Identification and characterization of SaRpAMP, a 60S ribosomal protein L27-derived antimicrobial peptide from amur catfish, Silurus asotus.

Aquatic freshwater fish like catfish, Silurus asotus, lives in microbe-rich environments, which enable this fish to develop necessary defense mechanisms. Antimicrobial peptides, along with other innate immune factors, are regarded as an important group in this defense. An antimicrobial peptide, which was isolated from the skin of S. asotus, was identified as a C-terminal fragment of 60S ribosomal protein L27 from S. asotus. The peptide was, then, designated Silurus asotus 60S ribosomal protein L27-derived antimicrobial peptide, SaRpAMP. Primary structure analyses and cDNA cloning revealed that SaRpAMP was 4185.36 Da and composed of 33 amino acids (AAs). Its precursor had a total of 136 AAs containing a pro-sequence of 103 AAs encoded by the nucleotide sequence of 512 bp that comprises a 5' untranslated region (UTR) of 32 bp, an open reading frame (ORF) of 411 bp, and a 3' UTR of 69 bp. Secondary structure analyses showed that SaRpAMP had two α-helices with turns and coils and an amphiphilic structure, a finding consistent with the 3D model of the peptide. SaRpAMP exhibited potent antibacterial activity comparable to piscidin 1, a powerful positive control. Its antimicrobial activity against fungus C. albicans was relatively weak. The antimicrobial activity of SaRpAMP was not diminished by heat treatment and changes in pH but was abolished by proteolytic enzyme digestion. Membrane permeability assays suggested that SaRpAMP interacts with both the outer and inner bacterial membranes. This was consistent with the results of lipid titration and quenching of Trp fluorescence that demonstrated SaRpAMP's interaction with acidic liposomes. Collectively, these findings suggest that the identified peptide, SaRpAMP, was the first antimicrobial peptide reported to be derived from the C-terminal region of 60S ribosomal protein L27. The findings also suggest that the action mechanism of SaRpAMP involved the interaction of the peptide with the bacterial membranes.

Biochemical and molecular identification of a novel hepcidin type 2-like antimicrobial peptide in the skin mucus of the pufferfish Takifugu pardalis.

Fish skin mucus is considered to act as the first line of defense against waterborne pathogens and to be potential source of novel antimicrobial components. Here we report the purification and characterization of a novel hepcidin type 2-like antimicrobial peptide (TpHAMP2) from the skin mucus of the pufferfish Takifugu pardalis. The purified TpHAMP2 comprised of 23 amino acids (AAs) with eight Cys residues that form four intramolecular disulfide bonds. The TpHAMP2 gene shared overall structural characteristics with all known hepcidins, which have a tripartite exon-intron gene organization and three structural signatures in the precursor protein. Phylogenetically, TpHAMP2 was classified as HAMP2 class in acanthopterygian fish. Interestingly, the AA sequence of TpHAMP2 did not contain a proprotein cleavage site (RXXR motif) that conserved in most hepcidins and showed a highly positive charged (RKR-) short N-terminus and Val18 and Gly22 residues, which are distinctive structures compared to other known active hepcidins. Recombinant TpHAMP2 identical to the native form exhibited a broad spectrum and potent antimicrobial activity against tested gram-positive and -negative bacteria. Expression of TpHAMP2 mRNA was predominant in the liver and was upregulated in the liver, the spleen, the intestine, and the skin of T. pardalis post immune challenge. Thus, our findings suggests that TpHAMP2 might be of importance in the framework of discovering the fish hepcidins, especially type 2s, and provide noteworthy insight into its gene structure and expression and in the innate immunity as well as the mucosal immunity in regard to hepcidins' evolutionary history in fish species.

Biochemical, Anatomical, and Pharmacological Characterization of Calcitonin-Type Neuropeptides in Starfish: Discovery of an Ancient Role as Muscle Relaxants.

Calcitonin (CT) is a peptide hormone released by the thyroid gland that regulates blood Ca2+ levels in mammals. The CT gene is alternatively spliced, with one transcript encoding CT and another transcript encoding the CT-like neuropeptide calcitonin-gene related peptide (α-CGRP), which is a powerful vasodilator. Other CT-related peptides in vertebrates include adrenomedullin, amylin, and intermedin, which also act as smooth muscle relaxants. The evolutionary origin of CT-type peptides has been traced to the bilaterian common ancestor of protostomes and deuterostomes and a CT-like peptide (DH31) has been identified as a diuretic hormone in some insect species. However, little is known about the physiological roles of CT-type peptides in other invertebrates. Here we characterized a CT-type neuropeptide in a deuterostomian invertebrate-the starfish Asterias rubens (Phylum Echinodermata). A CT-type precursor cDNA (ArCTP) was sequenced and the predicted structure of the peptide (ArCT) derived from ArCTP was confirmed using mass spectrometry. The distribution of ArCTP mRNA and the ArCT peptide was investigated using in situ hybridization and immunohistochemistry, respectively, revealing stained cells/processes in the nervous system, digestive system, and muscular organs, including the apical muscle and tube feet. Investigation of the effects of synthetic ArCT on in vitro preparations of the apical muscle and tube feet revealed that it acts as a relaxant, causing dose-dependent reversal of acetylcholine-induced contraction. Furthermore, a muscle relaxant present in whole-animal extracts of another starfish species, Patiria pectinifera, was identified as an ortholog of ArCT and named PpCT. Consistent with the expression pattern of ArCTP in A. rubens, RT-qPCR revealed that in P. pectinifera the PpCT precursor transcript is more abundant in the radial nerve cords than in other tissues/organs analyzed. In conclusion, our findings indicate that the physiological action of CT-related peptides as muscle relaxants in vertebrates may reflect an evolutionarily ancient role of CT-type neuropeptides that can be traced back to the common ancestor of deuterostomes.

Isolation of an invertebrate-type lysozyme from the nephridia of the echiura, Urechis unicinctus, and its recombinant production and activities.

Invertebrates, unlike vertebrates which have adaptive immune system, rely heavily on the innate immune system for the defense against pathogenic bacteria. Lysozymes, along with other immune effectors, are regarded as an important group in this defense. An invertebrate-type (i-type) lysozyme, designated Urechis unicinctus invertebrate-type lysozyme, Uu-ilys, has been isolated from nephridia of Urechis unicinctus using a series of high performance liquid chromatography (HPLC), and ultrasensitive radial diffusion assay (URDA) as a bioassay system. Analyses of the primary structure and cDNA cloning revealed that Uu-ilys was approximately 14 kDa and composed of 122 amino acids (AAs) of which the precursor had a total of 160 AAs containing a signal peptide of 18 AAs and a pro-sequence of 20 AAs encoded by the nucleotide sequence of 714 bp that comprises a 5' untranslated region (UTR) of 42 bp, an open reading frame (ORF) of 483 bp, and a 3' UTR of 189 bp. Multiple sequence alignment showed Uu-ilys has high homology to i-type lysozymes from several annelids. Relatively high transcriptional expression levels of Uu-ilys was detected in nephridia, anal vesicle, and intestine. The native Uu-ilys exhibited comparable lysozyme enzymatic and antibacterial activities to hen egg white lysozyme. Collectively, these data suggest that Uu-ilys, the isolated antibacterial protein, plays a role in the immune defense mechanism of U. unicinctus. Recombinant Uu-ilys (rUu-ilys) produced in a bacterial expression system showed significantly decreased lysozyme lytic activity from that of the native while its potency on radial diffusion assay detecting antibacterial activity was retained, which may indicate the non-enzymatic antibacterial capacity of Uu-ilys.

Identification of a novel antimicrobial peptide from the sea star Patiria pectinifera.

Antimicrobial peptides (AMPs) are components of innate immunity found in many forms of life. However, there have been no reports of AMPs in sea star (Phylum Echinodermata). Here we report the isolation and characterization of a novel antimicrobial peptide from the coelomic epithelium extract of the sea star Patiria pectinifera. The isolated peptide comprises 38 amino acid residues, is cationic (pI 9.2), has four cysteine residues that form two disulfide bonds (C1-C3 and C2-C4), is amidated at the C-terminus, and is designated P. pectinifera cysteine-rich antimicrobial peptide (PpCrAMP). Synthetic PpCrAMP identical to the native peptide exhibited the most potent antimicrobial activity compared to analogs with different disulfide bond configurations. Expression analysis of PpCrAMP precursor transcripts revealed constitutive expression in the coelomic epithelium and tube feet of P. pectinifera. Analysis of genomic DNA and cDNA encoding the PpCrAMP precursor protein revealed that an intron splits the coding region of the mature peptide into a positively charged N-terminal domain and a C-terminal domain harboring four cysteine residues and a glycine for C-terminal amidation. No significant homology with other known AMPs was observed, while orthologs of PpCrAMP were found in other echinoderm species. These findings indicate that PpCrAMP is the prototype of a family a novel cysteine-rich AMPs that participate in mechanisms of innate immunity in echinoderms. Furthermore, the discovery of PpCrAMP may lead to the identification of related AMPs in vertebrates and protostome invertebrates.

Identification of evolutionarily conserved residues required for the bioactivity of a pedal peptide/orcokinin-type neuropeptide.

Pedal peptides and orcokinins are structurally related neuropeptides that were first discovered in protostomian invertebrates - mollusks and arthropods, respectively. Recently, pedal peptide/ocokinin (PP/OK)-type neuropeptides were discovered in a deuterostomian phylum, the echinoderms, indicating that the evolutionary origin of this neuropeptide family can be traced back to the common ancestor of bilaterian animals. Sequences comparison of PP/OK-type neuropeptides reveals several conserved residues, including N- and C-terminally located hydrophobic residues that are important for the bioactivity of orcokinin. Here we report the first comprehensive analysis of the structure-activity relationships of a PP/OK-type neuropeptide - starfish myorelaxant peptide (SMP; FGKGGAYDPLSAGFTD) from the starfish Patiria pectinifera (Phylum Echinodermata). Comparison of the bioactivity of SMP with N-terminally and/or C-terminally truncated and alanine-substituted SMP analogs revealed a core peptide (GAYDPLSAGF; SMP(5-14)) that retains the muscle-relaxing activity of SMP, albeit with reduced potency and efficacy. Within this core peptide, alanine-substitution of several residues resulted in complete or partial loss of bioactivity, whilst loss or substitution of the N-terminal phenylalanine residue of SMP also caused a substantial reduction in bioactivity. Furthermore, analysis of the bioactivity of other SMP-like peptides derived from the same precursor as SMP revealed that none of these were more potent/effective than SMP as muscle relaxants. In conclusion, we have identified key residues required for the bioactivity of a PP/OK-type neuropeptide (SMP), including hydrophobic residues located in the N- and C-terminal regions that are conserved in PP/OK-type peptides from other phyla as well as core residues that are conserved in echinoderm PP/OK-type peptides.

Transcriptomics reveals tissue/organ-specific differences in gene expression in the starfish Patiria pectinifera.

Starfish (Phylum Echinodermata) are of interest from an evolutionary perspective because as deuterostomian invertebrates they occupy an "intermediate" phylogenetic position with respect to chordates (e.g. vertebrates) and protostomian invertebrates (e.g. Drosophila). Furthermore, starfish are model organisms for research on fertilization, embryonic development, innate immunity and tissue regeneration. However, large-scale molecular data for starfish tissues/organs are limited. To provide a comprehensive genetic resource for the starfish Patiria pectinifera, we report de novo transcriptome assemblies and global gene expression analysis for six P. pectinifera tissues/organs - body wall (BW), coelomic epithelium (CE), tube feet (TF), stomach (SM), pyloric caeca (PC) and gonad (GN). A total of 408 million high-quality reads obtained from six cDNA libraries were assembled de novo using Trinity, resulting in a total of 549,598 contigs with a mean length of 835 nucleotides (nt), an N50 of 1473nt, and GC ratio of 42.5%. A total of 126,136 contigs (22.9%) were obtained as predicted open reading frames (ORFs) by TransDecoder, of which 102,187 were annotated with NCBI non-redundant (NR) hits, and 51,075 and 10,963 were annotated with Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) using the Blast2GO program, respectively. Gene expression analysis revealed that tissues/organs are grouped into three clusters: BW/CE/TF, SM/PC, and GN, which likely reflect functional relationships. 2408, 8560, 2687, 1727, 3321, and 2667 specifically expressed genes were identified for BW, GN, PC, CE, SM and TF, respectively, using the ROKU method. This study provides a valuable transcriptome resource and novel molecular insights into the functional biology of different tissues/organs in starfish as a model organism.

Defensin-neurotoxin dyad in a basally branching metazoan sea anemone.

Recent studies suggest that vertebrate and invertebrate defensins have evolved from two independent ancestors, and that both defensins could share origins with animal toxins. Here, we purified novel sea anemone neurotoxin (BDS)-like antimicrobial peptides (AMPs)-Crassicorin-I and its putative homolog (Crassicorin-II)-from the pharynx extract of an anthozoan sea anemone (Urticina crassicornis). Based on structural analyses and cDNA cloning, mature Crassicorin-I represents a cationic AMP likely generated from a precursor and comprising 40 amino acid residues, including six cysteines forming three intramolecular disulfide bonds. Recombinant Crassicorin-I produced in a heterologous bacterial-expression system displayed antimicrobial activity against both a gram-positive bacterium (Bacillus subtilis) and gram-negative bacteria (Escherichia coli and Salmonella enterica). The Crassicorin-I transcript was upregulated by immune challenge, suggesting its involvement in defense mechanisms against infectious pathogens in sea anemone. Sequence alignment and three-dimensional molecular modeling revealed that Crassicorin-I exhibits high degrees of structural similarity to sea anemone neurotoxins that share ß-defensin fold which is found in vertebrate defensins and invertebrate big-defensins. Consistent with its structural similarity to neurotoxins, Crassicorin-I exhibited paralytic activity toward a crustacean. These findings motivated our investigation and subsequent discovery of antimicrobial activity from other known sea anemone neurotoxins, such as APETx1 and ShK. Collectively, our work signified that Crassicorin-I is the first AMP identified from a sea anemone and provided evidence of a functional linkage between AMPs and neurotoxins in a basally branching metazoan.

The novel model peptide, αAL14, regulates angiogenesis by inhibiting VEGFR 2-mediated signaling in HUVECs.

Inhibition of angiogenesis has been focused on as a strategy for treating several diseases including cancer. In this study, a novel model peptide αAL14 was synthesized and used to identify its inhibitory effects on angiogenesis. The anti-angiogenic effects of αAL14 were investigated using vascular endothelial cells, HUVECs. αAL14 inhibited critical angiogenic processes including tubule formation, cell migration and cell invasion with no influence on cell proliferation in HUVECs. Activity of VEGFR2 was inhibited by αAL14 treatment in HUVECs. Additionally, activities of major subsequent downstream factors of VEGFR2 such as ERK, FAK and Akt were decreased. αAL14 affected expression of Rac1, Cdc42, Arp2 and WAVE2 which are involved in formation of lamellipodia. Moreover, αAL14 reduced NF-κB that can promote expression of several genes relating to cell invasion such as MMP2 and MMP9. Therefore, the results suggest that αAL14 has a potential to be developed as anti-angiogenic drug for treating diseases driven by abnormal angiogenesis.

Novel pentapeptide, PALAL, derived from a bony fish elicits contraction of the muscle in starfish Patiria pectinifera.

A bioactive peptide mimicking peptide-signaling molecules has been isolated from the skin extract of fish Channa argus which caused contraction of the apical muscle of a starfish Patiria pectinifera, a deuterostomian invertebrate. The primary structure of the isolated pentapeptide comprises amino acid sequence of H-Pro-Ala-Leu-Ala-Leu-OH (PALAL) with a molecular mass of 483.7 Da. Pharmacological activity of PALAL, dosage ranging from 10-9 to 10-5 M, revealed concentration-dependent contraction of the apical muscles of P. pectinifera and Asterias amurensis. However, PALAL was not active on the intestinal smooth muscle of the goldfish Carassius auratus and has presumably other physiological roles in fish skin. Investigation of structure-activity relationship using truncated and substituted analogs of PALAL demonstrated that H-Ala-Leu-Ala-Leu-OH was necessary and should be sufficient to constrict apical muscle of P. pectinifera. Furthermore, the second alanine residue was required to display the activity, and the fifth leucine residue was responsible for its potency. Comparison with PALAL's primary structure with those of other known bioactive peptides from fish and starfish revealed that PALAL does not have any significant homology. Consequently, PALAL is a bioactive peptide that elicits a muscle contraction in starfish, and the isolation of PALAL may lead to develop other bioactive peptides sharing its similar sequence and/or activity. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Hemerythrin-related antimicrobial peptide, msHemerycin, purified from the body of the Lugworm, Marphysa sanguinea.

A ∼1.7 kDa antimicrobial peptide was purified from the acidified body extract of the Lugworm, Marphysa sanguinea, by preparative acid-urea-polyacrylamide gel electrophoresis and C18 reversed-phase high performance liquid chromatography (HPLC). The identified peptide is composed of 14 amino acids with the N-terminal acetylation. Comparison of the identified amino acid sequences and molecular weight of this peptide with those of other known proteins or peptides revealed that this peptide had high identity to the N-terminus of hemerythrin of marine invertebrates and named the msHemerycin. The full-length hemerythrin cDNA of Lugworm was contained 1027-bp, including a 5'-untranslated region (UTR) of 60-bp, a 3'-UTR of 595-bp, and an open reading frame of 372-bp encoding 123 amino acids including the msHemerycin at the N-terminus. Tissue distribution of the msHemerycin mRNA suggests that it is constitutively expressed as a non-tissue-specific manner, however, a relatively higher expression level was observed in muscle (6.8-fold) and brain (6.3-fold), and the lowest level in digestive gland. The secondary structural prediction and homology modeling studies indicate that the msHemerycin might form an unordered structure and might act via unconventional mechanism. Our results suggest that the msHemerycin might be an innate immune component related to the host defenses in the Lugworm. This is the first report on the antimicrobial function of the peptide derived from the N-terminus of hemerythrin in the Lugworm, Marphysa sanguinea.

Antimicrobial peptide, hdMolluscidin, purified from the gill of the abalone, Haliotis discus.

A 4.7 kDa antimicrobial peptide was purified from the acidified gill extract of the Abalone, Haliotis discus, by cation-exchange and C18 reversed-phase high performance liquid chromatography (HPLC). Comparison of the amino acid sequences and molecular weight of this peptide with those of other known antimicrobial peptides revealed that this antimicrobial peptide have high sequence homology with that of cgMolluscidin and was designated hdMolluscidin. hdMolluscidin is composed of 46 amino acid residues containing several dibasic residue repeats like KK or K-R. hdMolluscidin showed potent antimicrobial activity against both Gram-positive bacteria including Bacillus subtilis and Staphylococcus aureus (minimal effective concentrations [MECs]; 0.8-19.0 µg/mL) and Gram-negative bacteria including Aeromonas hydrophila, Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, Shigella flexneri, and Vibrio parahemolyticus ([MECs]; 1.0-4.0 µg/mL) without hemolytic activity. However, hdMolluscidin did not show any significant activity against Candida albicans. The secondary structural prediction suggested that hdMolluscidin might not form an ordered or an amphipathic structure. hdMolluscidin did not show membrane permeabilization or leakage ability. The full-length hdMolluscidin cDNA contained 566-bp, including a 5'-untranslated region (UTR) of 63-bp, a 3'-UTR of 359-bp, and an open reading frame of 144-bp encoding 47 amino acids (containing Met). cDNA study of hdMolluscidin suggests that it is expressed as a mature peptide. Our results indicate that hdMolluscidin could relate to the innate immune defenses in abalone and it may not act directly on bacterial membrane.

Identification of a novel starfish neuropeptide that acts as a muscle relaxant.

Neuropeptides that act as muscle relaxants have been identified in chordates and protostomian invertebrates but little is known about the molecular identity of neuropeptides that act as muscle relaxants in deuterostomian invertebrates (e.g. echinoderms) that are 'evolutionary intermediates' of chordates and protostomes. Here, we have used the apical muscle of the starfish Patiria pectinifera to assay for myorelaxants in extracts of this species. A hexadecapeptide with the amino acid sequence Phe-Gly-Lys-Gly-Gly-Ala-Tyr-Asp-Pro-Leu-Ser-Ala-Gly-Phe-Thr-Asp was identified and designated starfish myorelaxant peptide (SMP). Cloning and sequencing of a cDNA encoding the SMP precursor protein revealed that it comprises 12 copies of SMP as well as 3 peptides (7 copies in total) that are structurally related to SMP. Analysis of the expression of SMP precursor transcripts in P. pectinifera using qPCR revealed the highest expression in the radial nerve cords and lower expression levels in a range of neuromuscular tissues, including the apical muscle, tube feet and cardiac stomach. Consistent with these findings, SMP also caused relaxation of tube foot and cardiac stomach preparations. Furthermore, SMP caused relaxation of apical muscle preparations from another starfish species - Asterias amurensis. Collectively, these data indicate that SMP has a general physiological role as a muscle relaxant in starfish. Interestingly, comparison of the sequence of the SMP precursor with known neuropeptide precursors revealed that SMP belongs to a bilaterian family of neuropeptides that include molluscan pedal peptides (PP) and arthropodan orcokinins (OK). This is the first study to determine the function of a PP/OK-type peptide in a deuterostome. Pedal peptide/orcokinin (PP/OK)-type peptides are a family of structurally related neuropeptides that were first identified and functionally characterised in protostomian invertebrates. Here, we report the discovery of starfish myorelaxant peptide (SMP), a novel member of the PP/OK-type neuropeptide identified in the starfish Patiria pectinifera (phylum Echinodermata). SMP is the first PP/OK-type neuropeptide to be functionally characterised in a deuterostome.

Two myomodulins isolated from central nervous system of Northwest Pacific Sea Hare, Aplysia kurodai, and their activities on other mollusks.

The central nervous system (CNS) of Aplysia is a fascinating source to identify and characterize neuropeptides and neurotransmitters because of offering many useful divergent and convergent neuronal aggregates. Here, two neuropeptides were isolated from the extract of CNS of the northwest pacific sea hare, Aplysia kurodai, using HPLC system for fractionation and the anterior byssus retractor muscle (ABRM) of the Mytilis edulis as the bioassay system. Purified peptides, myomodulin A (MMA) and E (MME), were determined by amino acid sequencing and molecular mass analysis. MMA showed a potentiating effect at 100 nM or lower, on the contrary, an inhibitory effect at higher doses from 100 nM on phasic contraction elicited by repetitive electrical stimulation on the ABRM of Mytilus. However, MME only inhibited phasic contraction with all examined concentrations. MME revealed 100 times more potent activity than that of MMA on the relaxing catch-tension of ABRM stimulated by acetylcholine. Both MMA and MME potently stimulated a response on the crop and penial retractor muscle of the African giant snail, Achatina fulica, compared with other known mollusks neuropeptides. These results suggest that MMA and MME may be broadly distributed in CNS of Aplysia to function a neuromodulatory role controlled via excitatory and inhibitory neurons, and may be involved in the digestive and reproductive activity in other mollusk.

Antimicrobial function of SHßAP, a novel hemoglobin ß chain-related antimicrobial peptide, isolated from the liver of skipjack tuna, Katsuwonus pelamis.

A 2.3 kDa of antimicrobial peptide was purified from an acidified liver extract of skipjack tuna, Katsuwonus pelamis, by preparative acid-urea-polyacrylamide gel electrophoresis and C18 reversed-phase HPLC. A comparison of the amino acid sequence of the purified peptide with those of other known polypeptides revealed high homology with the C-terminus of hemoglobin ß-chain; thus, this peptide was designated as the Skipjack Hemoglobin ß chain-related Antimicrobial Peptide (SHßAP). SHßAP showed potent antimicrobial activity against Gram-positive bacteria, such as Bacillus subtilis, Staphylococcus aureus, and Streptococcus iniae (minimal effective concentrations [MECs], 6.5-57.0 µg/mL), Gram-negative bacteria, such as Escherichia coli D31, Pseudomonas aeruginosa, Salmonella enterica, Shigella sonnei, and two Vibrio parahaemolyticus species (MECs, 2.0-19.0 µg/mL), and against Candida albicans (MEC; 12.0 µg/mL) without significant hemolytic activity. Antimicrobial activity of this peptide was heatstable and pH resistant but is sensitive to proteases and salt. SHßAP did not show membrane permeabilization and killing ability. The secondary structural prediction and the homology modeling expected that this peptide formed an amphipathic α-helical structure. This is the first report the purification of a novel antimicrobial peptide related to the C-terminus of hemoglobin ß-chain from marine fish.

Antimicrobial function of the GAPDH-related antimicrobial peptide in the skin of skipjack tuna, Katsuwonus pelamis.

A 3.4 kDa of antimicrobial peptide was purified from an acidified skin extract of skipjack tuna, Katsuwonus pelamis, by preparative acid-urea-polyacrylamide gel electrophoresis and C18 reversed-phase HPLC. A comparison of the N-terminal amino acid sequence of the purified peptide with that of other known polypeptides revealed high sequence homology with the YFGAP (Yellowfin tuna Glyceraldehyde-3-phosphate dehydrogenase-related Antimicrobial Peptide); thus, this peptide was identified as the skipjack tuna GAPDH-related antimicrobial peptide (SJGAP). SJGAP showed potent antimicrobial activity against Gram-positive bacteria, such as Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus, and Streptococcus iniae (minimal effective concentrations [MECs], 1.2-17.0 µg/mL), Gram-negative bacteria, such as Aeromonas hydrophila, Escherichia coli D31, and Vibrio parahaemolyticus (MECs, 3.1-12.0 µg/mL), and against Candida albicans (MEC, 16.0 µg/mL) without significant hemolytic activity. Antimicrobial activity of this peptide is heat-stable but salt-sensitive. According to the secondary structural prediction and the homology modeling, this peptide consists of three secondary structural motifs, including one α-helix and two parallel ß-strands, and forms an amphipathic structure. This peptide showed neither membrane permeabilization ability nor killing ability, but did display a small degree of leakage ability. These results suggest that SJGAP acts through a bacteriostatic process rather than bactericidal one. SJGAP is another GAPDH-related antimicrobial peptide isolated from skipjack tuna and likely plays an important role for GAPDH in the innate immune defense of tuna fish.

Molecular cloning, expression, and enzymatic analysis of cathepsin X from starfish (Asterina pectinifera).

Cathepsin X, also known as cathepsin Z, is referred to as a "lysosomal proteolytic enzyme" and a member of the peptidase C1 family, which is involved in various biological processes such as immune response, cell adhesion, and proliferation. In the present study, the cDNA of starfish (Asterina pectinifera), which is known to cause serious damage to commercial shellfish mariculture, cathepsin X (ApCtX) was isolated through the combination of homology molecular cloning and rapid amplification of cDNA ends (RACE) methods for the application to find a way to reduce/control starfish densities. The full-length of ApCtX gene was determined to consist of the 2,240 bp nucleotide sequence, which encoded for a preproprotein of 296 amino acids with a molecular mass of about 32.7 kDa. The tissue type expression of ApCtX was determined in various tissues of A. pectinifera and was shown most abundantly in the liver. The cDNA encoding pro-mature enzyme of ApCtX was expressed in Escherichia coli BL21 (DE3) using the pGEX-4T-1 expression vector. Its activity was quantified by cleaving the synthetic peptide Z-Phe-Arg-AMC. The optimal pH for the protease activity was 6.5. The enzymatic activity of proApCtX was reduced by antipain, NEM, EDTA, EGTA, and 1,10-phenanthroline, and the proApCtX enzyme was significantly inhibited by CuSO4, HgCl2, CoCl2, and SDS whereas Triton X-100 and Brij 35 might have potentially acted as an activator. Here, we demonstrated for the first time that the structural features and enzymatic characteristics of Echinoderms cathepsin X are similar to those of the other mammalian and piscine cathepsin X except its pH optimum, and the results of tissue-specific expression might explain their importance in food digestion by hepatic cecain starfish.

Purification and antimicrobial function of ubiquitin isolated from the gill of Pacific oyster, Crassostrea gigas.

An antimicrobial polypeptide was purified from an acidified gill extract of Pacific oyster (Crassostrea gigas) by C(18) reversed-phase HPLC. The purified polypeptide had a molecular weight of 8471Da containing 74 amino acid residues. Comparison of the obtained N-terminal sequences with those of others revealed that it was identical to ubiquitin reported from other species and named cgUbiquitin. cgUbiquitin showed broad potent antimicrobial activity against Gram-positive and -negative bacteria including Streptococcus iniae and Vibrio parahemolyticus (minimal effective concentrations, 7.8 and 9.8µg/mL), respectively, without hemolytic activity. The cgUbiquitin cDNA was identified from an expressed sequence tag (EST) library of oyster gill as a precursor form, encoding ubiquitin consisting of 76 amino acids fused to ribosomal protein of S27. Although the cgUbiquitin precursor mRNA was expressed at the intermediate level in the gill, the mRNA was significantly up-regulated at 48h post injection with Vibrio sp. Analysis of the cgUbiquitin C-terminus by carboxypeptidase B treatment and comparison of the retention times revealed that cgUbiquitin lacks the terminal Gly-Gly doublet and ends in an C-terminal Arg residue which might be related to antimicrobial activity. Study of the kinetics of killing and membrane permeabilization showed that this peptide was not membrane permeable and acted through a bacteriostatic process. According to the homology modeling, this peptide is composed of three secondary structural motifs including three α-helices and four ß-strands separated by 7 loops regions. Our results indicate that cgUbiquitin might be related to the innate immune defenses in the Pacific oyster and this is the first report for antimicrobial function of ubiquitin isolated from any oyster species.

Cloning, heterologous expression, and enzymatic characterization of cathepsin L from starfish (Asterina pectinifera).

To determine the role of cathepsin L in Echinoderms, starfish (Asterina pectinifera) cathepsin L (ApCtL) was cloned. The results of RT-PCR analysis indicated that the expression of ApCtL in all of the tissues. The pro-mature enzyme of ApCtL, proApCtL, was expressed in Escherichia coli, and cathepsin activity was detected by cleaving of synthetic fluorogenic peptide substrates and gelatin zymography.

Purification and characterization of YFGAP, a GAPDH-related novel antimicrobial peptide, from the skin of yellowfin tuna, Thunnus albacares.

A 3.4 kDa of antimicrobial peptide was purified from an acidified skin extract of the yellowfin tuna, Thunnus albacares, by preparative acid-urea-polyacrylamide gel electrophoresis and C(18) reversed-phase HPLC. A comparison of the N-terminal amino acid sequence of the purified peptide with that of other known polypeptides revealed high homology with the N-terminus of glyceraldehyde-3-phosphate dehydrogenase (GAPDH); thus, this peptide was designated as the yellowfin tuna GAPDH-related antimicrobial peptide (YFGAP). YFGAP showed potent antimicrobial activity against Gram-positive bacteria, such as Bacillus subtilis, Micrococcus luteus, and Streptococcus iniae (minimal effective concentrations [MECs], 1.2-17.0 µg/mL), and Gram-negative bacteria, such as Aeromonas hydrophila, Escherichia coli D31, and Vibrio parahaemolyticus (MECs, 3.1-12.0 µg/mL) without significant hemolytic activity. According to the secondary structural prediction and the homology modeling, this peptide forms an amphipathic structure and consists of three secondary structural motifs including one α-helix and two parallel ß-strands. This peptide did not show membrane permeabilization ability and its activity was bacteriostatic rather than bactericidal. This is the first report of the isolation of an antimicrobial peptide from a tuna species and the first description of the antimicrobial function of the N-terminus of GAPDH of an animal species.