-1 and 1 : 5 for P-2 and P-3. After digestion, the
reaction was stopped by adding 0.1% trifluoroacetic acid at
a final volume of 0.1 mL.
Amino-acid sequence analysis
The amino-acid sequence of the three purified peptides, P-1,
P-2 and P-3 (1.5–2.9 lg), was determined on a Hewlett–
Packard G1005A protein sequencing system (Palo Alto,
CA, USA) by analyzing the data calibrated with 10 pmol
phenylthiohydantoin amino-acid standards.
Tricine/SDS/PAGE
The molecular mass of the sample was estimated by Tricine/
SDS/PAGE using a 16.5% separating gel, 10% spacer gel
and 4% stacking gel in the presence of 2-mercaptoethanol
[23], and the protein/peptide bands were stained with a silver
staining II kit from Wako.
Determination of peptide concentration
The concentrations of the samples from purification steps
were measured with the DC-protein assay kit using BSA as
standard. The concentration of purified and synthetic
chrysophsin-1 and chrysophsin-2 was obtained from the
A

%helix ¼À100ðH
222
þ 3000Þ=33000
where Q
222
is the CD at 222 nm.
Bactericidal assay
Bactericidal activity was routinely tested using Bacillus
subtilis ATCC6633 or Escherichia coli WP-2. After growth
in tryptic soy broth at 37 °C to exponential phase, bacteria
were washed twice with 0.85% NaCl and diluted in 50 m
M
Hepes/NaOH buffer (pH 7.4) to give % 2 · 10
5
colony-
forming unitsÆmL
)1
(CFUÆmL
)1
). Aliquots (25–100 lL) of
the fractions at each purification step from the acid extract
of the gills were lyophilized and dissolved in 0.1 mL 1 m
M
citric acid/sodium citrate buffer (pH 4.0). Solutions were
mixed with an equal volume of bacterial suspension and
incubated at 37 °C for 60 min. In a control experiment, the
cells were incubated with the same solvent as used for the
preparation of each fraction. After appropriate dilution of
the mixture with 50 m
M

.
The bacterial suspensions (each 0.1 mL) were incubated at
20–25 °C for 1 h with equal volumes of twofold serial
dilutions of the three synthetic chrysophsins in 1 m
M
citric
acid/sodium citrate buffer (pH 4.0) containing 2% NaCl.
After 100-fold dilution of the mixture, 0.1 mL aliquots were
spread on tryptic soy agar plates and incubated at 20–25 °C
for 18–24 h. Then, MLC was determined as described above.
Hemolytic assay
Before use, freshly collected human blood was washed with
50 m
M
phosphate buffer (pH 7.4) containing 0.14
M
NaCl
(NaCl/P
i
) until the supernatant was colorless. A suspension
was made of 1% packed cells in NaCl/P
i
containing 2%
glucose. Synthetic chrysophsin 1, 2 or 3, melittin or synthetic
magainin 2 was dissolved in 50% dimethyl sulfoxide at a
concentration of 1 m
M
and was serially diluted with NaCl/
P
i

i
by subcutaneous injection. At 4 days after the
final injection, blood was collected, and the resulting
antiserum was stored at )80 °C.Thetiteroftheantisera
was determined by ELISA using chrysophsin-1-Cys as an
antigen and peroxidase-labeled affinity-purified antibody to
rabbit IgG (H + L) as a secondary antibody.
Chrysophsin-1-Toyopearl was used for the purification of
anti-(chrysophsin-1) IgG. Chrysophsin-1-Cys (1 mg) was
coupled to Toyoperal AF-Epoxy-650M (1 g) according to
the manufacturer’s protocol. Anti-(chrysophsin-1) serum
(3 mL) was filtered through a 0.45-lm filter (Dismic-13,
Advantec, Tokyo, Japan) and applied to the chrysophsin-
1-Toyopearl column. The column was washed with 20 m
M
Tris/HCl (pH 7.4) containing 150 m
M
NaCl (Tris/NaCl)
and 20 m
M
Tris/HCl (pH 7.5) containing 1
M
NaCl and
1% Triton X-100, respectively, and specific antibody was
eluted from the column with 0.1
M
glycine/HCl (pH 2.5).
The eluted antibody was immediately neutralized with 1
M
Trisandstoredat)80 °C.

NaN
3
overnight at room temperature, and
washed 3 times with NaCl/P
i
. The sections were incubated
with the secondary antibody for 30 min at room tempera-
ture and washed with NaCl/P
i
. The color was developed in a
Simple Stain DAB solution. To determine the type of
immunoreactive cells in the gills, neighboring 5-lmserial
sections of the gills were stained with chrysophsin-1-specific
antibody or hematoxylin and eosin. As a negative control,
chrysophsin-1-specific antibody preabsorbed with chryso-
phsin-1 (mol ratio of antibody to chrysophsin-1 ¼
1 : 5000) was used as a primary antibody.
Results
Peptide purification and primary structure
After fractionation of the acid-extracted gill powder on SP-
sephadex C-25, fraction SP-3 showed the most bactericidal
activity (MLC 1.2 lgÆmL
)1
) and was further separated by
gel-filtration HPLC (Fig. 2). Bactericidal activities against
B. subtilis were detected in almost all of the fractions eluted
from the column. The molecular mass of fractions eluted
between 48 and 81min, showing high antibacterial activity,
was determined by Tricine/SDS/PAGE. As this study
focuses exclusively on bactericidal peptides of less than

molecular mass of His (Table 1). On replacing the unknown
amino acids of fragment 3 with His, the theoretical average
molecular mass (1365.62 Da) of fragment 3 calculated from
the amino-acid sequence was almost the same as that of
fragment 3 deduced by ESI-ITMS (1364.8 Da). Finally,
amino-acid sequences of P1, P2 and P3 were determined as
follows. P1, FFGWLIKGAIHAGKAIHGLIHRRRH;
P2, FFGWLIRGAIHAGKAIHGLIHRRRH; P3, FIG
LLISAGKAIHDLIRRRH. The theoretical average
molecular masses of P1 (2892.43 Da), P2 (2920.45 Da)
and P3 (2286.76 Da) matched the deduced average mole-
cular masses with a difference of 1 Da. This discrepancy
(1 Da) suggests amidation of the C-terminal histidine.
Therefore, elution profiles of synthetic nonamidated pep-
tides named P1-COOH, P2-COOH and P3-COOH, and
synthetic amidated peptides named P1-CONH
2
,
P2-CONH
2
and P3-CONH
2
were superimposed on
those of native P1, P2 and P3 by HPLC. Native P1 and
P3 were recognized as single peaks by RP-HPLC (data not
shown) and also by anion-exchange HPLC (Fig. 4A,C).
P1-COOH and P1-CONH
2
could not be separated by
RP-HPLC; however, they were clearly separated into

by RP-HPLC (data not shown). However, native P2
separated into two peaks, P2-1 and P2-2 (Fig. 4B), and
P2-2 was coeluted with P2-CONH
2
on anion-exchange
HPLC (Fig. 5C,D). P2-1 and P2-2 were separated by anion-
exchange HPLC and further analyzed by RP-HPLC
followed by ESI-ITMS. As the mean molecular mass of
P2-2 (2919.3 ± 0.4 Da) was identical with that of P2-
CONH
2
, P2-2 was also an amidated peptide. Two peptides,
2925 ± 0.7 Da and 2907 ± 0.3 Da, were detected in the
P2-1 fraction. From these results, the primary structures of
three novel bactericidal peptides, P1, P2-2 and P3, were
determined, and we decided to call them chrysophsin-1,
chrysophsin-2 and chrysophsin-3 based on the genus of red
sea bream, C. major. An alignment of these three peptides
with other antimicrobial peptides is shown in Fig. 6.
Chrysophsin-1, chrysophsin-2 and chrysophsin-3 are
C-terminally amidated, 25, 25 and 20 amino acids in length,
and rich in cationic residues (9/25, 9/25 and 6/20, respect-
ively). Chrysophsin-2 corresponds to the chrysophsin-1
isoform differing by a single residue at position 7 (lysine or
arginine). Interestingly, the characteristic C-terminal cat-
ionic tetrapeptide, RRRH, is conserved in chrysophsins, in
addition to the C-terminal amidation.
Secondary structure of chrysophsins
Schiffer–Edmunson helical wheel modeling was used to
predict hydrophobic and hydrophilic regions within the

Table 1. Mass measurement of peptide fragments of P1, P2 and P3.
Partial sequence
ESI-ITMS
M
r
Peptide fragment
ESI-ITMS
M
r
Calc.
M
r
His (·)
PI FFGWLIKGAI
XAGKAIXGLIXRRRX 2891.2 FFGWLIK 909.6 910.10
GAI
XAGK 652.5 515.61 137.1
AI
XGLIXRRRX 1364.8 954.20 3 ·137.1
P2 FFGWLIRGAI
XAGKAIXGLIXRRRX 2919.3 FFGWLIRGAIXAGK 1573.0 1435.71 137.1
AIXGLI
XRRRX 1364.8 954.20 3 ·137.1
P3 FIGLLISAGKAIXDLIRRRX 2285.7 FIGLLISAGK 1017.8 1018.26 –
AIXDLIRRR
X 1285.3 1012.24 2 · 137.1
Fig. 4. Cation-exchange HPLC of P1, P2 and P3. Aliquots of P1 (A),
P2 (B) and P3 (C) were loaded on a PolySulfoethyl Aspartamide col-
umn and eluted with a linear gradient of KCl in 5 m
M

Chrysophsins were hemolytic for human red blood cells,
but they were less hemolytic than melittin and more
hemolytic than magainin (Fig. 10).
Fig. 5. Cation-exchange HPLC of native P1, P2-2 and P3, and their synthetic peptides. Mixtures of synthetic P1-COOH and P1-CONH
2
(A), native
P1 and P1-CONH
2
(B), P2-COOH and P2-CONH
2
(C), native P2 and P2-CONH
2
(D), P3-COOH and P3-CONH
2
(E), and native P3 and
P3-CONH
2
(F) were loaded on a PolySulfoethyl Aspartamide column and eluted with a linear gradient of KCl in 5 m
M
KH
2
PO
4
/H
3
PO
4
(pH 3.0)
containing 25% acetonitrile at a flow rate of 0.8 mLÆmin
)1

cationic peptides without cysteine (Fig. 6). Searches of
sequence databases show 70% identity between chryso-
phsin-1 and the mature peptide sequence predicted from
the nucleotide sequence of winter flounder pleurocidin-like
genomic clone (WF3), which has not yet been purified as
a mature peptide [14]. However, chrysophsin-1 shows low
identity (24–36%) with other fish antimicrobial peptides,
such as piscidins [16], moronecidins [17] and pleurocidin
[12,30]. The C-terminal amino acid was amidated in all
three chrysophsins, similarly to those in marine animals,
such as solitary ascidians (styelin D) [31] and hybrid
striped bass (Morone saxatilis x M. chrysops) (moroneci-
din/piscidin) [16,17]. It has been proposed that amphi-
pathic a-helical peptides show antimicrobial activity by
interacting electrostatically with the anionic bacterial
membrane, adopting an amphipathic a-helical conforma-
tion that allows them to insert the hydrophobic face into
the lipid bilayers and form a pore [1,8,22]. The amphi-
pathic a-helical structure of chrysophsins was predicted by
the Schiffer–Edmundson wheel analysis (Fig. 7). All three
chrysophsins form an a-helical structure (> 80% helical
content) in the structure-forming solvent, trifluoroethanol,
but not in phosphate buffer (Fig. 8). This suggests that
random-coiled chrysophsins in the water environment will
form amphipathic a-helical conformation after contacting
with the bacterial membrane. Thus, chrysophsins will
show antimicrobial activity in a similar way to other
previously studied amphipathic a-helical antimicrobial
peptides. Interestingly, the identity in amino-acid sequence
between chrysophsin and misgurin is low (16%), but

0.625
M
NaCl [12]. On the other hand, hybrid striped
bass wb-moronecidin retained bacteriostatic activity
against Staphylococcus aureus even in the presence of
1.28
M
NaCl; however, it remains unclear whether
wb-moronecidin is bactericidal or not in 1.28
M
NaCl
[17]. The net charge of chrysophsin-1 (pI 12.64) and
chrysophsin-2 (pI 12.79) is slightly higher than that of
wb-moronecidin (piscidin 2) (pI 12.60). In addition, the
C-terminal amino acids of chrysophsins and wb-moro-
necidin were both amidated. These findings may indicate
that the difference in salt tolerance between chrysophsins
and wb-moronecidin is partly due to the difference in
bacteria, Gram-negative E. coli and Gram-positive
S. aureus used in the experiment. Antimicrobial peptides
must pass the lipopolysaccharide-rich external leaflet of
the outer membrane to interact with the inner membrane
of Gram-negative bacteria such as E. coli; however, they
can directly interact with the anionic cytoplasmic mem-
brane of Gram-positive bacteria such as S. aureus.Itis
necessary to compare the bacteriostatic and bactericidal
activities of chrysophsins against Gram-positive bacteria,
Fig. 8. CD spectrum for synthetic chrysophsins. The spectra of syn-
thetic chrysophsin-1 (A), chrysophsin-2 (B) and chrysophsin-3 (C)
were obtained in 20 m

Vibriopenaeicida KHA 10 5 10
Vibrio harveyi HUFP911 1 (ATCC 14126) 2.5 5 5
Vibrio vulnificus ET-7617(ATCC 33148) 5 2.5 10
Aeromonas hydrophila ET-4 R R R
Aeromonas salmonicida NCMB 1102 10 5 10
Pseudomonas putida ATCC 12633 40 R 40
Edwardsiella tarda ET-82016 R R R
Table 2. Bactericidal activity (minimal lethal concentration) of native and synthetic chrysophsins compared with that of magainin-2. Minimal lethal
concentrations of peptide are given in l
M
and are the concentration of substance required necessary to kill % 99% of the bacteria.
Chrysophsin-1 Chrysophsin-2 Chrysophsin-3
Strains of bacteria Native Synthetic Native Synthetic Native Synthetic Magainin2
Bacillus subtilis ATCC 6633 0.25 0.125 0.25 0.25 0.25 0.25 0.985
Escherichia coli WT-2 0.25 0.25 0.25 0.25 0.25 0.25 0.985
Fig. 9. Effect of NaCl on bactericidal activity of chrysophsins. Bacte-
ricidal activity of synthetic chrysophsin-1 (s), chrysophsin-2 (h)and
chrysophsin-3 (n)at0.5l
M
was determined with various concentra-
tions of NaCl ranging from 0 to 1.28
M
.
Fig. 10. Hemolytic activity of chrysophsins. Synthetic chrysophsin-1
(s), chrysophsin-2 (h) and chrysophsin-3 (n), synthetic magainin 2
(j), an antimicrobial peptide from the aquatic frog Xenopus laevis and
melittin (d), a peptide from bee venom cytotoxic to human erythro-
cytes, were incubated with a 1% suspension of washed human eryth-
rocytes for 30 min at 37 °C.
684 N. Iijima et al.(Eur. J. Biochem. 270) Ó FEBS 2003

It remains unclear which chrysophsin isoforms exist in
certain cells of secondary lamellae and EGC-like cells of the
primary lamellae of red sea bream gills. We are currently
trying to obtain cDNAs encoding chrysophsins from the
gills of red sea bream, and we aim to investigate the gene
expression of the chrysophsin isoforms in these cells by
in situ hybridization.
Acknowledgements
We thank Dr S. Ohta (Instrument Center for Chemical Analysis,
Hiroshima University) and Professor K. Gekko (Graduate School of
Science, Hiroshima University) for the CD analysis. This work was
supported in part by a grant-in-aid from the Ministry of Education,
Science, Sports, and Culture of Japan.
References
1. Zasloff, M. (2002) Antimicrobial peptides of multicellular organ-
isms. Nature (London) 415, 389–395.
2. Hancock, R.E. & Scott, M.G. (2000) The role of antimicrobial
peptides in animal defenses. Proc. Natl. Acad. Sci. USA 97, 8856–
8861.
3. Hancock, R.E. & Diamond, G. (2000) The role of cationic anti-
microbial peptides in innate host defences. Trends Microbiol. 8,
402–410.
4. Evelyn, T.P.T. (1996) Infection and disease. In The Fish Immune
System: Organism, Pathogen, and Environment (Iwama, G. &
Nakanishi, T., eds), pp. 339–366. Academic Press, Inc, San Diego.
5. Yano, T. (1996) The nonspecific immune system: humoral defense.
In The Fish Immune System: Organism, Pathogen, and Environ-
ment (Iwama, G. & Nakanishi, T., eds), pp. 105–157. Academic
Press, Inc, San diego.
6.Lemaitre,C.,Orange,N.,Saglio,P.,saint,N.,Gagnon,J.&

from moses sole fish Pardachirus marmoratus. Eur. J. Biochem.
237, 303–310.
12. Cole, A.M., Weis, P. & Diamond, G. (1997) Isolation and char-
acterization of pleurocidin, an antimicrobial peptide in the skin
secretions of winter flounder. J. Biol. Chem. 272, 12008–12013.
13. Park, I.Y., Park, C.B., Kim, M.S. & Kim, S.C. (1998) Parasin I, an
antimicrobial peptide derived from histone H2A in the catfish,
Parasilurus asotus. FEBS Lett. 437, 258–262.
14. Douglas,S.E.,Gallant,J.W.,Gong,Z.&Hew,C.(2001)Cloning
and developmental expression of a family of pleurocidin-like
antimicrobial peptides from winter flounder, Pleuronectes ameri-
canus (Walbaum). Dev. Comp. Immunol. 25, 137–147.
15. Park, C.B., Lee, J.H., Park, I.Y., Kim, M.S. & Kim, S.C. (1997) A
novel antimicrobial peptide from the loach, Misgurnus anguilli-
caudatus. FEBS Lett. 411, 173–178.
16. Silphaduang, U. & Noga, E.G. (2001) Peptide antibiotics in mast
cells of fish. Nature (London) 414, 268–269.
17. Lauth, X., Shike, H., Burns, J.C., Westerman, M.E., Ostland,
V.E.,Carlberg,J.M.,VanOlst,J.C.,Nizet,V.,Taylor,S.W.,
Shimizu, C. & Bulet, P. (2002) Discovery and characterization of
two isoforms of moronecidin, a novel antimicrobial peptide from
hybrid striped bass. J.Biol.Chem.277, 5030–5039.
18. Shike,H.,Lauth,X.,Westerman,M.E.,Ostland,V.E.,Carlberg,
J.M., Van Olst, J.C., Shimizu, C., Bulet, P. & Burns, J.C. (2002)
Bass hepcidin is a novel antimicrobial peptide induced by bacterial
challenge. Eur. J. Biochem. 269, 2232–2237.
19. Zeng, R., Xu, Q., Shao, X X., Wang, K Y. & Xia, Q C. (1999)
Characterization and analysis of a novel glycoprotein from snake
venom using liquid chromatography-electrospray mass spectro-
metry and edman degradation. Eur. J. Biochem. 266, 352–358.

neutrophils. Arch. Biochem. Biophys. 328, 219–226.
28. Amiche, M., Seon, A., Wroblewski, H. & Nicolas, P. (2000) Iso-
lation of dermatoxin from frog skin, an antibacterial peptide
encoded by a novel member of the dermaseptin gene family. Eur.
J. Biochem. 267, 4583–4592.
29. Uchiyama, S., Fujikawa, Y., Uematsu, K., Matsuda, H., Aida, S.
& Iijima, N. (2002) Localization of group IB phospholipase A
2
isoform in the gills of the red sea bream, Pagrus (Chrysophrys)
major. Comp. Biochem. Physiol. 132B, 671–683.
30. Cole, A.M., Darouiche, R.O., Legarda, D., Connell, N. &
Diamond, G. (2000) Characterization of a fish antimicrobial
peptide: gene expression, subcellular localization, and spectrum of
activity. Antimicrob. Agents Chemother. 44, 2039–2045.
31. Taylor, S.W., Craig, A.G., Fischer, W.H., Park, M. & Lehrer,
R.I. (2000) Styelin D, an extensively modified antimicrobial
peptide from ascidian hemocytes. J. Biol. Chem. 275,
38417–38426.
32. Vogel, H. & Jahnig, F. (1986) The structure of melittin in mem-
branes. Biophys. J. 50, 573–582.
33. Perez-Paya, E., Dufourcq, J., Braco, L. & Abad, C. (1997)
Structural characterisation of the natural membrane-bound state
of melittin: a fluorescence study of a dansylated analogue. Bio-
chim. Biophys. Acta 1329, 223–236.
34. Ladokhin, A.S. & White, S.T. (1999) Folding amphipathic
a-helices on membranes: energetics of helix formation by melittin.
J. Mol. Biol. 285, 1363–1369.
35. Reite, O.B. (1997) Mast cells/eosinophilic granule cells of salmo-
nids: staining properties and responses to noxious agents. Fish
Shellfish Immunol. 7, 567–584.