Cloning and characterization of integrin alpha subunits from the solitary ascidian, Halocynthia roretzi.
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Recent molecular and biochemical analysis has revealed the presence of an opsonic complement system in the solitary ascidian, Halocynthia roretzi, composed of at least C3, two mannan binding protein-associated serine proteases, and factor B. To elucidate further the structure and function of this apparently primitive complement system in the urochordates, we looked for the ascidian complement receptor type 3 (CR3), or type 4 (CR4), which are members of the leukocyte integrin family in mammals. Using degenerate primers, we isolated two integrin alpha subunits (alpha(Hr1) and alpha(Hr2)) from the hemocyte mRNA of H. roretzi, by RT-PCR, and the entire coding sequence of alpha(Hr1) was determined from cDNA clones. alpha(Hr1) contains an I domain, the inserted domain characteristic of a subset of mammalian alpha subunits, including the leukocyte integrin family. A phylogenetic tree constructed for the alpha subunits also supports the ancestral position of alpha(Hr1) in the monophyletic cluster of I domain-containing alpha integrins. The alpha(Hr1) gene shows hemocyte-specific expression on Northern blot analysis. Western blot analysis and immunocytochemical staining of the hemocytes of H. roretzi using anti-alpha(Hr1) Ab showed that alpha(Hr1) subunits exist on the surface of a subpopulation of phagocytic hemocytes. Furthermore, anti-alpha(Hr1) Ab inhibited C3-dependent phagocytosis, but not basic phagocytosis, of yeast cells by ascidian hemocytes. These observations strongly suggest that alpha(Hr1) constitutes an integrin molecule on the hemocytes of H. roretzi that functions as an ancestral form of CR3 and CR4 and mediates phagocytosis in the primitive complement system of the ascidian. (+info)
The maternal transcript for truncated voltage-dependent Ca2+ channels in the ascidian embryo: a potential suppressive role in Ca2+ channel expression.
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Ca2+ entry during electrical activity plays several critical roles in development. However, the mechanisms that regulate Ca2+ influx during early embryogenesis remain unknown. In ascidians, a primitive chordate, development is rapid and blastomeres of the muscle and neuronal lineages are easily identified, providing a simple model for studying the expression of voltage-dependent Ca2) channels (VDCCs) in cell differentiation. Here we isolate an ascidian cDNA, TuCa1, a homologue of the alpha(1)-subunit of L-type class Ca2+ channels. We unexpectedly found another form of Ca2+ channel cDNA (3-domain-type) potentially encoding a truncated type which lacked the first domain and a part of the second domain. An analysis of genomic sequence suggested that 3-domain-type RNA and the full-length type have alternative transcriptional start sites. The temporal pattern of the amount of 3-domain-type RNA was the reverse of that of the full-length type; the 3-domain type was provided maternally and persisted during early embryogenesis, whereas the full-length type was expressed zygotically in neuronal and muscular lineage cells. Switching of the two forms occurred at a critical stage when VDCC currents appeared in neuronal or muscular blastomeres. To examine the functional roles of the 3-domain type, it was coexpressed with the full-length type in Xenopus oocyte. The 3-domain type did not produce a functional VDCC current, whereas it had a remarkable inhibitory effect on the functional expression of the full-length form. In addition, overexpression of the 3-domain type under the control of the muscle-specific actin promoter in ascidian muscle blastomeres led to a significant decrease in endogenous VDCC currents. These findings raise the possibility that the 3-domain type has some regulatory role in tuning current amplitudes of VDCCs during early development. (+info)
Cloning and characterization of novel ficolins from the solitary ascidian, Halocynthia roretzi.
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Ficolins are animal lectins with collagen-like and fibrinogen-like domains. They are involved in the first line of host defense against pathogens. Human ficolin/P35 as well as mannose-binding lectin (MBL) activates the complement lectin pathway in association with MBL-associated serine proteases. To elucidate the origin and evolution of ficolins, we separated approximately 40 kDa (p40) and approximately 50 kDa (p50) N-acetylglucosamine-binding lectins from hemolymph plasma of the solitary ascidian. Binding assays revealed that p40 recognizes N-acetyl groups in association with a pyranose ring and that p50 recognizes N-acetylglucosamine alone. Based on the amino acid sequences of the proteins, we isolated two clones each of p40 and p50 from the ascidian hepatopancreas cDNA and determined the entire coding sequences of these clones. Because all of the clones contained both collagen-like and fibrinogen-like domains, we concluded that these were homologs of the mammalian ficolin family and designated ascidian ficolins (AsFCNs). The fibrinogen-like domain of the AsFCNs shows 45.4-52.4% amino acid sequence identity with the mammalian ficolin family. A phylogenetic tree of the fibrinogen-like sequences shows that all the fibrinogen-like domains may have evolved from a common ancestor that branched off an authentic fibrinogen. These results suggest that AsFCNs play an important role with respect to ascidian hemolymph lectin activity and the correlation of different functions with binding specificity. (+info)
Molecular patterning of the oikoplastic epithelium of the larvacean tunicate Oikopleura dioica.
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Appendicularia are protochordates that rely on a complex mucous secretion, the house, to filter food particles from seawater. A monolayer of cells covering the trunk of the animal, the oikoplastic epithelium, secretes the house. This epithelium contains a fixed number of cells arranged in characteristic patterns with distinct sizes and nuclear morphologies. Certain house structures appear to be spatially related to defined, underlying groups of cells in the epithelium. We show that the house is composed of at least 20 polypeptides, a number of which are highly glycosylated, with glycosidase treatments resulting in molecular mass shifts exceeding 100 kDa. Nanoelectrospray tandem mass spectrometric microsequencing of house polypeptides was used to design oligonucleotides to screen an adult Oikopleura dioica cDNA library. This resulted in the isolation of cDNAs coding for three different proteins, oikosin 1, oikosin 2, and oikosin 3. The latter two are novel proteins unrelated to any known data base entries. Oikosin 1 has 13 repeats of a Cys domain, previously identified as a subunit of repeating sequences in some vertebrate mucins. We also find one repeat of this Cys domain in human cartilage intermediate layer protein but find no evidence of this domain in any invertebrate species, including those for which entire genomes have been sequenced. The three oikosins show distinct and complementary expression patterns restricted to the oikoplastic epithelium. This easily accessible epithelium, with differential gene expression patterns in readily identifiable groups of cells with distinctive nuclear morphologies, is a highly attractive model system for molecular studies of pattern formation. (+info)
cDNA cloning and functional analysis of ascidian sperm proacrosin.
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cDNA cloning and functional analysis of proacrosin from the ascidian Halocynthia roretzi were undertaken. The isolated cDNA of the ascidian preproacrosin consists of 2367 nucleotides, and an open reading frame encodes 505 amino acids, which corresponds to the molecular mass of 55,003 Da. The mRNA of proacrosin was found to be specifically expressed in the gonad by Northern blotting and in the spermatocytes or spermatids by in situ hybridization. The amino acid sequences around His(76), Asp(132), and Ser(227), which make up a catalytic triad, showed high homology to those of the trypsin family. Ascidian acrosin has paired basic residues (Lys(56)-His(57)) in the N-terminal region, which is one of the most characteristic features of mammalian acrosin. This region seems to play a key role in the binding of (pro)acrosin to the vitelline coat, because the peptide containing the paired basic residues, but not the peptide substituted with Ala, was capable of binding to the vitelline coat. Unlike mammalian proacrosin, ascidian proacrosin contains two CUB domains in the C-terminal region, in which CUB domain 1 seems to be involved in its binding to the vitelline coat. Four components of the vitelline coat that are capable of binding to CUB domain 1 in proacrosin were identified. In response to sperm activation, acrosin was released from sperm into the surrounding seawater, suggesting that ascidian acrosin plays a key role in sperm penetration through the coat. These results indicate that ascidian sperm contains a mammalian acrosin homologue, a multi-functional protein working in fertilization. (+info)
Fragmentation of a novel marine peptide, plicatamide, involves an unusual gas-phase intramolecular rearrangement.
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During our characterization of plicatamide 1, a modified octapeptide: Phe-Phe-His-Leu-His-Phe-His-dc deltaDOPA (where dc deltaDOPA = decarboxy-(E)-alpha,beta-dehydro-3,4-dihydroxyphenylalanine) from the blood cells of the ascidian Styela plicata, we noted a series of fragment ions from the [M + H]+ ion which could not be assigned. There was no evidence in the 1H NMR spectrum to support an alternative molecular structure and the series of fragment ions were not present in the tandem mass spectrometry analysis of the [M + Na]+ ion. In addition, there was no evidence that the sample was a mixture of isobaric compounds. We propose that an unusual C-terminal to N-terminal rearrangement is responsible for the series of fragment ions from the [M + H]+ ion. This rearrangement was not observed in peptide analogs of plicatamide which did not contain the dc deltaDOPA at the C-terminus suggesting that this moiety is critical for the rearrangement. The proposed reaction is analogous to that recently reported by Vachet et al. involving a fragment ion formed from leucine enkephalin. (+info)
Characterization of cytochrome b(5) in the ascidian Polyandrocarpa misakiensis and budding-specific expression.
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A cDNA for cytochrome b(5) was cloned from a cDNA library of buds of the ascidian, Polyandrocarpa misakiensis, by a hybridization method involving a digoxigenin-labeled cDNA probe of human soluble cytochrome b(5). The nucleotide sequence of the cDNA for the ascidian cytochrome b(5) (Pmb5) consisted of about 1,800 base pairs including 5'- and 3'-noncoding regions, and a coding sequence of 405 base pairs. The amino acid sequence of 135 residues deduced from the coding nucleotide sequence exhibited 54% identity and 76% similarity to chicken cytochrome b(5). A highly conserved amino acid sequence was observed in the amino-terminal domain of 96 residues containing two heme-binding histidine residues. The putative soluble form of the recombinant Pmb5 expressed in Escherichia coli was purified to homogeneity by column chromatographies on an anion-exchanger and gel filtration. The purified Pmb5 showed the typical absorption spectrum of cytochrome b(5) with an asymmetric peak at 556 nm and a shoulder at 560 nm upon reduction with NADH and NADH-cytochrome b(5) reductase. The low temperature spectrum of the dithionite-reduced form of the protein contained the split peaks at 551 and 555 nm, this spectrum being very similar to that of mammalian liver cytochrome b(5). Expression of Pmb5 in the ascidian was examined immunohistochemically with a monoclonal antibody against the Pmb5. Apparently high level expression of Pmb5 was found in the developing buds, but the levels of cytochrome b(5) in the parents and juvenile adults were very low. This is the first report on the characterization of Pmb5, and the increased expression of Pmb5 in the ascidian. (+info)
Simultaneous measurement of intracellular nitric oxide and free calcium levels in chordate eggs demonstrates that nitric oxide has no role at fertilization.
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At fertilization in sea urchin, the free radical nitric oxide (NO) has recently been suggested to cause the intracellular Ca(2+) rise responsible for egg activation. The authors suggested that NO could be a universal activator of eggs and the present study was set up to test this hypothesis. Intracellular NO and Ca(2+) levels were monitored simultaneously in eggs of the mouse or the urochordate ascidian Ascidiella aspersa. Eggs were either fertilized or sperm extracts microinjected. Sperm-induced Ca(2+) rises were not associated with any global, or local, change in intracellular NO, although we were able to detect NO produced by the addition of a NO donor. Furthermore, the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester had no effect on sperm-induced Ca(2+) release but did block completely ionomycin-induced NO synthase activation. Therefore, we suggest that the current data provide evidence that NO has no role in the fertilization of these two chordate eggs. (+info)