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.

D-Amino Acids in Peptides from Animals, Including Human: Occurrence, Structure, Bioactivity and Pharmacology.

All life forms typically possess homochirality, with rare exceptions. In the case of peptides and proteins, only L-amino acids are known to be encoded by genes. Nevertheless, D-amino acids have been identified in a variety of peptides, synthesized by animal cells. They include neuroexcitatory and neuroprotective peptides, cardioexcitatory peptides, hyperglycemic hormones, opioid peptides, antimicrobial peptides, natriuretic and defensin-like peptides, and fibrinopeptides. This article is a review of their occurrence, structure and bioactivity. It further explores the pharmacology and potential medical applications of some of the peptides.

Natural and synthetic chiral isoforms of crustacean hyperglycemic hormone from the crayfish Astacus leptodactylus: hyperglycemic activity and hemolymphatic clearance.

In the crayfish Astacus leptodactylus, as in several crustacean species, the crustacean hyperglycemic hormone is present as two isoforms differing by the chirality of the third residue, a phenylalanine. In the present work, isoforms synthesized full length by solid-phase peptide synthesis have been purified, refolded, the location of the disulfide bridges has been checked, their immunoreactivity against different antibodies have been analyzed and their hyperglycemic activity tested, to ensure the identity of the synthetic peptides with their natural homologs. Different parameters of the hyperglycemic activity of both isoforms were studied. In addition to a difference in the kinetics of hyperglycemia, already known from other studies, it was observed that the dose-response was different depending on the season where experiments were performed, the response being stronger in spring than in autumn, especially for the d-Phe containing isoform. A dosage method based on sandwich enzyme linked immunosorbent assay (ELISA) has been developed to measure hemolymphatic levels of the isoforms after spiking of the animals with one isoform or the other. It was found that hemolymphatic clearance was identical for both isoforms, indicating that their differential effect is not linked to their different lifetime in the hemolymph but may rather rely on other mechanisms such as their binding to different target tissues.

Novel protocol for the chemical synthesis of crustacean hyperglycemic hormone analogues--an efficient experimental tool for studying their functions.

The crustacean Hyperglycemic Hormone (cHH) is present in many decapods in different isoforms, whose specific biological functions are still poorly understood. Here we report on the first chemical synthesis of three distinct isoforms of the cHH of Astacus leptodactylus carried out by solid phase peptide synthesis coupled to native chemical ligation. The synthetic 72 amino acid long peptide amides, containing L- or D-Phe³ and (Glp¹, D-Phe³) were tested for their biological activity by means of homologous in vivo bioassays. The hyperglycemic activity of the D-isoforms was significantly higher than that of the L-isoform, while the presence of the N-terminal Glp residue had no influence on the peptide activity. The results show that the presence of D-Phe³ modifies the cHH functionality, contributing to the diversification of the hormone pool.

Inhibition of coagulation activation and inflammation by a novel Factor Xa inhibitor synthesized from the earthworm Eisenia andrei.

We have cloned an earthworm-derived Factor Xa (FXa) inhibitor, with an excellent inhibitory specificity from the midgut of the Eisenia andrei. We designate this inhibitor eisenstasin. An eisenstasin-derived small peptide (ESP) was synthesized and we examined whether ESP played an essential role in FXa inhibition. Compared to antistasin-derived small peptides (ASP) originating from leech, ESP primarily exhibited a high level of FXa inhibition in chromogenic peptide substrate assays and revealed an approximately 2-fold greater inhibition of FXa cleavage of a target protein than ASP. This suggests that ESP could be an effective anti-coagulant that targets FXa during the propagation step of coagulation. ESP also inhibited proteinase-activated receptor 2-mediated FXa activation, which may trigger endothelial inflammation. Endothelial nitric oxide (NO) was significantly reduced by ESP (p<0.0001), indicating that protease-activated receptor-2 (PAR-2) was effectively inactivated. We also found that ESP reduced the expressions of pro-inflammatory cytokines (IL-1alpha, IL-1beta, IL-8, IL-16, MCP-1, MIP-1alpha and MIP-1beta) by cultured cells treated with both ESP and FXa. Our results provide the first evidence that ESP might interrupt coagulation cascades by inhibiting FXa, and thereby may effectively control the bidirectional alternation between coagulation and inflammation.

Design, synthesis and structure-activity relationship of a series of fragment analogues of antistasin (ATS) and ghilantens (GLS).

A series of low molecular weight peptide inhibitors of Factor Xa, fragment analogues of ATS and GLS, was designed and synthesized by the SPPS method. The new analogues included different basic amino acids in 109 position. In order to investigate the role of these factors, the newly synthesized peptides were tested for anticoagulant activity. To investigate the change in anticoagulant activity, new peptides were synthesized by replacement of the C-terminal COOH function with CONH2. The biological activity of all compounds was measured in respect to APTT (activated partial thromboplastin time) and IC50 values (the concentrations for doubling APTT clotting times of human plasma) were determined.

Synthesis and structure-activity relationship of new analogues of antistasin.

Intensive investigation connected with the development of new anticoagulant agents for the treatment of cardiovascular diseases was carried out. Direct and specific inhibition of thrombin and Factor Xa-like serine proteases in the coagulation cascade has been the focus of many efforts to design novel anticoagulants over the past decade. This work reports the synthesis and biological activity of new anticoagulant peptide analogues of natural isoforms 2 and 3 of antistasin. In addition they include different tripeptide sequences in their molecules, which are highly active inhibitors of different serine proteases such as plasmin, trypsin, thrombin and Factor Xa.

Antipeptide antibodies for detecting crab (Callinectes sapidus) molt-inhibiting hormone.

In crustaceans, the synthesis of ecdysteroid molting hormones is regulated by molt-inhibiting hormone (MIH), a neuropeptide produced by an eyestalk neuroendocrine system, the X-organ/sinus gland complex. Using sequence analysis software, two regions of the blue crab (Callinectes sapidus) MIH peptide were selected for antibody production. Two 14-mer peptides were commercially synthesized and used to generate polyclonal antisera. Western blot analysis revealed that each antiserum bound to proteins of the predicted size in extracts of C. sapidus sinus glands, and lysates of insect cells containing recombinant MIH. Thin section immunocytochemistry using either antiserum showed specific immunoreactivity in X-organ neurosecretory cell bodies, their associated axons and collaterals, and their axon terminals in the sinus gland.

The molt-inhibiting hormone in the American crayfish Procambarus clarkii: its chemical synthesis and biological activity.

The molt-inhibiting hormone of the American crayfish Procambarus clarkii (Prc-MIH), a 75-residue polypeptide containing three disulfide bridges, was synthesized by chemical ligation of two peptides, i.e., synthetic Prc-MIH(1-39) and Prc-MIH(40-75)-NH(2), and by subsequent folding to form the native disulfide-containing peptide molecule. The synthetic peptide was comparable to the natural Prc-MIH in inhibiting ecdysteroid secretion by in vitro bioassay and shared features with the natural Prc-MIH in some biochemical analyses. These results indicate that the chemical ligation method can be used for the synthesis of Prc-MIH. Furthermore, it was demonstrated that synthetic Prc-MIH has hyperglycemic activity, although the activity was weaker than that of the authentic crustacean hyperglycemic hormone in the American crayfish. To examine the structural requirement of the Prc-MIH for eliciting biological activity, an antibody raised against the C-terminal region (residues 55-75) and two synthetic peptides, i.e., a core region (residues 1-54) containing three disulfide bridges and the C-terminal region, were utilized. It is suggested that Prc-MIH exerts its activities through coordination between the core region and the C-terminal region.

N-acylation of Aplysia egg-laying hormone with biotin. Characterization of bioactive and inactive derivatives.

Chemical modification of the egg-laying hormone (ELH) of Aplysia by reaction with the N-hydroxysuccinimide ester of biotin, which contained 6-aminohexanoic acid as spacer, yielded seven distinct derivatives that were readily separated by reversed-phase high performance liquid chromatography. The derivatives were chemically characterized by amino acid compositional analysis, sequence analysis, and mass spectrometry. The seven derivatives resulted from combinations of differential modification of the three amino groups in the ELH molecule located at Ile1 (alpha-NH2), Lys8, and Lys36. Of the seven derivatives formed, only one, monobiotinyl Lys36-ELH, was biologically active in eliciting egg-laying activity and altering the electrophysiological activity of the abdominal ganglion neuron R15 and LB and LC cluster neurons. In addition, evaluation of the time course of biotinylation of ELH revealed that the relative rate of amino group reactivity was epsilon-NH2-Lys36 greater than epsilon-NH2-Lys8 much greater than alpha-NH2-Ile1. The slow rate of reaction of the terminal alpha-amino group suggested that it was relatively inaccessible to biotinylation, possibly due to conformational factors or to ion-pair formation with an unidentified carboxyl group. Loss of bioactivity of ELH monobiotinylated on the alpha-amino group, coupled with the unusually low reactivity of the alpha-amino group, provided strong evidence for the importance of the alpha-amino group in ELH function. Furthermore, the development and availability of a bioactive ELH probe should greatly facilitate the isolation, characterization, and localization of the ELH receptor.

Primary structure and relative potency of an analog of beta-PDH (pigment-dispersing hormone) from the crayfish Procambarus clarkii.

A pigment-dispersing hormone (PDH) from eyestalks of the crayfish Procambarus clarkii was purified by gel filtration, cation-exchange chromatography, partition chromatography, and reversed-phase HPLC. Based on automated sequencing and by the identical chromatographic behavior of the native PDH and the synthetic amidated form of the deduced sequence, the primary structure of Procambarus PDH has been established as: Asn-Ser-Glu-Leu-Ile-Asn-Ser-Ile-Leu-Gly-Leu-Pro-Lys-Val-Met-Asn-Glu-Ala- NH2. This peptide differs from beta-PDH of the fiddler crab Uca pugilator at a single position, Glu17 in place of Asp17. Because of this substitution, Procambarus PDH was 4 to 7-fold less potent than beta-PDH in causing pigment dispersion in the erythrophores, leucophores, and melanophores of Uca. In contrast, Procambarus PDH was 4-fold more potent than beta-PDH in eliciting pigment dispersion in the erythrophores of Procambarus. These peptides displayed less marked differences in potency in triggering leucophore pigment dispersion and light-adaptational distal eye pigment movement in Procambarus. These findings indicate that the structural requirements for PDH-receptor interactions vary with the species and with the target cell type within a given species.

Invertebrate neuropeptides resembling vasotocin and some analogues: synthesis and pharmacological properties.

The solid phase synthesis of three invertebrate vasopressin-oxytocin homologs: AVP-like factor, F1(1), ([Leu2, Thr4] AVT)2 isolated from subesophageal and thoracic ganglia of Locusta migratoria3, Arg-conopressin-S4. ([Ile2, Arg4] AVT), Lys-conopressin-G4 ([Phe2, Arg4] LVT), both isolated from the venom of fish-hunting marine snails of the genus Conus and six of their analogues is reported. These analogues are: [Arg4] AVT, [Ile2] AVT, [Leu2] AVT, [Phe2, Arg4] AVT, [Arg4] LVT and [Ile2, Arg4] LVT. All peptides were tested for antidiuretic and vasopressor activities.

Position 3 analogues of a crustacean pigment-dispersing hormone: synthesis and biological activity.

In an effort to explain the difference in potencies between the two characterized crustacean pigment-dispersing hormones (alpha-PDH; beta-PDH) and to define a role for residue 3 in these octadecapeptide hormones, we have synthesized and purified seven position 3 alpha-PDH analogues ([Ala3], [Ile3], [Asn3], [Gln3], [Asp3], [Glu3], and [Lys3]alpha-PDH). When tested for melanophore pigment-dispersing activity in destalked Uca, [Glu3]alpha-PDH was found to be 325% more potent than alpha-PDH. Reduced potencies were observed for the [Asp3] (58%), [Asn3] (26%), [Gln3] (11%), and [Ala3] (8%) derivatives. Much lower potencies were displayed by the [Lys3] and [Ile3] analogues (0.73% and 0.66%, respectively). These results suggest that the position 3 side chain carboxylate anion of [Glu3]alpha-PDH stabilizes the active receptor-bound conformer through a charge-charge interaction.

Effects of synthetic bag cell and atrial gland peptides on identified nerve cells in Aplysia.

We have examined the effects of peptides on the neuroendocrine bag cells, the R2 neuron and the left upper quadrant (LUQ) neurons of the abdominal ganglion of Aplysia californica. Peptides include those extracted from the atrial gland, a reproductive organ; those released by an afterdischarge of the bag cells; and 2 synthetic peptides: the amidated 9-amino acid C-terminal portion of atrial gland peptides A/B/ERH (B26-34), and the 8-amino acid alpha-bag cell peptide (alpha-BCP1-8). Peptides were applied by superfusion, arterial perfusion, pressure ejection from micropipettes, or by inducing a bag cell afterdischarge. Both alpha-BCP1-8 and B26-34 are able to produce a bag cell afterdischarge when applied to the abdominal ganglion but are not as effectively able to trigger the bag cells when applied selectively to the ganglia of the head ring. Peptides released by the bag cells inhibit R2 and LUQ neurons; whereas atrial gland extract mildly excites LUQ neurons and powerfully excites R2. The inhibitory effect of the LUQ cells and R2 following an afterdischarge of the bag cells is mimicked by alpha-BCP1-8. The excitatory effect of the atrial gland extract cannot be duplicated with B26-34. Rather, instead of having an excitatory effect on R2 and LUQ cells, B26-34 seems to mimick alpha-BCP1-8 and inhibit these neurons. Both peptides produce a membrane conductance increase in R2 and LUQ cells.

Synthesis of shrimp red pigment-concentrating hormone analogs and their biological activity in locusts.

Two analogs of the red pigment-concentrating hormone (RPCH) have been synthesized by the solid-phase method: [Thr6]-RPCH (I) and [Tyr4, Thr6]-RCPH (II). Analog I has the same amino acid composition as the second adipokinetic hormone (AKH-II) isolated from locust corpora cardiaca. Bioassay for lipid-mobilizing activity in adult male locusts gave the following increases in hemolymph lipid content: AKH-I, 3.5; I, 2.4; II, 2.9. The biological response shown by I lends support to the conclusion that its sequence is that of the presumptive AKH-II. Replacement of Phe in position 4 by Tyr does not reduce the adipokinetic response.

Crustacean color-change hormone: amino acid sequence and chemical synthesis.

The blanching hormone of the prawn, Pandalus borealis, is pGlu-Leu-Asn-Phe-Ser-Pro-Gly-Trp-NH(2). Its structure was settled by a combination of mass spectrometry and Edman-dansyl analysis of a thermolysin fragment. Confirmation of the structure was obtained by chemical synthesis from amino acids. This neurosecreted hormone is active in picogram amounts when tested in shrimps.