Although the antibacterial peptides described above are highly heterogeneous with respect to their Keywords antibacterial protein; innate immunity; Sebastes schlegeli; skin mucus Corresp
Trang 1oxidase in the skin mucus of rockfish Sebastes schlegeli
Yoichiro Kitani1, Chihiro Tsukamoto1, GuoHua Zhang1, Hiroshi Nagai2, Masami Ishida2,
Shoichiro Ishizaki1, Kuniyoshi Shimakura1, Kazuo Shiomi1and Yuji Nagashima1
1 Department of Food Science and Technology, Tokyo University of Marine Science and Technology, Japan
2 Department of Ocean Science, Tokyo University of Marine Science and Technology, Japan
Fish have humoral factors elaborated in a nonspecific
defense system against infectious agents [1–3] The
mucus layer covering the surface of fish has a
mechan-ical protective function and also contains a variety of
biologically active substances, such as complements,
immunoglobulins, lectins, protease inhibitors and lytic
enzymes including lysozyme, that may act as defense
substances [4–6] Antimicrobial agents are thought to
play an especially important role in the innate
immu-nity of fish, as fish are in intimate contact with the
aquatic environment, which is rich in pathogenic
viru-lence Indeed, the skin and skin mucus of fish have been shown to contain antibacterial peptides, including pardaxin (a 33-residue peptide of Moses sole fish Pardachirus marmoratus) [7], pleurocidin (a 25-residue peptide of winter flounder Pleuronectes americanus) [8,9], grammistins (12–28-residue peptides of soapfishes Grammistes sexlineatus and Pogonoperca punctata) [10,11] and moronecidin (a 22-residue peptide of hybrid striped bass Morone chrysops· Morone saxatri-lis) [12] Although the antibacterial peptides described above are highly heterogeneous with respect to their
Keywords
antibacterial protein; innate immunity;
Sebastes schlegeli; skin mucus
Correspondence
Y Nagashima, Department of Food Science
and Technology, Tokyo University of Marine
Science and Technology, Konan 4-5-7,
Minato, Tokyo 108-8477, Japan
Fax: +81 35463 0614
Tel: +81 35463 0604
E-mail: yujicd@kaiyodai.ac.jp
(Received 30 August 2006, revised 19
October, accepted 6 November 2006)
doi:10.1111/j.1742-4658.2006.05570.x
Fish skin mucus contains a variety of antimicrobial proteins and peptides that seem to play a role in self defense We previously reported an antibac-terial protein in the skin secretion of the rockfish, Sebastes schlegeli, which showed selective antibacterial activity against Gram-negative bacteria This study aimed to isolate and structurally and functionally characterize this protein The antibacterial protein, termed SSAP (S schlegeli antibacterial protein), was purified to homogeneity by lectin affinity column chromato-graphy, anion-exchange HPLC and hydroxyapatite HPLC It was found to
be a glycoprotein containing N-linked glycochains and FAD Its molecular mass was estimated to be 120 kDa by gel filtration HPLC and 53 kDa by SDS⁄ PAGE, suggesting that it is a homodimer On the basis of the partial amino-acid sequence determined, a full-length cDNA of 2037 bp including
an ORF of 1662 bp that encodes 554 amino-acid residues was cloned by 3¢ RACE, 5¢ RACE and RT-PCR A blast search showed that a mature protein (496 residues) is homologous to l-amino acid oxidase (LAO) family proteins SSAP was determined to have LAO activity by the H2O2 -genera-tion assay and substrate specificity for only l-Lys with a Kmof 0.19 mm It showed potent antibacterial activity against fish pathogens such as Aero-monas hydrophila, Aeromonas salmonicida and Photobacterium damselae ssp piscicida The antibacterial activity was completely lost on the addition
of catalase, confirming that H2O2 is responsible for the growth inhibition This study identifies SSAP as a new member of the LAO family and reveals LAO involvement in the innate immunity of fish skin
Abbreviations
Sebastes schlegeli antibacterial protein.
Trang 2primary structure, they are all positively charged, are
mostly amphipathic, and can form a-helical or b-sheet
structures in membrane-like environments, leading to
membrane destabilization and channel formation in
bacterial cells Recently, the following histones and
derived peptides have also been identified as
antimicro-bial polypeptides in fish skin secretion: histone H2A
and oncorhycin II (histone H1 C-terminal fragment, a
69-residue peptide) from rainbow trout,
Oncorhyn-chus mykiss [13,14], histone H2B from Atlantic cod,
Gadus morhua [15], histone H2B-like protein from
channel catfish, Ictalurus punctatus [16], parasin I
(N-terminus of histone H2A, a 19-residue peptide) from
catfish, Parasilurus asotus [17], hipposin (histone H2A
fragment, a 51-residue peptide) from Atlantic halibut,
Hippoglossus hippoglossus [18] and SAMP H1
(N-ter-minus of histone H1, a 30-residue peptide) from
Atlan-tic salmon, Salmo salar [19] In addition, other kinds
of antibacterial peptides derived from ribosomal and
chromosomal proteins have been detected in the skin
secretions of rainbow trout, O mykiss and Atlantic
cod, G morhua [15,20,21] The antibacterial peptides
from cytosolic or nuclear proteins appear to kill a wide
range of Gram-positive and Gram-negative bacteria,
although their mode of action is not fully understood
We have found a potent antibacterial protein with
strict selectivity against Gram-negative bacteria from
the skin secretion of rockfish, Sebastes schlegeli [22]
It is of particular interest that this protein is selective
against water-borne pathogenic bacteria such as
Aeromonas hydrophila, Aeromonas salmonicida,
Photo-bacterium damselae ssp piscicida and Vibrio
parahae-molyticus but not against enteric bacteria such as
Escherichia coli and Salmonella typhimurium,
suggest-ing the importance of the antibacterial protein as a
primary innate immune strategy in the rockfish skin
In a previous study [22], we obtained the antibacterial
protein by lectin affinity column chromatography and
gel filtration HPLC and reported it to be a
glycopro-tein with a molecular mass of 150 kDa However,
dur-ing subsequent purification, the antibacterial activity
was found only in the later part of the symmetrical
peak obtained by gel filtration HPLC Also, the
con-tent of the antibacterial protein in the symmetrical
peak was found to be very low compared with that of
the 150-kDa protein, leading to our previous
misidenti-fication of the 150-kDa protein as an antibacterial
pro-tein Therefore, the antibacterial protein was again
purified by a revised method to clarify its structure
and functional features in detail We report here that
the antibacterial protein purified from the skin mucus
of S schlegeli (SSAP) is a 120-kDa glycoprotein, being
a new member of the l-amino acid oxidase (LAO)
family, and that its antibacterial action is elicited by
H2O2generated from l-Lys as substrate
Results
Purification and physicochemical properties of SSAP
SSAP was purified by three steps of column chroma-tography It was retained on the concanavalin A (ConA)–Sepharose column and recovered in the manno-pyranoside eluate fraction as reported previously [22]
0 0.1 0.2 0.3 0.4 0.5
0 20 40 60 80 100 120
Retention time (min)
0.1 0.2 0.3 0.4
0 5 10 15
20
Retention time (min)
A
B
40
10 20 30
0
400
100 200 300
0 500
Fig 1 Purification of antibacterial protein, SSAP, by Mono Q HR
(A) Antibacterial fractions obtained by ConA–Sepharose column
buf-fer (pH 6.8) for 10 min and then with a linear gradient method to
minute and used to measure antibacterial activity.
Trang 3(data not shown) In anion-exchange HPLC on a
Mono Q HR5⁄ 5 column, SSAP was eluted between
retention times of 47 and 52 min (Fig 1A) Finally, it
was purified by HPLC using a CHT5-I hydroxyapatite
column, in which it appeared between retention times
of 47 and 49 min (Fig 1B) Purified SSAP afforded a single peak at a retention time of 17.0 min as analyzed
by gel filtration HPLC on a TSKgel G3000SW column (Fig 2A) In RP-HPLC, it gave a major peak at a retention time of 47.3 min along with a minor one at 23.1 min (Fig 2B) The absorbance spectrum, in which the major peak exhibited absorption maxima at 213 and 279 nm and the minor peak at 223, 267, 371 and
447 nm (data not shown), suggested the major peak to
be a proteinaceous component, and the minor peak a flavin-like chromophore In native and SDS⁄ PAGE, purified SSAP afforded only one band (Fig 3A,B) These results demonstrate that the purified SSAP was homogeneous As summarized in Table 1, 1.7 mg SSAP was obtained from 150 mL crude skin mucus extract containing 3890 mg protein The recovery of antibacterial activity was 28%, and 645-fold purifica-tion was achieved on the basis of the minimum inhibi-tory concentration (MIC)
Judging from the chromatography results, SSAP is likely to be an acidic glycoprotein with a molecular
1 2 3 4
0 30 60 90
Retention time (min)
Retention time (min)
200 300
0 100 100
0
50
Absorbance at 280 nm (mU) Absorbance at 220 nm (mU) Concentration of acetonitrile (%)
Fig 2 HPLC of antibacterial protein, SSAP,
on a TSKgel G3000SW column (A) and a
Chromolith Performance RP-18e column (B).
(A) SSAP was subjected to gel filtration
HPLC on a TSKgel G3000SW column,
proteins were used as a reference; 1,
ferr-itin (440 kDa); 2, aldolase (158 kDa); 3,
albu-min (67 kDa); 4, ovalbualbu-min (43 kDa) (B)
SSAP was subjected to RP-HPLC on a
Chromolith Performance RP-18e column,
eluted with a linear gradient of acetonitrile
(0–90% in 60 min) in 0.1% trifluoroacetic
Fig 3 SSAP on native PAGE (A) and SDS/PAGE (B) (A) SSAP was
subjected to native PAGE using a PhastGel homogeneous 20 (B)
containing 1% SDS and 6% 2-mercaptoethanol and subjected to
SSAP was detected using a PhastGel protein silver staining kit.
Table 1 Purification of SSAP MIC is defined as the lowest con-centration that inhibited the growth of P damselae ssp piscicida ATCC 51736.
Step
Protein (mg)
MIC
Total activity
Yield (%)
Trang 4mass of 120 kDa SDS⁄ PAGE analysis revealed a
molecular mass of 53 kDa (Fig 3B), suggesting that
SSAP has a dimeric conformation It was found to
contain 1.6% (w⁄ w) of d-mannose and 0.6% (w ⁄ w) of
N-acetyl-d-glucosamine by HPLC after derivatization
with 4-aminobenzoic acid ethyl ester A sugar moiety
was effectively liberated by digestion of SSAP with
gly-copeptidase F As seen in Fig 4, the digest migrated
ahead of the intact SSAP and gave no detectable band
when the ECL glycoprotein detection module was
used, indicating the presence of an N-linked
carbohy-drate side chain in SSAP
Purified SSAP was yellow-colored, suggesting the
involvement of flavin in the molecule, as supported by
the RP-HPLC result As illustrated in Fig 5A, the
chromophore dissociated from SSAP on heating with
SDS showed absorption maxima at 263, 371 and
447 nm, consistent with those of the FAD standard
Figure 5B shows the mass spectrum of the
chromo-phore from SSAP ESI-TOF-MS in the positive mode
gave the main peak at m⁄ z 786.32 and the ion peaks at
m⁄ z 439.60 and 348.63 The former corresponded to
the parent ion peak of [M + H]+of FAD (molecular
mass 785.56 Da), and the latter ion peaks at m⁄ z
439.60 and 348.63 were assignable to fragment A
(a dehydro ion of adenosine monophosphate, C10H13
N5O7P) and fragment B (a dehydroxy ion of flavin
mononucleotide, C17H20N4O8P), respectively,
accord-ing to a rule of mass shift in fragmentation [23] These
results provide evidence that FAD is the chromophore
of SSAP
The CD spectrum of SSAP is shown in Fig 6 SSAP
clearly gave two negative peaks at 208 and 222 nm in
0.5 m NaCl⁄ 0.01 m Tris ⁄ HCl buffer (pH 8.0), indicating
B
50
37
150
100
75
M
(kDa)
A
membrane (B) before and after treatment with glycopeptidase F.
(A) Protein was detected using a PhastGel protein silver staining
kit (B) Glycoprotein was visualized using an ECL glycoprotein
detection module Lane M, molecular mass marker; lane 1, SSAP;
lane 2, SSAP treated with glycopeptidase F.
B
A
Wave length (nm)
FAD standard Chromophore from SSAP
A
B FAD standard
348.62
439.60
786.30 100
100
348.63
439.60
786.32
m/z
m/z
Chromophore from SSAP
0
0
Fig 5 Absorption spectra (A) and mass spectra (B) of FAD stand-ard and the chromophore derived from SSAP The chromophore was obtained by boiling SSAP in 1% SDS for 10 min, ultrafiltration using an Ultrafree-MC, and RP-HPLC on a Chromolith Performance RP-18e column with a linear gradient of acetonitrile (5–25% in
FAD standard was obtained by application of FAD sodium salt hydrate to RP-HPLC under the same conditions as the chromo-phore The inset in (B) illustrates the structure of FAD.
Trang 5a-helical conformation The a-helical content of SSAP
in the buffer was found to be 20% and increased to 28%
on the addition of SDS irrespective of concentration in
the range 0.1–2.5%
cDNA cloning and sequence analysis of SSAP
The N-terminal amino-acid sequence of SSAP was
determined to be ISLRDNLAD Of the peptide
frag-ments isolated from the lysyl endopeptidase digest by
RP-HPLC, three (fragments 1–3) were randomly
selec-ted and sequenced as follows: SADELLQHALQK for
fragment 1, EGWYAELGAMRIPS for fragment 2, and
SYTWSDDSLLFLGASDED for fragment 3 A cDNA
fragment was successfully amplified by 3¢ RACE using
the degenerate primers designed from the amino-acid
sequence of fragment 1 On the basis of the nucleotide
sequence of this cDNA fragment, the remaining 5¢
region sequence was determined by 5¢ RACE Thus, the
nucleotide sequence of the full-length SSAP cDNA
(2037 bp) was elucidated (DDBJ accession number
AB218876) The accuracy of this sequence was verified
by recloning experiments The 5¢-untranslated region
contains stop codons TAA and TGA at nucleotides 4–6
and 31–33, respectively, upstream of the initial codon
ATG present at nucleotides 46–48 In the
3¢-untrans-lated region, a polyadenylation signal AATAAA is
observed at nucleotides 1997–2002, and a poly(A) tail
at nucleotide 2027 An ORF is composed of 1662 bp, encoding a precursor protein of 554 amino-acid residues from the putative initiating Met to the putative last Leu The amino-acid sequences of the N-terminal portion and the lysyl endopeptidase fragments 1–3 determined
by protein sequencing are all found at positions 59–67 (N-terminal portion), 138–151 (fragment 2), 210–221 (fragment 1) and 436–453 (fragment 3) of the deduced molecule (Fig 7)
signalp version 3.0 (http://www.cbs.dtu.dk/services/ SignalP/) predicted that SSAP consists of a signal pep-tide (Met1–Ala58) and a mature protein starting with Ile59, in accord with the result of N-terminal protein sequence analysis It is likely that the mature protein is composed of 496 amino acids with a calculated molecular mass of 55 260.63 Da A blast homology search showed the similarity of the deduced amino-acid sequence of SSAP to LAOs Amino-amino-acid sequence alignment analysis using clustalw (version 1.83) revealed that SSAP shows a weak homology to anti-bacterial LAOs, such as aplysianin A (11%), achacin (12%) and escapin (12%), and a moderate homology
to Pseudechis australis snake venom LAO (42%) The highest identity (76%) was observed with the apopto-sis-inducing protein (AIP) derived from the viscera of mackerel infected with the nematode, Anisakis simplex [24] (Fig 7)
LAO activity of SSAP SSAP exhibited high LAO activity, with a specific activity of 10.2 UÆmg)1 by the H2O2-generation method SSAP catalyzed oxidation of only l-Lys and was ineffective with any of the other proteinaceous amino acids tested No LAO activity was detected when l-Lys was replaced with d-Lys (Fig 8) From a Lineweaver–Burk plot of LAO activity of SSAP (data not shown), Kmand kcatwere evaluated to be 0.19 mm and 20.4 s)1, respectively
Antibacterial action of SSAP
As shown in Table 2, SSAP specifically inhibited the growth of Gram-negative bacteria, being most active against A salmonicida with an MIC of 0.078 lgÆmL)1, followed by P damselae ssp piscicida,
A hydrophila and V parahaemolyticus with MIC of 0.16, 0.31 and 0.63 lgÆmL)1, respectively LAOs have been reported to show antibacterial activity through
H2O2 generated from amino-acid substrates, which is markedly diminished in the presence of H2O2 scaven-gers such as catalase and peroxidase [25–27] In the present study therefore the inhibitory effect of
Fig 6 CD spectra of SSAP CD analyses were performed using a
represents the mean of three measurements in the range 200–
Trang 6catalase on the antibacterial activity of SSAP was
examined The addition of catalase almost completely
abolished the antibacterial activity of SSAP,
indica-ting that H2O2 is the mediator of the activity of
SSAP
Discussion
This study is the first to discover an LAO with anti-bacterial activity in the skin mucus of a teleost and to reveal the involvement of LAO in the innate immunity
Fig 7 Amino-acid sequence alignment of SSAP (DDBJ accession number AB218876) with AIP from mackerel Scomber japonicus (DDBJ accession number AJ400871), PA-LAO from Pseudechis australis snake venom (DDBJ accession number DQ088992), aplysianin A from Aplysia kurodai (DDBJ accession number D83255), escapin from Aplysia californica (DDBJ accession number AY615888) and achacin from Acatina fulica (DDBJ accession number X64584) Identical amino acids are shaded Potential N-glycosylation sites are boxed Gaps intro-duced into the sequences to optimize the alignment are represented by dashes The N-terminal sequence (59–67) and intrapeptide frag-ments 1 (210–221), 2 (138–151) and 3 (436–453) of SSAP are thick underlined.
Trang 7of fish skin In a previous report [22], the antibacterial
protein was obtained from the skin secretion of
rock-fish S schlegeli by lectin affinity chromatography and
gel filtration HPLC and misidentified as a 150-kDa
glycoprotein consisting of 75-kDa subunits by SDS⁄
PAGE, partly because of the scarcity of the protein of
interest In this study therefore we isolated the
antibac-terial protein, SSAP, from the skin mucus of S
schle-geli by a combination of three different types of
column chromatography and carefully examined the
degree of purity by HPLC as well as PAGE
Further-more, we elucidated the primary structure of SSAP by
cDNA cloning and identified SSAP to be a new
mem-ber of the LAO family by physicochemical and
bio-chemical analyses LAOs (EC 1.4.3.2) catalyze the
stereospecific oxidative deamination of an l-amino-acid substrate to a corresponding a-oxol-amino-acid with the production of H2O2 and ammonia, via an imino-acid intermediate It is well known that these enzymes are widely distributed across diverse phyla from bacteria
to mammals LAOs in micro-organisms appear to be involved in the utilization of ammonia as a nitrogen source, and those in animals have been characterized
as showing distinct biological and physiological effects such as apoptosis, cytotoxicity, hemolysis, platelet aggregation, hemorrhage, edema and antimicrobial activity [28] Since Skarnes [29] first found antibacterial activity in an LAO from snake (Crotalus adamanteus) venom, antibacterial LAOs have been reported from snake venoms of Ps australis [30], Trimeresurus jerdo-nii [31] and Bothrops alternatus [32], the body surface mucus of the giant African snail, Achatina fulica Fe´russac (termed achacin) [26], the albumen gland of the sea hare, Aplysia kurodai (termed aplysianin A) [27], and the ink of the sea hare, A californica (termed escapin) [33]
LAO family members possess in common flavin as a coenzyme and two motifs, a dinucleotide-binding motif comprising b-strand⁄ a-helix ⁄ b-strand of the secondary structure, and a GG motif (R-x-G-G-R-x-x-T⁄ S) shortly after the dinucleotide-binding motif [34] In the case of SSAP, FAD was identified as a coenzyme Moreover, the dinucleotide-binding motif to which FAD binds was certainly recognized at amino-acid resi-dues His93–Glu121, and the GG motif at amino-acid residues Arg125–Thr132 (Fig 7) A blast search found that SSAP shares the highest sequence identity (76%) with AIP from Anisakis-infected mackerel The secon-dary structures of SSAP and AIP appear to be similar
to each other The a-helix content of SSAP was deter-mined to be 28.2% in 2.5% SDS solution by CD spectrometry and calculated to be 31.6% by predator (http://bioweb.pasteur.fr/seqanal/interfaces/predator-simple.html), and that of AIP was estimated to be 26.9% by predator In addition, both SSAP and AIP have strict specificity with respect to the substrate, cata-lyzing the oxidation of only l-Lys These results suggest structural similarity between their substrate-binding sites
SSAP and AIP have two and five potential N-glyco-sylation sites, respectively One site at residues 89–91 is common to both LAOs SSAP was determined to con-tain 2 mol d-mannose and 5 mol N-acetyl-d-glucosa-mine per mol of subunit Treatment of SSAP with glycopeptidase F resulted in deglycosylation (Fig 4), but did not reduce antibacterial activity, suggesting
no or little involvement of the sugar moiety in the antibacterial effect of SSAP It has been reported that
Fig 8 Dependence of substrate concentration on LAO activity of
Table 2 Antibacterial spectrum of SSAP MIC is defined as the
lowest concentration that inhibited the growth of bacteria.
Bacterium
SSAP
SSAP with catalase Gram-positive
Gram-negative
Trang 8glycosylation is not essential for the antibacterial
activ-ity of escapin, which has one putative glycosylation
site, as recombinant escapin expressed in bacteria is as
active as the native one [33] On the other hand, Geyer
et al [35] examined the structure of the glycan moiety
of the LAO from the Malayan pit viper,
Calloselas-ma rhodostoCalloselas-ma, and assumed that putative binding of
the LAO to sialic acid-binding Ig superfamily lectins
via its sialylated glycan moiety results in the
produc-tion of locally high concentraproduc-tions of H2O2 in or near
the binding interface The role of the glycosyl
substitu-ents of LAOs in biological activities remains to be
elucidated
It should be noted that SSAP only has potent
antibac-terial activity against specific Gram-negative bacteria,
including fish pathogens (A hydrophila, A salmonicida
and P damselae ssp piscicida) and a marine bacterium
(V parahaemolyticus), but not against enteric
Gram-negative bacteria (E coli and S typhimurium) In
contrast, other antibacterial LAOs, such as achacin,
aplysianin A and escapin, broadly show an inhibitory
effect on both Gram-negative (E coli) and
Gram-posit-ive bacteria (Bacillus subtilis and Staphylococcus
aure-us) The antibacterial activities of these LAOs, including
SSAP, are significantly decreased in the presence of
cat-alase, confirming that H2O2generation by LAOs brings
about an oxidative burst in cells that is responsible for
cell death Shur & Kim [36] reported that the LAO from
snake (Agkistrodon halys) venom attaches to the cell
surface of mouse lymphocytic leukemia L1210, inducing
apoptosis in a cell-selective manner Similarly, achacin
from the giant African snail binds to the cell wall of
E coli, and its LAO activity is inhibited by
N-acetylneu-raminic acid [26,37] Therefore, it is possible that the
cell-specific antibacterial activity of SSAP is associated
with its binding to the bacterial cell surface Further
investigation is in progress to elucidate the bacterium
selectivity and the mode of action of SSAP Finally,
SSAP is likely to be important in the innate host defense
on the skin of rockfish and might be useful as a
chemo-therapeutic agent, especially in the field of aquaculture
because of its selective cytotoxicity against water-borne
virulent pathogens
Experimental procedures
Materials
Specimens of S schlegeli ranging from 28.0 to 32.5 cm in
body length were obtained from Minami-Sanriku Marine
Center, Motoyoshi, Miyagi Prefecture, Japan, and
trans-ported in oxygen-saturated water to our laboratory
Purification of SSAP The rockfish skin mucus was gently scraped off with a spat-ula, combined, and centrifuged at 18 800 g for 30 min using
an angle rotor (SCR18B; Hitachi, Tokyo, Japan) The result-ing supernatant was applied to a ConA–Sepharose column (2.0· 32.0 cm; GE Healthcare Bio-Science, Piscataway, NJ, USA), which was washed with 1 mm CaCl2⁄ 1 mm MnCl2⁄ 0.5 m NaCl ⁄ 0.02 m Tris ⁄ HCl buffer (pH 7.4) and then eluted with 0.5 m methyl-a-d-mannopyranoside⁄ 0.5 m NaCl⁄ 0.02 m Tris ⁄ HCl buffer (pH 7.4) as reported previ-ously [22] Antibacterial fractions were pooled and subjected
to HPLC on a Mono Q HR5⁄ 5 column (0.5 · 5.0 cm; GE Healthcare Bio-Science) The column was developed by a linear gradient of NaCl (0–0.5 m in 60 min) in 0.01 m Tris⁄ HCl buffer (pH 7.4) at a flow rate of 0.5 mLÆmin)1 Finally, the antibacterial protein was purified by HPLC on a CHT5-I hydroxyapatite column (1.0· 6.4 cm; Bio-Rad Laboratories, Hercules, CA, USA) with a linear gradient elution of 0.01–0.4 m phosphate buffer (pH 6.8) in 60 min at
a flow rate of 0.5 mLÆmin)1 At each chromatographic step, proteins were monitored by recording A280and the antibac-terial protein by growth inhibition against P damselae ssp piscicida The purified antibacterial protein was SSAP For purity determination, SSAP was subjected to either gel filtration HPLC on a TSKgel G3000SW column (0.78· 30 cm; Tosoh, Tokyo, Japan) with 0.5 m NaCl ⁄ 0.01 m Tris⁄ HCl buffer (pH 8.0) at a flow rate of 0.5 mLÆ min)1 or RP-HPLC on a Chromolith Performance RP-18e column (0.46· 10 cm; Merck, Darmstadt, Germany) with a linear gradient of acetonitrile (0–90% in 60 min) in 0.1% trifluoroacetic acid at a flow rate of 1.0 mLÆmin)1 SSAP was monitored by recording A220or A280with a photodiode array detector SSAP was analyzed for its homogeneity by PAGE with a PhastSystem apparatus (GE Healthcare Bio-Science) according to the manufacturer’s instructions Native PAGE was carried out on a PhastGel homogeneous
20 with PhastGel native buffer strips, and SDS⁄ PAGE on a PhastGel gradient 8–25 with PhastGel SDS buffer strips Before SDS⁄ PAGE, SSAP was dissolved in 0.01 m Tris ⁄ HCl buffer (pH 6.8) containing 1% SDS and 6% 2-mercaptoeth-anol and heated in a boiling-water bath for 5 min After being run, the gel was stained with a PhastGel protein silver staining kit (GE Healthcare Bio-Science)
Analysis of amino-acid sequence Amino-acid sequence analyses of SSAP and its peptide fragments produced by digestion with lysyl endopeptidase (EC 3.4.21.50; Wako Pure Chemical Industries, Osaka, Japan) were performed with an automatic gas-phase sequencer (LF-3400D TriCart with high sensitivity chem-istry; Beckman Coulter, Fullerton, CA, USA) To produce peptide fragments, SSAP (60 lg) was denatured in 400 lL
Trang 94 m urea, diluted twofold with 0.1 m Tris⁄ HCl buffer
(pH 9.3) and digested with 1 lg lysyl endopeptidase at
37C for 24 h The digest was subjected to RP-HPLC on a
Chromolith Performance RP-18e column (0.46· 10 cm;
Merck) with a linear gradient of acetonitrile (0–70% in
120 min) in 0.1% trifluoroacetic acid at a flow rate of
0.5 mLÆmin)1 Peptides were monitored at 220 nm with a
photodiode array detector
cDNA cloning
Total RNA was extracted from the skin of a live specimen
with TRIzol reagent (Invitrogen, Carlsbad, CA, USA)
First-strand cDNA was synthesized from 5 lg total RNA using a
3¢ RACE System for Rapid Amplification of cDNA Ends
Kit (Invitrogen) following the manufacturer’s instructions
Oligonucleotide primers were designed on the basis of the
determined amino-acid sequences of peptide fragments The
degenerate primer 5¢-CIGAYGARYTIYTICARAYGCIYT
IC-3¢ (forward; corresponding to 211
ADELLQHAL219) and the abridged universal amplification primer (AUAP) 5¢-GG
CCACGCGTCGACTAGTAC-3¢ (reverse) were used for the
first 3¢ RACE, and the degenerate primer 5¢-AYGARYTIY
TICARCAYGCIYTICARAA-3¢ (forward; corresponding to
212
DELLQHALQK221) and the AUAP (reverse) for the
sec-ond 3¢ RACE Amplification was carried out using rTaq
polymerase (Takara, Otsu, Japan) under the following
condi-tions: 94C for 5 min; 35 cycles of 94 C for 30 s, 55 C for
30 s and 72C for 90 s; and 72 C for 5 min The secondary
PCR products were subcloned into the pT7Blue T-vector
(Novagen, Darmstadt, Germany), and their nucleotide
sequences were analyzed with a Thermo Sequenase Cy5 Dye
Terminator Sequencing Kit (GE Healthcare Bio-Science)
and a Long-Read Tower DNA Sequencer (GE Healthcare
Bio-Science) The remaining 5¢-terminal sequence was
ana-lyzed by 5¢ RACE as follows First-strand cDNA was
syn-thesized from 5 lg total RNA using a 5¢ RACE System for
Rapid Amplification of cDNA Ends Kit (Invitrogen) and the
gene-specific primer
(5¢-CCTTCTTCTTTCAGATAATCC-3¢, corresponding to244KDYLKEEG251) The first 5¢ RACE
reaction was completed using the gene-specific primer
(5¢-AGAGTAACGGTCATATTTTAGC-3¢, corresponding to
235
LLKYDRYS242) and the abridged anchor primer (AAP)
5¢-GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGI
IG-3¢, followed by reamplification of the PCR products using
the gene-specific primer (5¢-CCACCTCATCTTGCACCT
TC-3¢, corresponding to220
QKVQDEVE227) and the AUAP
Amplification conditions were the same as described above
The secondary PCR products were subcloned into the
pT7Blue T-vector and sequenced The nucleotide sequence
of the full-length SSAP cDNA was confirmed by RT-PCR
using the forward primer 5¢-ATAACTTTGGAGACGG
AGTTC-3¢ and the reverse primer 5¢-TGGAGGAACATTA
GTGGTCC-3¢
Measurement of LAO activity LAO activity was assayed in a 96-well microtiter plate by the peroxidase⁄ o-phenylenediamine method [24] with slight modifications For the standard assay, 25 lL SSAP, 25 lL
20 mm l-Lys, 25 lL peroxidase (0.2 UÆmL)1; Type VI;
EC 1.11.1.7; Sigma-Aldrich Corp., St Louis, MO, USA) and 25 lL o-phenylenediamine (0.5 mgÆmL)1) were added sequentially to the well After incubation at 37C for
150 min, the reaction was terminated by adding 100 lL 1 m
H2SO4, and A490was measured The substrate specificity of SSAP was examined using Gly, 19 l-amino acids and
d-Lys as the substrates To determine kinetic parameters, various concentrations (0–5 mm) of l-Lys were used Km was evaluated using a Lineweaver–Burk plot by measuring the rate of H2O2production
Measurement of antibacterial activity Antibacterial activity was measured by a liquid growth-inhi-bition assay in a 96-well microtiter plate, as previously repor-ted [22] The following nine species of bacteria were used: three species of Gram-positive bacteria, B subtilis IAM1026, Micrococcus luteus IAM1056 and Staph aureus IAM1011; six species of Gram-negative bacteria, A hydrophila IAM12337, A salmonicida JCM7874, E coli JCM1649,
P damselae ssp piscicida ATCC51736, S typhimurium SH-1 isolated from fish meal, and V parahaemolyticus NBRC12711 During the isolation procedure of the antibac-terial protein, P damselae ssp piscicida was used because of its high sensitivity to SSAP [22] Briefly, a mixture of 50 lL sample solution and 50 lL bacterial suspension (1· 105
col-ony-forming unitsÆmL)1), both of which were made in Muller-Hinton broth medium (Difco Laboratories, Detroit,
MI, USA), was incubated at 25C for 24 h After incuba-tion, bacterial growth was observed with the unaided eye When the culture medium was completely transparent or no precipitate was recognized, inhibition of bacteria growth was judged to have occurred The reciprocal of the maximum inhibitory dilution was used to calculate arbitrary units (AU) per ml The MIC was defined as the lowest concentration that inhibited the growth of bacteria To examine the inhibi-tory effect of catalase on the antibacterial activity of SSAP,
50 U catalase (EC 1.11.1.6; from bovine liver; Wako Pure Chemical Industries) was added to the culture medium con-taining 5 lgÆmL)1SSAP and bacterial cells, and the mixture was incubated under the same condition as described above
Determination of molecular mass The molecular mass of the native SSAP was determined
by gel filtration HPLC on a TSKgel G3000SW column (0.78· 30 cm; Tosoh) as described above Four reference
Trang 10proteins, ferritin (440 kDa), aldolase (158 kDa), albumin
(67 kDa) and ovalbumin (43 kDa), were used to calibrate
the column The molecular mass of the denatured SSAP
was determined by SDS⁄ PAGE on a PhastGel gradient
8–25 with PhastGel SDS buffer strips (GE Healthcare
Bio-Science) as described above Precision Plus
Pro-tein standard (Bio-Rad Laboratories) was used as a
refer-ence
Analysis of FAD
An aliquot of SSAP (5 lg) was boiled in 200 lL 1% SDS
for 10 min and filtered with an Ultrafree-MC (molecular
mass cut off 10 kDa; Millipore, Bedford, MA, USA) The
filtrate was chromatographed on a Chromolith Performance
RP-18e column (0.46· 10 cm; Merck) with a linear
gradi-ent of acetonitrile (5–25% in 10 min) in 0.1%
trifluoroace-tic acid at a flow rate of 1.0 mLÆmin)1, with monitoring by
recording A447 The eluate containing the chromophore was
collected and subjected to spectrophotometry and MS The
MS⁄ MS measurement of the chromophore was performed
using a Q-tof Ultimate API (Micromass, Altrincham, UK)
in the positive-ion mode under the following conditions:
capillary voltage, 3200 V; cone voltage, 100 V; desolvation
temperature, 150C; source temperature, 80 C; collision
gas, argon
CD spectrometry
SSAP (30 lg) was dissolved in 3 mL 0.5 m NaCl⁄ 0.01 m
Tris⁄ HCl buffer (pH 8.0) containing 0%, 0.1%, 0.5% or
2.5% SDS and subjected to CD analysis with a CD
tropolarimeter (J-720; Jasco, Tokyo, Japan) The CD
spec-tra were measured at 25C with 40 scans per min in the
range 200–250 nm at 0.5-nm intervals a-Helical contents
were calculated by the method of Greenfield & Fasman
[38]
Carbohydrate analysis
SSAP (2 lg) was deglycosylated by incubation with
glyco-peptidase F (25· 10)3U; EC 3.5.1.52; Sigma-Aldrich Corp.)
in 10 lL 0.05 m Tris⁄ HCl buffer (pH 8.6) at 37 C for 18 h
and subjected to SDS⁄ PAGE The proteins separated by
SDS⁄ PAGE were electrotransferred from the gel to a
poly(vinylidene difluoride) membrane and visualized by the
chemical luminescence method using an ECL glycoprotein
detection module (GE Healthcare Bio-Science) following the
manufacturer’s instructions Total sugar composition
analy-sis was performed using the ABEE (4-aminobenzoic acid
ethyl ester) labeling kit plus S (Seikagaku Corporation,
Tokyo, Japan) and HPLC on a Honenpak C18
(0.46· 7.5 cm; Seikagaku Corporation), as reported
previ-ously [22]
Determination of protein Protein was determined by the micro bicinchoninic acid method [39] using BSA as a standard protein
Acknowledgements
This study was partly supported by a Grant-in Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan We thank Dr
Y Shida, Tokyo College of Pharmacy, for MS analysis for FAD, and Mr T Katsukura and Mr H Oikawa, Minami-Sanriku Marine Center, for provision of the rockfish specimens
References
1 Iwama G & Nakanishi T (1996) The Fish Immune System Academic press, San Diego, CA
2 Bayne CJ & Gerwick L (2001) The acute phase response and innate immunity of fish Dev Comp Immunol 25, 725–743
3 Ellis AE (2001) Innate host defense mechanisms of fish against viruses and bacteria Dev Comp Immunol 25, 827–839
4 Alexander JB & Ingram GA (1992) Noncellular nonspecific defense mechanism of fish Annu Rev Fish Dis 2, 249–279
5 Kozineko II, Isaeva NM & Balakhnin IA (1999) Humoral factors of nonspecific defense in fish
J Ichthyol 39, 322–328
6 Maganadottir B (2006) Innate immunity of fish (overview) Fish Shellfish Immunol 20, 137–151
7 Oren Z & Shai Y (1996) A class of highly potent antibacterial peptides derived from pardaxin, a pore-forming peptide isolated from Moses sole fish Pardachirus marmoratus Eur J Biochem 237, 303–310
8 Cole AM, Weis P & Diamond G (1997) Isolation and characterization of pleurocidin, an antimicrobial peptide
in the skin secretions of winter flounder J Biol Chem
272, 12008–12013
9 Saint N, Cadiou H, Bessin Y & Molle G (2002) Antibacterial peptide pleurocidin forms ion channel
in planner lipid bilayer Biochim Biophys Acta 1564, 359–364
10 Yokota H, Nagashima Y & Shiomi K (2001) Interac-tion of grammistins with lipids and their antibacterial activity Fish Sci 67, 928–933
11 Sugiyama N, Araki M, Ishida M, Nagashima Y & Shiomi K (2005) Further isolation and characterization
of grammistins from the skin secretion of the soapfish Grammistes sexlineatus Toxicon 45, 595–601