Yoichiro Kitani

Identification of an antibacterial protein as L-amino acid oxidase in the skin mucus of rockfish Sebastes schlegeli

Fish skin mucus contains a variety of antimicrobial proteins and peptides that seem to play a role in self defense. We previously reported an antibacterial 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 chromatography, 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‐generation assay and substrate specificity for only l‐Lys with a Km of 0.19 mm. It showed potent antibacterial activity against fish pathogens such as Aeromonas 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.

Isolation and cDNA cloning of an antibacterial L-amino acid oxidase from the skin mucus of the great sculpin Myoxocephalus polyacanthocephalus

The skin mucus of the great sculpin Myoxocephalus polyacanthocephalus showed both antibacterial and L-amino acid oxidase (LAO) activities. Antibacterial LAOs were purified from the skin mucus of the M. polyacanthocephalus by column chromatography and named MPLAO1, MPLAO2, and MPLAO3, based on the order of elution by ion-exchange high performance liquid chromatography. cDNA cloning of MPLAO3 revealed that the full-length cDNAwas 2659 bp and encoded the signal peptide (Met1-Ala26) and the mature protein (Val28-Phe520). A homology search using the BLAST program revealed that MPLAO3 shared sequence identity with LAO family proteins, and had 74% identity with the antibacterial LAO from the skin mucus of the rockfish Sebastes schlegeli. MPLAO3 catalyzed the oxidation of only L-lysine with a Km of 0.16 mM. MPLAO3 exhibited potent antibacterial activity against both Gram-positive and Gramnegative bacteria, and was most active against Aeromonas salmonicida JCM7874 with a minimum inhibitory concentration of 0.02 microg/mL. The antibacterial activity was attributable to H2O2, because the activity was completely lost in the presence of catalase. The antibacterial LAOs may be involved in the innate immunity of the great sculpin M. polyacanthocephalus skin.

Discovery of serum L-amino acid oxidase in the rockfish Sebastes schlegeli: isolation and biochemical characterization.

Fish have a complex innate defense mechanism against microbial invasion. In particular, epidermal mucus and serum in fish play important roles in innate immunity and contain a variety of bioactive substances such as complements, lectins and lysozymes, involved in host defense. Recently, L-amino acid oxidases (LAOs) with antibacterial activity were isolated from the skin and/or gill mucous secretions of rockfish, great sculpin and flounder, and were identified to be a novel type of antibacterial protein in the integument of fish. In the present study, we found LAO activity in the serum of rockfish Sebastes schlegeli. The LAO was isolated from the serum by sequential column chromatography of Con-A lectin affinity chromatography, anion exchange HPLC, hydroxyapatite HPLC and gel filtration HPLC, and characterized. The LAO (a molecular mass of 160 kDa) comprised subunits with a molecular mass of 53 kDa and showed strict substrate specificity, catalyzing only L-lysine with Km 0.37 mM and kcat 57.1s(-1). The serum LAO exhibited a broad antibacterial activity against Gram positive and negative bacteria, most potently against Aeromonas hydrophila and Aeromonas salmonicida with a minimum inhibitory concentration of 0.078 μg/mL. This is the first report of LAO in the serum of fish and its involvement in innate immunity in the rockfish body.

Homozygous and heterozygous GH transgenesis alters fatty acid composition and content in the liver of Amago salmon (Oncorhynchus masou ishikawae)

Summary Growth hormone (GH) transgenic Amago (Oncorhynchus masou ishikawae), containing the sockeye GH1 gene fused with metallothionein-B promoter from the same species, were generated and the physiological condition through lipid metabolism compared among homozygous (Tg/Tg) and heterozygous GH transgenic (Tg/+) Amago and the wild type control (+/+). Previously, we have reported that the adipose tissue was generally smaller in GH transgenic fish compared to the control, and that the &Dgr;-6 fatty acyl desaturase gene was down-regulated in the Tg/+ fish. However, fatty acid (FA) compositions have not been measured previously in these fish. In this study we compared the FAs composition and content in the liver using gas chromatography. Eleven kinds of FA were detected. The composition of saturated and monounsaturated fatty acids (SFA and MUFA) such as myristic acid (14:0), palmitoleic acid (16:1n-7), and cis-vaccenic acid (cis-18:1n-7) was significantly (P<0.05) decreased in GH transgenic Amago. On the other hand, the composition of polyunsaturated fatty acids (PUFAs) such as linoleic acid (18:2n-6), arachidonic acid (20:4n-6), and docosapentaenoic acid (22:5n-3) was significantly (P<0.05) increased. Levels of serum glucose and triacylglycerol were significantly (P<0.05) decreased in the GH transgenics compared with +/+ fish. Furthermore, 3′-tag digital gene expression profiling was performed using liver tissues from Tg/Tg and +/+ fish, and showed that Mid1 interacting protein 1 (Mid1ip1), which is an important factor to activate Acetyl-CoA carboxylase (ACC), was down-regulated in Tg/Tg fish, while genes involved in FA catabolism were up-regulated, including long-chain-fatty-acid–CoA ligase 1 (ACSL1) and acyl-coenzyme A oxidase 3 (ACOX3). These data suggest that liver tissue from GH transgenic Amago showed starvation by alteration in glucose and lipid metabolism due to GH overexpression. The decrease of serum glucose suppressed Mid1ip1, and caused a decrease of de novo FA synthesis, resulting in a decrease of SFA and MUFA. This induced expression of ACSL1 and ACOX3 to produce energy through &bgr;-oxidation in the GH transgenic Amago.

Intra-tissue localization of an antibacterial L-amino acid oxidase in the rockfish Sebastes schlegeli.

The rockfish Sebastes schlegeli skin mucus contains a potent antibacterial protein, SSAP (S. schlegeli antibacterial protein), a novel l-amino acid oxidase with strict substrate specificity that acts against water-borne Gram-negative bacteria. We previously demonstrated that SSAP distributes in the skin and gills. Here we investigated the intra-tissue localization of SSAP in the tissues by in situ hybridization. Skin and gill sections were hybridized with digoxigenin-conjugated SSAP-specific RNA probe. SSAP mRNA-positive cells located near the basal membrane of skin epidermis and the gill epithelium. Furthermore, skin section was analyzed by immunohistochemistry and reacted with anti-SSAP antiserum as a primary antibody. The mucus layer and mucous cells in the skin were immunopositive. Skin and gill extracts produced hydrogen peroxide, responsible for antibacterial activity, in the presence of l-lysine. These results suggested that SSAP functions locally as a humoral defense factor in S. schlegeli skin and gills and prevents pathogenic bacterial invasion.

Histological and MS spectrometric analyses of the modified tissue of bulgy form tadpoles induced by salamander predation

Summary The rapid induction of a defensive morphology by a prey species in face of a predation risk is an intriguing in ecological context; however, the physiological mechanisms that underlie this phenotypic plasticity remain uncertain. Here we investigated the phenotypic changes shown by Rana pirica tadpoles in response to a predation threat by larvae of the salamander Hynobius retardatus. One such response is the bulgy morph phenotype, a relatively rapid swelling in size by the tadpoles that begins within 4 days and reaches a maximum at 8 to 10 days. We found that although the total volume of bodily fluid increased significantly (P<0.01) in bulgy morph tadpoles, osmotic pressure was maintained at the same level as control tadpoles by a significant increase (P<0.01) in Na and Cl ion concentrations. In our previous report, we identified a novel frog gene named pirica that affects the waterproofing of the skin membrane in tadpoles. Our results support the hypothesis that predator-induced expression of pirica on the skin membrane causes retention of absorbed water. Midline sections of bulgy morph tadpoles showed the presence of swollen connective tissue beneath the skin that was sparsely composed of cells containing hyaluronic acid. Mass spectrographic (LC-MS/MS) analysis identified histone H3 and 14-3-3 zeta as the most abundant constituents in the liquid aspirated from the connective tissue of bulgy tadpoles. Immunohistochemistry using antibodies against these proteins showed the presence of non-chromatin associated histone H3 in the swollen connective tissue. Histones and 14-3-3 proteins are also involved in antimicrobial activity and secretion of antibacterial proteins, respectively. Bulgy tadpoles have a larger surface area than controls, and their skin often has bite wounds inflicted by the larval salamanders. Thus, formation of the bulgy morph may also require and be supported by activation of innate immune systems.

Gene expression profiles in Rana pirica tadpoles following exposure to a predation threat

BackgroundRana pirica tadpoles show morphological changes in response to a predation threat: larvae of the dragonfly Aeshna nigroflava induce heightened tail depth, whereas larval salamander Hynobius retardatus induce a bulgy morphology with heightened tail depth. Although both predators induce similar tail morphologies, it is possible that there are functional differences between these tail morphs.ResultsHere, we performed a discriminant microarray analysis using Xenopus laevis genome arrays to compare tail tissues of control and predator-exposed tadpoles. We identified 9 genes showing large-scale changes in their expression profile: ELAV-like1, methyltransferase like 7A, dolichyl-phosphate mannosyltransferase, laminin subunit beta-1, gremlin 1, BCL6 corepressor-like 1, and three genes of unknown identity. A further 80 genes showed greater than 5 fold differences in expression after exposure to dragonfly larvae and 81 genes showed altered expression after exposure to larval salamanders. Predation-threat responsive genes were identified by selecting genes that reverted to control levels of expression following removal of the predator. Thirteen genes were induced specifically by dragonfly larvae, nine others were salamander-specific, and sixteen were induced by both. Functional analyses indicated that some of the genes induced by dragonfly larvae caused an increase in laminins necessary for cell adhesion in the extracellular matrix. The higher expression of gremlin 1 and HIF1a genes after exposure to dragonfly larvae indicated an in vivo hypoxic reaction, while down-regulation of syndecan-2 may indicate impairment of angiogenesis. Exposure to larval salamanders caused down-regulation of XCIRP-1, which is known to inhibit expression of adhesion molecules; the tadpoles showed reduced expression of cα(E)-catenin, small muscle protein, dystrophin, and myosin light chain genes.ConclusionThe connective tissue of tadpoles exposed to larval salamanders may be looser. The differences in gene expression profiles induced by the two predators suggest that there are functional differences between the altered tail tissues of the two groups of tadpoles.

The constant threat from a non-native predator increases tail muscle and fast-start swimming performance in Xenopus tadpoles

ABSTRACT Predator-induced phenotypic plasticity is the ability of prey to adapt to their native predator. However, owing to environmental changes, encounters with unknown predators are inevitable. Therefore, study of prey and non-native predator interaction will reveal the primary stages of adaptive strategies in prey-predator interactions in the context of evolutionary processes. Here, Xenopus tadpoles exposed to a non-native predator, a larval salamander, showed a significant increase in body weight and tail length to body length ratio. The Tmax2 test indicated a significant enhancement of the tail muscle and decrease in the relative ventral fin height in tadpoles exposed to predation risk, leading to significantly higher average swimming speeds. The analysis of muscle-related metabolites revealed that sarcosine increased significantly in tadpoles exposed to non-native predators. Multiple linear regression analysis of the fast-start swimming pattern showed that the fast-start swimming speed was determined by the time required for a tadpole to bend its body away from the threat (C-start) and the angle at which it was bent. In conclusion, morphological changes in tadpoles were functionally adaptive and induced by survival behaviors of Xenopus tadpoles against non-native predators. Summary: Xenopus tadpoles exposed to a non-native predator increase their tail length, muscle tissue and swimming speed, all of which are functionally adaptive and induced by tadpole survival behavior.