Protein kinase A activity leads to the extension of the acrosomal process in starfish sperm.

Acrosomal vesicles (AVs) of sperm undergo exocytosis during the acrosome reaction, which is immediately followed by the actin polymerization-dependent extension of an acrosomal process (AP) in echinoderm sperm. In the starfish Asterias amurensis, a large proteoglycan, acrosome reaction-inducing substance (ARIS), together with asteroidal sperm-activating peptide (asterosap) and/or cofactor for ARIS, induces the acrosome reaction. Asterosap induces a transient elevation of intracellular cGMP and Ca2+ levels, and, together with ARIS, causes a sustained increase in intracellular cAMP and Ca2+ . Yet, the contribution of signaling molecules downstream of cAMP and Ca2+ in inducing AV exocytosis and AP extension remain unknown. A modified acrosome reaction assay was used here to differentiate between AV exocytosis and AP extension in starfish sperm, leading to the discovery that Protein kinase A (PKA) inhibitors block AP extension but not AV exocytosis. Additionally, PKA-mediated phosphorylation of target proteins occurs, and these substrates localize at the base of the AP, demonstrating that PKA activation regulates an AP extension step during the acrosome reaction. The major PKA substrate was further identified, from A. amurensis and Asterias forbesi sperm, as a novel protein containing six PKA phosphorylation motifs. This protein, referred to as PKAS1, likely plays a key role in AP actin polymerization during the acrosome reaction.

Molecular Genetic Markers of Intra- and Interspecific Divergence within Starfish and Sea Urchins (Echinodermata).

A fragment of the mitochondrial COI gene from isolates of several echinoderm species was sequenced. The isolates were from three species of starfish from the Asteriidae family (Asterias amurensis and Aphelasterias japonica collected in the Sea of Japan and Asterias rubens collected in the White Sea) and from the sea urchin Echinocardium cordatum (family Loveniidae) collected in the Sea of Japan. Additionally, regions including internal transcribed spacers and 5.8S rRNA (ITS1 - 5.8S rDNA - ITS2) were sequenced for the three studied starfish species. Phylogenetic analysis of the obtained COI sequences together with earlier determined homologous COI sequences from Ast. forbesii, Ast. rubens, and Echinocardium laevigaster from the North Atlantic and E. cordatum from the Yellow and North Seas (GenBank) placed them into strictly conspecific clusters with high bootstrap support (99% in all cases). Only two exceptions - Ast. rubens DQ077915 sequence placed with the Ast. forbesii cluster and Aph. japonica DQ992560 sequence placed with the Ast. amurensis cluster - were likely results of species misidentification. The intraspecific polymorphism for the COI gene within the Asteriidae family varied within a range of 0.2-0.9% as estimated from the genetic distances. The corresponding intrageneric and intergeneric values were 10.4-12.1 and 21.8-29.8%, respectively. The interspecific divergence for the COI gene in the sea urchin of Echinocardium genus (family Loveniidae) was significantly higher (17.1-17.7%) than in the starfish, while intergeneric divergence (14.6-25.7%) was similar to that in asteroids. The interspecific genetic distances for the nuclear transcribed sequences (ITS1 - 5.8S rDNA - ITS2) within the Asteriidae family were lower (3.1-4.5%), and the intergeneric distances were significantly higher (32.8-35.0%), compared to the corresponding distances for the COI gene. These results suggest that the investigated molecular-genetic markers could be used for segregation and identification of echinoderm species.

Amino acid sequence analysis and characterization of a ribonuclease from starfish Asterias amurensis.

The aim of this study was to phylogenetically characterize the location of the RNase T2 enzyme in the starfish (Asterias amurensis). We isolated an RNase T2 ribonuclease (RNase Aa) from the ovaries of starfish and determined its amino acid sequence by protein chemistry and cloning cDNA encoding RNase Aa. The isolated protein had 231 amino acid residues, a predicted molecular mass of 25,906 Da, and an optimal pH of 5.0. RNase Aa preferentially released guanylic acid from the RNA. The catalytic sites of the RNase T2 family are conserved in RNase Aa; furthermore, the distribution of the cysteine residues in RNase Aa is similar to that in other animal and plant T2 RNases. RNase Aa is cleaved at two points: 21 residues from the N-terminus and 29 residues from the C-terminus; however, both fragments may remain attached to the protein via disulfide bridges, leading to the maintenance of its conformation, as suggested by circular dichroism spectrum analysis. The phylogenetic analysis revealed that starfish RNase Aa is evolutionarily an intermediate between protozoan and oyster RNases.

Structural identification and proteolytic effects of the hatching enzyme from starfish Asterias amurensis.

Hatching enzyme (HE) is secreted from the blastula stage during fertilization and can cleave the egg membrane. The structural identification and proteolytic effects on the collagen and fibrinogen were investigated in this study. Approximate 20 kDa of Asn-linked oligosaccharides were attached to the HE. Five peptide fragments of the starfish HE were homogenous to those of the coat matrix protein of starfish Patiria pectinifera. Amino acids of the starfish HE consisted of mainly Leu (10.0%), Asp (12.5%), and Glu (12.8%). Collagenolytic and fibrinolytic activities of the starfish HE were weaker than those of collagenase and α-chymotrypsin. The degree values of hydrolysis for collagenase and α- chymotrypsin were significantly higher than those of HE in a dose- and time-dependent manner. The peptide mappings of the starfish HE on the collagenolysis (110.7, 84.7, and 20.8 kDa) and fibrinogenolysis (34, 30, and 29 kDa) were different from those of collagenase and α-chymotrypsin. Based on the proteolytic effects on the collagen and fibrinogen, the starfish hatching enzyme might have the potential application to remove the matrix composition in scar or keloid tissue.

A novel hatching enzyme from starfish Asterias amurensis: purification, characterization, and cleavage specificity.

Hatching enzyme (HE) is of importance to degrade egg membrane to let the larvae be free. HE was purified and characterized from starfish blastula. The specific activity and the purification ratio of the purified HE with 110.9 kDa of molecular weight were 449.62 U/mg and 7.42-fold, respectively. Its optimal pH and temperature for activity were pH 8.0 and 30 °C, respectively. This enzyme was relatively stable in the range of pH 4.0-6.0 and 30-40 °C. This enzyme was inhibited by ethylene diamine tetraacetic acid (EDTA) and ethylene glycol tetraacetic acid, and also done moderately by Leupeptin, tosyl-lysine chloromethyl ketone, tosyl-phenylalanine chloromethyl ketone, and phenyl-methanesulfonyl fluoride. Zn(2+) ion activated HE activity strongly and recovered the EDTA-pretreated activity more than did Ca(2+), Mg(2+), and Cu(2+). Based on the results above, the starfish HE was classified as a zinc metallo- and trypsin-like serine protease. The values of Km, Vmax, and Kcat of the starfish HE on dimethyl casein were 0.31 mg/ml, 0.17 U/ml, and 122.70 s(-1), respectively, whereas 1.09 mg/ml, 0.12 U/ml, and 771.98 s(-1) on type I collagen. Therefore, the starfish HE could be a potential cosmeceutical because of its strong cleavage specificity on type I collagen.

Purification and biochemical characterization of a lysosomal α-fucosidase from the deuterostomia Asterias rubens.

In vertebrates, mannose 6-phosphate receptors [MPR300 (Mr 300 kDa) and MPR46 (Mr 46 kDa)] are highly conserved transmembrane glycoproteins that mediate transport of lysosomal enzymes to lysosomes. Our studies have revealed the appearance of these putative receptors in invertebrates such as the molluscs and deuterostomes. Starfish tissue extracts contain several lysosomal enzyme activities and here we describe the affinity purification of α-fucosidase. The purified enzyme is a glycoprotein that exhibited a molecular mass of ∼56 kDa in SDS-PAGE under reducing conditions. It has also cross-reacted with an antiserum to the mollusc enzyme suggesting antigenic similarities among the two invertebrate enzymes. LC-MS/MS analysis of the proteolytic peptides of the purified enzyme in combination with de novo sequencing allowed us to do partial amino acid sequence determination of the enzyme. These data suggest that this invertebrate enzyme is homologous to the known mammalian enzyme. The purified enzyme exhibited a mannose 6-phosphate dependent interaction with the immobilized starfish MPR300 protein. Our results demonstrate that the lysosomal enzyme targeting pathway is conserved even among the invertebrates.

Isolation, affinity purification and biochemical characterization of a lysosomal cathepsin D from the deuterostome Asterias rubens.

Cathepsin D (EC 3.4.23.5) is one of the lysosomal enzymes responsible for proteolytic degradation in cells. By virtue of its mannose 6-phosphate residues, shortly after its synthesis, it is recognized by the receptors in the trans-Golgi network that mediate its transport to the lysosomes. The mammalian enzyme has been extensively characterized and several forms of cathepsin have also been identified. Cathepsins have also been isolated from other vertebrates and invertebrates and recent studies suggest that the lysosomal sorting machinery is evolutionarily conserved from fish to mammals. We recently characterized the putative mannose 6-phosphate receptors from the invertebrate starfish (Asterias rubens). In the present study we affinity purified the cathepsin D from this animal and biochemically characterized the same. Purified enzyme migrated as a single band on SDS-PAGE corresponding to a molecular mass of 45 kDa. The protein bound specifically to Con A-Sepharose gel and is glycosylated. The deglycosylated enzyme showed a molecular mass of ~40 kDa. Furthermore, an antibody raised for the purified enzyme in a rabbit recognizes the crude, the purified enzyme as well as the deglycosylated product in a western blot experiment. The enzyme in the extracts of different tissues can also be quantified by ELISA. We have further evaluated the binding of purified starfish cathepsin D with its receptor, MPR 300 (mannose 6-phosphate receptor) by immunoprecipitation. Cross-linking experiments using purified cathepsin D and MPR 300 revealed a cross-linked product that migrated with a higher molecular mass (345 kDa) compared to the enzyme (45 kDa). Furthermore the specificity of this interaction was also tested in a ligand blot experiment.

Effects of the Erika oil spill on the common starfish Asterias rubens, evaluated by field and laboratory studies.

Impacts of the Erika oil spill on the common starfish Asterias rubens were investigated in the field and using laboratory experiments based on contamination via food at different stages of the starfish reproductive cycle. Two months after the shipwreck, levels of hydrocarbons characteristic of Erika fuel were significantly higher in pyloric ceca and body wall of A. rubens from a contaminated site, compared with control animals from an unpolluted reference area. Concomitant immunological responses and detoxification enzyme activity (CYP1A) were enhanced in the impacted starfish, suggesting rapid biotransformation processes. This was confirmed by laboratory experiments which showed a fast PAH uptake during the 10 first days of contamination and the start of biotransformation processes from the third day. Our study confirms benzo(a)pyrene hydroxylase activity (BPH) in A. rubens and demonstrates the influence of CYP1A in the conversion of insoluble PAHs into soluble derivatives in this species for the first time. The rapidity of decontamination could explain why starfish growth, level of motile activity, reproductive investment, energy storage, and larval development were not significantly affected by these contaminants.

Identification of guanylate cyclases and related signaling proteins in sperm tail from sea stars by mass spectrometry.

Marine invertebrates employ external fertilization to take the advantages of sexual reproduction as one of excellent survival strategies. To prevent mismatching, successful fertilization can be made only after going though strictly defined steps in the fertilization. In sea stars, the fertilization process starts with the chemotaxis of sperm followed by hyperactivation of sperm upon arriving onto the egg coat, and then sperm penetrate to the egg coat before achieving the fusion. To investigate whether the initiation of chemotaxis and the following signaling has species specificity, we conducted comparative studies in the protein level among sea stars, Asterias amurensis, A. forbesi, and Asterina pectinifera. Since transcription of messenger ribonucleic acid (mRNA) has been suppressed in gamete, the roles of sperm proteins during the fertilization cannot be investigated by examining the mRNA profile. Therefore, proteomics analysis by mass spectrometry was used in this study. In sea stars, upon receiving asteroidal sperm-activating peptide (asterosap), the receptor membrane-bound guanylate cyclases in the sperm tail trigger sperm chemotaxis. We confirmed the presence of membrane-bound guanylate cyclases in the three sea star species, and they all had the same structural domains including the extracellular domain, kinase-like domain, and guanylate cyclase domain. The majority of peptides recovered were from alpha-helices distributed on the solvent side of the protein. More peptides were recovered from the intracellular domains. The transmembrane domain has not been recovered. The functions of the receptors seemed to be conserved among the species. Furthermore, we identified proteins that may be involved in the guanylate cyclase-triggered signaling pathway.

Peptidoglycan recognition proteins with amidase activity in early deuterostomes (Echinodermata).

Despite the ecological and evolutionary importance of echinoderms, very little is known about the immune mechanisms in this group especially regarding humoral immunity. In this paper, we screened for proteins putatively involved in immunity in the common European seastar Asterias rubens using a mass spectrometry-based proteomic approach. Two proteins showed striking sequence similarities with peptidoglycan recognition proteins (PGRPs). The two seastar proteins were identified as a single protein, termed PGRP-S1a, occurring in two forms in the coelomic plasma, one of 20kDa and another of 22kDa. We also cloned and sequenced a second member of the PGRP family, termed PGRP-S2a. It has a calculated molecular mass of 21.3kDa and is expressed in circulating phagocytes. Both the S1a-cDNA from coelomic epithelium RNA and the S2a-cDNA from phagocytes code for the amino acid residues necessary for peptidoglycan degradation. PGRP-S1a did not affect the phagocytic activity of seastar immune cells towards Micrococcus luteus but inhibited their production of reactive oxygen species (ROS). A recombinant, His-tagged, PGRP-S2a degrades peptidoglycan and increases the phagocytosis of M. luteus cells by seastar phagocytes.