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The complement system of Botryllus schlosseri
Full text linkAmong the various effector mechanisms involved in immune responses, the complement system is one of the most ancient, deeply-rooted and important for its ability to orchestrate different cells and factors of both innate and adaptive immunity. The comprehension of its roots in the evolution is useful to understand how the main complement-related proteins had changed in order to adapt to new environmental conditions and life-cycles or, in the case of vertebrates, to interact with the adaptive immunity. In this context, data from organisms evolutionary close to vertebrates, such as tunicates, are of primary importance for a better understanding of the changes in immune responses associated with the invertebratevertebrate transition. In our model tunicate Botryllus schlosseri we have described a lectin and alternative pathway of complement system activation very similar to those of Vertebrates. All the complement-related genes such as c3, bf, ficolin, mbl and masp are transcribed by morula cells, the immunocytes in immunomodulation and cytotoxic responses. Functional data suggest a complement-related cross-talk between morula cells and phagocytes immunocyte during the immune response. When B. schlosseri hemocytes are incubated with yeast (Saccharomyces cerevisiae) cells, there is an overexpression of C3 by morula cell that led to increase of phagocytosis that is prevented in the presence of the C3 inhibitor, compstatin. In the next future, we will focus our efforts on the regulation of complement system in tunicates to shed new light on the complement system function in a pre-adaptive immunity scenario
The C1q domain-containing protein from the ascidian Botryllus schlosseri manifests a cytokine-like behavior.
No full textGenes encoding complement component 3 (C3) have been extensively investigated in invertebrate genomes and traced back in evolutionary history to the early metazoan radiation. However, other components of the complement system, such as those related to the classical activation pathway, are still not much investigated. Currently, the genes encoding for proteins with a C1q domain, probably the main components of the classical pathway, have been only partially investigated from an evolutionary perspective. These genes exist in many of the sequenced genomes, from both vertebrates and invertebrates and functions have been described for some of the corresponding proteins. A C1q-like gene have been identified in the medicinal leech Hirudo medicinalis where a C1q-like peptide elicits a chemotactic behavior that could be blocked using a human antibody against the gC1q receptor. C1q-like genes have also been found in the urochordate Ciona intestinalis and the cephalocordate Branchiostoma floridae where it has been demonstrated that the globular domain is able to recognize and bind mammalian antibodies initiating the classical pathway of complement activation. The globular head C1q domain is a lectin domain present in transcriptomes of amphioxus, lamprey, and several teleost fishes. Few of these putative C1q-like proteins have been characterized; however, they can bind to a variety of carbohydrates. In Botryllus schlosseri we have found, in our EST collection, a single transcript with C1q characteristics (BsC1q-like). The deduced protein contains two globular head C1q domains, a feature unknown in invertebrates. As regard Vertebrates, we can find a similar architecture only in mammals, in the so called C1q/TNF-related Protein 4 (CTRP4). This protein is very poorly studied and seems to be expressed in the hypothalamus and contribute to the modulation of food intake and body weight. Our data, from the colonial ascidian, suggest a role for the BsC1q-like protein as mediator of the activation and degranulation of the cytotoxic hemocytes. Both ISH and ICC demonstrate that both cytotoxic morula cells and phagocytes express the BsC1q-like mRNA and protein; functional analyses demonstrate that the human antibody against globular head C1q is able to inhibit morula cell degranulation after bacterial challenge. It is not yet clear if it is possible to considered this molecule as member of the complement system in Botryllus but future analyses will be directed to the study of the functional relationships between BsC3 and BsC1q-like as well as of the binding capabilities of the latter
Individuation of a new metallothionein from the urochordate Ciona intestinalis.
No full textMetallothioneins (MTs) are metal-chelating proteins occurring in animals, plants and prokaryotes, involved in detossification and immunity. In 2001, Canpolat and Lynes showed that exogenous MT can affect cell proliferation macrophage and cytotoxic T lymphocyte function, and humoral immunity to T-dependent antigens in mice. These evidences strengthen the hypothesis that MTs have an active role in extracellular environment as immunomodulatory proteins. Up to now, there are no descriptions of MTs in invertebrate Chordates althought it seems that the vertebrate structure is maintained also in other deuterostomes such as the echinoderms. As we are interested in studing the involvement and role of MTs ascidian immune responses, we undertook a preliminary investigation amied to identify these molecules in Ciona intestinalis, the genome of which has been fully sequenced but no MTs have been annotated. We have cloned the transcript and characterized the gene of a new MT, codifying for 39 amino acids, including 12 cys residues (30% of total amino acids, in accordance with other MTs). Moreover, the typical organization of cysteine residues in C-X-C motifs is conserved. The gene is composed of two introns (one inside the coding region and the other inside the 3’ UTR region) and three exons. The 5’ untrascribed region contains several cis elements similar to those found in vertebrate MT genes such as: metal responding elements (MRE) involved in constitutive and metal-related induction, antioxidant responding elements, important for ROS-dependent MT expression and STAT3, having a role in cytokine-related induction. The amino acid sequence of C. intestinalis MT shows only limited similarity with other known MTs: Mytilus edulis MT (28,8% identity), Strongylocentrotus purpuratus MT (23,4% identity) and Sparus aurata MT (36,7% identity)
BsTLR: a new member of the TLR family of recognition proteins from the colonial ascidian Botryllus schlosseri.
Full text linkToll-like receptors (TLRs) represent a well-known family of conserved pattern recognition receptors the importance of which, in non-self recognition, was demonstrated in both vertebrates and invertebrates. Tunicates represent the vertebrate sister group and, as invertebrates, they rely only on innate immunity for their defense. As regards TLRs, two transcripts have been described and characterized in the solitary species Ciona robusta, referred to as CiTLR1 and CiTLR2. Using the Ciona TLR nucleotide sequences, we examined the available transcriptomes of Botryllus schlosseri looking for similar sequences. We were able to identify a sequence, with similarity to CiTLR2 and, through in silico transduction and subsequent sequence analysis, we studied the domain content of the putative protein. The sequence, called BsTLR, has a TIR and a transmembrane domain, four LLR and two LRR-CT domains. In addition, we analised bstlr transcription in vivo and in vitro, under various experimental conditions and in different phases of the Botryllus blastogenetic cycle. Our data show that, in different phases, there is a change in gene transcription and mRNA location, according to the blastogenetic phase
Lectins and immunity in compound ascidians
No full textLectins are proteins able to recognize and bind specific glycoconjugates, widely distributed among plants and animals. Most of them have agglutinating activity towards vertebrate erythrocytes and other animal cells, due to the presence of multiple carbohydrate recognition domains which bind to cell surface sugars.
A great number of invertebrate lectins have been described in the last two decades: they show different specificities, sizes and physico-chemical properties and are believed to be involved in various processes, such as cell-cell interaction, fertilisation, morphogenesis and defence reactions.
Ascidians are invertebrate chordates phylogenetically close to vertebrates and the study of their immune responses can contribute to a better understanding of the complex immune system of vertebrates.
In compound ascidians, lectins play an important role in opsonisation of foreign particles or cells having entered the organism. They can also induce cell proliferation and enhance the recruitment of immunocytes to the infection area.
In the compound ascidian Botryllus schlosseri, our model organism, we recently identified a rhamnose-binding lectin (BsRBL)which can recruit phagocytes, activate their respiratory burst with the consequent production of microbicidal reactive-oxygen species, and stimulate phagocytosis of foreign target cell by opsonising them and inducing cytoskeletal changes in phagocytes. In addition, BsRBL induces the synthesis and release, by cytotoxic morula cells, of cytokines recognised by anti-IL-1 and anti-TNF antibodies, with chemotactic activity towards cytotoxic immunocytes. It also triggers the degranulation of morula cells with the consequent release of the cytotoxic enzyme phenoloxidase. Results suggest an important role of BsRBL in Botryllus immunobiology and support the existence of a cross-talk between B. schlosseri immunocytes
Cellular aspects of allorecognition in the compound ascidian Botrylloides simodensis
No full textWhen colonies of the compound ascidian Botrylloides simodensis contact each other at their cut surfaces, either fusion or rejection occurs. Contact between genetically compatible colonies leads to the complete fusion of their tunics and vasculature within 24 h. Conversely, the rejection reaction between incompatible colonies is characterized by the appearance of a melanic, necrotic band along the contact border. In the case of fusion, limited crowding of cytotoxic morula cells (MCs) was observed in the ampullae near the contact border. In rejection, limited tunic fusion occurred in the contact region and MCs were selectively recruited inside the ampullae near the cut surface: most of them leaked into the tunic where they changed their morphology and contributed to the formation of the necrotic region. Granular amebocytes, like MCs, have granules well stained by eosin and were also seen inside the ampullae involved in the rejection reaction and along the contact border between incompatible colonies. Immunohistochemical analysis using antibodies raised against Botryllus schlosseri phenoloxidase (PO) and mammalian IL-1-α and TNF-α indicate that MCs were the only cells recognized by the anti-PO antibody; they resulted immunopositive also to the anti-cytokine antibodies in both fusion and rejection, whereas granular amoebocytes were recognized by the latter antibodies only during the rejection reactio
Nanos homologue expression in haemocytes of two botryllid ascidians (Botryllus primigenus and Botryllus schlosseri)
No full textNanos and nanos-related genes encode RNA-binding proteins and are expressed by stem cells of many metazoans. In the Japanese colonial ascidian Botryllus primigenus, a nanos homologue of 1.3 kb, Bpnos, has been recently identified (Sunanaga et al., 2008), containing two nanos-like CCHC zinc finger motifs. The gene in expressed in germ stem cells, immature and mature male germ cells, as well as in a fraction of circulating haemocytes. Using an anti-Bpnos monoclonal antibody, we confirmed that haemoblasts, i.e., circulating undifferentiated cells, are the only haemocytes recognised by the antibody in both B. primigenus and the Mediterranean species Botryllus schlosseri. In both species, the frequency of haemoblasts reaches the maximum value just after the take-over phase, when the generation change occurs and gradually decreases during the rest of the cycle. The number of circulating haemoblasts is much higher in the Japanese than in the Mediterranean species and this can be related to the occurrence of spontaneous vascular budding in B. primigenus. We also identified a nanos-related gene in B. schlosseri, sharing the nanos domain with two CCHC motifs, which we propose to call Bsnos. We are now trying to get the full cDNA sequence and the deduced amino acid sequence in order to get probes for Bsnos expression studies
First evidences of a complement system lytic pathway in invertebrates: data from the compound ascidian Botryllus schlosseri.
No full textThe complement system is well studied in mammals, where more than 30 proteins have been described, involved in the activation and regulation of this important humoral effector. However, the evolutionary history of the complement system is not yet fully elucidated and, in recent years, it has been widely demonstrated that complement system is more than just a defender against intruders. For instance, it is important for the clearance of apoptotic cells and corpses. Botryllus schlosseri is a cosmopolitan ascidian, belonging to the phylum Chordata, considered a model organism for the studies of the evolution of the immune system. Studying the complexity of the complement system in Botryllus, we identified a transcript, in our EST collection, coding a protein containing the MACPF (membrane attack complex/perforin) domain, shared by both most of the proteins involved in the lytic pathway and perforins. Invertebrates, we know that perforins are produced by T-lymphocytes and natural killer cells, whereas the activity of the proteins, with the MACPF domain is regulated by the complement component C3/C5.Comparing the domain topology of vertebrateC9 and our protein, called Botryllus C9-like protein (BsC9), a high level of similarity results. To demonstrate that our C9-like protein can be considered a part of the complement system in B. schlosseri we evaluated the expression of BsC9 with respect to C3 activity. Our previous data demonstrates that B. schlosseri C3 is activated by zymosan. We combined the microinjection of zymosan with and without a validated anti-C3 antibody (against human C3) to block the activity of C3. With this approach we studied in, time course, the expression of both BsC3 and BsC9 demonstrating that the anti-C3 antibody is able to inhibit the expression of BsC9. These results are confirmed in both ISH and ICC using the same antibody, and in vitro with the C3 inhibitor compstatin. In addition, a significant (p < 0.05) decrement of labeling with BsC9 antisense riboprobe and validated antibody anti hsC9 is observed when hemocytes are incubated with zymosan and compstatin. Collectively, these results argue in favor of the presence of components of the lytic pathway in our model organism
Botryllin, a Novel Antimicrobial Peptide from the Colonial Ascidian Botryllus schlosseri
Full text linkBy mining the transcriptome of the colonial ascidian Botryllus schlosseri, we identified a transcript for a novel styelin-like antimicrobial peptide, which we named botryllin. The gene is constitutively transcribed by circulating cytotoxic morula cells (MCs) as a pre-propeptide that is then cleaved to mature peptide. The synthetic peptide, obtained from in silico translation of the transcript, shows robust killing activity of bacterial and unicellular yeast cells, causing breakages of both the plasma membrane and the cell wall. Specific monoclonal antibodies were raised against the epitopes of the putative amino acid sequence of the propeptide and the mature peptide; in both cases, they label the MC granular content. Upon MC degranulation induced by the presence of nonself, the antibodies recognise the extracellular nets with entrapped bacteria nearby MC remains. The obtained results suggest that the botryllin gene carries the information for the synthesis of an AMP involved in the protection of B. schlosseri from invading foreign cells
Effects of cadmium on the functionality of haemocytes from the compound ascidian Botryllus schlosseri
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