Fish & Shellfish Immunology 26 (2009) 864–870

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Fish & Shellfish Immunology

journal homepage: www.elsevier.com/locate/fsi

Isolation and characterization of a hepcidin peptide from the head

kidney of large yellow croaker, Pseudosciaena crocea
Junjie Zhang a, b, Qingpi Yan a, *, Rongxing Ji a, Wenzheng Zou a, Guojun Guo c
a
Key Laboratory of Science and Technology for Aquaculture and Food Safety, Fisheries College, Jimei University, Xiamen, Fujiang 361021, PR China
b
College of Animal Science, Xinjiang Agricultural University, Urmuqi 830052, PR China
c
Zhengzhou College of Animal Husbandry Engineering, Zhengzhou 450011, PR China

a r t i c l e i n f o a b s t r a c t

Article history: Large yellow croaker (Pseudosciaena crocea) is one of the most important marine cultured fish in China.
Received 11 November 2008 Acidic extracts of five tissues of large yellow croaker showed strong anti-Vibrio alginolyticus activity. Acidic
Received in revised form extract of head kidney tissue was subjected to heat-treatment in boiling water, and solid-phase extraction
19 March 2009
on Sep-Pak C18 cartridge. It was found that the antibacterial substances were heat stable, and 20%
Accepted 23 March 2009
Available online 1 April 2009
acetonitrile effluent exhibited strong antibacterial activity. Active extract was further applied to Sephadex
G-25 gel permeation chromatography and StableBond C18 RP-HPLC. An antibacterial peptide with a single
peak was obtained. The results of amino acid sequencing and MALDI-TOF MS suggested that the peptide
Keywords:
Large yellow croaker (Pseudosciaena crocea) was RCRFCCRCCPRMRGCGICCRF with an observed molecular mass of 2523.2 Da. BLAST searching sug-
Vibrio alginolyticus gested that the purified antibacterial peptide was the mature peptide section of the hepcidin pre-
Head kidney proprotein presumed from cDNA of large yellow croaker, thus designated hepcidin-Pl. Hepcidin-P1
RP-HPLC exhibited strong antibacterial activity against four marine vibrios.
MALDI-TOF MS Ó 2009 Elsevier Ltd. All rights reserved.
Amino acid sequencing
Antibacterial peptide
Hepcidin

1. Introduction
Large yellow croaker, Pseudosciaena crocea (Richardson), is
mainly distributed between the southern Yellow Sea and the
northern Southern China Sea [1,2]. Today, large yellow croaker is
one of the most important maricultured fish in China with the
highest annual yield. The most significant factor that affects large
yellow croaker culture is the incidence of various diseases, espe-
cially vibriosis. Some studies on prevention and cure of the disease
of large yellow croaker have been carried out [3,4]. However,
information on immune mechanisms is sparse and no study on the
purification and identification of antibacterial peptides from this
fish has been done. The adaptive immune response in teleosts is
feebler and short-lived, when compared to that of mammals [5].
Previous studies of our laboratory showed that it took large yellow
croaker at least 2 weeks to produce specific antibody [6]. So the
innate defenses play important roles in teleosts.
Antibacterial peptides are widely distributed throughout the
animal and plant kingdoms [7]. They are regarded as important
* Corresponding author. Tel.: þ86 592 6180204; fax: þ86 592 6181476.
E-mail address: yanqp@jmu.edu.cn (Q. Yan).
components of the host innate immune system and play crucial
roles in host defense against bacterial invasion [8]. However, only
a relatively smaller number of antibacterial peptides has been
found so far from teleosts such as pleurocidin [9], paradaxin [10],
parasin [11], misgurin [12], piscidins [13], moronecidin [14], hep-
cidin [15], chrysophsin [16], oncorhyncin [17], and cathelicidin [18].
In this paper, we reported the isolation of a potent antibacterial
peptide from large yellow croaker, P. crocea. The amino acid
sequence and antibacterial activity of the peptide were analyzed.
The results indicated that this antibacterial peptide was the mature
peptide of large yellow croaker hepcidin.
2. Materials and methods
2.1. Experimental animals and sample collection
Ice-cooled adult large yellow croakers weighing 400–450 g were
obtained from Zhongpu aquatic product wholesale market (Xia-
men, Fujiang, China). They were just carried from commercial net
cage farms (Ningde, Fujiang, China) in approximately 10 h.
Seven kinds of tissue were collected from four individuals on
ice-cooled dish. Skin mucus was gently scraped from the dorso-
lateral surfaces of the fish with a steel spatula and mixed with

1050-4648/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fsi.2009.03.014
 J. Zhang et al. / Fish & Shellfish Immunology 26 (2009) 864–870
25 mM phosphate buffer (PB), pH 7.0, supplemented with 1.5 mM
aprotinin. The excised gills were immersed in the PB solution to
prepare gill mucus by scraping the gills. Following excision of
gastrointestinal tract, the content of them was washed into PB. The
tissue of head kidney, liver and gonad, as well as the gastrointes-
tinal tissue was homogenized in PB solution using a homogenizer.
2.2. Peptide extractions
All of the seven samples in PB were extracted by shaking for 2 h
at 0  C. After centrifugation at 10 000 g for 10 min at 4  C, the
supernatants were collected as PB extracts. The precipitates were
homogenized in 10% acetic acid supplemented with 1.5 mM apro-
tinin, stirred and centrifuged in the same manner. The supernatants
were collected as acidic extracts. These PB extracts and acidic
extracts were lyophilized, resuspended in NH4Ac solution (10 mM,
pH 6.6), then the antibacterial activity and protein concentration
were determined.
Head kidney was chosen as the source of antibacterial peptide
for further purification. To obtain enough extract and simplify the
extraction procedure, the head kidney tissue of 25 individuals was
extracted directly with 600 ml of 10% acetic acid supplemented
with 1.5 mM aprotinin to prepare the acidic extract of head kidney.
The acidic extract was subjected to boiling water bath for 15 min
with continuous agitation, and then cooled immediately in ice.
After centrifugation, the supernatant was lyophilized, resuspended
in 10 mM NH4Ac solution, pH 6.6, and the antibacterial activity was
determined.
2.3. Solid-phase extraction
Acidic extract of head kidney tissue from 25 individuals was
acidified with an equal volume of 0.2% TFA in deionized water, then
subjected to solid-phase extraction on six Sep-Pak Vac 1g C18
cartridges (Waters Associates, USA), which were previously condi-
tioned with 15 ml of methanol and equilibrated with 15 ml of
acidified water (0.1% TFA in deionized water), respectively. Following
elution with 15 ml of acidified water, each cartridge was eluted by
15 ml of 15, 20, 25, 30 and 50% acetonitrile in acidified water,
respectively. All the same fractions from six cartridges were pooled,
lyophilized and reconstituted in NH4Ac solution (10 mM, pH 6.6) for
antibacterial activity determination.
2.4. Gel permeation chromatography
Gel permeation chromatography was performed on a Pharmacia
ÄKTA Protein Purifier system (Amersham Pharmacia Biotech,
Sweden). The 20% acetonitrile effluent was applied to a 1.6  60 cm
Sephadex G-25 (Amersham Pharmacia Biotech, Sweden) column
equilibrated with NH4Ac solution (10 mM, pH 6.6). Gel column was
eluted with the same buffer at a flow rate of 1 ml min1. Absor-
bance was measured at 280 nm and 2-ml fractions were collected.
The antibacterial activity of each fraction was determined directly.
2.5. RP-HPLC
The active fractions denoted by the bar in Fig. 1 were pooled
from each time of G-25 elution, lyophilized and redissolved in 1 ml
acidified water. An aliquot (0.1 ml) was subjected to C18 reverse-
phase high-performance liquid chromatography (RP-HPLC) on an
analytical StableBond 300SB column (particle size 5 mm,
4.6 mm  250 mm, Agilent, USA) equilibrated with acidified water
every time. Elution was performed with a linear biphasic gradient
elution of ACN (0–15% over 10 min/15–23% over 55 min/23–50%
over 5 min) in acidified water at 28  C at a flow rate of 1 ml min1.
865
11 180
10
160
9
Inhibition zone area(mm2)
140
8
A280nm(mAU)
120
7
6 100
5 80
4
60
3
40
2
1 20
0 0
0 4 8 12 16 20 24 28 32 36 40
Fractions
Fig. 1. Purification by gel permeation chromatography on Sephadex G-25 of the 20%
acetonitrile effluent of head kidney acidic extract. The histogram represents the inhi-
bition zone areas. The fractions shown by the bar were pooled and subjected to further
purification by reverse-phase HPLC.
The absorbance was measured at 280 nm, and 1-ml fractions were
collected by hand. Each fraction was lyophilized, reconstituted in
0.2 ml deionized water for antibacterial activity determination.
The active fractions eluted between 58 and 60 min were pooled,
lyophilized, reconstituted in acidified water and further purified to
homogeneity by a second round of C18 RP-HPLC on the same
column using a lower linear gradient (0–20% over 5 min/20–23%
over 60 min/23–50% over 5 min). Fractions were collected and
tested for antibacterial activity. The active fractions eluted between
30 and 32 min were pooled, lyophilized, reconstituted in deionized
water for chemical characterization and antibacterial spectra assay.
2.6. Bacterial strains
Six strains of Gram-negative bacteria, Escherichia coli, Aero-
monas hydrophila, Vibrio alginolyticus, Vibrio harveyi, Vibrio fluvialis,
and Vibrio parahaemolyticus, and three strains of Gram-positive
bacteria, Staphylococcus aureus, Bacillus subtilis, Micrococcus lyso-
deikticus were obtained from the China General Microbiological
Culture Collection Center (Beijing, China) and listed in Table 3.
V. alginolyticus was used as the main test strain throughout the
purification procedure. Other strains were used to determine the
antibacterial spectra of the purified peptide.
Marine bacteria were inoculated on marine nutrient agar (2%
NaCl) and grown for 18 h at 28  C. Other strains were grown on
normal nutrient agar (0.5% NaCl) for 18 h at 36  C. After washing of the
bacterial cells into sterile 0.85% (w/v) NaCl, the bacterial suspensions
were adjusted to a final density of approximately 1  106 cfu ml1.
2.7. Antibacterial assay
Inhibition zone assay was used to determine the antibacterial
activity of fractionated samples at each purification step. 20 ml of
1% (w/v) agarose in 1/10 strength nutrient broth was seeded with
1  106 bacterial cells and poured into Petri dishes. Aliquots (18 ml)
of the samples were added into 3 mm diameter wells punched in
the agarose, and incubated at 4  C for 1 h before a 24 h incubation at
28  C (marine bacteria) or 37  C (other bacteria). Negative controls
comprised 10 mM NH4Ac solution, pH 6.6. Antibacterial activity
was identified as a clear zone around the well. The diameters of
these zones were measured and activity was expressed as clear
zone area (in mm2) minus the area of the well.
866 J. Zhang et al. / Fish & Shellfish Immunology 26 (2009) 864–870

Table 1 extracts and acidic extracts. The results of inhibition zone assay
Anti-V. alginolyticus activities and protein concentrations of acetic acidic extracts of showed that acidic extracts of five tissues exhibited strong anti-
different tissues from large yellow croaker (Pseudosciaena crocea).
bacterial activity against V. alginolyticus, while skin and gill mucus
Tissue source Inhibition zone Protein acidic extracts, as well as PB extracts of seven tissues had no anti-
area (mm2) concentration (mg ml1)
V. alginolyticus activity. The inhibition zone areas and protein
Skin mucus 0 0.04 concentrations of the acidic extracts are listed in Table 1.
Gill mucus 0 0.24
Gastrointestinal tissue acidic extract displayed the highest
Liver tissue 147 0.50
Gastrointestinal tissue 194 0.08 antibacterial activity, and then head kidney tissue and liver tissue
Gastrointestinal content 126 0.58 acidic extracts took the second place. Gastrointestinal content and
Head kidney tissue 147 0.81 gonad tissue acidic extract also displayed considerable activity.
Gonad tissue 126 0.64
10 mM NH4Ac solution, pH 6.6 0 0

3.2. Purification of antibacterial peptide from head kidney

2.8. Protein quantification
Protein concentration of samples was estimated by the method of
Bradford using bovine serum albumin (Amresco, USA) as standard [19].
2.9. MALDI-MS
The molecular mass of the purified antibacterial peptide was
determined by Matrix-Assisted Laser Desorption/Ionization Time-
of-Flight Mass Spectrometry (MALDI-TOF) on a Voyager DE-PRO
Biospectrometry (Applied Biosystem, USA). Sample was mixed with
a-cyano-4-hydroxycinnamic acid matrix and deposited onto
a target. Assay was performed in a linear mode at Genecorn Co. Ltd,
Shanghai, China.
2.10. Amino acid sequence analysis
Amino acid sequence of the homogeneous peptide was deter-
mined by automated Edman degradation using an automatic
peptide sequencer (ABI PROCISEÔ492cLC) at Genecorn Co. Ltd
(Shanghai, China). Online HPLC detection of phenylthiohydantoin-
coupled amino acids (Pth-Xaa) was performed under gradient
elution conditions. The kinds of amino acids were determined
according to the chromatogram of standard amino acids.
Amino acid sequence obtained was blasted with the NCBI (National
Center for Biotechnology Information), using search for short and
nearly exact matches program [20]. Multiple sequence alignment was
performed using Clustal X (2) [21]. The phylogenetic tree was drawn by
the neighbour-joining method [22] and analyzed with Mega 4 [23].
The theoretical isoelectric point (pI) and molecular mass were esti-
mated by ExPASy (http://www.expasy.ch/tools/peptide-mass.html).
3. Results
3.1. Antibacterial activities of different tissue extracts
600 ml of acidic extract, which contained 2.95 g dry substance, was
obtained from head kidney tissue of 25 individuals directly. After
removing some deposit by boiling water bath, the acidic extract
maintained its strong antibacterial activity (data not shown). Following
centrifugation, lyophilization and redissolution, 160 ml of concen-
trated extract, which contained 1.951 g dry substance, was obtained.
After being acidified with 0.2% TFA, 120 ml of the above
concentrated extract was subjected to solid-phase extraction by six
C18 Sep-Pak cartridges, while the other 40 ml was used in the
pre-experiment of solid-phase extraction, gel permeation chro-
matography and ion exchange fractionation. All the effluents at the
same concentration of acetonitrile from six cartridges were pooled,
lyophilized and reconstituted in NH4Ac solution, and 20 or 30 ml
of concentrated effluents was obtained. Protein concentration,
proportion and inhibition zone area of each effluent are listed in
Table 2. The 20% acetonitrile effluent, possessing 3.4% of total
protein, displayed the strongest antibacterial activity.
The 20% acetonitrile effluent (20 ml) was loaded on Sephadex G-
25 column for five times. The elution profile of every time exhibited
two major peaks (Fig. 1). The fractions mainly distributing in the
second peak showed the antibacterial activity to V. alginolyticus,
and the 23rd fraction had the strongest antibacterial activity.
The fractions 23 and 24 (denoted by the bar in Fig. 1) were
selected, pooled (total 8 ml), lyophilized, reconstituted in 2 ml of
acidified water as loading sample, and the C18 reversed-phase HPLC
was performed for sixteen times. The elution profile of every time
had two active peaks, eluting from 58 to 60 min and from 65 to
67 min (Fig. 2, labeled A and B).
In the present study, we focused our attention on the peak
labeled A. Sixteen times of fractions 58–60 were pooled (total
DAD1 A, Sig=280,16 Ref=360,100 (ANTI\ANTI0007.D)
mAU
30
A B
25 50
-----
----- A280nm

Seven kinds of tissue from four individuals were extracted by PB

20
solution and 10% acetic acid successively to prepare their PB 40
Acenotrile

15 30
Table 2
Protein concentration, proportion and inhibition zone area of each effluent of heat- 10 20
treated head kidney acidic extract after solid-phase extraction on C18 Sep-Pak cartridge.
5 10
Sample or fractions Protein Effluent Protein Inhibition
concentration volume proportion zone area 0 0
(mg ml1) (ml) (%) (mm2) m
0 10 20 30 40 50 60
Loading sample 3.42932 119.5 100 88
0% Acetonitrile effluent 11.7194 30 84.4329 43 Retention time
15% Acetonitrile effluent 0.18852 20 0.90545 0
Fig. 2. Purification profile of the pooled gel permeation chromatography fractions by
20% Acetonitrile effluent 0.70846 20 3.40276 170
the first RP-HPLC. The RP-HPLC was performed on an analytical 300SB C18 column
25% Acetonitrile effluent 0.60223 20 2.89253 31
using a biphasic water/acetonitrile gradient (dashed line). Absorbance was monitored
30% Acetonitrile effluent 0.61509 20 2.95429 13
at 280 nm (solid line). The fractions from peak A denoted by the bar were pooled, and
50% Acetonitrile effluent 1.12682 20 5.4121 0
subjected to further purification.
 J. Zhang et al. / Fish & Shellfish Immunology 26 (2009) 864–870
DAD1 A, Sig=280,16 Ref=360,100 (ANTI\ANTI0015.D)
mAU
6 data reported in this paper will appear in the UniProt knowledgebase
5 50
-----
4 40
----- A280nm
Acenotrile
3 30
2 20
1
10
0 0
m
0 10 20 30 40 50 60
Retention time
Fig. 3. Purification profile of the pooled 58–60th fractions by the second RP-HPLC. It was
performed on the same column using a slower biphasic gradient (dashed line). Absor-
bance was monitored at 280 nm (solid line). The fractions shown by the bar were pooled
and submitted for further chemical characterization and antibacterial spectra assay.
48 ml), lyophilized, reconstituted in 1.6 ml of acidified water used
as the second loading sample, and the second C18 reverse-phase
HPLC was performed for twelve times, but with a slower water/
acetonitrile gradient. A single active peak with a retention time of
between 30 and 32 min was yielded from the elution profile of
every time (Fig. 3). All twelve times of active fractions 30–32 were
pooled (total 36 ml), lyophilized, reconstituted in 0.3 ml of acidified
water. The ultimately purified peptide solution at the concentration
of circa 96 mg ml1 was submitted for further chemical character-
ization and antibacterial spectra assay.
3.3. Structural characterization
To identify the antibacterial peptide, the purified sample was
subjected to exact molecule mass measuring by MALDI-TOF MS and
N-terminal amino acid sequencing. The result of MALDI-TOF MS
showed that the purified peptide had an observed molecular mass of
2523.20 Da (Fig. 4). According to the results of N-terminal amino acid
sequencing, the sequence of the antibacterial peptide was RCRFCCR
CCPRMRGCGICCRF. The sequence had a theoretic molecular mass of
2522.18 Da, assuming that eight cysteine residues were engaged in
four intramolecular disulfide bridges. It was well consistent with the
observed molecular mass of MALDI-TOF MS. The sequence consists of
Fig. 4. Mass spectrogram obtained by MALDI-TOF-MS analysis of the purified antibacterial peptide from large yellow croaker head kidney tissue. Values are indicated in m/z. The
transformed spectrum shows a mass (MHþ) of 2524.20 Da.
867
21 residues including eight cysteines and six arginines. The calculated
isoelectric point (pI) of the purified peptide was 9.22. The sequence
under the accession numbers A1Z0M0.
3.4. Sequence alignment and phylogenetic analysis
Homologies of the sequences of the purified peptide with hep-
cidins or hepcidin-like proteins of mammals and other fish species
were analyzed. The highest identity was 100.0% with residues 65–85
of hepcidin precursor presumed from cDNA of large yellow croaker
(ABL96317). The sequence also had high identity (58–85%) with the
mature peptide sequence of hepcidin precursor predicted from
Japanese seabass (AAS55063, AAT09138), turbot (CAJ34592, AAX
92670), red sea bream (AAR28077, AAV34605, AAS66305), channel
catfish (AAX39713), Atlantic salmon (Q801Y3), Japanese flounder
(BAE06233), zebrafish (NP_991146), house mouse (Q80T19), black
porgy (AAU00795, AAU00797). Especially, it had comparatively high
identity with the hepcidin sequence purified from hybrid striped bass
(P82951, 75%) and human (P81172, 55%). All of these mature peptide
sequences shared nine identical residues, especially eight cysteines,
at the identical conserved positions (Fig. 5A). These results suggested
that the purified antibacterial peptide was the mature peptide
section of the hepcidin preproprotein from large yellow croaker, thus
designated as hepcidin-P1.
Phylogenetic analysis of these selected hepcidin sequences
indicated that there were two main clusters: mammalian and fish
hepcidins. Hepcidin-P1 was in the same branch with red sea bream,
black porgy, and hybrid striped bass hepcidins. It was obvious that
the hepcidin-P1 is more similar to red sea bream hepcidin
(AAR28077) than those of other species (Fig. 5B).
3.5. Antibacterial spectra
Hepcidin-P1 exhibited antibacterial activity against all Gram-
positive strains and Gram-negative strains tested, but more effec-
tive to marine Vibrio at a concentration of 96 mg ml1 (Table 3).
4. Discussion
Here we reported the isolation and characterization of hepcidin-
P1, a 21-amino acid-residue peptide containing eight cysteines and
six arginines, from head kidney tissue of large yellow croaker.
868 J. Zhang et al. / Fish & Shellfish Immunology 26 (2009) 864–870

B 76 Pseudosciaena crocea A1Z0M0

61 Chrysophrys major AAR28077

14 Pagrus major AAV34605

Morone chrysops x M. saxatilis P82951

25 53 Acanthopagrus schlegelii AAU00795

69 Pagrus major AAS66305
Lateolabrax japonicus2 AAT09138

56 34 Lateolabrax japonicus1 AAS55063

49 Acanthopagrus schlegelii4 AAU00797
Danio rerio NP 991146
Salmo salar Q801Y3
69 Ictalurus punctatus AAX39713
Scophthalmus maximus1 CAJ34592
54
59 Scophthalmus maximus AAX92670
50 Paralichthys olivaceus BAE06233
human P81172
Mus musculus Q80T19

0.30 0.25 0.20 0.15 0.10 0.05 0.00

Fig. 5. Alignment of the sequence of the Pseudosciaena crocea hepcidin-P1 with hepcidins or hepcidin-like proteins of mammals and other fish species and phylogenetic analysis.
(A) Identical amino acid residues were represented by same colour and conserved amino acids were indicated by asterisk above the alignment. Gaps showed by dashes (–) were
inserted to obtain maximum homology. Those mature hepcidin sequences, which were predicted from cDNA, were indicated by [p] following them. Sequences used for comparison
and their GenBank accession numbers were as follows: Japanese seabass (AAS55063, AAT09138), turbot (CAJ34592, AAX92670), red sea bream (AAR28077, AAV34605, AAS66305),
channel catfish (AAX39713), Atlantic salmon (Q801Y3), Japanese flounder (BAE06233), zebrafish (NP_991146), house mouse (Q80T19), black porgy (AAU00795, AAU00797), hybrid
striped bass (P82951) and human (P81172). The alignment of the amino acid sequences of hepcidin proteins was performed using Clustal X [21]. The phylogenetic tree (B) was
drawn by the neighbour-joining method [22] and analyzed with Mega 4 [23]. (For interpretation of the references to colour in this figure legend, the reader is referred to the web
version of this article.)

Table 3
Antibacterial spectra of hepcidin-P1 purified from the head kidney of large yellow croaker (Pseudosciaena crocea Richardson).

Bacterium Identification code (CGMCC) Gram reaction Source Area of inhibition (mm2)
M. lysodeikticus 1.634 Positive Non-marine 34.56
S. aureus 1.879 Positive Non-marine 47.12
B. subtilis 1.308 Positive Non-marine 28.86
E. coli 1.90 Negative Non-marine 34.56
A. hydrophila 1.927 Negative Non-marine 31.66
V. alginolyticus 1.1833 Negative Marine 118.06
V. harveyi 1.1600 Negative Marine 113.10
V. fluvialis 1.1608 Negative Marine 123.11
V. parahaemolyticus 1.1614 Negative Marine 94.25
 J. Zhang et al. / Fish & Shellfish Immunology 26 (2009) 864–870
It is documented that antibacterial peptides are widely distrib-
uted in various tissues [13,15,24]. Acetic acid extracts of five tissues
showed strong anti-V. alginolyticus activity in the present study,
while PB extracts of the seven kinds of tissue had not exhibited
anti-V. alginolyticus activity. Head kidney, an important immune
organ in teleosts [25,26] and proved to express antibacterial
substances [18,27], was chosen as the source of purification for
antibacterial peptide. Though several antibacterial peptides had
been isolated from skin or gill mucus of teleost [13,15,24], the skin
and gill mucus extract of large yellow croaker exhibited no
antibacterial activity against V. alginolyticus.
After a series of isolation procedures, an antibacterial peptide
with a single peak was obtained from acidic extract of head kidney
tissue. Heat-treatment in boiling water suggested that the anti-
bacterial peptide was heat stable. 29 mg hepcidin peptide was
obtained from 3 g head kidney tissue, so the concentration of
hepcidin in head kidney tissue should be higher than 9.6 mg g1. At
the same time the antibacterial assay showed that the active
fractions after the second RP-HPLC had an inhibition zone of
26 mm2 against V. alginolyticus at a concentration of circa 4 mg g1.
So we also could justify that hepcidin-P1 not only was a potent
antibacterial peptide in vitro but also had the capability to function
as an antibacterial substance in head kidney tissue directly.
In addition to eight cysteines, the presence of six arginine
residues in hepcidin-P1 made it to have a high isoelectric point (pI),
and become a highly basic peptide. This was consistent with its
behavior in ion exchange fractionation and two rounds of RP-HPLC.
After gel permeation chromatography the active substances
showed a very strong affinity to HiTrap SP FF, a kind of strong cation
ion exchange column. Even 2 M NH4Ac solution (pH 9) could not
elute them. The StableBond column used in RP-HPLC was not
blocked, and its silanol in the surface could adsorb basic substance,
resulting in a linear tailing peak.
The first hepcidin, also known as liver-expressed antimicrobial
peptide (LEAP-1), was initially purified from human urine and
plasma ultrafiltrates. It was confirmed to be a 25-residue peptide
containing four disulfide bonds [28]. Bass hepcidin was the first fish
hepcidin peptide isolated from the gills of hybrid striped bass [15].
Hepcidin genes have been detected in various fish, such as winter
flounder and Atlantic salmon [29], zebrafish [30], red sea bream [31],
channel catfish [32], black porgy [33], gilthead sea bream [34] and so
on, and similar hepcidin preproproteins have been predicted. Until
now, bass hepcidin is the only published hepcidin peptide isolated
from teleosts [15]. Hepcidin-P1, provided in this work, would be the
second hepcidin peptide isolated from teleosts and the first one from
large yellow croaker by protein isolation procedure. So the present
work would further confirm the presence of hepcidin in various fish.
Genomic organizations of all these hepcidins, obtained from fish
and human, are formed by 3 exons and 2 introns in a surprisingly
conservative mode [15,28,30,32–34]. Furthermore, there are at last
two copies of hepcidin gene existing in some teleost fish [29,30].
Hepcidin is always expressed in the liver, heart, head kidney,
spleen, intestine and stomach, especially predominantly expressed
in the liver [15,33].
It was the antibacterial activity that resulted in the discovery of
hepcidin peptide. Many studies, including the present study, have
revealed the in vitro inhibitory activity of synthetic or native
hepcidin peptides to many microorganisms, especially some
pathogenic bacteria [15,28,34]. At the same time, the expression of
these hepcidins could be up-regulated significantly by bacterial
challenges [29,30,34]. All these indirect proofs indicated that
hepcidin probably played an important role in the innate immune
response of animals. But so far there wasn’t direct proof to reveal
the real antibacterial activity and defense function of the hepcidin
in vivo, as endogenous antibiotics [35].
869
After human hepcidin was isolated as an antibacterial peptide,
hepcidin gene was found to be over expressed in mouse during iron
overload [36], which provoked many studies on the functions of
hepcidin in the iron regulation of animals, including mammals and
fish [35,37]. Hepcidin could bind to ferroportin and induced the
internalization and degradation of the latter, which resulted in the
decrease of cellular iron export and the degradation of extracellular
ferric concentration [38]. At the same time iron is an essential
element for all organisms. The ferric uptake regulator (Fur) protein is
a master regulator of iron metabolism in Gram-negative bacteria.
Highly conserved sequence of the Fur protein included an iron-
binding motif with rich histidines. But such ‘Fur-box’ was not iden-
tified in the fur Gene of several marine Gram-negative bacteria, such
as V. alginolyticus, Vibrio vulnificus [39]. The present study showed
that several strains of vibrios, marine Gram-negative bacteria were
more sensitive to hepcidin-P1 than non-marine bacteria tested,
including Gram-negative and Gram-positive bacteria. The mecha-
nism involved is worthy of further study.
In summary, five kinds of tissue acidic extracts of large yellow
croaker showed strong anti-V. alginolyticus activity. A heat stable,
homogenous peptide with strong antibacterial activity was purified
from acidic extract of head kidney tissue. Marine vibrios exhibited
more sensitivity to the antibacterial peptide. The peptide’s amino
acid sequence was RCRFCCRCCPRMRGCGICCRF with a molecular
mass of 2523.2 Da, which has 100% identity to partial peptide
sequence of hepcidin preproprotein presumed from cDNA of large
yellow croaker, thus designated hepcidin-P1.
Acknowledgements
This work was supported by grants from National Hi-Tech
Research and Development Program of China (863 Program) under
contract No. 2007AA09Z115, and Technology Program of Xiamen
under contract No. 3502Z20073019. We are thankful to Xu JS and
Liu AY for assistance in high-performance liquid chromatography,
Chen Q and Yi JP for sampling assistance, as well as to Huang ZY and
Huang WS for assistance in sample analysis.
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