A Novel Hemocyte-Derived Peptide and Its Possible Roles in Immune Response of Ciona intestinalis Type A.

A wide variety of bioactive peptides have been identified in the central nervous system and several peripheral tissues in the ascidian Ciona intestinalis type A (Ciona robusta). However, hemocyte endocrine peptides have yet to be explored. Here, we report a novel 14-amino-acid peptide, CiEMa, that is predominant in the granular hemocytes and unilocular refractile granulocytes of Ciona. RNA-seq and qRT-PCR revealed the high CiEma expression in the adult pharynx and stomach. Immunohistochemistry further revealed the highly concentrated CiEMa in the hemolymph of the pharynx and epithelial cells of the stomach, suggesting biological roles in the immune response. Notably, bacterial lipopolysaccharide stimulation of isolated hemocytes for 1-4 h resulted in 1.9- to 2.4-fold increased CiEMa secretion. Furthermore, CiEMa-stimulated pharynx exhibited mRNA upregulation of the growth factor (Fgf3/7/10/22), vanadium binding proteins (CiVanabin1 and CiVanabin3), and forkhead and homeobox transcription factors (Foxl2, Hox3, and Dbx) but not antimicrobial peptides (CrPap-a and CrMam-a) or immune-related genes (Tgfbtun3, Tnfa, and Il17-2). Collectively, these results suggest that CiEMa plays roles in signal transduction involving tissue development or repair in the immune response, rather than in the direct regulation of immune response genes. The present study identified a novel Ciona hemocyte peptide, CiEMa, which paves the way for research on the biological roles of hemocyte peptides in chordates.

Evidence for Conservation of the Calcitonin Superfamily and Activity-regulating Mechanisms in the Basal Chordate Branchiostoma floridae: INSIGHTS INTO THE MOLECULAR AND FUNCTIONAL EVOLUTION IN CHORDATES.

The calcitonin (CT)/CT gene-related peptide (CGRP) family is conserved in vertebrates. The activities of this peptide family are regulated by a combination of two receptors, namely the calcitonin receptor (CTR) and the CTR-like receptor (CLR), and three receptor activity-modifying proteins (RAMPs). Furthermore, RAMPs act as escort proteins by translocating CLR to the cell membrane. Recently, CT/CGRP family peptides have been identified or inferred in several invertebrates. However, the molecular characteristics and relevant functions of the CTR/CLR and RAMPs in invertebrates remain unclear. In this study, we identified three CT/CGRP family peptides (Bf-CTFPs), one CTR/CLR-like receptor (Bf-CTFP-R), and three RAMP-like proteins (Bf-RAMP-LPs) in the basal chordate amphioxus (Branchiostoma floridae). The Bf-CTFPs were shown to possess an N-terminal circular region typical of the CT/CGRP family and a C-terminal Pro-NH2. The Bf-CTFP genes were expressed in the central nervous system and in endocrine cells of the midgut, indicating that Bf-CTFPs serve as brain and/or gut peptides. Cell surface expression of the Bf-CTFP-R was enhanced by co-expression with each Bf-RAMP-LP. Furthermore, Bf-CTFPs activated Bf-CTFP-R·Bf-RAMP-LP complexes, resulting in cAMP accumulation. These results confirmed that Bf-RAMP-LPs, like vertebrate RAMPs, are prerequisites for the function and translocation of the Bf-CTFP-R. The relative potencies of the three peptides at each receptor were similar. Bf-CTFP2 was a potent ligand at all receptors in cAMP assays. Bf-RAMP-LP effects on ligand potency order were distinct to vertebrate CGRP/adrenomedullin/amylin receptors. To the best of our knowledge, this is the first molecular and functional characterization of an authentic invertebrate CT/CGRP family receptor and RAMPs.

Peptidomic analysis of the central nervous system of the protochordate, Ciona intestinalis: homologs and prototypes of vertebrate peptides and novel peptides.

The phylogenetic position of ascidians as the chordate invertebrates closest to vertebrates suggests that they might possess homologs and/or prototypes of vertebrate peptide hormones and neuropeptides as well as ascidian-specific peptides. However, only a small number of peptides have so far been identified in ascidians. In the present study, we have identified various peptides in the ascidian, Ciona intestinalis. Mass spectrometry-based peptidomic analysis detected 33 peptides, including 26 novel peptides, from C. intestinalis. The ascidian peptides are largely classified into three categories: 1) prototypes and homologs of vertebrate peptides, such as galanin/galanin-like peptide, which have never been identified in any invertebrates; 2) peptides partially homologous with vertebrate peptides, including novel neurotesin-like peptides; 3) novel peptides. These results not only provide evidence that C. intestinalis possesses various homologs and prototypes of vertebrate neuropeptides and peptide hormones but also suggest that several of these peptides might have diverged in the ascidian-specific evolutionary lineage. All Ciona peptide genes were expressed in the neural complex, whereas several peptide gene transcripts were also distributed in peripheral tissues, including the ovary. Furthermore, a Ciona neurotensin-like peptide, C. intestinalis neurotensin-like peptide 6, was shown to down-regulate growth of Ciona vitellogenic oocytes. These results suggest that the Ciona peptides act not only as neuropeptides in the neural tissue but also as hormones in nonneuronal tissues and that ascidians, unlike other invertebrates, such as nematodes, insects, and sea urchins, established an evolutionary origin of the peptidergic neuroendocrine, endocrine, and nervous systems of vertebrates with certain specific molecular diversity.

Gene expression profile of Ci-VSP in juveniles and adult blood cells of ascidian.

VSP is a transmembrane protein whose cytoplasmic region shows significant similarity to phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Notably, VSP exhibits a unique ability to transduce electrical signals into phosphoinositide turnover by coupling a transmembrane voltage sensor domain to the PTEN-like phosphoinositide phosphatase domain. Moreover, VSP gene is known to be widely conserved among deuterostome genomes, though the function of VSP in vivo remains largely unknown. In the present study, the expression pattern of ascidian VSP(Ci-VSP) was examined in embryos and juveniles of a marine invertebrate chordate, Ciona intestinalis. RT-PCR showed that Ci-VSP is expressed at the larval stage and that expression persists in juveniles. Whole mount in situ hybridization showed that Ci-VSP is expressed in cells of the stomach, intestine and blood cells of 2- to 3-week-old juveniles. Moreover, double staining blood cells from 2-month-old adults with Ci-VSP and Ci-PTEN probes showed that Ci-VSP-positive cells are a distinct population, separate from cells expressing Ci-PTEN. These findings suggest that in addition to its previously suggested roles in testis or sperm, Ci-VSP plays a key role in voltage-induced signal transduction in cells of the digestive system and blood.

Troponin in both smooth and striated muscles of Ascidian Ciona intestinalis functions as a Ca2+-dependent accelerator of actin−myosin interaction.

Troponin, a Ca2+-dependent regulator of muscle contraction, acts as an inhibitor of the actin−myosin interaction in the absence of Ca2+ during contraction in vertebrate striated muscle. However, variation has been observed in the mode of troponin-dependent regulation among the animals belonging to Protochordata, the taxon most closely related to Vertebrata. Although troponin in striated muscle of a cephalochordate amphioxus functions as an inhibitor in the absence of Ca2+ as in vertebrates [Dennisson, J. G., et al. (2010) Zool. Sci. 27, 461−469], troponin in the smooth muscle of a urochordate ascidian (Halocynthia roretzi) regulates actin−myosin interaction as an activator in the presence of Ca2+ and not an inhibitor in the absence of Ca2+ as in vertebrates [Endo, T., and Obinata, T. (1981) J. Biochem. 89, 1599−1608]. In this study, to further clarify the functional diversity of troponin, we examined the role of troponin in Ca2+-dependent regulation of the actin−myosin interaction in striated and smooth muscles in another member of Ascidiacea (Ciona inetestinalis) using three recombinant troponin components, TnT, TnI, and TnC, produced using an Escherichia coli expression system. On the basis of actomyosin ATPase assays, we show here that troponins in both smooth and striated muscles of ascidian function as a Ca2+-dependent activator of the actin−myosin interaction and TnT is the component responsible for this activation. These results indicate that troponin of ascidian has evolved in a manner different from that of amphioxus and vertebrates in terms of function.

Functional characteristics of amphioxus troponin in regulation of muscle contraction.

Troponin regulates contraction of vertebrate striated muscle in a Ca(2+)-dependent manner. More specifically, it acts as an inhibitor of actin-myosin interaction in the absence of Ca(2+) during contraction. In vertebrates, this regulatory mechanism is unlike that in some less highly derived taxa. Troponin in the smooth muscle of the protochordate ascidian species Halocynthia roretzi regulates actinmyosin contraction as an activator in the presence of Ca(2+), not as an inhibitor in the absence of Ca(2+) as is the case in vertebrates. In this study, contractile regulation of striated muscle from another protochordate, the amphioxus Branchiostoma belcheri, was analyzed using recombinant troponin components TnT, TnI, and TnC that were produced in an Escherichia coli expression system to further elucidate their roles in Ca(2+)-dependent regulation of the actin-myosin interaction. Combination of these troponin components in an actin-myosin ATPase activity assay showed that troponin in amphioxus striated muscle functions in a similar manner to troponin in vertebrate striated muscle, and differently from ascidian smooth muscle troponin. Thus, troponin function appears to have evolved differently in different protochordate muscles.

A novel inhibitory gonadotropin-releasing hormone-related neuropeptide in the ascidian, Ciona intestinalis.

The gonadotropin-releasing hormone (GnRH) family peptides are most widely distributed neuropeptides and/or neurophysial hormones. GnRH is involved in diverse neuroendocrine, paracrine, autocrine, and neurotransmitter/neuromodulatory functions in the central and peripheral nervous system as well as peripheral tissues. In the present study, we show the identification of a novel GnRH-related peptide, Ci-GnRH-X, in the ascidian, Ciona intestinalis. Intriguingly, Ci-GnRH-X possesses a unique primary sequence consisting of 16 amino acids, although typical GnRH family peptides are composed of 10 amino acids. On the other hand, Ci-GnRH-X shares the GnRH consensus motifs, including the N-terminal pQHWS ('pQ' indicates a pyro-glutamic acid) and C-terminal Gly-amide. Reverse transcription (RT)-PCR analysis shows that the Ci-GnRH-X gene is expressed exclusively in the central nervous system. Moreover, in situ hybridization demonstrated that the Ciona GnRH-1 gene encoding Ciona GnRHs (t-GnRH-3, -5 and -6) was co-expressed with the Ci-GnRH-X gene in neurons of the cerebral ganglion. Of particular interest is that Ci-GnRH-X exhibited moderate (10-50%) inhibitory activity against t-GnRHs at their cognate receptors. Ci-GnRH-X repressed the elevation of the intracellular calcium and cAMP production by t-GnRH-6 at Ci-GnRHR-1, and cAMP production by t-GnRH-3, and t-GnRH-5 via Ci-GnRHR-3 was also inhibited by Ci-GnRH-X. In contrast, no inhibitory effect of Ci-GnRH-X at Ci-GnRHR-2 was observed. The localization and biochemical assays revealed that Ci-GnRH-X acts as an endogenous antagonist for the Ciona GnRHergic system. This is the first molecular and functional characterization of an endogenous inhibitor of GnRHs in an animal species.

Toll-like receptors of the ascidian Ciona intestinalis: prototypes with hybrid functionalities of vertebrate Toll-like receptors.

Key transmembrane proteins in the innate immune system, Toll-like receptors (TLRs), have been suggested to occur in the genome of non-mammalian organisms including invertebrates. However, authentic invertebrate TLRs have been neither structurally nor functionally investigated. In this paper, we originally present the structures, localization, ligand recognition, activities, and inflammatory cytokine production of all TLRs of the ascidian Ciona intestinalis, designated as Ci-TLR1 and Ci-TLR2. The amino acid sequence of Ci-TLR1 and Ci-TLR2 were found to possess unique structural organization with moderate sequence similarity to functionally characterized vertebrate TLRs. ci-tlr1 and ci-tlr2 genes were expressed predominantly in the stomach and intestine as well as in hemocytes. Ci-TLR1 and Ci-TLR2 expressed in HEK293 cells, unlike vertebrate TLRs, were localized to both the plasma membrane and endosomes. Intriguingly, both Ci-TLR1 and Ci-TLR2 stimulate NF-kappaB induction in response to multiple pathogenic ligands such as double-stranded RNA, and bacterial cell wall components that are differentially recognized by respective vertebrate TLRs, revealing that Ci-TLRs recognize broader pathogen-associated molecular patterns than vertebrate TLRs. The Ci-TLR-stimulating pathogenic ligands also induced the expression of Ci-TNFalpha in the intestine and stomach where Ci-TLRs are expressed. These results provide evidence that the TLR-triggered innate immune systems are essentially conserved in ascidians, and that Ci-TLRs possess "hybrid" biological and immunological functions, compared with vertebrate TLRs. Moreover, it is presumed that chordate TLR ancestors also acquired the Ci-TLR-like multiple cellular localization and pathogen-associated molecular pattern recognition.

M-Ras evolved independently of R-Ras and its neural function is conserved between mammalian and ascidian, which lacks classical Ras.

The Ras family small GTPases play a variety of essential roles in eukaryotes. Among them, classical Ras (H-Ras, K-Ras, and N-Ras) and its orthologues are conserved from yeast to human. In ascidians, which phylogenetically exist between invertebrates and vertebrates, the fibroblast growth factor (FGF)-Ras-MAP kinase signaling is required for the induction of neural system, notochord, and mesenchyme. Analyses of DNA databases revealed that no gene encoding classical Ras is present in the ascidians, Ciona intestinalis and Halocynthia roretzi, despite the presence of classical Ras-orthologous genes in nematode, fly, amphioxus, and fish. By contrast, both the ascidians contain single genes orthologous to Mras, Rras, Ral, Rap1, and Rap2. A single Mras orthologue exists from nematode to mammalian. Thus, Mras evolved in metazoans independently of other Ras family genes such as Rras. Whole-mount in situ hybridization showed that C. intestinalis Mras orthologue (Ci-Mras) was expressed in the neural complex of the ascidian juveniles after metamorphosis. Knockdown of Ci-Mras with morpholino antisense oligonucleotides in the embryos and larvae resulted in undeveloped tails and neuronal pigment cells, abrogation of the notochord marker brachyury expression, and perturbation of the neural marker Otx expression, as has been shown in the experiments of the FGF-Ras-MAP kinase signaling inhibition. Mammalian Ras and M-Ras mediate nerve growth factor-induced neuronal differentiation in rat PC12 cells by activating the ERK/MAP kinase pathway transiently and sustainedly, respectively. Activated Ci-M-Ras bound to target proteins of mammalian M-Ras and Ras. Exogenous expression of an activated Ci-M-Ras in PC12 cells caused ERK activation and induced neuritogenesis via the ERK pathway as do mammalian M-Ras and Ras. These results suggest that the ascidian M-Ras orthologue compensates for lacked classical Ras and plays essential roles in neurogenesis in the ascidian.

Characterization of a novel vasopressin/oxytocin superfamily peptide and its receptor from an ascidian, Ciona intestinalis.

The vasopressin (VP)/oxytocin (OT) superfamily peptides are one of the most widely distributed neuropeptides and/or neurohypophysial hormones, but have ever not been characterized from any deuterostome invertebrates including protochordates, ascidians. In the present study, we show the identification of a novel VP/OT superfamily peptide and its receptor in the ascidian, Ciona intestinalis. Intriguingly, the Ciona VP/OT-related peptide (Ci-VP), unlike other 9-amino acid and C-terminally amidated VP/OT superfamily peptides, consists of 13 amino acids and lacks a C-terminal amidation. Mass spectrometry confirmed the presence of the 13-residue Ci-VP in the neural complex. Furthermore, 10 of 14 cysteines are conserved in the neurophysin domain, compared with other VP/OT counterparts. These results revealed that the VP/OT superfamily is conserved in ascidians, but the Ci-VP gene encodes an unprecedented VP/OT-related peptide and neurophysin protein. Ci-VP was also shown to activate its endogenous receptor, Ci-VP-R, at physiological concentrations, confirming the functionality of Ci-VP as an endogenous ligand. The Ci-VP gene was expressed exclusively in neurons of the brain, whereas the Ci-TK-R mRNA was distributed in various tissues including the neural complex, alimentary tract, gonad, and heart. These expression profiles suggest that Ci-VP, like other VP/OT superfamily peptides, serves as a multifunctional neuropeptides. Altogether, our data revealed both evolutionary conservation and specific divergence of the VP/OT superfamily in protochordates. This is the first molecular characterization of a VP/OT superfamily peptide and its cognate receptor from not only ascidians but also deuterostome invertebrates.

Identification of thirty-four transcripts expressed specifically in hemocytes of Ciona intestinalis and their expression profiles throughout the life cycle.

The innate immunity of ascidian hemocytes is considered to be a prototype of that in vertebrates. In this study, we identified as many transcripts as possible that were expressed specifically in hemocytes of Ciona intestinalis, a ubiquitous species of ascidian. Using a large-scale whole-mount in situ hybridization (WISH) technique and young adult specimens of C. intestinalis, 34 such transcripts were identified. Three of these appeared to encode immunity-related polypeptides, whereas 23 encoded hypothetical and/or new genes. Interestingly, different sets of transcripts appeared to be expressed in different subsets of hemocytes, as revealed by double-colored WISH. The 34 genes were categorized into two major subgroups based on their expression patterns during the C. intestinalis life cycle. Based on the gene expression profiles, we speculate that C. intestinalis hemocytes may exert more pleiotropic effects in immunity than previously believed.

Tachykinin and tachykinin receptor of an ascidian, Ciona intestinalis: evolutionary origin of the vertebrate tachykinin family.

Tachykinins (TKs) are the most prevalent vertebrate brain/gut peptides. In this study, we originally identified authentic TKs and their receptor from a protochordate, Ciona intestinalis. The Ciona TK (Ci-TK) precursor, like mammalian gamma-preprotachykinin A (gamma-PPTA), encodes two TKs, Ci-TK-I and -II, including the -FXGLM-NH(2) vertebrate TK consensus. Mass spectrometry of the neural extract revealed the production of both Ci-TKs. Ci-TK-I contains several Substance P (SP)-typical amino acids, whereas a Thr is exceptionally located at position 4 from the C terminus of Ci-TK-II. The Ci-TK gene encodes both Ci-TKs in the same exon, indicating no alternative generation of Ci-TKs, unlike the PPTA gene. These results suggested that the alternative splicing of the PPTA gene was established during evolution of vertebrates. The only Ci-TK receptor, Ci-TK-R, was equivalently activated by Ci-TK-I, SP, and neurokinin A at physiological concentrations, whereas Ci-TK-II showed 100-fold less potent activity, indicating that the ligand selectivity of Ci-TK-R is distinct from those of vertebrate TK receptors. Ci-TK-I, like SP, also elicited the typical contraction on the guinea pig ileum. The Ci-TK gene was expressed in neurons of the brain ganglion, small cells in the intestine, and the zone 7 in the endostyle, which corresponds to the vertebrate thyroid gland. Furthermore, the Ci-TK-R mRNA was distributed in these three tissues plus the gonad. These results showed that Ci-TKs play major roles in sexual behavior and feeding in protochordates as brain/gut peptides and endocrine/paracrine molecules. Taken together, our data revealed the biochemical and structural origins of vertebrate TKs and their receptors.

Drug eruption due to peplomycin: an unusual form of Stevens-Johnson syndrome with pustules.

A rare case of Stevens-Johnson syndrome (SJS) due to peplomycin in a 48-year-old man is described. The patient had squamous cell carcinoma on the scalp and underwent preoperative neoadjuvant chemotherapy with peplomycin. On the fifth day of the chemotherapy, he developed a fever and multiple dusky violaceous erythematous areas and pustules on his trunk, thighs, and palms. Erosive erythema and erosions also developed on his soles, scrotum, and oral mucosa. A biopsy specimen taken from the eruption on the thigh revealed marked liquefaction degeneration of the basal layer of the epidermis. Laboratory examinations demonstrated aggravation of liver function. Additionally, the patient developed conjunctivitis and corneal erosions. Although he had some subcorneal pustules, we diagnosed the case as an unusual form of SJS because of severe mucous membrane involvement. Oral prednisolone was administered, and the symptoms subsided. Then the patient underwent wide local excision. One month after surgery, we performed patch tests and a lymphocyte stimulation test with negative results. Then we re-administered peplomycin starting with 1/20 of a daily dose and gradually increasing the dose each day. After administration of the regular daily dose, the patient had a relapse of fever, eruptions, stomatitis, corneal erosions, and liver dysfunction. Therefore, a definite diagnosis of drug eruption due to peplomycin was made.