Expression of steroidogenic enzymes and metabolism of steroids in COS-7 cells known as non-steroidogenic cells.

The COS-7 (CV-1 in Origin with SV40 genes) cells are known as non-steroidogenic cells because they are derived from kidney cells and the kidney is defined as a non-steroidogenic organ. Therefore, COS-7 cells are used for transfection experiments to analyze the actions of functional molecules including steroids. However, a preliminary study suggested that COS-7 cells metabolize [3H]testosterone to [3H]androstenedione. These results suggest that COS-7 cells are able to metabolize steroids. Therefore, the present study investigated the expression of steroidogenic enzymes and the metabolism of steroids in COS-7 cells. RT-PCR analyses demonstrated the expressions of several kinds of steroidogenic enzymes, such as cytochrome P450 side-chain cleavage enzyme, 3ß-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase, cytochrome P450 7α-hydroxylase, cytochrome P450 17α-hydroxylase/17,20-lyase, 17ß-hydroxysteroid dehydrogenase, 5α-reductase, cytochrome P450 21-hydroxylase, cytochrome P450 11ß-hydroxylase, and cytochrome P450 aromatase in COS-7 cells. In addition, steroidogenic enzymes 3ß-HSD, P4507α, 5α-reductase, P450c17, P450c21, P450c11ß, and 17ß-HSD actively metabolized various steroids in cultured COS-7 cells. Finally, we demonstrated that 17ß-HSD activity toward androstenedione formation was greater than other steroidogenic enzyme activities. Our results provide new evidence that COS-7 cells express a series of steroidogenic enzyme mRNAs and actively metabolize a variety of steroids.

Oct-GnRH, the first protostomian gonadotropin-releasing hormone-like peptide and a critical mini-review of the presence of vertebrate sex steroids in molluscs.

In protostome and deuterosome invertebrates, neurosecretory cells play major roles in the endocrine system. The optic glands of cephalopods are indicators of sexual maturation. In mature octopuses, optic glands enlarge and secrete a gonadotropic hormone. A peptide with structural features similar to that of vertebrate gonadotropin-releasing hormone (GnRH) was isolated from the octopus, Octopus vulgaris, and was named oct-GnRH. The discovery of oct-GnRH has triggered structural determinations and predictions of other mollusc GnRH-like peptides in biochemical and in silico studies. Interestingly, cephalopods studied so far are characterized by a single molecular form of oct-GnRH with a C-terminal -Pro-Gly-NH2 sequence, which is critical for gonadotropin-releasing activity in vertebrates. Other molluscan GnRH-like peptides lack the C-terminal -Pro-Gly-NH2 sequence but have -X-NH2 or -Pro-Gly although all protostome GnRH-like peptides have yet to be sequenced. In marine molluscs, relationships between GnRH-like peptides and sex steroids have been studied to verify the hypothesis that molluscs have vertebrate-type sex steroid system. However, it is currently questionable whether such sex steroids are present and whether they play endogenous roles in the reproductive system of molluscs. Because molluscs uptake and store steroids from the environment and fishes release sex steroids into the external environment, it is impossible to rule out the contamination of vertebrate sex steroids in molluscs. The function of key enzymes of steroidogenesis within molluscs remains unclear. Thus, evidence to deny the existence of the vertebrate-type sex steroid system in molluscs has been accumulated. The elucidation of substances, which regulate the maturation and maintenance of gonads and other reproductive functions in molluscs will require rigorous and progressive scientific study.

Osmotic/ionic status of body fluids in the euryhaline cephalopod suggest possible parallel evolution of osmoregulation.

Acclimation from marine to dilute environments constitutes among the dramatic evolutionary transitions in the history of life. Such adaptations have evolved in multiple lineages, but studies of the blood/hemolymph homeostasis mechanisms are limited to those using evolutionarily advanced Deuterostome (chordates) and Ecdysozoa (crustaceans). Here, we examined hemolymph homeostasis in the advanced Lophotrochozoa/mollusc, the other unexplored taxa, and its possible regulation by the vasopressin/oxytocin superfamily peptides known to be implicated in fluid homeostasis in Chordata and Arthropoda. The hemolymph osmotic and ionic status in the euryhaline cephalopod (Octopus ocellatus) following transfer from 30-ppt normal seawater to 20 ppt salinity indicate hyperosmo- and hyperionoregulatory abilities for more than 1 week, as in crustaceans and teleost fish. While ventilation frequency decreased by 1 day, Na(+)/K(+)-ATPase activity, which has been generally implicated in ion transport, was induced in two of the eight posterior gills after 1 week. In addition, the octopuses were intravenously injected with 1 or 100 ng/g octopressin or cephalotocin, which are Octopus vasopressin/oxytocin orthologs. After 1 day, octopressin, but not cephalotocin, decreased the hemolymph osmolality and Ca concentrations, as well as urinary Na concentrations. These data provide evidence for possible parallel evolution in hyperionoregulatory mechanisms and coordination by conserved peptides.

Molecular cloning of precursors for TEP-1 and TEP-2: The GGNG peptide-related peptides of a prosobranch gastropod, Thais clavigera.

TEP (Thais excitatory peptide)-1 and TEP-2 are molluscan counterparts of annelidan GGNG-peptides, identified in a neogastropod, Thais clavigera (Morishita et al., 2006). We have cloned two cDNAs encoding TEP-1 and TEP-2 precursor protein, respectively, by the standard molecular cloning techniques. Predicted TEP-1 precursor protein consists of 161 amino acids, while predicted TEP-2 precursor protein has 118 amino acids. Only a single copy of TEP was found on the respective precursor. The semi-quantitative RT-PCR showed that expression of TEP-1 was high in sub-esophageal, pleural, pedal and visceral ganglia, while it was low in supra-esophageal ganglion. By contrast, expression level of TEP-2 was high in pedal and visceral ganglia. In situ hybridization visualized different subsets of TEP-1 and TEP-2 expressing neurons in Thais ganglia. For example, supra-esophageal ganglion contained many TEP-2 expressing neuron, but not TEP-1 expressing ones. These results suggest that expression of TEP-1 and TEP-2 is differently regulated in the Thais ganglia.

Oxytocin/vasopressin and gonadotropin-releasing hormone from cephalopods to vertebrates.

Recent advances in peptide search methods have revealed two peptide systems that have been conserved through metazoan evolution. Members of the oxytocin/vasopressin-superfamily have been identified from protostomian and deuterostomian animals, indicating that the oxytocin/vasopressin hormonal system represents one of the most ancient systems. In most protostomian animals, a single member of the superfamily shares oxytocin-like and vasopressin-like actions. Co-occurrence of two members has been discovered in modern cephalopods, octopus, and cuttlefish. We propose that cephalopods have developed two peptides in the molluscan evolutionary lineage like vertebrates have established two lineages in the oxytocin/vasopressin superfamily. The existence of gonadotropin-releasing hormone (GnRH) in protostomian animals was initially suggested by immunohistochemical analysis using chordate GnRH antibodies. A peptide with structural features similar to those of chordate GnRHs was originally isolated from octopus, and an identical peptide has been characterized from squid and cuttlefish. Novel forms of GnRH-like molecules from other molluscs, an annelid, arthropods, and nematodes demonstrate somewhat conserved structures at the N-terminal regions; but structures of the C-terminal regions critical to gonadotropin-releasing activity are diverse. These findings may be important for the study of the molecular evolution of GnRH in protostomian animals.

Identification, localization, and function of a novel avian hypothalamic neuropeptide, 26RFa, and its cognate receptor, G protein-coupled receptor-103.

Several neuropeptides with the C-terminal RFamide sequence have been identified in the hypothalamus of a variety of vertebrates. Among the RFamide peptide groups, however, only LPXRFamide peptides, including gonadotropin-inhibitory hormone, have been characterized in the avian brain. In the present study, we sought for the presence of other RFamide peptides in the avian hypothalamus. We identified a cDNA encoding an RFamide peptide orthologous to 26RFa (also referred to as QRFP) in the hypothalamus of the Japanese quail. The deduced quail 26RFa precursor consisted of 120-amino-acid residues, encoding one RFamide peptide with 27 amino acids. This RFamide peptide was flanked at the N terminus by a dibasic amino acid cleavage site and at the C terminus by a glycine amidation signal. Quantitative RT-PCR analysis demonstrated specific expression of quail 26RFa mRNA in the diencephalon including the hypothalamus. Furthermore, mass spectrometry analysis revealed the presence of a peptide exhibiting the mass of mature 26RFa, indicating that the peptide is actually produced from the precursor in the diencephalon. 26RFa-producing cell bodies were localized in the anterior hypothalamic nucleus in the brain. Synthetic 26RFa increased intracellular Ca(2+) concentration in HEK293T cells transfected with the chicken G protein-coupled receptor GPR103. Intracerebroventricular injection of 26RFa in broiler chicks stimulated feeding behavior. These data provide the first evidence for the occurrence of the peptide 26RFa in the avian hypothalamus and indicate that this peptide exerts orexigenic activity.

Identification of human GnIH homologs, RFRP-1 and RFRP-3, and the cognate receptor, GPR147 in the human hypothalamic pituitary axis.

The existence of a hypothalamic gonadotropin-inhibiting system has been elusive. A neuropeptide named gonadotropin-inhibitory hormone (GnIH, SIKPSAYLPLRF-NH(2)) which directly inhibits gonadotropin synthesis and release from the pituitary was recently identified in quail hypothalamus. Here we identify GnIH homologs in the human hypothalamus and characterize their distribution and biological activity. GnIH homologs were isolated from the human hypothalamus by immunoaffinity purification, and then identified as MPHSFANLPLRF-NH(2) (human RFRP-1) and VPNLPQRF-NH(2) (human RFRP-3) by mass spectrometry. Immunocytochemistry revealed GnIH-immunoreactive neuronal cell bodies in the dorsomedial region of the hypothalamus with axonal projections to GnRH neurons in the preoptic area as well as to the median eminence. RT-PCR and subsequent DNA sequencing of the PCR products identified human GnIH receptor (GPR147) mRNA expression in the hypothalamus as well as in the pituitary. In situ hybridization further identified the expression of GPR147 mRNA in luteinizing hormone producing cells (gonadotropes). Human RFRP-3 has recently been shown to be a potent inhibitor of gonadotropin secretion in cultured sheep pituitary cells by inhibiting Ca(2+) mobilization. It also directly modulates GnRH neuron firing. The identification of two forms of GnIH (RFRP-1 and RFRP-3) in the human hypothalamus which targets human GnRH neurons and gonadotropes and potently inhibit gonadotropin in sheep models provides a new paradigm for the regulation of hypothalamic-pituitary-gonadal axis in man and a novel means for manipulating reproductive functions.

Structures and diverse functions of frog growth hormone-releasing peptide (fGRP) and its related peptides (fGRP-RPs): a review.

A new Arg-Phe-NH(2) (RFamide) peptide has been discovered in the amphibian hypothalamus. The cell bodies and terminals containing this peptide were localized in the suprachiasmatic nucleus and median eminence, respectively. This peptide was further revealed to have a considerable growth hormone (GH)-releasing activity in vitro and in vivo and hence designated as frog GH-releasing peptide (fGRP). Molecular cloning of cDNA encoding the fGRP precursor polypeptide revealed that it encodes fGRP and its putative gene-related peptides (fGRP-RP-1, -RP-2, and -RP-3). Subsequently, we identified these putative fGRP-RPs as mature peptides and analyzed their hypophysiotropic activities. Only fGRP-RP-2 stimulated the release of GH and prolactin (PRL) in vitro and in vivo. Thus, in addition to fGRP, fGRP-RP-2 acts as a hypothalamic factor on the frog pituitary to stimulate the release of GH and PRL.

Na+ /Ca2+ exchanger contributes to asterosap-induced elevation of intracellular Ca2+ concentration in starfish spermatozoa.

Asterosap, a group of equally active isoforms of sperm-activating peptides from the egg jelly of the starfish Asterias amurensis, functions as a chemotactic factor for sperm. It transiently increases the intracellular cGMP level of sperm, which in turn induces a transient elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)). Using a fluorescent Ca(2+)-sensitive dye, Fluo-4 AM, we measured the changes in sperm [Ca(2+)](i) in response to asterosap. KB-R7943 (KB), a selective inhibitor of Na(+)/Ca(2+) exchanger (NCX), significantly inhibited the asterosap-induced transient elevation of [Ca(2+)](i), suggesting that asterosap influences [Ca(2+)](i) through activation of a K+-dependent NCX (NCKX). An NCKX activity of starfish sperm also shows K(+) dependency like other NCKXs. Therefore, we cloned an NCKX from the starfish testes and predicted that it codes for a 616 amino acid protein that is a member of the NCKX family. Pharmacological evidence suggests that this exchanger participates in the asterosap-induced Ca(2+) entry into sperm.

Isolation and characterization of a novel small cardioactive peptide-related peptide from the brain of Octopus vulgaris.

A novel small cardioactive peptide (SCP)-related peptide (oct-SCPRP: Ser-Asn-Gly-Tyr-Leu-Ala-Leu-Pro-Arg-Gln-NH2) was isolated from the brain of the octopus (Octopus vulgaris). cDNA encoding this precursor protein was cloned. Oct-SCPRP was shown to evoke contraction in the radula protractor muscle, and the precursor protein was highly homologous to the SCP family in the Mollusk but did not encode a related peptide, SCPB. The expression of oct-SCPRP mRNA was present not only in the peripheral nervous system (PNS) which is a motor center for the control of feeding, but also in the central nervous system (CNS) which is capable of complex analysis, learning, and controls behaviors.

Molecular and functional characterization of a novel gonadotropin-releasing-hormone receptor isolated from the common octopus (Octopus vulgaris).

GnRH (gonadotropin-releasing hormone) plays a pivotal role in the regulation of reproduction in vertebrates through interaction with a specific receptor. Previously, we isolated a GnRH homologue, oct-GnRH, from the common octopus (Octopus vulgaris). In the present study, we have identified a GnRH receptor (oct-GnRHR) specific for oct-GnRH from Octopus brain. Oct-GnRHR includes domains and motifs typical of vertebrate GnRH receptors. The intron-inserted positions are conserved between oct-GnRHR and the chordate GnRHR genes. The oct-GnRHR expressed in Xenopus (South African clawed frog) oocytes was responsive to oct-GnRH, but not to any other HPLC fractions of the Octopus brain extract. These results show that oct-GnRHR is an authentic receptor for oct-GnRH. Southern blotting of reverse-transcription PCR products revealed that the oct-GnRHR mRNA was widely distributed in the central and peripheral nervous systems and in several peripheral tissues. In situ hybridization showed that oct-GnRHR mRNA was expressed in some regions involved in autonomic functions, feeding, memory and movement. Oct-GnRH was shown to induce steroidogenesis of testosterone, progesterone and 17beta-oestradiol in Octopus ovary and testis, where oct-GnRHR was abundantly expressed. These results suggest that oct-GnRH, like its vertebrate counterparts, acts as a multifunctional neurotransmitter, neuromodulator and hormone-like factor, both in Octopus central nervous system and peripheral tissues, and that both structure and functions of the GnRH family are, at least partially, evolutionarily conserved between octopuses and chordates.

Asterosap-induced elevation in intracellular pH is indispensable for ARIS-induced sustained increase in intracellular Ca2+ and following acrosome reaction in starfish spermatozoa.

In the starfish, Asterias amurensis, the cooperation of three components of the egg jelly, namely ARIS (acrosome reaction-inducing substance), Co-ARIS and asterosap, is responsible for the induction of acrosome reaction. For the induction, ARIS alone is enough in high-Ca2+ or high-pH seawater, but, besides ARIS, the addition of either Co-ARIS or asterosap is required in normal seawater. Asterosap transiently increased both the intracellular pH (pHi) and Ca2+ ([Ca2+]i), while ARIS slightly elevated the basal level of [Ca2+]i. However, a sustained elevation of [Ca2+]i and acrosome reaction occurred if sperm were simultaneously treated with ARIS and asterosap. EGTA inhibited the sustained [Ca2+]i elevation and acrosome reaction. The sustained [Ca2+]i elevation and acrosome reaction were highly susceptible to SKF96365 and Ni2+, specific blockers of the store-operated Ca2+ channel (SOC). These results suggest that sustained [Ca2+]i elevation, mediated by the SOC-like channel, is a prerequisite for the acrosome reaction. In high-pH seawater, ARIS alone induced a prominent [Ca2+]i increase and acrosome reaction, which were similarly sensitive to SKF96365. The acrosome reaction was effectively induced by ARIS alone when pHi was artificially increased to more than 7.7. Asterosap increased pHi from 7.6 +/- 0.1 to 7.7 +/- 0.1. Furthermore, the sustained [Ca2+]i elevation and acrosome reaction, induced by a combination of ARIS and asterosap, were drastically inhibited by a slight reduction in pHi. Taking these results into account, we suggest that an asterosap-induced pHi elevation is required for triggering the ARIS-induced sustained [Ca2+]i elevation and consequent acrosome reaction.

Novel evolutionary lineages of the invertebrate oxytocin/vasopressin superfamily peptides and their receptors in the common octopus (Octopus vulgaris).

The common octopus, Octopus vulgaris, is the first invertebrate species that was shown to possess two oxytocin/vasopressin (OT/VP) superfamily peptides, octopressin (OP) and cephalotocin (CT). Previously, we cloned a GPCR (G-protein-coupled receptor) specific to CT [CTR1 (CT receptor 1)]. In the present study, we have identified an additional CTR, CTR2, and a novel OP receptor, OPR. Both CTR2 and OPR include domains and motifs typical of GPCRs, and the intron- exon structures are in accord with those of OT/VP receptor genes. CTR2 and OPR expressed in Xenopus oocytes induced calcium-mediated inward chloride current in a CT- and OP-specific manner respectively. Several regions and residues, which are requisite for binding of the vertebrate OT/VP receptor family with their ligands, are highly conserved in CTRs, but not in OPR. These different sequences between CTRs and OPR, as well as the amino acid residues of OP and CT at positions 2-5, were presumed to play crucial roles in the binding selectivity to their receptors, whereas the difference in the polarity of OT/VP family peptide residues at position 8 confers OT and VP with the binding specificity in vertebrates. CTR2 mRNA was present in various peripheral tissues, and OPR mRNA was detected in both the nervous system and peripheral tissues. Our findings suggest that the CT and OP genes, similar to the OT/VP family, evolved through duplication, but the ligand-receptor selectivity were established through different evolutionary lineages from those of their vertebrate counterparts.

Tachykinin and tachykinin receptor of an ascidian, Ciona intestinalis: evolutionary origin of the vertebrate tachykinin family.

Tachykinins (TKs) are the most prevalent vertebrate brain/gut peptides. In this study, we originally identified authentic TKs and their receptor from a protochordate, Ciona intestinalis. The Ciona TK (Ci-TK) precursor, like mammalian gamma-preprotachykinin A (gamma-PPTA), encodes two TKs, Ci-TK-I and -II, including the -FXGLM-NH(2) vertebrate TK consensus. Mass spectrometry of the neural extract revealed the production of both Ci-TKs. Ci-TK-I contains several Substance P (SP)-typical amino acids, whereas a Thr is exceptionally located at position 4 from the C terminus of Ci-TK-II. The Ci-TK gene encodes both Ci-TKs in the same exon, indicating no alternative generation of Ci-TKs, unlike the PPTA gene. These results suggested that the alternative splicing of the PPTA gene was established during evolution of vertebrates. The only Ci-TK receptor, Ci-TK-R, was equivalently activated by Ci-TK-I, SP, and neurokinin A at physiological concentrations, whereas Ci-TK-II showed 100-fold less potent activity, indicating that the ligand selectivity of Ci-TK-R is distinct from those of vertebrate TK receptors. Ci-TK-I, like SP, also elicited the typical contraction on the guinea pig ileum. The Ci-TK gene was expressed in neurons of the brain ganglion, small cells in the intestine, and the zone 7 in the endostyle, which corresponds to the vertebrate thyroid gland. Furthermore, the Ci-TK-R mRNA was distributed in these three tissues plus the gonad. These results showed that Ci-TKs play major roles in sexual behavior and feeding in protochordates as brain/gut peptides and endocrine/paracrine molecules. Taken together, our data revealed the biochemical and structural origins of vertebrate TKs and their receptors.

Characterization of crustacean cardioactive peptide as a novel insect midgut factor: isolation, localization, and stimulation of alpha-amylase activity and gut contraction.

The midgut, which plays a major role in the feeding behavior of insects, is believed to be controlled by various factors including neuropeptides. In the present study, we identified a neuropeptide crustacean cardioactive peptide (CCAP) as a novel gut factor in insects. CCAP was isolated from the midgut of the cockroach, Periplaneta americana. RT-PCR analysis detected the expression of the cockroach CCAP mRNA in the ventral nerve cord and the midgut, revealing the production of CCAP in the midgut as well as the central nervous system. Moreover, we observed expression of the CCAP gene in the endocrine cells of the midgut by in situ hybridization, and immunohistochemical analysis showed that CCAP was distributed around the lateral surfaces of the endocrine cells. Elevation of alpha-amylase activity was observed upon addition of CCAP to the midgut. These results suggest that CCAP is involved in digestion of carbohydrate in a paracrine manner. In situ hybridization and immunohistochemistry also revealed CCAP expression in the cell bodies of the ingluvial ganglion, which innervates the midgut muscle layer but not the epithelium, indicating that CCAP is produced in the ingluvial ganglion and then transported to the muscle layer through nerve fibers. Furthermore, CCAP was found to stimulate the contraction of the foregut, midgut, and hindgut in a dose-dependent manner. Taken together, our data indicate the multifunctionality of CCAP in the regulation of gut tissues as both a neuropeptide and a novel midgut factor.

Expression and distribution of octopus gonadotropin-releasing hormone in the central nervous system and peripheral organs of the octopus (Octopus vulgaris) by in situ hybridization and immunohistochemistry.

We recently purified a peptide with structural features similar to vertebrate gonadotropin-releasing hormone (GnRH) from the brain of Octopus vulgaris, cloned a cDNA encoding the precursor protein, and named it oct-GnRH. In the current study, we investigated the expression and distribution of oct-GnRH throughout the central nervous system (CNS) and peripheral organs of Octopus by in situ hybridization on the basis of the cDNA sequence and by immunohistochemistry using a specific antiserum against oct-GnRH. Oct-GnRH mRNA-expressing cell bodies were located in 10 of 19 lobes in the supraesophageal and subesophageal parts of the CNS. Several oct-GnRH-like immunoreactive fibers were seen in all the neuropils of the CNS lobes. The sites of oct-GnRH mRNA expression and the mature peptide distribution were consistent with each other as judged by in situ hybridization and immunohistochemistry. In addition, many immunoreactive fibers were distributed in peripheral organs such as the heart, the oviduct, and the oviducal gland. Modulatory effects of oct-GnRH on the contractions of the heart and the oviduct were demonstrated. The results suggested that, in the context of reproduction, oct-GnRH is a key peptide in the subpedunculate lobe and/or posterior olfactory lobe-optic gland-gonadal axis, an octopus analogue of the hypothalamo-hypophysial-gonadal axis. It may also act as a modulatory factor in controlling higher brain functions such as feeding, memory, movement, maturation, and autonomic functions

Identification of a novel receptor for an invertebrate oxytocin/vasopressin superfamily peptide: molecular and functional evolution of the oxytocin/vasopressin superfamily.

Annetocin is structurally related to an OT (oxytocin)/VP (vasopressin) family peptide, which has been isolated from the earthworm Eisenia foetida and has been shown to induce OT-like egg-laying behaviour. We now report the identification of an endogenous AnR (annetocin receptor). The deduced AnR precursor displays high sequence similarity with OT/VP receptors. Genomic analysis of the AnR gene revealed that the intron-inserted position is conserved between the AnR gene and the mammalian OT/VP receptor genes. These results indicate that AnR and mammalian OT/VP receptors share a common ancestor gene. Administration of annetocin to the AnR expressed in Xenopus oocytes induced a calcium-dependent signal transduction. Reverse transcriptase-PCR analysis and in situ hybridization showed that the AnR gene is expressed specifically in the nephridia located in the clitellum region, although the nephridia are distributed throughout the worm body. This result suggests that annetocin induces egg-laying behaviour through its action on the nephridia. This is the first description concerning the functional correlation between an invertebrate OT/VP-related peptide and egg-laying behaviour.

Crystallographic and biochemical investigations of kumamolisin-As, a serine-carboxyl peptidase with collagenase activity.

Kumamolisin-As (previously called ScpA) is the first known example of a collagenase from the sedolisin family (MEROPS S53). This enzyme is active at low pH and in elevated temperatures. In this study that used x-ray crystallographic and biochemical methods, we investigated the structural basis of the preference of this enzyme for collagen and the importance of a glutamate residue in the unique catalytic triad (Ser(278)-Glu(78)-Asp(82)) for enzymatic activity. Crystal structures of the uninhibited enzyme and its complex with a covalently bound inhibitor, N-acetyl-isoleucyl-prolyl-phenylalaninal, showed the occurrence of a narrow S2 pocket and a groove that encompasses the active site and is rich in negative charges. Limited endoproteolysis studies of bovine type-I collagen as well as kinetic studies using peptide libraries randomized at P1 and P1', showed very strong preference for arginine at the P1 position, which correlated very well with the presence of a negatively charged residue in the S1 pocket of the enzyme. All of these features, together with those predicted through comparisons with fiddler crab collagenase, a serine peptidase, rationalize the enzyme's preference for collagen. A comparison of the Arrhenius plots of the activities of kumamolisin-As with either collagen or peptides as substrates suggests that collagen should be relaxed before proteolysis can occur. The E78H mutant, in which the catalytic triad was engineered to resemble that of subtilisin, showed only 0.01% activity of the wild-type enzyme, and its structure revealed that Ser(278), His(78), and Asp(82) do not interact with each other; thus, the canonical catalytic triad is disrupted.

Guanylyl cyclase and cGMP-specific phosphodiesterase participate in the acrosome reaction of starfish sperm.

In the starfish, Asterias amurensis, the cooperation of three components of the egg jelly, i.e. ARIS (acrosome reaction-inducing substance), Co-ARIS and asterosap, is responsible for inducing the acrosome reaction. Experimentally, ARIS and asterosap are sufficient for the induction. However, when sperm are treated only with asterosap, they become unresponsive to the egg jelly to undergo the reaction. In this study, we analysed the mechanism of the acrosome reaction, using sperm inactivation by asterosap as a clue. Asterosap causes a rapid and transient increase in intracellular cGMP through the activation of the asterosap receptor, a guanylyl cyclase, and causes an increase in intracellular Ca(2+). When sperm were pretreated with asterosap, the guanylyl cyclase seemed to be inactivated irreversibly by dephosphorylation. They were still responsive to ARIS but no longer to asterosap. However, in the presence of IBMX or zaprinast, inhibitors against phosphodiesterases (PDEs), they retained their capacity to undergo the acrosome reaction in response to the egg jelly or ARIS alone. IBMX and zaprinast suppressed the intracellular catabolism of cGMP, but not of cAMP. These results suggest that guanylyl cyclase and cGMP-specific, IBMX- and zaprinast-susceptible PDEs are involved in the regulation of the acrosome reaction.

Cloning of Octopus cephalotocin receptor, a member of the oxytocin/vasopressin superfamily.

We reported that the common octopus, Octopus vulgaris, in common with vertebrates, possesses two members of the oxytocin/vasopressin superfamily: octopressin (OP) and cephalotocin (CT). This was the first observation of its kind in invertebrates. As OP and CT have different biological activities, the presence of specific receptors has been proposed. We cloned the cDNA of an orphan receptor from Octopus brain and found it to encode a polypeptide of 397 amino acids that displays sequences characteristic of G-protein coupled receptors. The orphan receptor showed high homology to receptors of the oxytocin/vasopressin superfamily and seemed to conserve the agonist-binding pocket common to the oxytocin and vasopressin receptors. Xenopus oocytes that express the orphan receptor responded to the application of CT by an induction of membrane Cl(-) currents coupled to the inositol phosphate/Ca(2+) pathway. OP and the other members of the oxytocin/vasopressin superfamily did not activate this receptor. HPLC fractionation of the Octopus brain extract combined with an oocyte assay yielded a single substance that was identical to CT. On the basis of these results, we conclude that the cloned receptor is the CT receptor (CTR). Expression of CTR mRNA in Octopus was detected in the central and the peripheral nervous systems, the pancreas, the oviduct and the ovary. This receptor may mediate physiological functions of CT in Octopus such as neurotransmission, reproduction and metabolism.