Chan-Hee Kim

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.

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 β‐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.

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.

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.

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

Graphical abstract Starfish myorelaxant peptide (SMP) belongs to a bilaterian family of pedal peptide/orcokinin (PP/OK)‐type neuropeptides that have evolutionarily conserved structural features. Here we report the first analysis of the structure‐activity relationships of SMP and identify key residues for SMP bioactivity, including hydrophobic residues located in the N‐ and C‐terminal regions that are conserved in PP/OK‐type peptides in other phyla as well as core residues that are only conserved in echinoderm PP/OK‐type peptides. Figure. No caption available. HighlightsHydrophobic residues (Phe1, Phe14, and Leu10) in SMP are important for bioactivity.The hydrophobic residues are evolutionarily conserved in bilaterian PP/OK‐type neuropeptides.Core residues (Tyr7, Asp8, and Pro9) in SMP are important for bioactivity.The core residues are key residues in deuterostomian PP/OK‐type neuropeptides. &NA; 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.

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

ABSTRACT 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 14kDa 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. HIGHLIGHTSAn invertebrate‐type lysozyme, Uu‐ilys, was isolated from nephridia of a marine annelid, Urechis unicinctus.Uu‐ilys exhibited muramidase and antibacterial activities that was comparable to activities of hen egg white lysozyme.Uu‐ilys is involved in the immune defense in nephridia of U. unicinctus.Recombinant Uu‐ilys exerted antibacterial activity by non‐enzymatic antibacterial mechanism.

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

Abstract 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. HighlightsA novel cysteine‐rich AMP (PpCrAMP) is identified from the starfish Patiria pectinifera.PpCrAMP adopts two disulfide bonds with Cys16‐C27 and Cys25‐Cys38 pairings.PpCrAMP transcripts are highly expressed in the tube feet and the coelomic epithelium.PpCrAMP gene contains an intron.PpCrAMP exhibits antimicrobial activity to different bacteria.

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 PALALs 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

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.

Antimicrobial, Antioxidant and Hemolytic Activity of Water-soluble Extract of Mottled Anemone Urticina crassicornis

We evaluated the biological activities of five water extracts of tissue of the mottled anemone Urticina crassicornis. Most extracts exhibited broad-spectrum antimicrobial activity as determined by ultrasensitive radial diffusion assay (URDA) against gram-positive and -negative bacteria, including a fish pathogen, Aeromonas hydrophila, but no activity against fungi. The activity of the extracts was abolished by tryptic digestion, indicating that protein compounds were responsible for the antimicrobial activity. Furthermore, in a 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical-scavenging activity assay, only the visceral tissue extract showed activity. However, no extract had hemolytic activity against human red blood cells. Consequently, this study suggests the water-soluble extract of mottled anemone to be a promising source of proteinaceous antimicrobial compounds that can be utilized for development of novel antibiotics.