The crown-of-thorns starfish genome as a guide for biocontrol of this coral reef pest.

The crown-of-thorns starfish (COTS, the Acanthaster planci species group) is a highly fecund predator of reef-building corals throughout the Indo-Pacific region. COTS population outbreaks cause substantial loss of coral cover, diminishing the integrity and resilience of reef ecosystems. Here we sequenced genomes of COTS from the Great Barrier Reef, Australia and Okinawa, Japan to identify gene products that underlie species-specific communication and could potentially be used in biocontrol strategies. We focused on water-borne chemical plumes released from aggregating COTS, which make the normally sedentary starfish become highly active. Peptide sequences detected in these plumes by mass spectrometry are encoded in the COTS genome and expressed in external tissues. The exoproteome released by aggregating COTS consists largely of signalling factors and hydrolytic enzymes, and includes an expanded and rapidly evolving set of starfish-specific ependymin-related proteins. These secreted proteins may be detected by members of a large family of olfactory-receptor-like G-protein-coupled receptors that are expressed externally, sometimes in a sex-specific manner. This study provides insights into COTS-specific communication that may guide the generation of peptide mimetics for use on reefs with COTS outbreaks.

Initiation of DNA replication after fertilization is regulated by p90Rsk at pre-RC/pre-IC transition in starfish eggs.

Initiation of DNA replication in eukaryotic cells is controlled through an ordered assembly of protein complexes at replication origins. The molecules involved in this process are well conserved but diversely regulated. Typically, initiation of DNA replication is regulated in response to developmental events in multicellular organisms. Here, we elucidate the regulation of the first S phase of the embryonic cell cycle after fertilization. Unless fertilization occurs, the Mos-MAPK-p90Rsk pathway causes the G1-phase arrest after completion of meiosis in starfish eggs. Fertilization shuts down this pathway, leading to the first S phase with no requirement of new protein synthesis. However, how and in which stage the initiation complex for DNA replication is arrested by p90Rsk remains unclear. We find that in G1-arrested eggs, chromatin is loaded with the Mcm complex to form the prereplicative complex (pre-RC). Inactivation of p90Rsk is necessary and sufficient for further loading of Cdc45 onto chromatin to form the preinitiation complex (pre-IC) and the subsequent initiation of DNA replication. However, cyclin A-, B-, and E-Cdk's activity and Cdc7 accumulation are dispensable for these processes. These observations define the stage of G1 arrest in unfertilized eggs at transition point from pre-RC to pre-IC, and reveal a unique role of p90Rsk for a negative regulator of this transition. Thus, initiation of DNA replication in the meiosis-to-mitosis transition is regulated at the pre-RC stage as like in the G1 checkpoint, but in a manner different from the checkpoint.

A single starfish Aurora kinase performs the combined functions of Aurora-A and Aurora-B in human cells.

Aurora, an essential mitotic kinase, is highly conserved during evolution. Most vertebrates have at least two Aurora kinases, Aurora-A and Aurora-B, which have distinct functions in the centrosome-spindle and inner centromere-midbody, respectively. However, some non-vertebrate deuterostomes have only a single Aurora. It remains to be verified whether the single Aurora performs the same functions as vertebrate Auroras A and B combined. We have isolated a cDNA of a single Aurora (ApAurora) from the echinoderm starfish, Asterina pectinifera, and show that ApAurora displays most features of both Aurora-A and Aurora-B in starfish oocytes and early embryos. Furthermore, ApAurora that is stably expressed in HeLa cells can substitute for both human Aurora-A and Aurora-B when either is reduced by RNAi. A single ApAurora thus has properties of both Aurora-A and Aurora-B in starfish eggs and HeLa cells. Together with phylogenetic analysis indicating that ApAurora forms a clade with all types of vertebrate Auroras and single Auroras of non-vertebrate deuterostomes, our observations support the idea that the single Aurora found in non-vertebrate deuterostomes represents the ancestor that gave rise to various types of vertebrate Auroras. This study thus provides functional evidence for phylogenetic considerations.

Cyclin B-cdk1 controls pronuclear union in interphase.

In sexual reproduction, the union of the male and female pronuclei occurs in fertilized eggs to mix genetic materials derived from both parents, thereby creating a new genome for the next generation [1-4]. The process leading to pronuclear union consists of pronuclear congression, which depends on astral microtubules derived from sperm centrosome [5-8], and the subsequent pronuclear fusion or karyogamy. The union process progresses in parallel with the first embryonic cell cycle, but the molecular mechanisms involved are poorly understood. Here, we devise a labeling method with Dendra2 to track both pronuclei individually in living starfish eggs. Although pronuclear union naturally proceeds while G1 arrest is released by fertilization and S phase progresses [9], we show that the cell-cycle resumption and progression are not prerequisites for pronuclear union. However, low levels of cyclin B- (but not cyclin A-) Cdk1 activity are detectable even in interphase, and are indispensable for pronuclear union, by contributing at least to pronuclear congression through formation of sperm aster. Pronuclear congression thus requires the activity of M-phase cell-cycle regulator in interphase, independently of the cell-cycle regulation. These findings not only provide a clue to the regulatory aspect of creation of new genome with fertilization, but also reveal a novel role for the M-phase Cdk1 during interphase.

Selective dephosphorylation by PP2A-B55 directs the meiosis I-meiosis II transition in oocytes.

Meiosis is a specialized cell cycle that requires sequential changes to the cell division machinery to facilitate changing functions. To define the mechanisms that enable the oocyte-to-embryo transition, we performed time-course proteomics in synchronized sea star oocytes from prophase I through the first embryonic cleavage. Although we found that protein levels were broadly stable, our analysis reveals that dynamic waves of phosphorylation underlie each meiotic stage. We found that the phosphatase PP2A-B55 is reactivated at the meiosis I/meiosis II (MI/MII) transition, resulting in the preferential dephosphorylation of threonine residues. Selective dephosphorylation is critical for directing the MI/MII transition as altering PP2A-B55 substrate preferences disrupts key cell cycle events after MI. In addition, threonine to serine substitution of a conserved phosphorylation site in the substrate INCENP prevents its relocalization at anaphase I. Thus, through its inherent phospho-threonine preference, PP2A-B55 imposes specific phosphoregulated behaviors that distinguish the two meiotic divisions.

Purification and characterization of alpha-N-acetylgalactosaminidases I and II from the starfish Asterina amurensis.

Two alpha-N-acetylgalactosaminidases, alpha-N-acetylgalactosaminidase (alpha-GalNAcase) I and II, were purified from the digestive organ of starfish. Purified alpha-GalNAcase I and II gave nearly single protein bands on SDS-polyacrylamide gel electrophoresis, individually. Even the final preparation of alpha-GalNAcase I contained alpha-galactosidase activity, while alpha-GalNAcase II was almost free from that activity with p-nitrophenyl and 4-methylumbelliferyl alpha-N-acetylgalactosaminides as substrates. alpha-GalNAcase I and II both hydrolyzed terminal alpha-N-acetylgalactosaminyl linkages of the natural compounds investigated: Forssman hapten glycolipid, blood group A active oligosaccharide and GalNAc-alpha1-O-serine. On the other hand, oligosaccharides, and glycolipid containing alpha-galactosyl terminals were hydrolyzed by alpha-GalNAcase I but not by alpha-GalNAcase II. The substrate specificities and other enzymatic properties of alpha-GalNAcase I were similar to those of human placental alpha-GalNAcase, but distinct from alpha-GalNAcase II.

Okadaic acid mimics a nuclear component required for cyclin B-cdc2 kinase microinjection to drive starfish oocytes into M phase.

G2-arrested oocytes contain cdc2 kinase as an inactive cyclin B-cdc2 complex. When a small amount of highly purified and active cdc2 kinase, prepared from starfish oocytes at first meiotic metaphase, is microinjected into Xenopus oocytes, it induces activation of the inactive endogenous complex and, as a consequence, drives the recipient oocytes into M phase. In contrast, the microinjected kinase undergoes rapid inactivation in starfish oocytes, which remain arrested at G2. Endogenous cdc2 kinase becomes activated in both nucleated and enucleated starfish oocytes injected with cytoplasm taken from maturing oocytes at the time of nuclear envelope breakdown, but only cytoplasm taken from nucleated oocytes becomes able thereafter to release second recipient oocytes from G2 arrest, and thus contains M phase-promoting factor (MPF) activity. Both nucleated and enucleated starfish oocytes produce MPF activity when type 2A phosphatase is blocked by okadaic acid. If type 2A phosphatase is only partially inhibited, neither nucleated nor enucleated oocytes produce MPF activity, although both do so if purified cdc2 kinase is subsequently injected as a primer to activate the endogenous kinase. The nucleus of starfish oocytes contains an inhibitor of type 2A phosphatase, but neither active nor inactive cdc2 kinase. Microinjection of the content of a nucleus into the cytoplasm of G2-arrested starfish oocytes activates endogenous cdc2 kinase, produces MPF activity, and drives the recipient oocytes into M phase. Together, these results show that the MPF amplification loop is controlled, both positively and negatively, by cdc2 kinase and type 2A phosphatase, respectively. Activation of the MPF amplification loop in starfish requires a nuclear component to inhibit type 2A phosphatase in cytoplasm.

Combining microinjection and immunoblotting to analyze MAP kinase phosphorylation in single starfish oocytes and eggs.

The starfish oocyte has proven useful for studies involving microinjection because it is relatively large (190 mum) and optically clear. These oocytes are easily obtained from the ovary arrested at prophase of meiosis I, making them useful as a model system for the study of cell cycle-related events. In this chapter, a method for combining microinjection with immunoblotting of single cells is described. Individual starfish oocytes are injected, removed from the microinjection chamber, and analyzed by immunoblotting for the dual-phosphorylated form of mitogen-activated protein kinase (MAPK). This method will allow for experiments testing the regulation of MAPK in single cells and for the manipulation of these cells by a quantitative microinjection technique.

Molecular cloning and expression of starfish protein kinase C isoforms.

To investigate the roles of protein kinase C (PKC) isoforms in Echinoderms, we cloned starfish cDNAs for novel, atypical, and conventional PKCs. They showed highest homology with PKCdelta, iota, and alpha isoforms respectively. It was predicted from the whole genome sequence and by RT-PCR that sea urchin has only one isoform of each PKC subgroups. It is thus likely that these isoforms are the prototypes or ancestors of the PKC subgroups. The phylogenetic tree suggests that atypical PKC was first formed by evolution from the common prototype of AGC protein kinase family, and novel and conventional PKCs next. RT-PCR analysis indicated that novel and atypical PKC mRNAs are expressed ubiquitously in all tissues of adult starfish, whereas conventional PKC mRNA is expressed mainly in the ovary and oocytes, and only slightly in the tube foot and stomach. Upon heterologous expression, only atypical PKC was expressed in the functional form in insect cells.

SGK regulates pH increase and cyclin B-Cdk1 activation to resume meiosis in starfish ovarian oocytes.

Tight regulation of intracellular pH (pHi) is essential for biological processes. Fully grown oocytes, having a large nucleus called the germinal vesicle, arrest at meiotic prophase I. Upon hormonal stimulus, oocytes resume meiosis to become fertilizable. At this time, the pHi increases via Na+/H+ exchanger activity, although the regulation and function of this change remain obscure. Here, we show that in starfish oocytes, serum- and glucocorticoid-regulated kinase (SGK) is activated via PI3K/TORC2/PDK1 signaling after hormonal stimulus and that SGK is required for this pHi increase and cyclin B-Cdk1 activation. When we clamped the pHi at 6.7, corresponding to the pHi of unstimulated ovarian oocytes, hormonal stimulation induced cyclin B-Cdk1 activation; thereafter, oocytes failed in actin-dependent chromosome transport and spindle assembly after germinal vesicle breakdown. Thus, this SGK-dependent pHi increase is likely a prerequisite for these events in ovarian oocytes. We propose a model that SGK drives meiotic resumption via concomitant regulation of the pHi and cell cycle machinery.

Biochemical Characterization of a Lysosomal α-Mannosidase from the Starfish Asterias rubens.

Acidic α-mannosidase is an important enzyme and is reported from many different plants and animals. Lysosomal α-mannosidase helps in the catabolism of glycoproteins in the lysosomes thereby playing a major role in cellular homeostasis. In the present study lysosomal α-mannosidase from the gonads of echinoderm Asterias rubens was isolated and purified. The crude protein sample from ammonium sulfate precipitate contained two isoforms of mannosidase as tested by the MAN2B1 antibody, which were separated by anion exchange chromatography. Enzyme with 75 kDa molecular weight was purified and biochemically characterized. Optimum pH of the enzyme was found to be in the range of 4.5-5 and optimum temperature was 37 °C. The activity of the enzyme was inhibited completely by swainsonine but not by 1-deoxymannojirimycin. Ligand blot assays showed that the enzyme can interact with both the lysosomal enzyme sorting receptors indicating the presence of mannose 6-phosphate in the glycan surface of the enzyme. This is the first report of lysosomal α-mannosidase in an active monomeric form. Its interaction with the receptors suggest that the lysosomal enzyme targeting in echinoderms might follow a mannose 6-phosphate mediated pathway similar to that in the vertebrates.

Effect of crude oil contaminated sediment exposure on cytochrome P450 enzymes in the Australian asteroid Coscinasterias muricata.

Levels of cytochrome P450 enzymes were measured in pyloric caeca microsomes of the asteroid Coscinasterias muricata following exposure to sediment with nominal concentrations of 0, 0.1 or 2 ml crude oil kg(-1) (dry weight) and subsequent depuration. No significant differences were observed in total cytochrome P450 levels or cytochrome P418 levels following the exposure period. However after five days of depuration, levels of total P450 in the pyloric caeca of C. muricata exposed to the highest oiled sediment concentration were significantly lower than in specimens exposed to the other treatments. Cytochrome P418 levels were inversely related to total P450 levels following exposure and subsequent depuration. Preliminary results show that levels of CYP1A-like immunopositive protein (CYP1A-like IPP) in exposed asteroids exhibited a concentration response relationship following the exposure period. Variations in CYP1A-like IPP levels observed during the depuration period may be influenced by the sublethal toxicity of hydrocarbons within the crude oil.

Purification of a carboxypeptidase B-like enzyme from the starfish Dermasterias imbricata.

A carboxypeptidase B-like enzyme which catalyses the hydrolysis of synthetic esters of lysine and arginine has been isolated from the starfish Dermasterias imbricata. This carboxypeptidase B-like enzyme has a molecular weight of approximately 34 000 and shares this and other properties with bovine pancreatic carboxypeptidase B. The existence of zymogen for this activity in the pyloric caeca of the starfish is demonstrated. This zymogen has a molecular weight near 40 000 and appears to be analogous to other monomeric procarboxypeptidases B. The zymogen possesses an intrinsic low-level activity toward synthetic substrates of carboxypeptidase B and is activated by trypsin.

Tyrosyl phosphorylation and activation of the myelin basic protein kinase p44mpk during sea star oocyte maturation.

The most prominent tyrosyl-phosphorylated protein in maturing sea star oocytes was identified as the 44 kDa myelin basic protein (MBP) kinase p44mpk. Immunoblotting studies with anti-phosphotyrosine PY-20 antibody and phosphoamino acid analysis of in vivo [32P]phosphate-labelled p44mpk showed that the tyrosyl phosphorylation of the kinase correlated with a greater than 10-fold stimulation of its MBP phosphotransferase activity. The activation of p44mpk was reversed almost completely by purified preparations of the protein-tyrosyl phosphatases CD45 and 1B. Purified p44mpk has previously been shown to undergo autophosphorylation in vitro on seryl residues and this was associated with further enhancement of its MBP phosphorylating activity (Sanghera et al. (1991) J. Biol. Chem. 266, 6700-6707). p44mpk also underwent seryl phosphorylation during oocyte maturation, and the protein-seryl/threonyl phosphatase 2A reversed partially the maturation-associated stimulation of its MBP kinase activity. The properties of p44mpk resemble the murine 42 kDa mitogen-activated protein kinase (p42mapk). While p44mpk may feature the phosphorylatable tyrosyl residue that is critical for activation in p42mapk, it lacks the upstream threonyl phosphorylation site that is also required for p42mapk activity (Payne et al. (1991) EMBO J: 10, 885-892). These findings indicate partial differences in the regulatory mechanisms that govern the activities of these isozymes.

Partial purification and properties of phospholipase A2 from the starfish, Asterina pectinifera.

1. Phospholipase A2 (phosphatide 2-acyl-hydrolase, EC 3.1.1.4) activity was shown to occur in the supernatant fraction of a freshly prepared homogenate from the pyloric caecum of starfish (Asterina pectinifera). 2. The phospholipase A2 has been isolated and purified 130-fold by ultracentrifugation, ammonium sulfate precipitation and column chromatographic procedures. 3. The purified enzyme was stable to heat at low pH values and the optimal pH was observed at approximately 9.0. 4. The enzyme activity was activated by Ca2+ and sodium deoxycholate, and was inhibited by EDTA.

Homology model of the CDK1/cyclin B complex.

We describe a refined homology model of a CDK1/cyclin B complex that was previously used for the structure-based optimization of the Paullone class of inhibitors. The preliminary model was formed from the homologous regions of the deposited CDK2/cyclin A crystal structure. Further refinement of the CDK1/cyclin B complex was accomplished using molecular mechanics and hydropathic analysis with a protocol of constraints and local geometry searches. For the most part, our CKD1/cyclin B homology model is very similar to the high resolution CDK2/cyclin A crystal structure regarding secondary and tertiary features. However, minor discrepancies between the two kinase structures suggest the possibility that ligand design may be specifically tuned for either CDK1 or CDK2. Our examination of the CDK1/cyclin B model includes a comparison with the CDK2/cyclin A crystal structure in the PSTAIRE interface region, connecting portions to the ATP binding domain, as well as the ATP binding site itself.

Human mesotrypsin defies natural trypsin inhibitors: from passive resistance to active destruction.

More than twenty years ago Rinderknecht et al. identified a minor trypsin isoform resistant to natural trypsin inhibitors in the human pancreatic juice. At the same time, Estell and Laskowski found that an inhibitor-resistant trypsin from the pyloric caeca of the starfish, Dermasterias imbricata rapidly hydrolyzed the reactive-site peptide bonds of trypsin inhibitors. A connection between these two seminal discoveries was made recently, when human mesotrypsin was shown to cleave the reactive-site peptide bond of the Kunitz-type soybean trypsin inhibitor, and degrade the Kazal-type pancreatic secretory trypsin inhibitor. These observations indicate that proteases specialized for the degradation of protease inhibitors are ubiquitous in metazoa, and prompt new investigations into their biological significance. Here we review the history and properties of human mesotrypsin, and discuss its function in the digestive degradation of dietary trypsin inhibitors and possible pathophysiological role in pancreatitis.

Purification and properties of hexokinase from the starfish, Asterias amurensis.

Hexokinase from pyloric caeca of the starfish, Asterias amurensis, was purified to a specific activity of 148 units/mg protein. The purified enzyme appeared to be homogeneous on SDS-polyacrylamide gel electrophoresis. The molecular weight determined by SDS polyacrylamide gel electrophoresis and Ultrogel AcA 34 gel filtration was about 50,000. The enzyme showed a broad pH optimum ranging from 7.4 to 9.5. The Km values for D-glucose, D-fructose, 2-deoxy-D-glucose, D-mannose, D-glucosamine and ATP were 0.045, 4, 0.21, 0.05, 0.35 and 0.3 mM, respectively. N-Acetyl-D-glucosamine, D-xylose and D-galactose were not phosphorylated. The enzyme was strongly inhibited by the reaction products, glucose 6-phosphate and ADP, but not by high levels of D-glucose. The starfish hexokinase thus resembled mammalian isozyme A with respect to kinetic properties.

Isolation and characterization of DNA polymerases from mature oocytes of the starfish, Asterina pectinifera.

Four distinctive DNA polymerases have been isolated from mature oocytes of the starfish, Asterina pectinifera, and characterized. The N-ethylmaleimide-resistant DNA polymerase activity was separated from the other three DNA polymerases by hydroxyapatite column chromatography. The enzyme, whose approximate sedimentation coefficient was 3.3S, effectively utilized poly(dA)-oligo(dT) as a template-primer and was completely inhibited by 10 microM 2', 3'-dideoxythymidine-5'-triphosphate but was not inhibited by aphidicolin. Therefore, this enzyme was classified as a eukaryotic DNA polymerase-beta. Three species of the N-ethylmaleimide-sensitive DNA polymerase were separated with a phosphocellulose column. Two (5.5S and 6.5S) of the enzymes eluted from the column preferred activated DNA as a template-primer. Both of the DNA polymerases were inhibited by aphidicolin but not by 2',3'-dideoxythymidine-5'-triphosphate. Judging from the above results, these two DNA polymerases were classified as eukaryotic DNA polymerase-alpha. The third N-ethylmaleimide-sensitive enzyme (approximately 7S) eluted from the phosphocellulose column utilized poly(rA)-oligo(dT) as well as poly(dA)-oligo(dT) as template-primers. The enzyme activity was resistant to aphidicolin but sensitive to 2',3'-dideoxythymidine-5'-triphosphate. This enzyme resembles eukaryotic DNA polymerase-gamma. Thus, it is concluded that the four species of DNA polymerase found in starfish oocytes resemble eukaryotic DNA polymerase-alpha, -beta, and -gamma.

Heterologous expression and catalytic properties of the C-terminal domain of starfish cdc25 dual-specificity phosphatase, a cell cycle regulator.

The 3'-terminal region of starfish Asterina pectinifera cdc25 cDNA encoding the C-terminal catalytic domain was overexpressed in Escherichia coli. The C-terminal domain consisted of 226 amino acid residues containing the signature motif HCxxxxxR, a motif highly conserved among protein tyrosine and dual-specificity phosphatases, and showed phosphatase activity toward p-nitrophenyl phosphate. The enzyme activity was strongly inhibited by SH inhibitors. Mutational studies indicated that the cysteine and arginine residues in the conserved motif are essential for activity, but the histidine residue is not. These results suggest that the enzyme catalyzes the reaction through a two-step mechanism involving a phosphocysteine intermediate like in the cases of other protein tyrosine and dual-specificity phosphatases. The C-terminal domain of Cdc25 activated the histone H1 kinase activity of the purified, inactive form of Cdc2.cyclin B complex (preMPF) from extracts of immature starfish oocytes. Synthetic diphosphorylated di- to nonadecapeptides mimicking amino acid sequences around the dephosphorylation site of Cdc2 still retained substrate activity. Phosphotyrosine and phosphothreonine underwent dephosphorylation in this order. This is the reverse order to that reported for the in vivo and in vitro dephosphorylation of preMPF. Monophosphopeptides having the same sequence served as much poorer substrates. As judged from the results with synthetic phosphopeptides, the presence of two phosphorylated residues was important for specific recognition of substrates by the Cdc25 phosphatase.