From the Fisheries Laboratory, the University of Tokyo, Shizuoka 431-0211, Japan
Received for publication, January 30, 2003
, and in revised form, March 20, 2003.
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ABSTRACT |
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INTRODUCTION |
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Fish species with lectins in the integumental mucus include the windowpane flounder Lophopsetta maculata (7), the ling Genypterus blacodes (8), the Arabian Gulf catfish Arius thalas-sinus (9), the Oriental catfish Plotosus lineatus (10), the conger eel Conger myriaster (11, 12), the dragonet Repomucenus rich-ardsonii (13), the loach Misgurnus anguillicaudatus (14), the kingklip Genypterus capensis (15), and the Japanese eel Anguilla japonica (16). Lectins are probably present in the skin mucus of many additional species, because the mucus of many fishes induces hemagglutination, a property typical of lectins (17). Despite this apparent ubiquity of mucus lectins, experimental data that elucidate their assumed function in pathogen defense are scarce.
On the basis of the structure of the carbohydrate recognition domain, animal lectins are divided into four main groups: C-type (20), galectins (21, 22), P-type (23), and I-type (24). So far, sequences of mucosal lectins have been determined in only two species of the Anguilliformes, namely the conger (congerin I and II) and the Japanese eel (AJL-2). Congerin I and II were classified as galectins (18, 19); this class probably dominates among the skin mucus lectins because many of them interact specifically with galactose or lactose. On the other hand, AJL-2 has a sequence typical of C-type lectins (16). Lectins of ling and kingklip might belong to the same class because of the Ca2+ dependence of their activity and their typical SDS-PAGE pattern (8, 15). However, it is questionable whether all skin mucus lectins belong to the galectins or the C-type. Unfortunately, the sound knowledge of lectin structure that is necessary to establish the biological role and the functional mechanisms of lectins in the fish integumental mucus is mostly lacking to date.
The pufferfish, Fugu rubripes, is a model species in which the genome sequencing project has reached an advanced stage (25). We have studied the mucus of the skin and intestine of this species and found interesting lectins that resemble those of monocotyledonous plants. Here we report the primary structure, tissue-specific expression, and biochemical characteristics of these lectins. In addition, we describe binding of the lectin to Heterobothrium okamotoi, a serious parasite of the Fugu (26).
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EXPERIMENTAL PROCEDURES |
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Hemagglutination TestsSerial 2-fold dilutions of the samples (25 µl) with PBS(+) buffer were made on microtiter plates with 96 wells. Twenty five µl of a 5% suspension of rabbit red blood cells purchased from commercial dealers were added to each well. After 1 h of incubation at room temperature, samples were checked for hemagglutination. The lectin titer was expressed as the greatest sample dilution in which agglutination could be observed.
Partial Purification by Ion-exchange Column ChromatographyThe crude skin mucus extract was analyzed by fast protein liquid chromatography (FPLC, Amersham Biosciences) on a Poros HQ/M (4.8 x 100 cm, PerSeptive Biosystems) ion-exchange column with a linear gradient of 01 M NaCl in 20 mM Tris-HCl buffer (pH 8.0) at a flow rate of 1 ml/min. The eluted proteins were monitored by absorbance at 280 nm, and hemagglutination activity was tested in each 1-ml fraction. Active fractions were combined and concentrated for determination of carbohydrate specificity.
Determination of Specific SugarsSugar-dependent inhibition of lectin-induced hemagglutination was determined to establish specific sugar-lectin interactions. Twenty five µl of the active fraction identified by FPLC was serially 2-fold diluted with PBS(+) buffer containing 200 mM of either D-glucose, D-galactose, D-mannose, D-fucose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, maltose, lactose, saccharose, or melibiose in microtiter plates. After incubation at 4 °C for 1 h, 25 µl of a 5% rabbit erythrocyte suspension was added to each well, and the lectin titer was determined after 1 h as detailed above.
Lectin Isolation by Affinity ChromatographyD-Mannose, which specifically interacted with the mucus lectin, was coupled to epoxy-activated Sepharose 6B (Amersham Biosystems) as a ligand according to the manufacturer's instructions. In brief, the epoxy-activated Sepharose 6B matrix was incubated with 3 mM D-mannose in NaOH solution containing 0.5 M NaCl (pH 11.5) at 40 °C for 16 h; then the remaining active sites on the gel were blocked with 1 M ethanolamine (pH 8.0). The crude skin mucus extract was pumped at 10 ml/h through a column (1 x 10 cm) packed with the gel that had been pre-equilibrated with PBS(+) buffer. Afterward, the column was washed with PBS(+) buffer to remove unabsorbed material. Then proteins were eluted from the column with PBS(+) buffer containing 50 mM D-mannose and were collected manually by monitoring the absorbance of the eluate at 280 nm.
ElectrophoresisSDS-PAGE was performed according to Laemmli (27) using 15% separating gel in the presence or absence, respectively, of 2-mercaptoethanol. Five µl of affinity-purified fractions were added to the same volume of SDS sample buffer containing 4% SDS with or without 10% 2-mercaptoethanol, respectively. The samples were loaded onto the gels together with a set of markers (Dalton Mark VII-L, Sigma), and gels were run at 20 mA for 1.5 h. After electrophoresis, protein bands were visualized by Coomassie Brilliant Blue R-250. To determine whether sugar residues were attached to the lectin detected, lectin purified by affinity chromatography was separated by SDS-PAGE and was blotted from the gel onto a polyvinylidene difluoride membrane (Atto Instruments) at 100 mA for 1 h. The membrane was stained with periodic acid-Schiff reagent using a periodic acid-Schiff staining kit (Muto Kagaku).
Isoelectric focusing was performed using 4% polyacrylamide isoelectric focusing gel containing 8 M urea and 2% ampholine (pH 3.59.5, Amersham Biosciences). A set of two-dimensional protein pI markers (Dai-ichi) was loaded onto the gel together with the sample. After focusing at 150 V for 5 h, the gel was stained with Coomassie Brilliant Blue R-250.
Molecular Mass DeterminationThe molecular mass of intact lectin was determined by gel filtration and carried out by FPLC on a column (1 x 30 cm) of Superose 6 (Amersham Biosciences) with 20 mM Tris-HCl buffer containing 150 mM NaCl (pH 8.0). The flow rate was maintained at 0.5 ml/min, and the absorbance at 280 nm of the eluate was monitored. Albumin (67 kDa), ovalbumin (43 kDa), and ribonuclease (13.7 kDa) were used as standards. The mass of the lectin subunit was also determined by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry (Voyager DE-STR, PerkinElmer Life Sciences). For this test, lectin purified by affinity chromatography was embedded in a sinapinic acid (5-dimethoxy-4-hydroxycinnamic acid) matrix.
Enzymatic Digestion and Amino Acid SequencingInitial amino acid sequencing attempts showed that the N terminus of the lectin was blocked. Therefore, internal amino acid sequences were determined following enzymatic digestion. Affinity-purified lectin was dissolved in 50 µl of 0.4 M Tris-HCl buffer (pH 9.0) containing 2 M urea and was incubated with lysyl endopeptidase (Wako) at an enzyme to substrate ratio of 1:20 (w/w) at 37 °C for 16 h. The digest was fractionated by high performance liquid chromatography (HPLC, Hitachi) on a TSKgel ODS-120T reversed-phase column (4.6 x 250 mm) (Tosoh) using a linear gradient of acetonitrile (080%) in 0.05% trifluoroacetic acid. The flow rate was 1 ml/min. Amino acid sequences of digested fragments were determined using a model 476A protein sequencer (PerkinElmer Life Sciences).
Molecular Cloning of cDNATotal RNA was extracted from the skin of three individuals using RNA extraction solution (Isogen, Nippon gene). First strand cDNA was synthesized from the total RNA with the SMARTTM RACE cDNA amplification kit (Clontech) for 5'- and 3'-rapid amplification of cDNA ends (RACE). Based on the amino acid sequences of the fragments LP-5 and LP-13 (described under "Results"), two degenerate primers were designed: LP-13F, 5'-GA(A/G)AT(A/C/T)GCI(C/T)TITTTCA-3', and LP-5R, 5'-CATICC(A/G)TG(A/G)TCIC(G/T)(A/G)TCI(C/G)(A/T)GTTC-3'. The first strand cDNA was amplified by PCR (i-Cycler, Bio-Rad) in 20 µl of a reaction mixture containing 5 units of Taq DNA polymerase (Takara), 2.5 µl of 10x Universal Primer Mix (Clontech), and 500 nM of LP-13F primer for 3'-RACE or LP-5R primer for 5'-RACE, respectively. Amplification was performed in 40 cycles consisting of 94 °C for 5 s, 60 °C for 10 s, and 72 °C for 2 min; the final elongation reaction was conducted at 72 °C for 10 min. To confirm independently the sequence of the 5'-untranslated region, which had been found to lack a signal peptide, first strand cDNA was synthesized from 1 µg of skin total RNA of another individual using the GeneRacerTM kit (Invitrogen) for 5'-RACE. Reverse transcription was performed at 60 °C for 90 min with ThermoScript (Invitrogen) to remove any RNA secondary structure. GeneRacerTM 5' primer and the specific primer FML-5'-RACE, 5'-CTCTTGAGCTCAGAGCCTTTCTC-3', which was synthesized on the basis of the determined sequence, were used for PCR amplification.
The nucleotide sequence obtained by 3'- and 5'-RACE over the coding region was confirmed by PCR amplification with the two specific primers FML-SP2 (5'-ATCTTCCTCCTTCTGCTTGCAGTCTA-3') and FML-AP4 (5'-GTGATGAGAGTTTGGCTTTC-3'). Denaturation was performed at 94 °C for 3 min, followed by 25 cycles of 94 °C for 30 s, 62 °C for 30 s, and 72 °C for 1 min. The resulting PCR product was also used as a probe for Northern blot analysis, and the above-mentioned primers were employed for the RT-PCR analysis described below.
Each PCR product generated was ligated into the pCR®2.1-TOPO vector (Invitrogen). The plasmid DNA was purified and sequenced with the 310 Genetic Analyzer (Applied Biosystems). Sequences were analyzed using the SeqEdTM version 1.0.3 software (Applied Biosystems). Analyses were repeated with at least five independent PCR amplifications to avoid PCR errors.
Homologs of the deduced amino acid sequence were searched with the BLAST 2.0. program accessed via GenomNet (blast.genome.ad.jp/). Multiple alignment of sequences was conducted using the ClustalW Multiple Sequence Alignment Program version 1.8 (clustalw.genome.ad.jp/).
Reverse Transcription-PCR AnalysisTotal RNA from muscle, liver, heart, kidney, head kidney, spleen, brain, gonad, gill, oral cavity wall, esophagus, intestine, and skin was isolated from three individuals as described above. Each total RNA sample was reverse-transcribed with the SMARTTM RACE cDNA amplification kit. PCR amplification was performed in a total volume of 20 µl with Taq DNA polymerase and specific primers, FML-SP2 and FML-AP4 (500 nM each), for 25 cycles of 94 °C for 30 s, 62 °C for 30 s, and 72 °C for 1 min. Alternatively, the following oligonucleotide primers were synthesized based on the sequence of the intestine-type isoform: FIL-SP1, 5'-CCATGTCCGTCACAGTCTTGGAGAAT-3'; and FIL-AP2, 5'-ATCGGAGTTCCAGAGCTGCTGGCA-3'. These were used to detect the expression of the intestine-type isoform. The protocol of PCR amplification was the same as described above. The PCR products were analyzed by 2.0% agarose gel electrophoresis and staining with ethidium bromide.
Northern Blot AnalysisTen µg of the same total RNA from 13 tissues as used in RT-PCR analysis were separated on a 1.5% formamide-agarose denaturing gel in 1x MOPS running buffer (20 mM MOPS, pH 7.0, 8 mM acetate, and 1 mM EDTA) and then blotted onto a Biodyne® nylon membrane (PALL) using 20x SSC (3 M NaCl, 0.3 M sodium citrate, pH 7.0) as transfer buffer. The membrane was UV cross-linked with a Tl-2000 Ultraviolet Translinker (Ultraviolet Products). A partial cDNA of the lectin amplified by PCR as described above was labeled with alkaline phosphatase using the AlkPhos Direct Labeling kit (Amersham Biosystems) according to the manufacturer's instructions and was used as a probe. Hybridization was conducted using the same kit at 55 °C for 16 h. After hybridization, the membrane was washed and then treated with CDP-Star chemiluminescent detection reagent (Amersham Biosystems). Signals were detected on a LAS-1000 plus image analyzer (Fuji Film) after exposure for 1 h. Sizes of RNA bands were estimated by comparison to a standard 0.161.77-kb RNA ladder (Invitrogen).
Characterization of Hemagglutination Activity of the LectinTo examine divalent cation requirements of the purified lectin, serial 2-fold dilutions of the lectin having hemagglutination titer values of 26 (25 µl) was made with PBS(-) buffer containing either 50 mM EDTA or 50 mM CaCl2. After 1 h incubation with 25 µl of 5% rabbit erythrocyte suspension, the lectin titer was measured as detailed above.
The inhibitory effects of saccharides on the hemagglutination activity of the lectin were assayed as follows. Twenty five µl of the saccharide solutions tested in this study were diluted 2-fold in series on microtiter plates with PBS(+) buffer and incubated with the same volume of lectin solution diluted to a titer of 26 for 1 h at room temperature. Then 50 µl of a 5% rabbit erythrocyte suspension were added to each well and incubated for 2 h at room temperature, and the concentration of each saccharide leading to complete inhibition was determined.
Agglutination Test with BacteriaFive strains of fish
pathogenic bacteria (provided from the National Research Institute of
Aquaculture, Fisheries Research Agency, Japan) were tested for agglutination
with affinity-purified lectin. The Gram-negative bacteria, Aeromonas
hydrophila FPC 866, Edwardsiella tarda FPC 499, and
Pseudomonas plecoglossicida FPC 337 were cultured in tryptic soy
broth (TSB), and Vibrio anguillarum FPC 675 was grown in TSB
containing 2% NaCl. The Gram-positive Streptococcus difficile FPC 576
was cultured in brain heart infusion with 0.5% glucose. The number of cells
was estimated by measuring colony-forming units/ml. The cells were collected
by centrifugation at 3,000 x g for 1 min, resuspended in 0.5%
(v/v) formalin, and shaken at 25 °C for 24 h. Formalin-killed cells were
collected and suspended at 108 colony-forming units/ml in
PBS(+) buffer. Aliquots of these cells were mixed with equal volumes of
affinity-purified lectin at a concentration equivalent to a titer of
1:25 as determined in hemagglutination tests with rabbit
erythrocytes. After incubation at room temperature for 1 h, agglutination was
observed under a light microscope BX60 (Olympus).
Binding Test with ParasiteFluorescence-labeled lectin was prepared using the Alexa Fluor® 488 protein labeling kit (Molecular Probes) according to the manufacturer's directions. By using tweezers, the adult H. okamotoi (about 1 cm long), which is a monogenean trematode that parasitizes Fugu (26), was excised from the gill chamber of infected fish. Parasites were fixed with 0.5% formalin in PBS(+) buffer for 5 min and washed with pure PBS(+) buffer. Fixed parasites were immersed in a blocking solution (PBS(+) buffer containing 1 mg/ml bovine serum albumin) at room temperature for 1 h to block nonspecific binding sites. Subsequently, they were incubated with a 1:4 dilution of Alexa-labeled lectin in blocking solution at room temperature for 1 h. As a control, fixed parasites were incubated with 1:4 dilution of Alexa-labeled lectin in blocking solution containing 1 M D-mannose. Finally, parasites were washed carefully with PBS(+) buffer to remove excess lectin and were placed on glass slides for observation under a fluorescence microscope BX60 (Olympus).
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RESULTS |
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Purification of Skin Mucus LectinA single protein peak was obtained from the crude skin mucus extract of Fugu by D-mannose affinity chromatography when the medium was changed to PBS(+) buffer containing 50 mM D-mannose (Fig. 2). The peak fraction showed hemagglutination activity (data not shown).
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In both unreduced and reduced SDS-PAGE, the affinity-purified fraction
yielded a protein band of 14 kDa, which was one of the major soluble
proteins in the skin mucus (Fig.
3). This protein, termed pufflectin, was negative to periodic
acid-Schiff staining (data not shown), suggesting that it is not glycosylated.
The isoelectric point of the lectin was 6.42 (data not shown).
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The molecular mass of pufflectin was determined to be 26 kDa by gel
filtration, whereas MALDI-TOF mass spectrometry suggested a mass of about
13.05 kDa (Fig. 4),
respectively. Together with the results from SDS-PAGE, these findings suggest
that native pufflectin is a dimer of identical 1314-kDa subunits, which
are non-covalently linked.
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Amino Acid Sequence AnalysisAffinity-purified pufflectin was subjected to Edman degradation, but no sequence could be determined, indicating that the N terminus was blocked. Therefore, we analyzed internal fragments obtained by enzymatic cleavage. Four fragments separated by reversed-phase HPLC (LP-2, -5, -10, and -13; Fig. 5) were scrutinized, and two internal amino acid sequences could be established (LP-5: NH2-Asp-Gly-His-Gln-Leu-Trp-Asn-Ser-Asp-Arg-Asp-His-Gly-Met-COOH; LP-13: NH2-Trp-Ile-Ala-Leu-Phe-Gln-COOH).
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Molecular CloningThe partial cDNAs encoding pufflectin
including the 5'- and 3'-untranslated regions were isolated by
5'- and 3'-RACE methods, respectively. The overlapping sequence
was confirmed by PCR amplification with two specific primers, FML-SP2 and
FML-AP4. The resulting pufflectin cDNA sequence consisted of 527 bp, including
59 bp of 5'-untranslated region, 348 bp of open reading frame, a stop
codon (TGA), and 120 bp of 3'-untranslated region containing a
polyadenylation signal (Fig.
6). The open reading frame encoded 116 amino acids with a
calculated molecular mass of 13,133 Da, which was in good agreement with the
conclusions drawn from the biochemical analyses discussed above. There were no
ATG translation start sites upstream of the open reading frame, indicating
that this lectin did not possess a signal peptide. This feature is the common
characteristic of the galectins, a super-family of animal lectins that bind to
-galactosides (21,
28).
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Homology AnalysisA data base search indicated that the amino acid sequence of pufflectin was not similar to other animal lectins but to mannose-binding lectins of monocotyledonous plants such as common snowdrop, Galanthus nivalis (29), Narcissus hybrid cultivar (GenBankTM accession M88120 [GenBank] ), Clivia miniata (30), leek, Allium prorrum (31), and garlic, Allium sativum (32) (Fig. 7). The highest degree of homology was found in the common snowdrop lectin, G. nivalis agglutinin (29.4% identity).
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RT-PCR AnalysisTo establish the organ specificity of pufflectin expression, total RNA from different tissues was reverse-transcribed into cDNA. The PCR amplification of the cDNA provided bands of the predicted size (432 bp) in gill, oral cavity wall, esophagus, and skin but not in muscle, liver, heart, kidney, head kidney, spleen, brain, gonads, and intestine (Fig. 8A). There were no differences in the expression patterns between the three individuals tested. The PCR products amplified from positive tissue samples were subcloned and sequenced and were confirmed to be the pufflectin cDNA partial sequences.
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Northern Blot AnalysisPufflectin mRNA was present in gill, oral cavity wall, esophagus, intestine, and skin but not in muscle, liver, heart, kidney, head kidney, spleen, brain, and gonads as detected by Northern blot analysis (Fig. 9). The relative level of gene expression in the skin was higher than in the other positive tissues. The expression pattern was identical in the three individuals tested.
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Isolation of IsoformsThe organ specificity of expression patterns determined by two alternative methods was similar except for the intestine, which was positive in the Northern blot analysis but negative in the RT-PCR-based approach. This result could be explained by the existence of an intestine-specific isoform of pufflectin. We attempted to isolate this isoform by 3'- and 5'-RACE using degenerate primers (LP-13F and LP-5R, respectively), and we were able to isolate the cDNA encoding the intestine-type pufflectin (Fig. 10). In its nucleotide sequence, adenine 9, guanine 31, and thymine 432 replaced cytosine 9, adenine 31, and cytosine 432, respectively, found in pufflectin (Fig. 10). Therefore, neither of the primers used in RT-PCR analysis (FML-SP2 or FML-AP4) could recognize this isoform. The deduced amino acid sequence of the isoform showed 91.4% identity with pufflectin.
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Expression of the intestine-type isoform gene in muscle, liver, heart, kidney, head kidney, spleen, brain, gonad, gill, oral cavity wall, esophagus, intestine, and skin was examined by RT-PCR analysis. However, the isoform was detected exclusively in the intestine (Fig. 8B).
Effects of Divalent Cation and Sugars on PufflectinThe hemagglutination activity of pufflectin was not altered by the addition of either EDTA or CaCl2, indicating that pufflectin-induced agglutination does not require calcium.
Inhibition of hemagglutination activity via the addition of sugars occurred with D-mannose at a concentration of 125 mM (Table II). Other saccharides showed no inhibition at concentrations of 500 mM.
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Bacterial AgglutinationAt a concentration equivalent to the titer of 25 that was fully effective in inducing rabbit erythrocyte hemagglutination, pufflectin showed no agglutination activity against any of the five bacterial species tested (see under "Experimental Procedures" for list of species).
Binding to Parasitic TrematodeH. okamotoi treated with Alexa-labeled pufflectin in blocking solution showed pronounced fluorescence on the surface of its body, which was reduced substantially in the presence of 1 M D-mannose (Fig. 11).
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DISCUSSION |
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In several mammalian and bird species, mannose-binding lectins (MBL) in the serum are known to activate the lectin pathway of the complement system (34, 35). Because cDNAs encoding MBL homologues were detected in the family Cyprinidae (36), bony fishes also are assumed to possess MBL and the lectin pathway (37). Pufflectin specifically binds mannose, but its amino acid sequence does not resemble that of any known MBL. Furthermore, the SDS-PAGE pattern of the plasma mannose-specific lectin of Fugu purified by the same method as pufflectin is different from that of pufflectin (data not shown). Thus, pufflectin is unlikely to be derived from the serum lectins.
Pufflectin mRNA was widely expressed in both internal and external surface tissues, i.e. gill, oral cavity wall, esophagus, and skin. An intestine-specific isoform was also identified in this study. The two putative mannose-binding sites are conserved in the intestine-type isoform as Gln30, Asp32, Asn34, Tyr38 and Gln54, Asp56, Asn58, Tyr62 (Fig. 10), suggesting that the isoform also possesses lectin activity. These facts are well explained by the duplication-degeneration-complementation model, which predicts that pairs of duplicated genes share the ancestral functions but are expressed differentially in the various domains in which the ancestral gene was expressed uniformly before the duplication (38). Previously, information on expression patterns of mucosal lectins has been limited to the Anguilliformes. Congerins in the conger eel are expressed in skin, gills, and the upper alimentary canal from the oral cavity to the esophagus, but not in stomach and intestine (39). In the Japanese eel, the lactose-binding lectin, AJL-2, is produced in the skin only (16), and the two galactose-specific C-type lectins, eCL-1 and eCL-2, are expressed exclusively in the gills (40). None of these lectins is present in the intestine. On the other hand, a lactose-specific lectin, GJL-I, was isolated from stomach and intestine of the toxic moray eel, Gymnothrax javanicus (41), but skin mucus lectins have not been studied in this species. Therefore, the intestine-type isoform of pufflectin is the first intestinal lectin shown to be homologous to a skin mucus lectin. This finding suggests that the skin and the integuments of digestive organs share common defense mechanisms in Fugu.
Pufflectin did not induce agglutination in any of the five bacterial species investigated in this study. A number of fish skin mucus lectins were reported to possess agglutination activity against bacteria, but the activity was specific for certain bacterial species. For instance, skin mucus lectins of windowpane flounder agglutinated Microcyclus marina and Metschnikowia reukaufii, but did not affect six other strains (7). Therefore, effects of pufflectin on a greater number of bacterial species including virulent and avirulent strains should be explored before general conclusions regarding the role of pufflectin in the defense against bacterial invasions are drawn.
Pufflectin is the first fish mucosal lectin that is demonstrated to bind to parasitic metazoans. Future functional studies therefore should take into account the possibility that the action of mucosal lectins is directed not only against bacteria but also against eucaryotic parasites. H. okamotoi is restricted to the gill chamber and is not observed on the skin in Fugu (42). This might be explained by the finding that pufflectin is less expressed in gills as compared with skin (Fig. 9). However, H. okamotoi frequently colonizes Fugu, indicating that it has evolved mechanisms that enable it to overcome the putative pufflectin-based defense system. In order to resolve this hypothesis, the mechanism of pufflectin activity will have to be investigated in comparative studies on various parasite and fish species.
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FOOTNOTES |
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* This work was supported by Grant-in-aid for Creative Basic Research
12NP0201 from the Ministry of Education, Science, Sports and Culture of Japan.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
Present address: Undergraduate Program for Bioinformatics and Systems
Biology, Faculty of Science, the University of Tokyo, Bunkyo, Tokyo 113-0633,
Japan.
To whom correspondence should be addressed. Tel.: 81-53-592-2821; Fax:
81-53-592-2822; E-mail:
ayuzuru{at}mail.ecc.u-tokyo.ac.jp.
1 The abbreviations used are: PBS, phosphate-buffered saline; MALDI-TOF,
matrix-assisted laser desorption ionization time of flight; RACE, rapid
amplification of cDNA ends; RT, reverse transcriptase; FPLC, fast protein
liquid chromatography; HPLC, high pressure liquid chromatography; MOPS,
4-morpholinepropanesulfonic acid; MBL, mannose-binding lectins.
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ACKNOWLEDGMENTS |
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REFERENCES |
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