Primary structure of ascidian trypsin inhibitors in the hemolymph of a solitary ascidian, Halocynthia roretzi

The amino acid sequences of trypsin inhibitors I and II from the hemolymph of a solitary ascidian, Halocynthia roretzi, were determined after reduction and S-pyridylethylation. The results indicated that inhibitor I consists of a single polypeptide chain with 55 amino acid residues and four intramolecular disulfide bridges, whereas inhibitor II is composed of two polypeptide chains corresponding to a form derived from inhibitor I by cleavage at the Lys16-Met17 bond. Lys16 may be the reactive-site residue of these inhibitors, because carboxypeptidase B treatment destroys most of the inhibitory activity of inhibitor II but not that of inhibitor I.     

Purification and characterization of beta-N-acetylhexosaminidase from the ascidian, Halocynthia roretzi

beta-N-Acetylhexosaminidase [EC 3.2.1.30] was purified 820-fold from the viscera of Halocynthia roretzi by Sephadex G-200 gel filtration and chromatography on columns of DEAE-Sephadex and CM-Sephadex. The final preparation was sufficiently free from alpha-N-acetylglucosaminidase, alpha-N-acetylgalactosaminidase, alpha- and beta-glucosidases, alpha- and beta-galactosidases, alpha- and beta-mannosidases, and alpha-L-fucosidase, and gave one protein band on disc gel electrophoresis. Two different molecular weight forms which depended upon the pH were observed on Sephadex gel filtration. At pH 7.0, a species with a molecular weight of 170,000 was observed, whereas at pH 4.5, an enzyme of 330,000 daltons was seen. The enzyme was active at pH 4.5 but inactive at pH 7.0. The optimum pH and the Km were pH 4.2 and 1.9 mM for p-nitrophenyl beta-N-acetylglucosaminide and pH 4.0 and 0.9 mM for p-nitrophenyl beta-N-acetylgalactosaminide. The terminal beta-N-acetylhexosamine of glycolipids such as globoside I, GM2, and asialo GM2 was cleaved by the ascidian beta-N-acetylhexosaminidase though GM2 was less susceptible to the enzyme.     

Galactose-specific lectin in the hemolymph of solitary ascidian, Halocynthia roretzi. Molecular, binding and functional properties

Galactose-specific lectin isolated from the hemolymph of solitary ascidian, Halocynthia roretzi, has been further characterized. The hemagglutinating activity of the lectin is Ca2+-dependent. The lectin has a large molecular form as revealed by gel-permeation chromatography, sedimentation equilibrium and velocity measurement, and electron microscopic observation. The lectin is adsorbed to columns of blue-Sepharose and phenyl-Sepharose, and eluted with ethylene glycol, not with lactose or high concentration of NaCl. The lectin shows a stimulatory effect on the superoxide anion production by guinea-pig polymorphonuclear leukocytes, and the effect is inhibited, among various sugars, most strongly by melibiose.     

Cloning and characterization of novel ficolins from the solitary ascidian, Halocynthia roretzi

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.     

Cloning and characterization of integrin alpha subunits from the solitary ascidian, Halocynthia roretzi

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.     

Purification and characterization of multicatalytic proteinase from eggs of the ascidian Halocynthia roretzi

A multicatalytic (high-molecular-weight) proteinase has been purified from eggs of the ascidian Halocynthia roretzi by a procedure including column chromatographies on DEAE-cellulose and hydroxylapatite and gel filtration on Sepharose 6B. The purified enzyme seemed to be homogeneous, as judged by disc-polyacrylamide gel electrophoresis, isoelectrofocusing, sedimentation velocity, and gel filtration. The molecular weight of the enzyme was estimated to be 610,000 by gel filtration. The isoelectric point and the sedimentation coefficient (S20,w) were 6.2 and 22.8S, respectively. The enzyme showed several protein bands with molecular weight ranging from 25,000 to 33,000 on SDS-polyacrylamide gel electrophoresis and a cylindrical or ring-like structure composed of several subunits under the electron microscope, indicating that the enzyme exists as a large molecule consisting of several protein components. The enzyme exhibited chymotrypsin-like and trypsin-like activities whose pH optima were both 7.0. Chymostatin and its analog, calpain inhibitor I, and elastatinal inhibited both activities, whereas leupeptin and antipain only inhibited the latter. The former activity was stimulated by a low concentration of SDS or fatty acid, whereas the latter was not. Thus, the properties of the enzyme purified from ascidian eggs are similar to those of multicatalytic proteinases from mammalian tissues.     

Establishment of self-sterility of eggs in the ovary of the solitary ascidian,Halocynthia roretzi

When unfertilized eggs (UFE) of the solitary ascidian, Halocynthia roretzi, are released naturally they are strictly self-sterile, whereas almost all ovarian eggs isolated after spawning are self-fertile. Self-sterile eggs are prepared within a relatively short period of several hours before the spawning. The morphological changes in ovarian eggs during late oogenesis were studied with special reference to the establishment of self-sterility. Four types of eggs at serial developmental stages were classified according to the morphology of their external envelopes. Self-sterility was established in the last stage, from the ovarian egg type 3 (OVE3) to UFE stages. Ovarian eggs which had become committed to UFE were denoted as full-grown ovarian eggs (FOE). FOE were able to differentiate into self-sterile UFE in vitro, whereas OVE3 could not. Several morphological differences between OVE3 and UFE were found. OVE3 had a germinal vesicle (GV), a type of vitelline coat (VC-OVE3) and no expanded perivitelline space, whereas UFE had completed germinal vesicle break down (GVBD), had another type of coat (VC-UFE) and showed an expanded perivitelline space. There were also some differences in the mode of fertilization between OVE3 and UFE. In UFE, sperm became bound firmly to the vitelline coat and passed through the coat with the help of follicle cells, whereas in OVE3, sperm did not bind so strongly and entered the perivitelline space without the aid of follicle cells. The relationships between the establishment of self-sterility and these morphological and functional changes in ovarian eggs are discussed.     

Extracellular ubiquitin system implicated in fertilization of the ascidian, Halocynthia roretzi: isolation and characterization

The ubiquitin-proteasome system is essential for intracellular protein degradation, but there are few studies of this system in the extracellular milieu. Recently, we reported that a 70-kDa sperm receptor, HrVC70, on the vitelline coat is ubiquitinated and then degraded by the sperm proteasome during fertilization of the ascidian, Halocynthia roretzi. Here, we investigated the mechanism of extracellular ubiquitination. The HrVC70-ubiquitinating enzyme activity was found to be released from the activated sperm during the fertilization process. This enzyme was purified from an activated sperm exudate, by chromatography on DEAE-cellulose and ubiquitin-agarose columns, and by glycerol density gradient centrifugation. The molecular mass of the enzyme was estimated to be 700 kDa. The purified enzyme requires CaCl2 and MgATP for activity, and is active in seawater. The purified enzyme preparation, but not the crude enzyme preparation, showed narrow substrate specificity to HrVC70. Moreover, ATP and ubiquitin are released from the activated sperm to the surrounding seawater during fertilization. These results indicate that ascidian sperm release a novel extracellular ubiquitinating enzyme system together with ATP and ubiquitin during penetration of the vitelline coat of the egg, which catalyzes the ubiquitination of the HrVC70, an essential component of ascidian fertilization.     

Study of color variation in the solitary ascidian Halocynthia roretzi, collected in the Inland Sea of Japan

In the vicinity of Yashiro Island in the Inland Sea of Japan, the solitary ascidian (tunicate) Halocynthia roretzi with tunics of various colors were collected. Samples of these animals were sorted into three groups on the basis of visual observation of tunic color. The red group includes animals with dark-red, light-red, or orange tunics. The pink group includes animals with tunic colors ranging between red and white. The white group includes only animals with completely white tunics. Animals in the white group lacked color internally, with the exception of the hepatopancreas and the gonads in breeding season; the epidermis and gill basket were white. In contrast, animals of both the red group and the pink group were colored internally, with red-orange epidermis and yellow gill basket. Alloreactivity was tested by mixed-hemocyte incubation between different animals belonging to the same color group and between animals belonging to different color groups. Alloreactivity between animals of the white group was 56.3%, between animals of the pink group was 60.0%, and between animals of the red group was 69.3%. The relatively high frequency of compatible combinations among the white animals is discussed.     

Self and non-self recognition between gametes of the ascidian,Halocynthia roretzi

Summary The self-sterility ofHalocynthia roretzi from Mutsu Bay, Japan, was examined. This sterility is strict and not a single egg can be fertilized in self-sterile animals. Less than 2% of the animals were self-fertile (with 100% cross-fertility). All heterologous sperm can fertilize all eggs, although there are pairs of individuals in which the coelomocytes recognize each other as self. Eggs deprived of follicle cells cannot be fertilized by either autologous or heterologous spermatozoa. Detached autologous or heterologous follicle cells can reattach to the chorion in calcium-enriched sea water and the reconstituted eggs recover their ability to be fertilized. A mosaic egg can therefore be obtained, which consists of oocyte, test cells and chorion originating from one individual and follicle cells from another. The mosaic egg was used to determine the site of recognition of self and non-self. The results indicate that the recognition resides in the chorion and/or test cells, probably the chorion. The relationship between somatic alloreactivity, previously found in coelomocytes ofH. roretzi, and gamete reactivity is discussed.     

Halocyamines: novel antimicrobial tetrapeptide-like substances isolated from the hemocytes of the solitary ascidian Halocynthia roretzi

Two novel antimicrobial tetrapeptide-like substances, halocyamine A and B, were isolated from the solitary ascidian Halocynthia roretzi by a procedure including extraction steps, chromatographies on coarse and fine HP-20 columns, and preparative reversed-phase high-performance liquid chromatography. The structures of halocyamine A and B were determined to be L-histidyl-L-6,7-dihydroxyphenylalanylglycyl-6-bromo-8,9-didehy drotryptamine and L-threonyl-L-6,7-dihydroxyphenylalanyl-L-histidyl-6-bromo-8,9- didehydrotryptamine, respectively, by spectral analyses and degradation studies. Besides antimicrobial activities against several kinds of bacteria and yeasts, both of them showed cytotoxic activities against neuronal cells cultured from rat fetal brain, mouse neuroblastoma N-18 cells, and human hepatoma Hep-G2 cells. They were only detected in the "morula"-like cells, which are of the most abundant cell type among the hemocytes of H. roretzi.     

Trypsin-like enzyme from eggs of the ascidian (protochordate), Halocynthia roretzi. Purification, properties, and physiological role

A trypsin-like enzyme has been purified to apparent homogeneity from eggs of the ascidian, Halocynthia roretzi, by a procedure including column chromatography on diethylaminoethyl-cellulose, phenyl-Sepharose, and soybean trypsin inhibitor-immobilized Sepharose 4B. The molecular weight of the enzyme was estimated to be 31,000 and 33,000 by gel electrophoresis in sodium dodecyl sulfate under the reducing and the nonreducing conditions, respectively. The isoelectric point of the enzyme was 4.8. The pH optimum of the activity was 8.4. The enzyme was stable between pH 6 and 9 in the presence of 0.005% Brij 35 as a stabilizer. Substrate specificity of the purified enzyme was broad toward various peptidyl-arginine (or -lysine) 4-methylcoumaryl-7-amides and was similar to that of a trypsin-like enzyme found in the fertilization product. The purified enzyme was inhibited by diisopropyl fluorophosphate and a variety of trypsin inhibitors including leupeptin, but not, or scarcely, inhibited by p-chloromercuribenzoic acid, pepstatin, chymostatin, bestatin, elastatinal, and tosyl-phenylalanyl-chloromethane. The rankings in the potencies of leupeptin and its six analogs as the inhibitors of the purified enzyme were well correlated with those found in their inhibitory effects on the expansion of perivitelline space. Thus, the trypsin-like enzyme possibly present in the fertilization product participates in the expansion of perivitelline space of the egg during fertilization of the ascidian.     

Developmental fates of larval tissues after metamorphosis in the ascidian, Halocynthia roretzi

Cell lineages during ascidian embryogenesis are invariant. Developmental fates of larval mesodermal cells after metamorphosis are also invariant with regard to cell type of descendants. The present study traced developmental fates of larval endodermal cells after metamorphosis in Halocynthia roretzi by labeling each endodermal precursor blastomere of larval endoderm. Larval endodermal cells gave rise to various endodermal organs of juveniles: endostyle, branchial sac, peribranchial epithelium, digestive organs, peripharyngeal band, and dorsal tubercle. The boundaries between clones descended from early blastomeres did not correspond to the boundaries between adult endodermal organs. Although there is a regular projection from cleavage stage and larval stage to juvenile stage, this varies to some extent between individuals. This indicates that ascidian development is not entirely deterministic. We composed a fate map of adult endodermal organs in larval endoderm based on a statistical analysis of many individual cases. Interestingly, the topographic position of each prospective region in the fate map was similar to that of the adult organ, indicating that marked rearrangement of the positions of endodermal cells does not occur during metamorphosis. These findings suggest that fate specification in endoderm cells during metamorphosis is likely to be a position-dependent rather than a deterministic and lineage-based process. Received: 16 June 1999 / Accepted: 16 August 1999     

Evidence for participation of sperm proteinases in fertilization of the solitary ascidian,Halocynthia roretzi: Effects of protease inhibitors

Effects of 15 proteinase inhibitors and an inhibitor against aminopeptidases on fertilization of the solitary ascidian, Halocynthia roretzi were studied in search of lysins. Fertilization of intact eggs was blocked by three trypsin inhibitors, leupeptin, antipain, and soybean trypsin inhibitor, and by two chymotrypsin inhibitors, chymostatin and potato proteinase inhibitor I. On the other hand, the fertilization of naked eggs was not blocked at all by leupeptin and was only partially blocked by chymostatin at the concentrations sufficient for blocking that of intact eggs. This indicates that spermatozoa utilize trypsin-like and chymotrypsin-like proteinases probably as lysins for penetrating through the chorion. The chymotrypsin-like activity appears to be also required for some step besides sperm penetration through the egg investments.     

Sperm-egg binding mediated by sperm alpha-L-fucosidase in the ascidian,Halocynthia roretzi

Spermatozoa bind to the vitelline coat in the ascidians and many other animals. The binding of sperm in Halocynthia roretzi is mediated by a sperm alpha-L-fucosidase and complementary-L-fucosyl residues of glycoproteins in the vitelline coat. cDNA clones for alpha-L-fucosidase were isolated from growing testis mRNA. It contained a 1398 bp full-length cDNA insert (HrFuc'ase) that encoded the 466 amino acid residues of H. roretzi sperm alpha-L-fucosidase. A putative signal peptide of 21 amino acid residues proceeded the sequence for the mature protein (M.W. 52.4 kDa). The coding sequence for HrFuc'ase showed 47.7% sequence identity to the human liver fucosidase sequence. The polyclonal antibody was prepared against a lacZ-HrFuc'ase fusion protein expressed in E. coli. The antibody crossed to a 54 kDa protein in sperm on western blotting and inhibited fertilization in a dose dependent manner. These data suggest that sperm-egg binding is mediated by the sperm alpha-L-fucosidase, HrFuc'ase in the ascidian, H. roretzi.     

The structural organization of ascidian Halocynthia roretzi troponin I genes

The organization of troponin I (TnI) genes from the ascidian Halocynthia roretzi have been determined. Halocynthia possesses roughly two types of TnI isoforms. One type is a single-copied adult TnI (adTnI) gene, which contains eight exons and seven introns. adTnI expresses two isoforms, the shorter body wall muscle TnI and the longer cardiac TnI, through alternative splicing. The mRNAs of these TnI isoforms may undergo trans-splicing of the 5'-leader sequences, like the TnI mRNA of another ascidian species, Ciona intestinalis. The other type comprises multi-copied larval TnI (laTnI) genes. Halocynthia has at least three laTnIs (alpha, beta, and gamma), which are composed of five exons and four introns, and two of them (alpha and gamma) are clustered in tandem. All laTnIs have B- and M-regions within their 5'-upstream regions, which have been discovered to be the regulatory elements of Halocynthia larval actin genes. The expression of Halocynthia laTnIs and larval actins may be regulated in the same manner. It is known that Ciona does not possess a larva-specific TnI isoform. The phylogenetic tree of ascidian TnIs suggests that laTnIs might have only been generated within the Pleurogona lineage after Enterogona/Pleurogona divergence, and this scenario well agrees with the absence of laTnIs in Ciona.     

Structure of multinucleated smooth muscle cells of the ascidian Halocynthia roretzi

Summary Cells isolated from ascidian smooth muscle were about 1.5–2 mm in length. Each contained 20–40 nucle in proportion to cell length. The cytoplasm was characterized by the presence of an enormous quantity of glycogen particles, tubular elements of sarcoplasmic reticulum coupled to the cell membrane, and conspicuous contractile elements. Thick and thin filaments had diameters of about 14–16 nm and 6–7 nm, respectively. The population density of the thick filaments was much higher (mean 270/m² filament area) than in vertebrate smooth muscles. The ratio of thick to thin filaments was about 16. All the thick filaments were surrounded by a single row of 5–9 thin filaments forming a rosette, and cross-bridges with periodicities of 14.5 and 29 nm were found between them. The contractile apparatus consisted of numerous myofibrils which were arranged nearly along the cell axis and were separated from each other by a network of 10-nm filaments. The myofibrils further consisted of many irregularly arranged sarcomerelike structures, each of which was comprised of a small group of thick and thin filaments with attached dense bodies.