Primary structure of histone H2B from gonads of the starfish Asterias rubens. Identification of an N-dimethylproline residue at the amino-terminal

The complete amino acid sequence (121 residues) of histone H2B from gonads of the starfish Asterias rubens has been established from structural data obtained essentially from large fragments generated by cleavage of histone H2B at aspartyl residues and by limited hydrolysis of the dimer H2A-H2B with mouse submaxillary gland protease. No real sequence homology can be found between the amino-terminal sequence (residues 1-21) of starfish and calf H2B. One non-conservative substitution (serine-32 in calf----lysine-28 in starfish) leads to the presence of a cluster of eight basic residues (sequence 23-30) and to the disappearance of a potential site of phosphorylation. A particular structural feature of starfish histone H2B is the presence of N-dimethylproline at its amino-terminal end. By comparison with N-terminal acetylation, which is commonly found in histones, N-terminal methylation is rarely observed. At the present time the functional significance of the N-terminal methylation as well as that of the proline-rich nature of the amino-terminal sequence of the starfish histone H2B remain to be defined.     

Histones from gonads of the star-fish Asterias rubens.

1. Histones were isolated from gonads of the star-fish Asterias rubens and characterized by their amino acid composition and their electrophoretic migration. 2. Comparative studies with calf thymus homologous histones show the highly conservative structure of the histones H3 and H4, and the variability of the other histones namely H1 and H2B.     

Analysis of monosaccharides, fatty constituents and rare O-acetylated sialic acids from gonads of the starfish Asterias rubens

A previous study (Bergwerff et al., Biochimie 74 (1992) 25-37) reported that sialic acids present in Asterias rubens gonads were essentially composed of 8-methyl-N-glycolylneuraminic acid (Neu5Gc8Me), a large part of it being acetylated in position 9. Using GC/MS of heptafluorobutyrate derivatives (Zanetta et al., Glycobiology 11 (2001) 663-676) on the chloroform/methanol soluble and insoluble fractions, we showed that most sialic acids were found in the latter and demonstrated that all sialic acids were derived from N-glycolylneuraminic acid, most of them being 8-methylated, but that the majority were also acetylated in position 4 or 7 (or both positions). GC/MS analyses of the constituents liberated using acid-catalysed methanolysis verified that major glycoprotein-bound glycans were N-linked and of the gluco-oligomannosidic type. Major fatty acids were poly-unsaturated (especially C20:4) and long-chain bases were C22:1 phytosphingosine and C22:2 6-hydroxysphingenine. Major monosaccharides found in the chloroform/methanol extract (quinovose and fucose) were derived from steroidal saponins.     

Isolation and characterization of a sialidase from the starfish Asterias rubens

The starfish Asterias rubens contains a soluble sialidase (1.4 mU/mg homogenate protein), which was purified over 500-fold to apparent homogeneity by ammonium sulfate precipitation, gel filtration and affinity chromatography on immobilized 2-deoxy-2,3-didehydroneuraminic acid. The native sialidase has a molecular mass of 230 kDa (gel filtration) and consists of 4 subunits of each 63 kDa, as determined by SDS-gel electrophoresis. Its isoelectric point is at pH 4.9, the activity is optimum at pH 4.2 and 37 degrees C, and it hydrolyses preferably 4-methylumbelliferyl-alpha-N-acetyl-neuraminic acid, followed by sialyllactose and glycoproteins. The hydrolysis rate is decreased or stopped by the presence of O-acetyl groups on the sialic-acid residue to be cleaved. N-Glycoloyl residues also retard enzyme action, as well as alpha(2-6) bonds when compared with alpha(2-3) linkages. This relatively stable enzyme is inhibited by mercury or copper ions, 2-deoxy-2,3-didehydro-N-acetylneuraminic acid and by the increase of ionic strength. The evolutionary significance of starfish sialidase is discussed.     

Isolation and characterization of cytidine-5'-monophosphate-N-acetylneuraminate hydroxylase from the starfish Asterias rubens

The sialic acid N-glycolylneuraminic acid (Neu5Gc) is formed by cytidine-5'-monophosphate-N-acetylneuraminic acid (CMP-Neu5Ac) hydroxylase (EC 1.14.13.45). The enzyme from mammals exhibits several unusual characteristics, raising questions about its evolution. Since echinoderms are the most primitive organisms possessing glycoconjugate-bound Neu5Gc, studies on the hydroxylase from members of this phylum may yield insights into the origin and development of the hydroxylase. Investigations on crude CMP-Neu5Ac hydroxylase in gonads from the starfish Asterias rubens revealed that it shares many properties with its mammalian counterpart. However, the echinoderm hydroxylase also exhibits fundamental differences, particularly its association with a membrane and a requirement for high ionic strength for optimal activity. Here, we describe the isolation of the CMP-Neu5Ac hydroxylase from A. rubens gonads using anion exchange chromatography and chromatography on immobilized cytochrome b(5). The enzyme was enriched 137-fold with a yield of 13%. The preparation exhibited a main polypeptide of 76 kDa, consistent with a cDNA sequence published earlier, and a minor protein of 64 kDa. A kinetic characterization showed that salt activation of this enzyme results from an increase in affinity for CMP-Neu5Ac. Evidence for the formation of a ternary complex of hydroxylase, CMP-Neu5Ac and cytochrome b(5) is also presented. The mechanistic and physiological significance of these results is discussed.     

Cloning and expression of a membrane-bound CMP-N-acetylneuraminic acid hydroxylase from the starfish Asterias rubens.

The sialic acid N-glycolylneuraminic acid (Neu5Gc) is synthesized by the action of CMP-Neu5Ac hydroxylase. The enzyme from various mammals has been purified, characterized and sequenced by cDNA cloning. Although functional sequence motifs can be postulated from comparisons with several enzymes, no global homologies to any other proteins have been found. The unusual characteristics of this hydroxylase raise questions about its evolution. As echinoderms are phylogenetically the oldest organisms possessing Neu5Gc, they represent a starting point for investigations on the origin of this enzyme. Despite many similarities with its mammalian counterpart, CMP-Neu5Ac hydroxylase from the starfish A. rubens exhibits fundamental differences, most notably its association with a membrane and a requirement for high ionic strength. In order to shed light on the structural basis for these differences, the primary structure of CMP-Neu5Ac hydroxylase from A. rubens has been determined by PCR and cDNA-cloning techniques, using initial sequence information from the mouse enzyme. The complete assembled cDNA contained an ORF coding for a protein of 653 amino acids with a molecular mass of 75 kDa. The deduced amino-acid sequence exhibited a high degree of homology with the mammalian enzyme, although the C-terminus was some 60 residues longer. This extension consists of a terminal hydrophobic region, which may mediate membrane-binding, and a preceding hydrophilic sequence which probably serves as a hinge or linker. The identity of the ORF was confirmed by expression of active CMP-Neu5Ac hydroxylase in E. coli at low temperatures.     

Anatomy of the ovaries of the starfish Asterias rubens (Echinodermata)

Summary The ovaries of the starfish Asterias rubens were studied histologically and ultrastructurally. The reproductive system in female specimens consists of ten separate ovaries, two in each ray. Each ovary is made up of a rachis with lateral primary and secondary folds: the acini maiores and acini minores. The ovarian wall is composed of an outer and an inner part, separated by the genital coelomic sinus. The ovarian lumen contains oocytes in various phases of oogenesis, follicle cells, nurse cells, phagocytosing cells and steroid-synthesizing cells.Oogenesis is divided into four phases: (i) multiplication phase of oogonia, (ii) initial growth phase of oocytes I, (iii) growth phase proper of oocytes I, and (iv) post-growth phase of oocytes I. The granular endoplasmic reticulum and the Golgi complex of the oocytes appear to be involved in yolk formation, while the haemal system, haemal fluid and nurse cells may also be important for vitellogenesis. The haemal system is discussed as most likely being involved in synchronizing the development of the ovaries during the annual reproductive cycle and in inducing, stimulating and regulating the function of the ovaries.Steroid-synthesizing cells are present during vitellogenesis; a correlation between the presence of these cells and vitellogenesis is discussed.     

Immunocompetent cells in the starfish Asterias rubens. An ultrastructural study

Cells from the axial organ of the starfish Asterias rubens were fractionated into two populations, adherent and non-adherent to nylon wool. In both populations the ultrastructural study revealed the presence of cells resembling the lymphocytes of the vertebrates, as well as phagocytic, peroxidase positive cells. The lymphocyte-like cells in the non-adherent population (average diameter 4 mu) have a high nucleo-cytoplasmatic ratio and are morphologically similar to Th lymphocytes while the adherent cells (average diameter 5.5 mu) are more similar to Bm lymphocytes. These observations are in line with the hypothesis that there exist, in the starfish, a primitive immune system with characteristics resembling those of the immune system of vertebrates.     

The lysozyme of the starfish Asterias rubens. A paradygmatic type i lysozyme.

On the basis of a partial N-terminal sequence, Jollès and Jollès previously proposed that the lysozyme from the starfish Asterias rubens represents a new form of lysozyme, called type i (invertebrate) lysozyme. Indeed, it differed from both the types c (chicken) and g (goose) known in other animals, as well as from plant and phage lysozymes. Recently, several proteins belonging to the same family have been isolated from protostomes. Here we report the complete mature protein sequence and cDNA sequence of the lysozyme from Asterias. These sequences vindicate the previously proposed homology between the starfish, a deuterostome, and protostome lysozymes. In addition, we present a structural analysis that allows us to postulate upon the function of several conserved residues.     

Biosynthesis and functions of eicosanoids generated by the coelomocytes of the starfish, Asterias rubens

Eicosanoids are a group of oxygenated fatty acid derivatives formed from C20 polyunsaturated fatty acids, including arachidonic and eicosapentaenoic acids. The potential of the coelomocytes of the starfish, Asterias rubens, to generate eicosanoids through the cyclooxygenase (COX) and lipoxygenase (LOX) pathways was investigated using reverse-phase high performance liquid chromatography, enzyme immunoassay and gas chromatography–mass spectrometry. The principal LOX product was identified as 8-hydroxyeicosatetraenoic acid (8-HETE) with 8-hydroxyeicosapentaenoic acid (8-HEPE) synthesised at significantly lower levels. No classical prostaglandins (PG), such as PGE₂ or PGD₂, were found to be generated by ionophore-challenged coelomocytes. Incubation of coelomocytes with lipopolysaccharides from either Escherichia coli or Salmonella abortus failed to induce an increase in generation of LOX products and the presence of 8-HETE (0–25 μM) had no significant effect on the in vitro phagocytic activity of Asterias coelomocytes. Neither indomethacin (a COX inhibitor) or esculetin (a LOX inhibitor) had any effect on the clearance of the bacterium, Vibrio splendidus, from the coelomic cavity of starfish suggesting that products of these enzymes are not involved in such coelomocyte responses to foreign particles.     

Identification of protein phosphatases-1 and 2A and inhibitor-2 in oocytes of the starfish Asterias rubens and Marthasterias glacialis

Protein phosphatases present in the particulate and soluble fractions of oocytes of the starfish Asterias rubens and Marthasterias glacialis have been classified according to the criteria used for these enzymes from mammalian cells. The major protein phosphatase activity in the particulate fraction had very similar properties to protein phosphatase-1 from mammalian tissues, including preferential dephosphorylation of the beta subunit of phosphorylase kinase, sensitivity to inhibitor-1 and inhibitor-2, inhibition of phosphorylase phosphatase activity by protamine and heparin, and retention by heparin-Sepharose. The major protein phosphatase in the soluble fraction had very similar properties to mammalian protein phosphatase-2A, including preferential dephosphorylation of the alpha subunit of phosphorylase kinase, insensitivity to inhibitors-1 and 2, activation by protamine and heparin, and exclusion from heparin-Sepharose. An acid-stable and heat-stable protein was detected in the soluble fraction of starfish oocytes, whose properties were indistinguishable from those of inhibitor-2 from mammalian tissues. It inhibited protein phosphatase-1 specifically, and its apparent molecular mass on SDS polyacrylamide gels was 31 kDa. Furthermore, an inactive hybrid formed between the starfish oocyte inhibitor and the catalytic subunit of mammalian protein phosphatase-1 could be reactivated by preincubation with MgATP and mammalian glycogen synthase kinase-3. The remarkable similarities between starfish oocyte protein phosphatases and their mammalian counterparts are indicative of strict phylogenetic conservation of these enzymes. The results will facilitate further analysis of the role of protein phosphorylation in the control of starfish oocyte maturation by the hormone 1-methyladenine.     

Environmental factors influencing the immune responses of the common European starfish (Asterias rubens)

The influence of handling, salinity, temperature, parasitism, and gender on the immune responses (reactive oxygen species (ROS) production and coelomic amoebocyte concentration (CAC) of the starfish Asterias rubens was investigated in experimental conditions. Additionally, a year-round monthly survey in two distant sites was conducted in order to understand which of these factors most influences the immunity of A. rubens in field conditions. All considered factors, except gender and handling stress, influenced the studied immune responses of A. rubens in experimental conditions. Amoebocyte ROS production was increased at low salinity and at the lowest temperature tested (6 degrees C). Amoebocyte concentration in the coelomic fluid was increased in starfish infested by the ciliate Orchitophrya stellarum. However, among all these factors, only temperature could be linked with the variability in ROS production measured in the field during the monthly survey. The variability in amoebocyte concentration in the field does not seem to be linked to any of the factors considered in this study; it appears to reflect mostly an inter-individual variation rather than seasonal fluctuations. Recommended periods and indicative values of immune responses are proposed for field studies using A. rubens.