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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 components of the host innate immune system and play crucial
roles in host defense against bacterial invasion [8]. However, only
Large yellow croaker, Pseudosciaena crocea (Richardson), is a relatively smaller number of antibacterial peptides has been
mainly distributed between the southern Yellow Sea and the found so far from teleosts such as pleurocidin [9], paradaxin [10],
northern Southern China Sea [1,2]. Today, large yellow croaker is parasin [11], misgurin [12], piscidins [13], moronecidin [14], hep-
one of the most important maricultured fish in China with the cidin [15], chrysophsin [16], oncorhyncin [17], and cathelicidin [18].
highest annual yield. The most significant factor that affects large In this paper, we reported the isolation of a potent antibacterial
yellow croaker culture is the incidence of various diseases, espe- peptide from large yellow croaker, P. crocea. The amino acid
cially vibriosis. Some studies on prevention and cure of the disease sequence and antibacterial activity of the peptide were analyzed.
of large yellow croaker have been carried out [3,4]. However, The results indicated that this antibacterial peptide was the mature
information on immune mechanisms is sparse and no study on the peptide of large yellow croaker hepcidin.
purification and identification of antibacterial peptides from this
fish has been done. The adaptive immune response in teleosts is 2. Materials and methods
feebler and short-lived, when compared to that of mammals [5].
Previous studies of our laboratory showed that it took large yellow 2.1. Experimental animals and sample collection
croaker at least 2 weeks to produce specific antibody [6]. So the
innate defenses play important roles in teleosts. Ice-cooled adult large yellow croakers weighing 400–450 g were
Antibacterial peptides are widely distributed throughout the obtained from Zhongpu aquatic product wholesale market (Xia-
animal and plant kingdoms [7]. They are regarded as important 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
* Corresponding author. Tel.: þ86 592 6180204; fax: þ86 592 6181476. ice-cooled dish. Skin mucus was gently scraped from the dorso-
E-mail address: yanqp@jmu.edu.cn (Q. Yan). 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) 864–870 865

25 mM phosphate buffer (PB), pH 7.0, supplemented with 1.5 mM 11 180


aprotinin. The excised gills were immersed in the PB solution to 10
160
prepare gill mucus by scraping the gills. Following excision of
9

Inhibition zone area(mm2)


gastrointestinal tract, the content of them was washed into PB. The 140
8
tissue of head kidney, liver and gonad, as well as the gastrointes-

A280nm(mAU)
120
tinal tissue was homogenized in PB solution using a homogenizer. 7
6 100
2.2. Peptide extractions 5 80
4
All of the seven samples in PB were extracted by shaking for 2 h 60
at 0  C. After centrifugation at 10 000 g for 10 min at 4  C, the 3
40
supernatants were collected as PB extracts. The precipitates were 2
homogenized in 10% acetic acid supplemented with 1.5 mM apro- 1 20
tinin, stirred and centrifuged in the same manner. The supernatants
0 0
were collected as acidic extracts. These PB extracts and acidic 0 4 8 12 16 20 24 28 32 36 40
extracts were lyophilized, resuspended in NH4Ac solution (10 mM, Fractions
pH 6.6), then the antibacterial activity and protein concentration
were determined. 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-
Head kidney was chosen as the source of antibacterial peptide
bition zone areas. The fractions shown by the bar were pooled and subjected to further
for further purification. To obtain enough extract and simplify the purification by reverse-phase HPLC.
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 absorbance was measured at 280 nm, and 1-ml fractions were
The acidic extract was subjected to boiling water bath for 15 min collected by hand. Each fraction was lyophilized, reconstituted in
with continuous agitation, and then cooled immediately in ice. 0.2 ml deionized water for antibacterial activity determination.
After centrifugation, the supernatant was lyophilized, resuspended The active fractions eluted between 58 and 60 min were pooled,
in 10 mM NH4Ac solution, pH 6.6, and the antibacterial activity was lyophilized, reconstituted in acidified water and further purified to
determined. 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%
2.3. Solid-phase extraction over 60 min/23–50% over 5 min). Fractions were collected and
tested for antibacterial activity. The active fractions eluted between
Acidic extract of head kidney tissue from 25 individuals was 30 and 32 min were pooled, lyophilized, reconstituted in deionized
acidified with an equal volume of 0.2% TFA in deionized water, then water for chemical characterization and antibacterial spectra assay.
subjected to solid-phase extraction on six Sep-Pak Vac 1g C18
cartridges (Waters Associates, USA), which were previously condi-
2.6. Bacterial strains
tioned with 15 ml of methanol and equilibrated with 15 ml of
acidified water (0.1% TFA in deionized water), respectively. Following
Six strains of Gram-negative bacteria, Escherichia coli, Aero-
elution with 15 ml of acidified water, each cartridge was eluted by
monas hydrophila, Vibrio alginolyticus, Vibrio harveyi, Vibrio fluvialis,
15 ml of 15, 20, 25, 30 and 50% acetonitrile in acidified water,
and Vibrio parahaemolyticus, and three strains of Gram-positive
respectively. All the same fractions from six cartridges were pooled,
bacteria, Staphylococcus aureus, Bacillus subtilis, Micrococcus lyso-
lyophilized and reconstituted in NH4Ac solution (10 mM, pH 6.6) for
deikticus were obtained from the China General Microbiological
antibacterial activity determination.
Culture Collection Center (Beijing, China) and listed in Table 3.
V. alginolyticus was used as the main test strain throughout the
2.4. Gel permeation chromatography
purification procedure. Other strains were used to determine the
antibacterial spectra of the purified peptide.
Gel permeation chromatography was performed on a Pharmacia
Marine bacteria were inoculated on marine nutrient agar (2%
ÄKTA Protein Purifier system (Amersham Pharmacia Biotech,
NaCl) and grown for 18 h at 28  C. Other strains were grown on
Sweden). The 20% acetonitrile effluent was applied to a 1.6  60 cm
normal nutrient agar (0.5% NaCl) for 18 h at 36  C. After washing of the
Sephadex G-25 (Amersham Pharmacia Biotech, Sweden) column
bacterial cells into sterile 0.85% (w/v) NaCl, the bacterial suspensions
equilibrated with NH4Ac solution (10 mM, pH 6.6). Gel column was
were adjusted to a final density of approximately 1  106 cfu ml1.
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. 2.7. Antibacterial assay

2.5. RP-HPLC Inhibition zone assay was used to determine the antibacterial
activity of fractionated samples at each purification step. 20 ml of
The active fractions denoted by the bar in Fig. 1 were pooled 1% (w/v) agarose in 1/10 strength nutrient broth was seeded with
from each time of G-25 elution, lyophilized and redissolved in 1 ml 1  106 bacterial cells and poured into Petri dishes. Aliquots (18 ml)
acidified water. An aliquot (0.1 ml) was subjected to C18 reverse- of the samples were added into 3 mm diameter wells punched in
phase high-performance liquid chromatography (RP-HPLC) on an the agarose, and incubated at 4  C for 1 h before a 24 h incubation at
analytical StableBond 300SB column (particle size 5 mm, 28  C (marine bacteria) or 37  C (other bacteria). Negative controls
4.6 mm  250 mm, Agilent, USA) equilibrated with acidified water comprised 10 mM NH4Ac solution, pH 6.6. Antibacterial activity
every time. Elution was performed with a linear biphasic gradient was identified as a clear zone around the well. The diameters of
elution of ACN (0–15% over 10 min/15–23% over 55 min/23–50% these zones were measured and activity was expressed as clear
over 5 min) in acidified water at 28  C at a flow rate of 1 ml min1. 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
600 ml of acidic extract, which contained 2.95 g dry substance, was
Protein concentration of samples was estimated by the method of obtained from head kidney tissue of 25 individuals directly. After
Bradford using bovine serum albumin (Amresco, USA) as standard [19]. 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-
2.9. MALDI-MS
trated extract, which contained 1.951 g dry substance, was obtained.
After being acidified with 0.2% TFA, 120 ml of the above
The molecular mass of the purified antibacterial peptide was
concentrated extract was subjected to solid-phase extraction by six
determined by Matrix-Assisted Laser Desorption/Ionization Time-
C18 Sep-Pak cartridges, while the other 40 ml was used in the
of-Flight Mass Spectrometry (MALDI-TOF) on a Voyager DE-PRO
pre-experiment of solid-phase extraction, gel permeation chro-
Biospectrometry (Applied Biosystem, USA). Sample was mixed with
matography and ion exchange fractionation. All the effluents at the
a-cyano-4-hydroxycinnamic acid matrix and deposited onto
same concentration of acetonitrile from six cartridges were pooled,
a target. Assay was performed in a linear mode at Genecorn Co. Ltd,
lyophilized and reconstituted in NH4Ac solution, and 20 or 30 ml
Shanghai, China.
of concentrated effluents was obtained. Protein concentration,
proportion and inhibition zone area of each effluent are listed in
2.10. Amino acid sequence analysis Table 2. The 20% acetonitrile effluent, possessing 3.4% of total
protein, displayed the strongest antibacterial activity.
Amino acid sequence of the homogeneous peptide was deter- The 20% acetonitrile effluent (20 ml) was loaded on Sephadex G-
mined by automated Edman degradation using an automatic 25 column for five times. The elution profile of every time exhibited
peptide sequencer (ABI PROCISEÔ492cLC) at Genecorn Co. Ltd two major peaks (Fig. 1). The fractions mainly distributing in the
(Shanghai, China). Online HPLC detection of phenylthiohydantoin- second peak showed the antibacterial activity to V. alginolyticus,
coupled amino acids (Pth-Xaa) was performed under gradient and the 23rd fraction had the strongest antibacterial activity.
elution conditions. The kinds of amino acids were determined The fractions 23 and 24 (denoted by the bar in Fig. 1) were
according to the chromatogram of standard amino acids. selected, pooled (total 8 ml), lyophilized, reconstituted in 2 ml of
Amino acid sequence obtained was blasted with the NCBI (National acidified water as loading sample, and the C18 reversed-phase HPLC
Center for Biotechnology Information), using search for short and was performed for sixteen times. The elution profile of every time
nearly exact matches program [20]. Multiple sequence alignment was had two active peaks, eluting from 58 to 60 min and from 65 to
performed using Clustal X (2) [21]. The phylogenetic tree was drawn by 67 min (Fig. 2, labeled A and B).
the neighbour-joining method [22] and analyzed with Mega 4 [23]. In the present study, we focused our attention on the peak
The theoretical isoelectric point (pI) and molecular mass were esti- labeled A. Sixteen times of fractions 58–60 were pooled (total
mated by ExPASy (http://www.expasy.ch/tools/peptide-mass.html).
DAD1 A, Sig=280,16 Ref=360,100 (ANTI\ANTI0007.D)
mAU
3. Results
30
3.1. Antibacterial activities of different tissue extracts 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) 864–870 867

DAD1 A, Sig=280,16 Ref=360,100 (ANTI\ANTI0015.D) 21 residues including eight cysteines and six arginines. The calculated
mAU
isoelectric point (pI) of the purified peptide was 9.22. The sequence
6 data reported in this paper will appear in the UniProt knowledgebase
under the accession numbers A1Z0M0.
5 50

-----
4 40 3.4. Sequence alignment and phylogenetic analysis
----- A280nm

Acenotrile
3 30
Homologies of the sequences of the purified peptide with hep-
2 20 cidins or hepcidin-like proteins of mammals and other fish species
1
were analyzed. The highest identity was 100.0% with residues 65–85
10
of hepcidin precursor presumed from cDNA of large yellow croaker
0 0 (ABL96317). The sequence also had high identity (58–85%) with the
m
mature peptide sequence of hepcidin precursor predicted from
0 10 20 30 40 50 60
Japanese seabass (AAS55063, AAT09138), turbot (CAJ34592, AAX
Retention time
92670), red sea bream (AAR28077, AAV34605, AAS66305), channel
Fig. 3. Purification profile of the pooled 58–60th fractions by the second RP-HPLC. It was catfish (AAX39713), Atlantic salmon (Q801Y3), Japanese flounder
performed on the same column using a slower biphasic gradient (dashed line). Absor- (BAE06233), zebrafish (NP_991146), house mouse (Q80T19), black
bance was monitored at 280 nm (solid line). The fractions shown by the bar were pooled porgy (AAU00795, AAU00797). Especially, it had comparatively high
and submitted for further chemical characterization and antibacterial spectra assay.
identity with the hepcidin sequence purified from hybrid striped bass
(P82951, 75%) and human (P81172, 55%). All of these mature peptide
48 ml), lyophilized, reconstituted in 1.6 ml of acidified water used sequences shared nine identical residues, especially eight cysteines,
as the second loading sample, and the second C18 reverse-phase at the identical conserved positions (Fig. 5A). These results suggested
HPLC was performed for twelve times, but with a slower water/ that the purified antibacterial peptide was the mature peptide
acetonitrile gradient. A single active peak with a retention time of section of the hepcidin preproprotein from large yellow croaker, thus
between 30 and 32 min was yielded from the elution profile of designated as hepcidin-P1.
every time (Fig. 3). All twelve times of active fractions 30–32 were Phylogenetic analysis of these selected hepcidin sequences
pooled (total 36 ml), lyophilized, reconstituted in 0.3 ml of acidified indicated that there were two main clusters: mammalian and fish
water. The ultimately purified peptide solution at the concentration hepcidins. Hepcidin-P1 was in the same branch with red sea bream,
of circa 96 mg ml1 was submitted for further chemical character- black porgy, and hybrid striped bass hepcidins. It was obvious that
ization and antibacterial spectra assay. the hepcidin-P1 is more similar to red sea bream hepcidin
(AAR28077) than those of other species (Fig. 5B).
3.3. Structural characterization
3.5. Antibacterial spectra
To identify the antibacterial peptide, the purified sample was
subjected to exact molecule mass measuring by MALDI-TOF MS and Hepcidin-P1 exhibited antibacterial activity against all Gram-
N-terminal amino acid sequencing. The result of MALDI-TOF MS positive strains and Gram-negative strains tested, but more effec-
showed that the purified peptide had an observed molecular mass of tive to marine Vibrio at a concentration of 96 mg ml1 (Table 3).
2523.20 Da (Fig. 4). According to the results of N-terminal amino acid
sequencing, the sequence of the antibacterial peptide was RCRFCCR 4. Discussion
CCPRMRGCGICCRF. The sequence had a theoretic molecular mass of
2522.18 Da, assuming that eight cysteine residues were engaged in Here we reported the isolation and characterization of hepcidin-
four intramolecular disulfide bridges. It was well consistent with the P1, a 21-amino acid-residue peptide containing eight cysteines and
observed molecular mass of MALDI-TOF MS. The sequence consists of six arginines, from head kidney tissue of large yellow croaker.

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.
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) 864–870 869

It is documented that antibacterial peptides are widely distrib- After human hepcidin was isolated as an antibacterial peptide,
uted in various tissues [13,15,24]. Acetic acid extracts of five tissues hepcidin gene was found to be over expressed in mouse during iron
showed strong anti-V. alginolyticus activity in the present study, overload [36], which provoked many studies on the functions of
while PB extracts of the seven kinds of tissue had not exhibited hepcidin in the iron regulation of animals, including mammals and
anti-V. alginolyticus activity. Head kidney, an important immune fish [35,37]. Hepcidin could bind to ferroportin and induced the
organ in teleosts [25,26] and proved to express antibacterial internalization and degradation of the latter, which resulted in the
substances [18,27], was chosen as the source of purification for decrease of cellular iron export and the degradation of extracellular
antibacterial peptide. Though several antibacterial peptides had ferric concentration [38]. At the same time iron is an essential
been isolated from skin or gill mucus of teleost [13,15,24], the skin element for all organisms. The ferric uptake regulator (Fur) protein is
and gill mucus extract of large yellow croaker exhibited no a master regulator of iron metabolism in Gram-negative bacteria.
antibacterial activity against V. alginolyticus. Highly conserved sequence of the Fur protein included an iron-
After a series of isolation procedures, an antibacterial peptide binding motif with rich histidines. But such ‘Fur-box’ was not iden-
with a single peak was obtained from acidic extract of head kidney tified in the fur Gene of several marine Gram-negative bacteria, such
tissue. Heat-treatment in boiling water suggested that the anti- as V. alginolyticus, Vibrio vulnificus [39]. The present study showed
bacterial peptide was heat stable. 29 mg hepcidin peptide was that several strains of vibrios, marine Gram-negative bacteria were
obtained from 3 g head kidney tissue, so the concentration of more sensitive to hepcidin-P1 than non-marine bacteria tested,
hepcidin in head kidney tissue should be higher than 9.6 mg g1. At including Gram-negative and Gram-positive bacteria. The mecha-
the same time the antibacterial assay showed that the active nism involved is worthy of further study.
fractions after the second RP-HPLC had an inhibition zone of In summary, five kinds of tissue acidic extracts of large yellow
26 mm2 against V. alginolyticus at a concentration of circa 4 mg g1. croaker showed strong anti-V. alginolyticus activity. A heat stable,
So we also could justify that hepcidin-P1 not only was a potent homogenous peptide with strong antibacterial activity was purified
antibacterial peptide in vitro but also had the capability to function from acidic extract of head kidney tissue. Marine vibrios exhibited
as an antibacterial substance in head kidney tissue directly. more sensitivity to the antibacterial peptide. The peptide’s amino
In addition to eight cysteines, the presence of six arginine acid sequence was RCRFCCRCCPRMRGCGICCRF with a molecular
residues in hepcidin-P1 made it to have a high isoelectric point (pI), mass of 2523.2 Da, which has 100% identity to partial peptide
and become a highly basic peptide. This was consistent with its sequence of hepcidin preproprotein presumed from cDNA of large
behavior in ion exchange fractionation and two rounds of RP-HPLC. yellow croaker, thus designated hepcidin-P1.
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 Acknowledgements
elute them. The StableBond column used in RP-HPLC was not
blocked, and its silanol in the surface could adsorb basic substance, This work was supported by grants from National Hi-Tech
resulting in a linear tailing peak. Research and Development Program of China (863 Program) under
The first hepcidin, also known as liver-expressed antimicrobial contract No. 2007AA09Z115, and Technology Program of Xiamen
peptide (LEAP-1), was initially purified from human urine and under contract No. 3502Z20073019. We are thankful to Xu JS and
plasma ultrafiltrates. It was confirmed to be a 25-residue peptide Liu AY for assistance in high-performance liquid chromatography,
containing four disulfide bonds [28]. Bass hepcidin was the first fish Chen Q and Yi JP for sampling assistance, as well as to Huang ZY and
hepcidin peptide isolated from the gills of hybrid striped bass [15]. Huang WS for assistance in sample analysis.
Hepcidin genes have been detected in various fish, such as winter
flounder and Atlantic salmon [29], zebrafish [30], red sea bream [31],
References
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