SGK phosphorylates Cdc25 and Myt1 to trigger cyclin B-Cdk1 activation at the meiotic G2/M transition.

The kinase cyclin B-Cdk1 complex is a master regulator of M-phase in both mitosis and meiosis. At the G2/M transition, cyclin B-Cdk1 activation is initiated by a trigger that reverses the balance of activities between Cdc25 and Wee1/Myt1 and is further accelerated by autoregulatory loops. In somatic cell mitosis, this trigger was recently proposed to be the cyclin A-Cdk1/Plk1 axis. However, in the oocyte meiotic G2/M transition, in which hormonal stimuli induce cyclin B-Cdk1 activation, cyclin A-Cdk1 is nonessential and hence the trigger remains elusive. Here, we show that SGK directly phosphorylates Cdc25 and Myt1 to trigger cyclin B-Cdk1 activation in starfish oocytes. Upon hormonal stimulation of the meiotic G2/M transition, SGK is activated by cooperation between the Gßγ-PI3K pathway and an unidentified pathway downstream of Gßγ, called the atypical Gßγ pathway. These findings identify the trigger in oocyte meiosis and provide insights into the role and activation of SGK.

Chemical and structural characterization of α-N-acetylgalactosaminidase I and II from starfish, asterina amurensis.

BACKGROUND: The marine invertebrate starfish was found to contain a novel α-N-acetylgalactosaminidase, α-GalNAcase II, which catalyzes removal of terminal α-N-acetylgalactosamine (α-GalNAc), in addition to a typical α-N-acetylgalactosaminidase, α-GalNAcase I, which catalyzes removal of terminal α-N-acetylgalactosamine (α-GalNAc) and, to a lesser extent, galactose. The interrelationship between α-GalNAcase I and α-GalNAcase II and the molecular basis of their differences in substrate specificity remain unknown. RESULTS: Chemical and structural comparisons between α-GalNAcase I and II using immunostaining, N-terminal amino acid sequencing and peptide analysis showed high homology to each other and also to other glycoside hydrolase family (GHF) 27 members. The amino acid sequence of peptides showed conserved residues at the active site as seen in typical α-GalNAcase. Some substitutions of conserved amino acid residues were found in α-GalNAcase II that were located near catalytic site. Among them G171 and A173, in place of C171 and W173, respectively in α-GalNAcase were identified to be responsible for lacking intrinsic α-galactosidase activity of α-GalNAcase II. Chemical modifications supported the presence of serine, aspartate and tryptophan as active site residues. Two tryptophan residues (W16 and W173) were involved in α-galactosidase activity, and one (W16) of them was involved in α-GalNAcase activity. CONCLUSIONS: The results suggested that α-GalNAcase I and II are closely related with respect to primary and higher order structure and that their structural differences are responsible for difference in substrate specificities.

Starase: A bi-functional fibrinolytic protease from hepatic caeca of Asterina pectinifera displays antithrombotic potential.

A bi-functional fibrinolytic serine protease, Starase exhibiting thrombolytic potency was purified from hepatic caeca of Asterina pectinifera. Starase showed a single band of approximately 48 kDa by SDS-PAGE and fibrin zymography. The N-terminal sequence of Starase was AIPTEFDARTKKHNN, which does not match with any known fibrinolytic enzyme. Starase had optimum amidolytic activity at 50 °C and pH 8.0 and the activity was inhibited by PMSF and APMSF. Starase showed the highest specificity toward the substrate H-D-Val-Leu-Lys-pNA for plasmin followed by pyroGlu-Gly-Arg-pNA for urokinase. The apparent Km and Vmax values of Starase toward a chromogenic substrate for plasmin H-D-Val-Leu-Lys-pNA were determined as 1.37 mM and 6.8 mM/min/mg respectively. The fibrinolytic activity of Starase by fibrin plate assay displayed that it could not only directly degrade fibrin clot but also activate plasminogen. Starase showed a strong fibrinogenolytic activity, cleaving all three major chains of fibrinogen rapidly. In addition, Starase with more than 1 µg could cleave extracellular matrix component type VII collagen, and plasma proteins such as bovine albumin and bovine gamma globulin. It could also inhibit factor Xa and thrombin activity. Starase at a dose of 0.8 mg/kg was devoid of hemorrhagic activity and it demonstrated antithrombotic effect in three animal models; FeCl2-induced carotid arterial thrombus model, carrageenan-induced tail thrombosis model and collagen and epinephrine induced pulmonary thromboembolism mice model. These results suggest that Starase has the potential to be a potent thrombolytic agent due to its bi-functional properties (containing both direct-acting and plasminogen-activating activities) and lack of hemorrhagic activity. Although Starase might interfere with the normal composition of the plasma proteins, it may be used not only for the treatment and prevention of thrombosis, but also in a number of biomedical applications.

Cyclin B-Cdk1 inhibits protein phosphatase PP2A-B55 via a Greatwall kinase-independent mechanism.

Entry into M phase is governed by cyclin B-Cdk1, which undergoes both an initial activation and subsequent autoregulatory activation. A key part of the autoregulatory activation is the cyclin B-Cdk1-dependent inhibition of the protein phosphatase 2A (PP2A)-B55, which antagonizes cyclin B-Cdk1. Greatwall kinase (Gwl) is believed to be essential for the autoregulatory activation because Gwl is activated downstream of cyclin B-Cdk1 to phosphorylate and activate α-endosulfine (Ensa)/Arpp19, an inhibitor of PP2A-B55. However, cyclin B-Cdk1 becomes fully activated in some conditions lacking Gwl, yet how this is accomplished remains unclear. We show here that cyclin B-Cdk1 can directly phosphorylate Arpp19 on a different conserved site, resulting in inhibition of PP2A-B55. Importantly, this novel bypass is sufficient for cyclin B-Cdk1 autoregulatory activation. Gwl-dependent phosphorylation of Arpp19 is nonetheless necessary for downstream mitotic progression because chromosomes fail to segregate properly in the absence of Gwl. Such a biphasic regulation of Arpp19 results in different levels of PP2A-B55 inhibition and hence might govern its different cellular roles.

Cloning, heterologous expression, and enzymatic characterization of cathepsin L from starfish (Asterina pectinifera).

To determine the role of cathepsin L in Echinoderms, starfish (Asterina pectinifera) cathepsin L (ApCtL) was cloned. The results of RT-PCR analysis indicated that the expression of ApCtL in all of the tissues. The pro-mature enzyme of ApCtL, proApCtL, was expressed in Escherichia coli, and cathepsin activity was detected by cleaving of synthetic fluorogenic peptide substrates and gelatin zymography.

Molecular cloning, expression, and characterization of starfish DNA (cytosine-5)-methyltransferases.

To determine whether and if so how a DNA methylation-dependent epigenetic mechanism for transcriptional gene silencing functions in Echinoderms, we cloned and sequenced dnmt1 and dnmt3 cDNAs of the starfish Asterina pectinifera. Since the Strongylocentrotus purpuratus genome has only two loci of DNA (cytosine-5)-methyltransferase genes encoding Dnmt1 and Dnmt3, they might constitute a sufficient set of dnmt genes in Echinoderms. The starfish Dnmt3 whose cDNA we cloned showed highest homology to a mammalian Dnmt3a2 splicing variant. Essentially all the characteristic motifs and sequences of the mammalian counterparts were found in the starfish Dnmts as well, except that a typical PCNA binding domain motif was lacking in the starfish Dnmt1. RT-PCR analysis indicated that the dnmt1 mRNA exists in both ovary and oocytes, but its levels in other tissues were very low or almost negligible. In contrast, the dnmt3 mRNA was detected only in the ovary, and not at all in the oocytes. The size of a dnmt1 transcript was about 6.5 kb on Northern blot analysis. On heterologous expression, the starfish Dnmt1 protein was expressed in insect cells in catalytically active form.

Enzymatic properties of starfish phospholipase A2 and its application.

Industrial phospholipase A2 (PLA2) mainly produced from porcine pancreas is used for production of lysolecithin which is well known as an excellent natural emulsifier for food, cosmetic, and pharmaceutical industries. Since the outbreak of bovine spongiform encephalopathy (BSE) or religious tradition, it is hoped that the new sources of PLA2, as well as other enzymes and proteins, will be developed instead of mammal. From these backgrounds, we studied for PLA2 from marine organisms and found that starfish Asterina pectinifera PLA2 possesses extremely high activity and characteristic polar-group specificity comparing with commercially available PLA2 from porcine pancreas. Therefore, it was suggested that the starfish A. pectinifera would be a potential source of PLA2, and the PLA2 can be utilized as alternative enzyme of mammalian PLA2.

Involvement of mitogen-activating protein kinase and intracellular pH in the duration of the metaphase I (MI) pause of starfish oocytes after spawning.

The metaphase I (MI) arrest of starfish oocytes is released after spawning. In this study using starfish Asterina pectinifera, the duration of MI after spawning was ~20 min and approximately 30 min in fertilized and unfertilized oocytes, respectively. This prolongation of MI in unfertilized oocytes, referred to as the MI pause, was maintained by mitogen-activating protein kinase (MAPK) as well as low intracellular pH (approximately 7.0). Contrary to previous reports, MI arrest was not maintained by MAPK, since it was inactive in the oocytes arrested at MI in the ovary and activated immediately after spawning. Also, cyclin B was not degraded at pH 6.7 in the cell-free preparation without MAPK activity, whereas it was degraded at pH 7.0, suggesting that MI arrest was solely maintained by lower pH (< 7.0). Normal development occurred when the spawned oocytes were fertilized before the first polar body formation, whereas fertilization after the first polar body formation increased the rate of abnormal development. Thus, due to MI pause and MI arrest, the probability for fertilization before the polar body formation might be increased, leading to normal development.

Identification and characterization of cathepsin D in a highly purified sialidase from starfish A. pectinifera.

A sialidase [EC 3.2.1.18] from the ovary of starfish Asterina pectinifera was isolated and highly purified by preparative PAGE. The SDS-PAGE separation of the purified enzyme revealed two natures of protein bands, upper (50 kDa) and a lower (47 kDa). To identify the protein, N-terminal amino acid sequence of the upper band was done. The sequence matched with the N-terminal amino acid sequence of human lysosomal mature cathepsin D and cathepsin D activity was also found in all the preparation steps. Protease inhibitor pepstatin A inhibited the proteolysis activity of cathepsin D against a synthetic substrate. The two enzymes sialidase and cathepsin D were separated from each other by using high-performance gel-filtration chromatography. The Western blot analysis and isoelectric focusing showed the co-purified cathepsin D is a 50 kDa protein with a PI value of 4.2.

[NADPH-diaphorase localization in the radial nerve cords of the starfish Patiria pectinifera]. / Lokalizatsiia NADPH-diaforazy v radial'nykh nervakh morskoi zvezdy Patiria pectinifera.

The presence and localization of NADPH-diaphorase (NADPH-d) in the radial nerve cords of Patiria pectonifera was shown by electron histochemistry. NADPH-d-positive structures were found in ectoneural and hyponeural regions of the radial nerve cord. Ultrastructural localization of NADPH-d was detected in neurons, sensory cells, supporting cells, and in the nerve plexus. The highest enzymatic activity in ectoneural region of the radial nerve cord is due, presumably, to the involvement of NADPH in sensory signal processing.