Identification, chemical synthesis, and receptor binding of a reptilian gecko ghrelin.

Ghrelin is known to be a gastrointestinal peptide hormone in vertebrates. It has a unique posttransrational modification, octanoylation, at the Ser side chain of the third position. In this study, we identified the genes encoding ghrelin and its receptor from the Schlegel's Japanese gecko Gekko japonicus. The C-terminal residue of gecko ghrelin was His, although the chemical synthesis method for the O-octanoyl peptide with a C-terminal His residue has not yet been well-established. Acyl-ghrelin has been synthesized using a Ser derivative without side chain protecting group in the solid-phase peptide synthesis, although this synthetic strategy has not yet been well-established. Here we show the efficient synthetic method with minimal side reactions, and G. japonicus ghrelin could be obtained in good yield. This would be useful and applicable to the synthesis of ghrelin from other animal species. The gecko ghrelin receptor was expressed in HEK 293 cells, which was fully responsive to the synthetic gecko ghrelin. These results indicate that the ghrelin system similar to mammals also exists in a reptilian gecko, G. japonicus.

Diselenide-bond replacement of the external disulfide bond of insulin increases its oligomerization leading to sustained activity.

Seleno-insulin, a class of artificial insulin analogs, in which one of the three disulfide-bonds (S-S's) of wild-type insulin (Ins) is replaced by a diselenide-bond (Se-Se), is attracting attention for its unique chemical and physiological properties that differ from those of Ins. Previously, we pioneered the development of a [C7UA,C7UB] analog of bovine pancreatic insulin (SeIns) as the first example, and demonstrated its high resistance against insulin-degrading enzyme (IDE). In this study, the conditions for the synthesis of SeIns via native chain assembly (NCA) were optimized to attain a maximum yield of 72%, which is comparable to the in vitro folding efficiency for single-chain proinsulin. When the resistance of BPIns to IDE was evaluated in the presence of SeIns, the degradation rate of BPIns became significantly slower than that of BPIns alone. Furthermore, the investigation on the intermolecular association properties of SeIns and BPIns using analytical ultracentrifugation suggested that SeIns readily forms oligomers not only with its own but also with BPIns. The hypoglycemic effect of SeIns on diabetic rats was observed at a dose of 150 µg/300 g rat. The strategy of replacing the solvent-exposed S-S with Se-Se provides new guidance for the design of long-acting insulin formulations.

Gonadotropic activity of a second relaxin-type peptide in starfish.

In starfish, a relaxin-like gonad-stimulating peptide (RGP) acts as a gonadotropin that triggers gamete maturation and spawning. In common with other relaxin/insulin superfamily peptides, RGP consists of an A- and a B-chain, with cross-linkages mediated by one intra- and two inter-chain disulfide bonds. In this study, a second relaxin-like peptide (RLP2) was identified in starfish species belonging to the orders Valvatida, Paxillosida, and Forcipulatida. Like RGP, RLP2 precursors comprise a signal peptide and a C-peptide in addition to the A- and B-chains. However, a unique cysteine motif [CC-(3X)-C-(10X)-C] is present in the A-chain of RLP2, which contrasts with the cysteine motif in other members of the relaxin/insulin superfamily [CC-(3X)-C-(8X)-C]. Importantly, in vitro pharmacological tests revealed that Patiria pectinifera RLP2 (Ppe-RLP2) and Asterias rubens RLP2 (Aru-RLP2) trigger shedding of mature eggs from ovaries of P. pectinifera and A. rubens, respectively. Furthermore, the potencies of Ppe-RLP2 and Aru-RLP2 as gonadotropic peptides were similar to those of Ppe-RGP and Aru-RGP, respectively, and the effect of RLP2 exhibited partial species-specificity. These findings indicate that two relaxin-type peptides regulate spawning in starfish and therefore we propose that RGP and RLP2 are renamed RGP1 and RGP2, respectively.

Chemical synthesis of insulin-like peptide 1 and its potential role in vitellogenesis of the kuruma prawn Marsupenaeus japonicus.

The insulin superfamily comprises a group of peptides with diverse physiological functions and is conserved across the animal kingdom. Insulin-like peptides (ILPs) of crustaceans are classified into four major types: insulin, relaxin, gonadulin, and androgenic gland hormone (AGH)/insulin-like androgenic gland factor (IAG). Of these, the physiological functions of AGH/IAG have been clarified to be the regulation of male sex differentiation, but those of the other types have not been uncovered. In this study, we chemically synthesized Maj-ILP1, an ILP identified in the ovary of the kuruma prawn Marsupenaeus japonicus, using a combination of solid-phase peptide synthesis and regioselective disulfide bond formation reactions. As the circular dichroism spectral pattern of synthetic Maj-ILP1 is typical of other ILPs reported thus far, the synthetic peptide likely possessed the proper conformation. Functional analysis using ex vivo tissue incubation revealed that Maj-ILP1 significantly increased the expression of the yolk protein genes Maj-Vg1 and Maj-Vg2 in the hepatopancreas and Maj-Vg1 in the ovary of adolescent prawns. This is the first report on the synthesis of a crustacean ILP other than IAGs and also shows the positive relationship between the reproductive process and female-dominant ILP.

Heme protein identified from scaly-foot gastropod can synthesize pyrite (FeS2) nanoparticles.

The scaly-foot gastropod (Chrysomallon squamiferum), which lives in the deep-sea zone of oceans around thermal vents, has a black shell and scales on the foot. Both the black shell and scales contain iron sulfide minerals such as greigite (Fe3S4) and pyrite (FeS2). Although pyrite nanoparticles can be used as materials for solar panels, it is difficult to synthesize stable and spherical nanoparticles in vitro. In this study, we extracted organic molecules that interact with nano-pyrite from the shell of the scaly-foot gastropod to develop a low-cost, eco-friendly method for pyrite nanoparticles synthesis. Myoglobin (csMG), a heme protein, was identified in the iron sulfide layer of the shell. We purified recombinant csMG (r-csMG) and demonstrated that r-csMG helped in the conversion of ferric ions, sulfide ions and sulfur into spherical shaped pyrite nanoparticles at 80°C. To reduce the effort and cost of production, we showed that commercially available myoglobin from Equus caballus (ecMG) also induced the in vitro synthesis of pyrite nanoparticles. Using structure-function experiments with digested peptides, we highlighted that the amino acid sequence of r-csMG peptides controlled the spherical shape of the nanoparticle while the hemin molecules, which the peptides interacted with, maintained the size of nanoparticles. Synthesized pyrite nanoparticles exhibited strong photoluminescence in the visible wavelength region, suggesting its potential application as a photovoltaic solar cell material. These results suggest that materials for solar cells can be produced at low cost and energy under eco-friendly conditions. STATEMENT OF SIGNIFICANCE: Pyrite is a highly promising material for photovoltaic devices because of its excellent optical, electrical, magnetic, and transport properties and high optical absorption coefficient. Almost all current pyrite synthesis methods use organic solvents at high temperature and pressure under reducing conditions. Synthesized pyrite nanoparticles are unstable and are difficult to use in devices. The scaly-foot gastropod can synthesize pyrite nanoparticles in vivo, meaning that pyrite nanoparticles can be generated in an aqueous environment at low temperature. In this study, we demonstrated the synthesis of pyrite nanoparticles using a heme protein identified in the iron sulfide layer of the scaly-foot gastropod shell. These results exemplify how natural products in organisms can inspire the innovation of new technology.

A Relaxin-Like Gonad-Stimulating Peptide Appears in the Early Development of the Starfish Patiria pectinifera.

Relaxin-like gonad-stimulating peptide (RGP) is a hormone with gonadotropin-like activity in starfish. This study revealed that spawning inducing activity was detected in an extract of brachiolaria larvae of Patiria pectinifera. Spawning inducing activity in the extract was due to P. pectinifera RGP (PpeRGP), not 1-methyladenine. The expression of PpeRGP mRNA was also found in brachiolaria. Immunohistochemical observation with specific antibodies for PpeRGP showed that PpeRGP was distributed in the peripheral adhesive papilla of the brachiolaria arms. In contrast, PpeRGP was not detected in the adult rudiment or ciliary band regions, which are present in the neural system. These findings strongly suggest that RGP exists in the larvae before metamorphosis. Because gonads are not developed in starfish larvae, it seems likely that RGP plays another role other than gonadotropic action in the early development of starfish.

The C-terminally amidated relaxin-like gonad-stimulating peptide in the starfish Astropecten scoparius.

A relaxin-like gonad-stimulating peptide (RGP) in starfish was the first identified invertebrate gonadotropin, consisting of A- and B-chain. Recently, an RGP ortholog (Asc-RGP) from Astropecten scoparius in the order Paxillosida was found to harbor an amidation signal (Gly-Arg) at the C-terminus of the B-chain (Mita et al., 2020a). Two cleavage sites were also predicted within the signal peptide of the Asc-RGP precursor. Thus, four kinds of analogs (Asc-RGP-NH2(S), Asc-RGP-GR(S), Asc-RGP- NH2(L), Asc-RGP-GR(L) were hypothesized as natural Asc-RGPs. To identify the natural Asc-RGP, an extract of radial nerve cords from A. scoparius was analyzed using reverse-phase high-performance liquid chromatography and MALDI-TOF-mass spectrometry. The molecular weight of Asc-RGP was 4585.3, and those of A- and B-chains were 2511.8 and 2079.8, respectively. This strongly suggests that natural RGP in A. scoparius is Asc-RGP-NH2(S). Asc-RGP-NH2(S) stimulated 1-methyladenine and cyclic AMP production in isolated ovarian follicle cells of A. scoparius. On the other hand, the concentrations of four synthetic Asc-RGP analogs required for the induction of spawning in 50% of ovarian fragments were almost the same. The size and C-terminal amidation of the B-chain might not be important for spawning-inducing activity. C-terminally amidated RGPs in the B-chain were also observed in other species of starfish belonging to the order Paxillosida, particularly the family Astropectinidae, but not the family Luidiidae.

Characterization and localization of relaxin-like gonad-stimulating peptide in the crown-of-thorns starfish, Acanthaster cf. solaris.

In starfish, a relaxin-like gonad-stimulating peptide (RGP) is the gonadotropin responsible for final gamete maturation. RGP comprises two different peptides, A- and B-chains with two interchain and one intrachain disulfide bonds. The existence of two isomers of RGP in the crown-of-thorns starfish, Acanthaster planci, has been reported previously, but it was recently shown that A. planci represents a species complex with four different species. Here we elucidated the authentic sequence of the Pacific species, Acanthaster cf. solaris, RGP (Aso-RGP). The Aso-RGP precursor encoded by a 354 base pair open reading frame was composed of 117 amino acids (aa). The amino acid identity of Aso-RGP to Patiria pectinifera RGP (Ppe-RGP) and Asterias amurensis RGP (Aam-RGP) was 74% and 60%, respectively. Synthetic Aso-RGP induced spawning of ovarian fragments from A. cf. solaris. Ppe-RGP and Aam-RGP also induced spawning by A. cf. solaris ovaries. In contrast, Ppe-RGP and Aso-RGP induced spawning by P. pectinifera ovaries, but Aam-RGP was inactive. Notably, anti-Ppe-RGP antibodies recognized Aso-RGP as well as Ppe-RGP. Localization of Aso-RGP was observed immunohistochemically using anti-Ppe-RGP antibodies, showing that Aso-RGP was mainly present in the radial nerve cords of A. cf. solaris. Aso-RGP was distributed not only in the epithelium of the ectoneural region but also in the neuropile of the ectoneural region. These results suggest that Aso-RGP is synthesized in the epithelium of the ectoneural region, then transferred to fibers in the neuropile of the ectoneural region in radial nerve cords.

Chemical synthesis and functional evaluation of the crayfish insulin-like androgenic gland factor.

Insulin-like androgenic gland factor (IAG) from the marbled crayfish Procambarus virginalis is an insulin-like heterodimeric peptide composed of A and B chains and has an Asn-linked glycan at the B chain. IAG is considered to be a male sex hormone inducing the sex differentiation to male in decapod crustacean, although there is no report on the function of IAG peptide in vivo. In order to characterize P. virginalis IAG, we chemically synthesized it and evaluated its biological function in vivo. A and B chains were prepared by the ordinary solid-phase peptide synthesis, and three disulfide bonds were formed regioselectively by dimethyl sulfoxide oxidation, pyridylsulfenyl-directed thiolysis and iodine oxidation reactions. An IAG disulfide isomer was also prepared by the same manner. Circular dichroism spectral analysis revealed that the disulfide bond arrangement affected the peptide conformation, which was similar to the other insulin-family peptides analyzed so far. On the other hand, the glycan moiety attached at the B chain had no effect on the peptide secondary structure. Injection of the synthetic IAG and its disulfide isomer to female crayfish did not induce male characteristics on the external morphology, but both peptides suppressed the oocyte maturation in vivo. These results suggest that IAG has a pivotal role on the suppression of female secondary sex characteristics.

A specific and sensitive enzyme-linked immunosorbent assay for measurement of relaxin-like gonad-stimulating peptide in the starfish Asterias rubens.

A relaxin-like gonad-stimulating peptide (RGP) acts as a gonadotropic hormone in starfish. In this study, antibodies to Asterias rubens RGP (AruRGP) were used for the development of a specific and sensitive enzyme-linked immunosorbent assay (ELISA) to measure AruRGP. Biotin-conjugated RGP (biotin-AruRGP) that binds to peroxidase-conjugated streptavidin was synthesized chemically so that it could be specifically detected using 3, 3', 5, 5'-tetramethylbenzidine (TMB)/hydrogen peroxide as a substrate. Similar to AruRGP, biotin-AruRGP bound to AruRGP antibodies. In binding experiments with biotin-AruRGP using wells coated with AruRGP antibodies, a displacement curve was obtained using serial dilutions of AruRGP. Using this ELISA system, AruRGP could be measured in the range 0.01-5.0 pmol per 50 µl test solution. Furthermore, 0.22 ± 0.03 and 0.20 ± 0.04 pmol AruRGP/mg wet weight tissue were detected in the radial nerve cords and circumoral nerve-rings of A. rubens, respectively. Smaller amounts of AruRGP were detected in tube feet, pyloric stomach and cardiac stomach but AruRGP was not detected in pyloric caeca, ovaries and testes. Analysis of the specificity of the AruRGP antibodies revealed that the A- and B-chains of AruRGP, Patiria pectinifera RGP, Aphelasterias japonica RGP, and human relaxin exhibit little or no cross-reactivity in the ELISA. We conclude, therefore, that we have successfully generated an ELISA system that is highly sensitive and specific for detection of AruRGP.

Sex Determination and Differentiation in Decapod and Cladoceran Crustaceans: An Overview of Endocrine Regulation.

Mechanisms underlying sex determination and differentiation in animals are known to encompass a diverse array of molecular clues. Recent innovations in high-throughput sequencing and mass spectrometry technologies have been widely applied in non-model organisms without reference genomes. Crustaceans are no exception. They are particularly diverse among the Arthropoda and contain a wide variety of commercially important fishery species such as shrimps, lobsters and crabs (Order Decapoda), and keystone species of aquatic ecosystems such as water fleas (Order Branchiopoda). In terms of decapod sex determination and differentiation, previous approaches have attempted to elucidate their molecular components, to establish mono-sex breeding technology. Here, we overview reports describing the physiological functions of sex hormones regulating masculinization and feminization, and gene discovery by transcriptomics in decapod species. Moreover, this review summarizes the recent progresses of studies on the juvenile hormone-driven sex determination system of the branchiopod genus Daphnia, and then compares sex determination and endocrine systems between decapods and branchiopods. This review provides not only substantial insights for aquaculture research, but also the opportunity to re-organize the current and future trends of this field.

Application of 2,2'-dipyridyl disulfide-mediated thiazolidine ring-opening reaction to glycoprotein synthesis: Total chemical synthesis of evasin-3.

Thiazolidine ring-opening reaction is one of the key steps in protein chemical synthesis via sequential native chemical ligation strategy. We recently developed a novel thiazolidine ring-opening reaction with 2,2'-dipyridyl disulfide (DPDS). In order to investigate the applicability of this reaction to glycoprotein synthesis, we synthesized evasin-3, a cysteine-rich glycoprotein with chemokine-binding ability originally found in tick saliva. The sequence of evasin-3 was divided into three segments, and these segments were separately synthesized with the ordinary solid-phase peptide synthesis method. After the first ligation of middle and C-terminal segments, thiazolidine used as a protecting group of Cys residue at the N-terminus of the middle segment was converted to Cys with DPDS. In this thiazolidine ring-opening reaction, DPDS treatment did not affect the N-linked glycan moiety. After the second ligation with the N-terminal segment and the refolding reaction, evasin-3 could be obtained in good yield. The synthetic evasin-3 showed the binding ability specifically to CXCL chemokines. These results clearly indicate that this DPDS method is useful for glycoprotein synthesis.

A relaxin-like gonad-stimulating peptide identified from the starfish Astropecten scoparius.

A relaxin-like gonad-stimulating peptide (RGP) in starfish was the first identified invertebrate gonadotropin responsible for final gamete maturation. An RGP ortholog was newly identified from Astropecten scoparius of the order Paxillosida. The A. scoparius RGP (AscRGP) precursor is encoded by a 354 base pair open reading frame and is a 118 amino acid (aa) protein consisting of a signal peptide (26 aa), B-chain (21 aa), C-peptide (47 aa), and A-chain (24 aa). There are three putative processing sites (Lys-Arg) between the B-chain and C-peptide, between the C-peptide and A-chain, and within the C-peptide. This structural organization revealed that the mature AscRGP is composed of A- and B-chains with two interchain disulfide bonds and one intrachain disulfide bond. The C-terminal residues of the B-chain are Gln-Gly-Arg, which is a potential substrate for formation of an amidated C-terminal Gln residue. Non-amidated (AscRGP-GR) and amidated (AscRGP-NH2 ) peptides were chemically synthesized and their effect on gamete shedding activity was examined using A. scoparius ovaries. Both AscRGP-GR and AscRGP-NH2 induced oocyte maturation and ovulation in similar dose-dependent manners. This is the first report on a C-terminally amidated functional RGP. Collectively, these results suggest that AscRGP-GR and AscRGP-NH2 act as a natural gonadotropic hormone in A. scoparius.

Salmon acyl-ghrelin increases food intake and reduces doxorubicin-induced myocardial apoptosis in rats, likely by anti-oxidative activity.

We had reported that orally administered ghrelin-containing salmon stomach extract prevents doxorubicin (DOX)-induced cardiotoxicity. In this study, we investigated the binding affinity of salmon ghrelin to rat ghrelin receptor and the cardioprotective effects of subcutaneous (sc) injected synthetic salmon ghrelin in rats with DOX-induced acute heart failure in order to clarify the potential efficacy of salmon ghrelin. Intracellular calcium mobilization assay was performed on rat GHS-R1a-expressing CHO cells to reveal ghrelin activity. Rats were divided into five groups; the normal control (I), and toxic control (II) groups were given saline (sc, twice daily), and the salmon acyl-ghrelin (sAG) (III), salmon unacylated-ghrelin (sUAG) (IV), and rat acyl-ghrelin (rAG) (V) groups were given corresponding synthetic ghrelins (sc, twice daily), respectively. After seven days of treatment, DOX (20 mg/kg BW) or saline was administered to the corresponding groups by intraperitoneal injection. The toxic control group was the negative control group for the DOX-induced cardiotoxicity groups. While sAG displayed similar affinity to rAG upon application to GHS-R1a-expressing cells, and also decreased DOX-induced apoptosis and increased food intake, sUAG did not. Both sAG and rAG improved DOX-induced deterioration, showing anti-oxidative activity. The anti-oxidative activity of sAG might contribute to the protective effects on cardiomyocytes. The results also suggest that, similar to rAG, sAG is a potent protectant against DOX-induced cardiotoxicity and a potential functional component in orally administered ghrelin-containing salmon stomach extract, which prevented DOX-induced cardiotoxicity in our previous study.

Relaxin-2 May Suppress Endometriosis by Reducing Fibrosis, Scar Formation, and Inflammation.

BACKGROUND: Relaxin (RLX)-2, produced by the corpus luteum and placenta, is known to be potentially effective in fibrotic diseases of the heart, lungs, kidneys, and bladder; however, its effectiveness in endometriosis has not yet been investigated. In the present study, we conducted a comprehensive study on the effect of RLX-2 on endometriosis. We checked the expressions of LGR-7, a primary receptor of RLX-2, in endometriomas using immunohistochemistry. Endometriotic stromal cells (ESCs) purified from surgical specimens were used in in vitro experiments. The effects of RLX-2 on ESCs were evaluated by quantitative-PCR, ELISA, and Western blotting. Gel contraction assay was used to assess the contraction suppressive effect of RLX-2. The effect of RLX-2 was also examined in the endometriosis mouse model. LGR-7 was expressed in endometriotic lesions. In ESCs, RLX-2 increased the production of cAMP and suppressed the secretion of interleukin-8, an inflammatory cytokine, by 15% and mRNA expression of fibrosis-related molecules, plasminogen activator inhibitor-1 (PAI-1), and collagen-I by approximately 50% (p < 0.05). In the gel contraction assay, RLX-2 significantly suppressed the contraction of ESCs, which was cancelled by removing RLX-2 from the medium or by adding H89, a Protein Kinase A (PKA) inhibitor. In ESCs stimulated with RLX-2, p38 MAPK phosphorylation was significantly suppressed. In the endometriosis mouse model, administration of RLX-2 significantly decreased the area of the endometriotic-like lesion with decreasing fibrotic component compared to non-treated control (p = 0.01). RLX-2 may contribute to the control of endometriotic lesion by suppressing fibrosis, scar formation, and inflammation.

Dioctatin Activates ClpP to Degrade Mitochondrial Components and Inhibits Aflatoxin Production.

Aflatoxin contamination of crops is a serious problem worldwide. Utilization of aflatoxin production inhibitors is attractive, as the elucidation of their modes of action contributes to clarifying the mechanism of aflatoxin production. Here, we identified mitochondrial protease ClpP as the target of dioctatin, an inhibitor of aflatoxin production of Aspergillus flavus. Dioctatin conferred uncontrolled caseinolytic capacity on ClpP of A. flavus and Escherichia coli. Dioctatin-bound ClpP selectively degraded mitochondrial energy-related proteins in vitro, including a subunit of respiratory chain complex V, which was also reduced by dioctatin in a ClpP-dependent manner in vivo. Dioctatin enhanced glycolysis and alcohol fermentation while reducing tricarboxylic acid cycle metabolites. These disturbances were accompanied by reduced histone acetylation and reduced expression of aflatoxin biosynthetic genes. Our results suggest that dioctatin inhibits aflatoxin production by inducing ClpP-mediated degradation of mitochondrial energy-related components, and that mitochondrial energy metabolism functions as a key determinant of aflatoxin production.

Effect of chimeric relaxin-like gonad-stimulating peptides on oocyte maturation and ovulation in the starfish Asterias rubens and Aphelasterias japonica.

A relaxin-like gonad-stimulating peptide (RGP), comprising two peptide chains (A- and B-chains) linked by two interchain bonds and one intrachain disulfide bond, acts as a gonadotropin in starfish. RGP orthologs have been identified in several starfish species, including Patiria pectinifera (PpeRGP), Asterias rubens (AruRGP) and Aphelasterias japonica (AjaRGP). To analyze species-specificity, this study examined the effects on oocyte maturation and ovulation in ovaries of A. rubens and A. japonica of nine RGP derivatives comprising different combinations of A- and B-chains from the three species. All nine RGP derivatives induced spawning in A. rubens and A. japonica ovaries. However, AruRGP, AjaRGP and their chimeric derivatives were more potent than peptides containing the A- or B-chain of PpeRGP. Three-dimensional models of the structures of the RGP derivatives revealed that residues in the B-chains, such as AspB6, MetB10 and PheB13 in PpeRGP and GluB7, MetB11, and TyrB14 in AruRGP and AjaRGP, respectively, are likely to be involved in receptor binding. Conversely, it is likely that ArgA18 in the A-chain of AruRGP and AjaRGP impairs binding of these peptides to the PpeRGP receptor in P. pectinifera. In conclusion, this study provides new insights into the structural basis of RGP bioactivity and RGP receptor activation in starfish.

Chemical synthesis of N-glycosylated insulin-like androgenic gland factor from the freshwater prawn Macrobrachium rosenbergii.

Crustacean insulin-like androgenic gland factor (IAG) of Macrobrachium rosenbergii, a heterodimeric peptide having both four disulfide bonds and an N-linked glycan, was synthesized by the combination of solid-phase peptide synthesis and the regioselective disulfide formation reactions. The disulfide isomer of IAG could also be synthesized by the same manner. The conformational analysis of these peptides by circular dichroism (CD) spectral measurement indicated that the disulfide bond arrangement affected the peptide conformation in IAG. On the other hand, the N-linked glycan attached at A chain showed no effect on CD spectra of IAG. This is the first report for the chemical synthesis of insulin-like heterodimeric glycopeptide having three interchain disulfides, and the synthetic strategy shown here might be useful for the synthesis of other glycosylated four-disulfide insulin-like peptides.

A novel approach for preparing disulfide-rich peptide-KLH conjugate applicable to the antibody production.

To produce the antiserum against a small peptide, the target peptide-keyhole limpet hemocyanine (KLH) conjugate is generally used as an antigen, although the disulfide-rich peptide-KLH conjugate is still difficult to prepare. In our previous study, we have developed a preparation method of the disulfide-rich peptide-KLH conjugate, and this method was applied to produce the antiserum against a relaxin-like peptide. However, this method is limited to the synthetic peptide antigen, and is not applicable to a native or a recombinant peptide. In this study, to expand the applicability of this method to wide variety of peptides, we newly designed a novel thiol probe enabling the conjugation between various peptides and KLH, and applied it to produce the antiserum against relaxin-like peptide of a starfish Asterias amurensis. The antiserum obtained here showed high antibody-titer and good specificity, strongly suggesting that the method developed in this study is applicable to various peptides.

Interaction of starfish gonadotropin with its receptor: Effect of chimeric relaxin-like gonad-stimulating peptides.

A relaxin-like gonad-stimulating peptide (RGP) of starfish Patiria (Asterina) pectinifera is the first identified invertebrate gonadotropin for final gamete maturation. Recently, we found three orthologs of RGP in the class Asteroida; PpeRGP in P. pectinifera, AamRGP in Asterias amurensis, and AjaRGP in Aphelasterias japonica. In this study, nine kinds of RGP derivatives with exchanged each A- and B-chain were synthesized chemically to analyze the interaction of RGP with its receptor. Among these RGP derivatives, PpeRGP and its chimeric RGPs with B-chains from AamRGP or AjaRGP could induce oocyte maturation and ovulation in P. pectinifera ovaries. In contrast, other RGP derivatives were failed to induce spawning in P. pectinifera ovaries. Circular dichroism spectra of PpeRGP were similar to those of chimeric RGPs with the B-chains from AamRGP or AjaRGP. Furthermore, the predicted three-dimensional structure models of the B-chains from RGP derivatives have almost the same conformation. These findings suggest that the B-chain of PpeRGP is involved in binding to its receptor. Thus, it is likely that the A-chain of AamRGP or AjaRGP disturbs the binding of the PpeRGP B-chain to its receptor.