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Gonadotropic hormones play important regulatory roles in reproduction. Relaxin-like gonad-stimulating peptide (RGP) is a gonadotropin-like hormone in starfish. However, a receptor for RGP remains to be identified. Here, we describe the identification of an authentic receptor for RGP (RGPR) in the starfish, Patiria pectinifera. A binding assay using radioiodinated P. pectinifera RGP (PpeRGP) revealed that RGPR was expressed in ovarian follicle cells. A RGPR candidate was identified by homology-searching of transcriptome data of P. pectinifera follicle cells. Based on the contig sequences, a putative 947-amino acid PpeRGPR was cloned from follicle cells. Like the vertebrate relaxin family peptide receptors (RXFP 1 and 2), PpeRGPR was a G protein-coupled receptor that harbored a low-density lipoprotein-receptor class A motif and leucine-rich repeat sequences in the extracellular domain of the N-terminal region. Sf9 cells transfected with Gαq16-fused PpeRGPR activated calcium ion mobilization in response to PpeRGP, but not to RGP of another starfish Asterias amurensis, in a dose-dependent fashion. These results confirmed the species-specific reactivity of RGP and the cognate receptor. Thus, the present study provides evidence that PpeRGPR is a specific receptor for PpeRGP. To the best of our knowledge, this is the first report on the identification of a receptor for echinoderm RGP., Competing Interests: The authors have declared that they have no competing interests.

Decapod crustaceans are important animal models for neurobiologists due to their relatively simple nervous systems with well-defined neural circuits and extensive neuromodulation by a diverse set of signaling peptides. However, biochemical characterization of these endogenous neuropeptides is often challenging due to limited sequence information about these neuropeptide genes and the encoded preprohormones. By taking advantage of sequence homology in neuropeptides observed in related species using a home-built crustacean neuropeptide database, we developed a semi-automated sequencing strategy to characterize the neuropeptidome of Panulirus interruptus, an important aquaculture species, with few known neuropeptide preprohormone sequences. Our streamlined process searched the high mass accuracy and high-resolution data acquired on a LTQ-Orbitrap with a flexible algorithm in ProSight that allows for sequence discrepancy from reported sequences in our database, resulting in the detection of 32 neuropeptides, including 19 novel ones. We further improved the overall coverage to 51 neuropeptides with our multidimensional platform that employed multiple analytical techniques including dimethylation-assisted fragmentation, de novo sequencing using nanoliquid chromatography-electrospray ionization-quadrupole-time-of-flight (nanoLC-ESI-Q-TOF), direct tissue analysis, and mass spectrometry imaging on matrix-assisted laser desorption/ionization (MALDI)-TOF/TOF. The high discovery rate from this unsequenced model organism demonstrated the utility of our neuropeptide discovery pipeline and highlighted the advantage of utilizing multiple sequencing strategies. Collectively, our study expands the catalog of crustacean neuropeptides and more importantly presents an approach that can be adapted to exploring neuropeptidome from species that possess limited sequence information.

Anti-lipopolysaccharide factors (ALF) are a group of small basic proteins which are released into the hemolymph as a result of rapid degranulation of hemocytes in response to bacterial lipopolysaccharide (LPS). In the present study, using a combined approach of degenerate and RACE PCR, the gene coding for Scylla serrata anti-lipopolysaccharide factor (SsALF) was cloned and characterized. The full-length SsALF cDNA sequence consists of 607 bp and encodes a polypeptide of 97 amino acids, constituting a molecular mass of 11172 Da with an estimated pI of 10.01. The SsALF protein showed upto 92% similarity with ALF from Scylla paramamosain and about 33-53% amino acid sequence identity with other known ALF sequences. SsALF protein sequence demonstrated the presence of two highly conserved cysteine residues and putative LPS binding domain. An in vivo expression study showed that SsALF mRNA was expressed predominantly in hemocytes, heart and muscle of healthy mud crabs. The recombinant form of SsALF protein (rSsALF) was expressed with a Histag, in Escherichia coli, using the pTriEx-4 Ek/LIC vector. The purified rSsALF protein demonstrated antimicrobial activity against both Gram-positive and Gram-negative bacteria. The recombinant protein was able to significantly neutralize LPS-induced expression on SsALF in vivo as demonstrated by real-time PCR. rSsALF was able to permeabilize artificial phospholipid membranes as demonstrated by calcein enclosed liposome model. These studies strongly suggest that SsALF is one among the important antimicrobial factors produced in the crab during a microbial invasion.

Gonad-stimulating substance-like molecule (GSSL) was isolated from the radial nerve of the sea cucumber, Apostichopus japonicus (Aj-GSSL), and its partial DNA and protein sequences were characterized. The smaller part of the molecule that also retains GSSL activity was estimated. Radial nerve extract (RNE) induced germinal vesicle breakdown (GVBD) at 3 mg/ml in 85% of immature ovarian oocytes. Similar intensity of GSSL activity to RNE was seen in a fraction that contained peptides between 3 kDa and 10 kDa (3-10 kDa-fraction) separated by ultrafiltlation membrane. MALDI-TOF MS analysis and silver-stained 18% SDS-PAGE slab gels identified a major peptide at around 4.6 kDa in a 3-10 kDa-fraction, and that was subjected to internal protein sequencing. The resulting 12-amino acid sequence was not found in the BLAST database to date. Immunohistochemistry using antiserum raised against the 12-amino acid peptide located the peptide to granular cells in the hyponeural part of the radial nerve and in the epineural sinus beneath the radial nerve. Sequence data was obtained using degenerate primers designed from the 12-amino acid sequence and 5 and 3 RACEs. These resulted in a 148 bp cDNA that coded a 43-amino acid sequence of H2N-VLSKQAHHHHHEGWSLPGVPAEIDDLAGNIDYNIFKEQREKIK-COOH. The synthetic 43-amino acid Aj-GSSL generated from this sequence induced GVBD in 50% of immature ovarian oocytes at 6 microM. An N-terminal 21-amino acid peptide of the synthetic partial Aj-GSSL (Aj-GSSL-P1) induced GVBD to 80% of immature ovarian oocytes at 12 microM. This indicated that Aj-GSSL-P1 is of sufficient length for GSSL activity.

Bursicon was identified in 1965 as a peptide neurohormone that initiates the tanning of the insect cuticle immediately after the shedding of the old one during the final stages of the molting process. Its molecular identity as an approximately 30 kDa bioactive heterodimer consisting of two cystine knot proteins resisted elucidation for 43 years. The sequence of the two bursicon subunits is highly conserved among arthropods, and this conservation extends even to echinoderms. We review the efforts leading to bursicon's characterization, the identification of its leucine-rich repeat-containing, G protein-coupled receptor (LGR2), and the progress towards revealing its various functions. It is now clear that bursicon regulates different aspects of wing inflation in Drosophila melanogaster besides being involved at various points in the cuticle tanning process in different insects. We also describe the current knowledge of the expression of bursicon in the central nervous system of different insects in large homologous neurosecretory cells, and the changes in its expression during the development of Manduca sexta and D. melanogaster. Although much remains to be learned, the elucidation of its molecular identity and that of its receptor has provided the breakthrough needed for investigating the diverse actions of this critical insect neurohormone.

The crustacean hyperglycemic hormone is the most abundant neuropeptide present in the eyestalk of Crustacea and its main role is to control the glucose level in the hemolymph. Our study was aimed at assessing the importance of C-terminal amidation for its biological activity. Two recombinant peptides were produced, Asl-rcHH-Gly with a free carboxyl terminus and Asl-rcHH-amide with an amidated C-terminus. Homologous bioassays performed on the astacid crayfish Astacus leptodactylus showed that the amidated peptide had a stronger hyperglycemic effect compared to the non-amidated peptide. To assess the relevance of amidation also in other decapods and how much the differences in the cHH amino acid sequence can affect the functionality of the peptides, we carried out heterologous bioassays on the cambarid Procambarus clarkii and palaemonid Palaemon elegans. The Asl-rcHH-amide elicited a good response in P. clarkii and in P. elegans. The injection of Asl-rcHH-Gly evoked a weak response in both species. These results prove the importance of C-terminal amidation for the biological activity of cHH in crayfish as well as the role of the peptide primary sequence for the species-specificity hormone-receptor recognition.

To characterize a receptor for molt-inhibiting hormone (MIH) in the kuruma prawn, Marsupenaeus japonicus, we performed a binding assay and chemical cross-linking experiments using radiolabeled recombinant MIH ([(125)I]rMIH). The specific binding of [(125)I]rMIH was found in the membrane fraction of the Y-organ (K(d) = 4.76 x 10(-10) M and B(max) = 5.51 x 10(-12) M). Chemical cross-linking between [(125)I]rMIH and the membrane fraction of the Y-organ revealed that the MIH receptor protein has a molecular size of approximately 70 kDa.

Recently, it was demonstrated by PCR amplification that an additional molt-inhibiting hormone (MIH)-like peptide was present in the kuruma prawn Marsupenaeus japonicus. In this study, a cDNA encoding this peptide designated Pej-MIH-B was cloned. The Pej-MIH-B gene was expressed strongly in the nerve cord, and weakly in the eyestalk. It was possible to isolate Pej-MIH-B from the sinus glands in the eyestalks. The recombinant Pej-MIH-B expressed in Escherichia coli showed low molt-inhibiting activity, but did not exhibit hyperglycemic activity. These results suggest that Pej-MIH-B does not function as MIH or CHH intrinsically, but may have some unknown functions.

Crustacean hyperglycemic hormone (CHH), molt-inhibiting hormone (MIH), and gonad-inhibiting hormone (GIH) are members of a major peptide family produced from the X-organ sinus gland complex in the eyestalk of crustaceans. This peptide family plays important roles in controlling several physiologic processes such as regulation of growth and reproduction. In this study the complementary DNA encoding a peptide related to the CHH/MIH/GIH family (so-called Pem-CMG) of the black tiger prawn Penaeus monodon was successfully expressed in the yeast Pichia pastoris under the control of the AOX1 promoter. The recombinant Pem-CMG was secreted into the culture medium using the alpha-factor signal sequence; of Saccharomyces cerevisiae without the Glu-Ala-Glu-Ala spacer peptide. The amino terminus of the recombinant Pem-CMG was correctly processed as evidenced by amino-terminal peptide sequencing. The recombinant Pem-CMG was purified by reverse-phase high-performance liquid chromotography and used in a biological assay for CHH activity. The final yield of the recombinant Pem-CMG after purification was 260 micro g/L of the culture medium. Both crude and purified recombinant Pem-CMG produced from P. pastoris showed the ability to elevate the glucose level in the hemolymph of eyestalk-ablated P. monodon, which demonstrates that Pem-CMG peptide functions as hyperglycemic hormone in P. monodon.

Production of recombinant Limulus endotoxin neutralizing protein (rENP) was attained with the GS115 methylotrophic strain of Pichia pastoris transformed with a plasmid, bearing multiple ENP gene copies. The synthetic gene for Limulus ENP was cloned into the integrative plasmid pAO815 under the control of a methanol-inducible promoter. Clones containing a single enp insert were used to construct cassettes bearing 2 and 3 tandem copies of enp. These were then integrated at the HIS locus of P. pastoris GS115 (his4). Clones were chosen for their ability to produce rENP upon methanol induction in shaker flasks, and then the 1x, 2x, and 3x-enp strains were analyzed by Southern blot for the presence of the ENP gene(s). Isolate 3 x 5q, containing a 3x-enp cassette, was the best producer of rENP. Under optimal conditions this strain grown in a fed-batch mode produced yields of >500 mg rENP/L with an average of 5.46 mg rENP/g DCW. Purification of rENP from the clarified broth resulted in a yield of 35% and a purity of >86%. Glycosylated rENP, the main contaminant, was removed with a concanavalin-A column and characterized. The pure rENP neutralized lipopolysaccharide and had the mass, amino-acid composition and N-terminal sequence expected from the cloned gene.

Bursicon is the final neurohormone released at the end of the molting cycle. It triggers the sclerotization (tanning) of the insect cuticle. Until now, its existence has been verified only by bioassays. In an attempt to identify this important neurohormone, bursicon was purified from homogenates of 2,850 nerve cords of the cockroach Periplaneta americana by using high performance liquid chromatography technology and two-dimensional gel electrophoresis. Bursicon bioactivity was found in four distinct protein spots at approximately 30 kDa between pH 5.3 and 5.9. The protein of one of these spots at pH 5.7 was subsequently microsequenced, and five partial amino acid sequences were retrieved. Evidence is presented that two of these sequences are derived from bursicon. Antibodies raised against the two sequences labeled bursicon-containing neurons in the central nervous systems of P. americana. One of these antisera labeled bursicon-containing neurons in the crickets Teleogryllus commodus and Gryllus bimaculatus, and the moth Manduca sexta. A cluster of four bilaterally paired neurons in the brain of Drososphila melanogaster was also labeled. In addition, this antiserum detected three spots corresponding to bursicon in Western blots of two-dimensional gels. The 12-amino acid sequence detected by this antiserum, thus, seems to be conserved even among species that are distantly related., (Copyright 2002 Wiley-Liss, Inc.)

To identify neuropeptides that have a broad spectrum of actions on the feeding system of Aplysia, we searched for bioactive peptides that are present in both the gut and the CNS. We identified a family of structurally related nonapeptides and decapeptides (enterins) that are present in the gut and CNS of Aplysia, and most of which share the HSFVamide sequence at the C terminus. The structure of the enterin precursor deduced from cDNA cloning predicts 35 copies of 20 different enterins. Northern analysis, in situ hybridization, and immunocytochemistry show that the enterins are abundantly present in the CNS and the gut of Aplysia. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry we characterized the enterin-precursor processing, demonstrated that all of the precursor-predicted enterins are present, and determined post-translational modifications of various enterins. Enterin-positive neuronal somata and processes were found in the gut, and enterins inhibited contractions of the gut. In the CNS, the cerebral and buccal ganglia, which control feeding, contained the enterins. Enterin was also present in the nerve that connects these two ganglia. Enterins reduced the firing of interneurons B4/5 during feeding motor programs. Such enterin-induced reduction of firing also occurred when excitability of B4/5 was tested directly. Because reduction of B4/5 activity corresponds to a switch from egestive to ingestive behaviors, enterin may contribute to such program switching. Furthermore, because enterins are present throughout the nervous system, they may also play a regulatory role in nonfeeding behaviors of Aplysia.

The medicinal leech Hirudo medicinalis produces various types of proteinase inhibitors: bdellins (inhibitors of trypsin, plasmin, and acrosin), hirustasin (inhibitor of tissue kallikrein, trypsin, alpha-chymotrypsin, and granulocyte cathepsin G), tryptase inhibitor, eglins (inhibitors of alpha-chymotrypsin, subtilisin, and chymasin and the granulocyte proteinases elastase and cathepsin G), inhibitor of factor Xa, hirudin (thrombin inhibitor), inhibitor of carboxypeptidase, and inhibitor of complement component C1s. This review summarizes data on their primary and tertiary structures, action mechanisms, and biological activities.

About 24 intrinsic neurosecretory neurons within the pericardial organs (POs) of the crab Carcinus maenas produce a novel crustacean hyperglycaemic hormone (CHH)-like peptide (PO-CHH) and two CHH-precursor-related peptides (PO-CPRP I and II) as identified immunochemically and by peptide chemistry. Edman sequencing and MS revealed PO-CHH as a 73 amino acid peptide (8630 Da) with a free C-terminus. PO-CHH and sinus gland CHH (SG-CHH) share an identical N-terminal sequence, positions 1-40, but the remaining sequence, positions 41-73 or 41-72, differs considerably. PO-CHH may have different precursors, as cDNA cloning of PO-derived mRNAs has revealed several similar forms, one exactly encoding the peptide. All PO-CHH cDNAs contain a nucleotide stretch coding for the SG-CHH(41-76) sequence in the 3'-untranslated region (UTR). Cloning of crab testis genomic DNA revealed at least four CHH genes, the structure of which suggest that PO-CHH and SG-CHH arise by alternative splicing of precursors and possibly post-transcriptional modification of PO-CHH. The genes encode four exons, separated by three variable introns, encoding part of a signal peptide (exon I), the remaining signal peptide residues, a CPRP, the PO-CHH(1-40)/SG-CHH(1-40) sequences (exon II), the remaining PO-CHH residues (exon III) and the remaining SG-CHH residues and a 3'-UTR (exon IV). Precursor and gene structures are more closely related to those encoding related insect ion-transport peptides than to penaeid shrimp CHH genes. PO-CHH neither exhibits hyperglycaemic activity in vivo, nor does it inhibit Y-organ ecdysteroid synthesis in vitro. From the morphology of the neurons it seems likely that novel functions remain to be discovered.

The biliverdin-binding protein from the larval hemolymph of the swallowtail butterfly, Papilio xuthus L., was purified and characterized. The crude biliverdin-binding protein, obtained by ammonium sulfate fractionation, was purified in two steps, the first one by gel filtration chromatography and the second one by ion-exchange chromatography. The molecular mass of the purified protein was analyzed by SDS-polyacrylamide gel electrophoresis and estimated to be 21 kDa. The Namino terminal sequence of P. xuthus biliverdin-binding protein analyzed up to the 19th residue showed that 42% of the amino acid sequence are sequence similarity to the bilin-binding protein from Pieris brassicae. These results suggest that the P. xuthus biliverdin-binding protein belongs to the insecticyanin-type.

We have isolated a peptide from extracts of sinus glands from a South African spiny lobster species, Jasus lalandii, by high-performance liquid chromatography (HPLC) and identified it as a putative molt-inhibiting hormone (MIH) by (i) an in vitro assay with J. lalandii Y-organs to measure the inhibition of ecdysteroid synthesis and (ii) an immunoassay using antiserum raised against MIH of the edible crab. The MIH of J. lalandii has 74 amino acid residues, a molecular mass of 9006 Da, a free N-terminus and an amidated C-terminus. The full primary sequence has been obtained from sequencing various digest fragments (tryptic, endoproteinase Asp-N, cyanogen bromide) of the unreduced (native) peptide: RFTFDCPGMMGQRYLYEQVEQVCDDCYNLYREEKIAVNCRENCFLNSWFTVCLQATMREHETPRFDIWR SIILKA-NH(2). Structural comparisons with other peptides show that the J. lalandii MIH belongs to the peptide family which includes the crustacean hyperglycemic hormone, molt-inhibiting hormone and vitellogenesis-inhibiting hormone (cHH/MIH/VIH). This novel peptide has 36-43% sequence identity to putative MIHs from other decapod crustaceans and 32-34% identity to the two cHH peptides previously identified in this spiny lobster species. This is the first report of a peptide with MIH activity in the Palinuridae infraorder.

The Y-organs of the xanthid crab Menippe mercenaria secrete the ecdysteroids, 3-dehydroecdysone (3DE) and lesser amounts of 3-dehydro (or 2-dehydro)-25-deoxyecdysone (3D25dE) in vitro. These ecdysteroids were identified by elution-time comparisons with authentic standards, mass spectrography, and, for 3D25dE, infrared spectrometry. Tissues were incubated 18 hr with [(3)H]3DE. Activities representing 3beta-reductase and 20-hydroxylase generally were present, evidenced by finding in the tissue/medium extract labeled ecdysone (E) and 20-hydroxyecdysone (20E). Labeled 3-dehydro-20-hydroxyecdysone (3D20E) also appeared to be present. Tissue blanks and hemolymph were devoid of activity. Muscle was low, hypodermis was intermediate, and hindgut and gonads were high in activity of the enzymes. Consistent with the presence of these enzymes in peripheral tissues, ecdysteroid products identified in the hemolymph were 20E, 3D20E, and 25-deoxy-20-hydroxyecdysone (25d20E; ponasterone A). Structures of 20E and 3D20E were confirmed by co-elution with authentic standards in high-performance liquid chromatography (HPLC), co-elution of derivatives in gas chromatography, and mass spectroscopy. Ponasterone A (identified by HPLC co-elution with the standard), like 20E is present in the hemolymph in prominent amounts. These data indicate that Menippe, among crustaceans thus far studied, secretes a unique combination of ecdysteroid hormones, namely, a 3- (or 2-) oxo compound and a 25-deoxy compound. This represents a different kind of branch point from 5beta-diketol in ecdysteroid biosynthesis, in which the intermediate, 5beta-ketodiol is bypassed. A result is the joint appearance in the circulation of the hormones, 20E and ponasterone A, which in other species are singly prominent.

The crustacean eyestalk CHH/MIH/GIH neurohormone gene family represents a unique group of neuropeptides identified mainly in crustaceans. In this study, we report the cloning and characterization of the cDNA and the gene encoding the hyperglycemic hormone (MeCHH-B) of the shrimp Metapenaeus ensis. The amino acid sequence of MeCHH-B shows 85% identity to that of MeCHH-A (formerly MeCHH-like neuropeptide). Two separate but identical MeCHH-B genes were identified in the genome of shrimp by library screening and they are located on different CHH gene clusters. The organization of the MeCHH-B gene is identical to other members of the CHH/MIH/GIH neurohormone family. MeCHH-B is expressed at a constant level in the eyestalks of juveniles and mature females. Unlike the MeCHH-A gene, a low level of MeCHH-B transcripts can also be detected in the central nervous system. Interestingly, the expression pattern of MeCHH-B in the eyestalk of vitellogenic females is reversed to that of the MeCHH-A gene. At the middle stage of gonad maturation, a minimum level of MeCHH-B transcript was recorded and a maximum level of MeCHH-A transcript was detected. Recombinant proteins for MeCHH-A and MeCHH-B were produced by a bacterial expression system. The hemolymph glucose level of bilaterally eyestalk-ablated shrimp increased two-fold 1 h after the rCHH injection and then returned to normal after 2 h. The hyperglycemic effect of these fusion proteins is comparable to that of de-stalked shrimp injected with crude extract from a single sinus gland.

Peptides belonging to the CHH/MIH/GIH-family of crustacean hormones were isolated from acetic acid extracts of sinus glands isolated from eyestalks of the shrimp, Penaeus vannamei. The peptides were isolated by chromatography and molecular weights determined by MALDI mass spectrometry. Peptides in the range of 7-9 kDa and containing three disulfide bridges were selected for amino acid sequence analysis. Three peptides with the requisite properties were present in sufficient amounts for sequence analysis. Two peptides had unique sequences similar to CHH/MIH/GIH peptides from other crustaceans. A third peptide seemed to be a truncated form of one of the previous sequences.

From a crude extract of the sinus glands of the shrimp Penaeus (litopenaeus) schmitti a peptide with hyperglycemic activity in a homologous bioassay was isolated and characterized by a combination of automatic Edman degradation, enzymatic digestions, TLC of dansyl-amino acids, and mass spectrometry. Its M(r) is 8359.4 Da by MS, which coincides with the deduced sequence. Its N-terminus is free and its C-terminus is amidated. It has 6 Cys residues in conserved positions compared with other known CHHs. This is the first sinus gland hormone from an Atlantic Ocean shrimp characterized to date. It has a remarkable 90% sequence similarity to the Indo-Pacific shrimp P. (marsupenaeus) japonicus Pej-VII hyperglycemic hormone.

In an effort to characterize the insect molting hormone bursicon from the cockroach, Periplaneta americana, amino acid sequences with high identity of Cu,Zn-superoxide dismutase (SOD) of Drosophila virilis were identified. Antisera against a conserved region of SOD, and a sequence unique to Periplaneta SOD were produced and used to test whether bursicon might be a form of SOD. Western blots of one- and two-dimensional gels revealed that the dimeric form of SOD and bursicon have a similar molecular mass (30 kDa). The two proteins can be separated, however, according to their different isoelectric points. Bursicon is identified in two-dimensional gels by elution from four unique spots not labeled by the anti-SOD antisera. In sections of Periplaneta nerve cords the antisera labeled glial material surrounding neuronal somata close to the neural sheath. Bursicon, however, is contained in unique cell pairs in the ganglia of the ventral nerve cord. These neurons were labeled with new antisera produced against novel sequences of one of the four above-mentioned bursicon active spots. The results show unequivocally that SOD and bursicon are distinctly different proteins. Furthermore, the anti-SOD antisera provided a tool to isolate and sequence bursicon.

Isolation of the crustacean hyperglycemic hormone (CHH) from the eyestalk of the giant freshwater prawn Macrobrachium rosenbergii was performed using 5,000 ground eye-stalks extracted in methanol-acetic acid-water (90:1:9). After the extract was partially purified using C18 cartridges, it was further purified by eight steps of RP-HPLC using four kinds of columns: C18, C8, cyano and phenyl, and three solvent systems: acetonitrile (ACN)/trifluoroacetic acid, ACN/heptafluoroacetic acid and ACN/triethylammonium acetate. The bioassay of CHH during purification was done by injection of eluate fractions into eyestalk-ablated prawns and determination of the ability of the fractions to elevate glucose in the haemolymph. A complete amino acid sequence analysis was performed on one isoform of CHH (Mar-CHH-1), consisting of 71 residues. The sequence of Mar-CHH-1 shows considerable similarity (45-68%) to CHHs reported in other crustaceans. It is expected that there might be more than one isoform of CHH in M. rosenbergii.

Three chromatophorotropic neuropeptide hormones were purified from an aqueous extract of the sinus glands of the kuruma prawn Penaeus japonicus by two steps of reverse-phase HPLC and their amino acid sequences determined. One of them was found to show pigment concentrating activity and to have an amino acid sequence identical with that of the known red pigment concentrating hormone (RPCH), and therefore it was named Pej-RPCH. The other two peptides showed pigment dispersing hormone (PDH) activity and were named Pej-PDH-I and -II. They both consisted of 18 amino acid residues with a free amino-terminus and an amidated carboxyl-terminus, the sequences of Pej-PDH-I and -II being NSELINSLLGIPKVMTDAamide and NSELINSLLGLPKFMIDAamide, respectively. Three amino acid residues at positions 11, 14, and 16 differed between the two PDHs. Pej-PDH-II was about 5-, 7-, and 10-fold more potent than Pej-PDH-I for erythrophores, xanthophores, and melanophores, respectively. The major reason for the difference in potency between the two PDHs was attributed to differences in residues at position 16. In addition, they were found to be produced by a single individual. The order of sensitivity of the four types of chromatophores to Pej-RPCH and both PDHs was found to be erythrophores = xanthophores > melanophores > leukophores., (Copyright 1999 Academic Press.)

We have isolated a peptide from extracts of sinus glands of Jasus lalandii, a South African spiny lobster, by high-performance liquid chromatography (HPLC) and identified it as crustacean hyperglycaemic hormone (cHH) by (i) a conspecific bioassay measuring glucose elevation in the haemolymph and (ii) an immunoassay using an antiserum raised against cHH of the American lobster. The J. lalandii peptide has a free N-terminus as evidenced by sequencing the first 30 amino acid residues of the intact peptide. Further primary structural data were obtained from sequencing HPLC-purified tryptic and Asp-N proteolytic digests and by cyanogen bromide cleavage of the native, unreduced peptide. In this way, less than 400 sinus glands were used to provide the full sequence. Mass spectrometric analysis in conjunction with inferences based on interspecies sequence homology of cHH molecules unequivocally assigned the complete primary structure of cHH in a member of the crustacean infraorder Palinura for the first time. Our results show 51-76% homology with cHHs known from other decapod infraorders, the major difference being a free N-terminus and several amino acid substitutions interspersed in the non-conserved regions of the molecule. The J. lalandii cHH sequence presented here differs from a partial cHH sequence previously reported from allegedly the same species.

A serine-protease inhibitor of plasma kallikrein was screened and purified from a native Korean leech species, Hirudo nipponia. The peptide, named piguamerin, potently inhibited plasma and tissue kallikreins, and trypsin. Sequence analyses by automated Edman degradation revealed 48 amino acid residues and a molecular mass for the peptide of 5090 Da. Piguamerin is similar to antistasin-type inhibitors with the same spacing of ten cysteine residues, but shows differences from hirustasin, antistasin and ghilanten at the residues surrounding Arg27, which is a common P1 reactive residue for these inhibitors. The purified inhibitor modulated plasma clotting in tests of activated partial thromboplastin time at nanomolar concentrations. The serine-protease inhibitor of this leech may be involved in leech hematophagia.

New data on proteolytic enzyme inhibitors and mechanisms of their interaction with the enzymes are reviewed. In recent years, a number of new inhibitors comprising families earlier unknown have been described such as proteins from the parasitic nematode Ascaris lumbricoides, ecotin from the periplasm of Escherichia coli, proteins PMP-C and PMP-D from locust Locusta migratoria, and hirustasin from the medicinal leech Hirudo medicinalis. At the same time, some proteins that may be assigned to inhibitors on the basis of their structures were found to perform other (not inhibitory) functions. Thus, the family of the Kunitz soybean trypsin inhibitor includes plant storage proteins and proteins whose synthesis is induced by stress factors. Numerous inhibitors interacting with the enzymes by mechanisms other than the substrate-like ones were identified, such as ornithodorin and anticoagulant peptide from tick Ornithodoros moubata (inhibitors of the blood clotting system proteases), an inhibitor from snake (Bothrops jararaca) venom, and ecotin, an inhibitor of serine proteases with an unusually broad specificity range. Special emphasis is placed on enzyme inhibition with propeptides and the mechanism of this process.

Two different biliverdin-binding proteins, designated BBP-I and BBP-II, were purified from the larval hemolymph of the Eri-silkworm, Samia cynthia ricini. These proteins were readily isolated from the hemolymph of fifth instar larvae using two chromatographic steps, hydrophobic interaction chromatography and ion exchange chromatography. Both BBPs were easily separated by Q-Sepharose HP column chromatography. BBP-I has an apparent molecular weight of 24 kDa, as determined by gel-filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Native BBP-II had a molecular weight of 48 kDa estimated by gel-filtration. SDS-PAGE revealed a single band with a molecular weight of 26 kDa. Moreover, the molecular weights of BBP-I and BBP-II were determined to be 20,468 and 22,708 by MALDI-TOF/MS (matrix-assisted laser desorption ionization-time of flight/mass spectrometry), respectively. On this basis, BBP-I and BBP-II molecules are assumed to be a monomer and a dimer, respectively. The blue color of BBPs collected from the hemolymph is attributed to the presence of biliverdin IX gamma, which is non-covalently and stoichiometrically bound to the apoprotein, based on absorbance maxima at 359 and 695 nm in methanol:HCl (95:5, v/v). One molecule of BBP-I contains one molecule of biliverdin IX gamma, whereas BBP-II contains two molecules of biliverdin IX gamma. The amino acid compositions of BBP-I and BBP-II are different, although the N-terminal sequences of both BBPs have a 48% identity. These BBPs were found in the hemolymph of fourth and fifth instar larvae. The newly molted fifth instar larvae had the highest concentration of BBP-I in the hemolymph. This gradually decreased during larval development. In contrast to BBP-I, the level of BBP-II was low, and increased slightly at the same developmental stage in S. cynthia ricini larvae.

Mass spectrometry (MS) was employed to detect and structurally characterize peptides in two functionally related neurons, named VD1 and RPD2, which form a network involved in the modulation of heartbeat in Lymnaea. Matrix-assisted laser desorption/ionization MS, directly applied to single neurons VD1 and RPD2, showed overlapping yet distinct mass profiles, with a subset of putative peptides specifically present in neuron VD1. Direct tandem MS of a single VD1 neuron revealed the primary structures of the VD1-specific peptides, which were identified as members of the family of small cardioactive peptides. Based on the tandem MS data, a degenerate oligonucleotide was made for use in a polymerase chain reaction strategy to isolate the cDNA encoding the precursor to the small cardioactive peptides from a brain-specific cDNA library. The calculated masses of the mature, posttranslationally modified peptides, as predicted from the corresponding cDNA, agreed with the measured masses of the actual peptides, as detected in single-cell MS analysis. In situ hybridization studies showed that the transcript encoding the precursor is present in VD1, but not in RPD2, thus corroborating the single-cell MS analysis. Finally, the small cardioactive peptides were shown to enhance the contractions of the auricle in vitro.

Blue biliprotein, insecticyanin (INS), has been purified from the hemolymph of the sweet potato hornworm, Agrius convolvuli. The protein was efficiently isolated from the hemolymph of fifth instar larvae using three successive column chromatographic techniques: hydrophobic interaction chromatography, ion exchange chromatography and gel-filtration. The purified INS showed a native molecular weight of approximately 59,000 by gel-filtration. SDS-PAGE revealed a single band with Mr of approximately 26,000. Moreover, the molecular mass of INS was 21,213 by MALDI-TOF/MS. Thus, the native A. convolvuli INS molecule was assumed to be a trimer in solution. The blue coloration of A. convolvuli INS from the hemolymph was attributed to the presence of biliverdin IX gamma, due to the absorbance maxima at 360 and 695 nm, which was non-covalently bound with the apoprotein. Amino acid composition and N-terminal sequence of A. convolvuli INS is similar to M. sexta INS. A. convolvuli INS represents one of the biliverdin-binding proteins in lepidopteran insects.

A molt-inhibiting hormone (Prc-MIH) was isolated from the sinus glands of the American crayfish, Procambarus clarkii, and its amino acid sequence was determined. It comprised 75 amino acid residues and had an amidated carboxyl terminus. The amino acid sequence was much more similar to MIH of the shore crab (Cam-MIH) than to MIH of the American lobster (Hoa-MIH).

Putative moult-inhibiting hormone (MIH) from sinus glands of the edible crab Cancer pagurus was characterized by high-performance liquid chromatography, followed by fractional bioassay (inhibition of ecdysteroid synthesis by Y-organs) and immunoassay (using antisera raised against Carcinus MIH). This peptide was fully sequenced by automated Edman degradation of endoproteinase-derived fragments. C. pagurus MIH is a 78 residue peptide (M(r) 9194), with free N- and C-termini and three intrachain disulphide bridges. Comparison with previously published MIH sequences confirms a high degree of sequence identity (c. 80%), supporting the view that brachyurans (crabs), possess distinct, structurally similar MIH neuropeptides.

A cysteine-rich serine protease inhibitor (Guamerin II) was isolated from the non-blood sucking leech Whitmania edentula. The new inhibitor was identified as a low molecular weight (6,012 Da) polypeptide with some sequence similarities to antistasin, hirustasin and guamerin. The inhibitor contained 56 amino acid residues with 76.8% sequence similarity to guamerin, 48.2% to hirustasin and 28.6% to the first domain of antistasin. This new inhibitor was the first completely sequenced serine protease inhibitor from a non-blood sucking leech. Analysis of the inhibitor revealed that it was active against neutrophil elastase and chymotrypsin, but had no activity against a variety of other proteases. The P1 reactive site residue was identified as methionine and the residues surrounding the P1 site were hydrophobic amino acids. The primary structure of the inhibitor showed no similarity to well-known elastase inhibitors from leeches such as eglin.

We are investigating peptidergic neurotransmission between ganglia in the Aplysia CNS. Neurons in the cerebral ganglia of Aplysia synthesize [35S]methionine-labeled peptides, which are transported to other central ganglia by fast axonal transport. We report the characterization of one of these peptides, termed cerebral peptide 1 (CP1). Transported [35S]methionine-labeled CP1 was used as a probe for the purification of CP1 from the pooled extracts of 1000 cerebral ganglia using three sequential modes of RP-HPLC. Amino acid sequence analysis by automated Edman degradation yielded the following sequence: Phe-Ser-Gly-Leu-Met-Ser-Glu-Gly-Ser-Ser-Leu-Glu-Ala. This sequence was consistent with data from amino acid composition analysis and FAB-MS of the purified peptide. In addition, a synthetic peptide with the proposed sequence coeluted with [35S]methionine-labeled native CP1 using the three RP-HPLC conditions used in the purification. Antisera raised against synthetic CP1 stained about 90 neuronal cell bodies in the cerebral ganglion including those of the asymmetric H cluster. Extracts of the H cluster dissected from ganglia incubated in [35S]methionine synthesized a radiolabeled peptide that coeluted with synthetic CP1, indicating that these neurons indeed synthesize authentic CP1. The other major ganglia each contained only a few CP1-immunoreactive neurons. The neuropil of each ganglion contains both smooth and varicose-immunoreactive fibers. Thus, CP1 is broadly distributed within the Aplysia CNS and may be a new peptide transmitter.

Ghilantens are a family of cysteine-rich inhibitors of the clotting enzyme, factor Xa, that are produced in the salivary glands of the South American leech Haementeria ghilianii. In this study, a gene, designed from the amino acid sequence of a specific ghilanten isoform, was assembled from eight double-stranded oligonucleotide fragments. A yeast expression plasmid, pPIC9HG-2, was constructed by making an inframe fusion of the ghilanten-coding sequences with the region encoding the pre-pro alpha-mating factor signal sequence for secretion. The expression of ghilanten in pPIC9HG-2 was under the control of the methanol-inducible, alcohol oxidase (AOX1) promoter. Pichia pastoris yeast strains KM 71 and SMD 1168 were transformed with linearized pPIC9HG-2 to target integration of the plasmid to the chromosomal 5'-AOX1 locus via homologous recombination. Both strains yielded His+ transformants that secreted a potent anticoagulant activity into the medium. Product yield was improved by using buffered media (pH 6.0) supplemented with either casamino acids or a mixture of yeast extract and peptone. The protease-deficient strain, SMD 1168, secreted about a twofold higher level of r-ghilanten than KM 71. Significant clonal variation in the expression of r-ghilanten was found among the His+ transformants. A high producing clone was selected for production at the 2-liter shake flask and 10-liter bioreactor scales. r-Ghilanten was recovered from the fermentation broths in a single step by heparin Sepharose affinity chromatography. Protein sequence analysis of the amino terminus showed that the correct processing to yield mature ghilanten varied with the fermentation conditions.

Annetocin, an oxytocin-related peptide recently isolated from the lumbricid earthworm Eisenia foetida, and putative transmitter substances were examined for their effects on rhythmic, spontaneous contractions of isolated gut preparations of the earthworm. Significant, dose-dependent effects of the following substances were observed: acetylcholine (ACh), gamma-aminobutyric acid (GABA), and dopamine were excitatory, while serotonin (5-HT) and octopamine were inhibitory. Annetocin, oxytocin, and vasotocin stimulated spontaneous contraction of the earthworm gut, annetocin being approximately 10-fold more potent than oxytocin or vasotocin. However, arginine-vasopressin (Arg-vasopressin), lysine-vasopressin (Lys-vasopressin), tocinoic acid (N-terminal hexapeptide fragment of oxytocin), and MSH release-inhibiting factor (MIF; C-terminal tripeptide fragment of oxytocin) did not show any effect on the earthworm gut motility. On the other hand, oxytocin, vasotocin, Arg-vasopressin, Lys-vasopressin, and tocinoic acid caused spontaneous contractions of isolated rat uterine preparations, where the potency was in this order, while annetocin and MIF exerted no oxytocic activity on the uterus. Dose-response relationship of the effects of annetocin and its related peptides on the annelid and mammalian systems shows that amino acid residue at the third position of these peptides is important for exertion of excitatory action on the smooth muscle systems. The results in the present study suggest that receptors for annetocin and for GABA on the earthworm gut, unlike those for ACh, desensitize during continuous exposure to these substances.

A new human leukocyte elastase inhibitor was extracted and purified from a Korean native leech Hirudo nipponia. The inhibitor, called guamerin, has a molecular weight of 6,110 and shows inhibition constant (Ki) of 8.1 x 10(-14) M. It is stable at a wide range of pH from 1 to 11 and heat-stable up to 90 degrees C. The complete amino acid sequence of guamerin reveals a cysteine-rich polypeptide of 57 amino acid residues that shows no similarity to any known elastase inhibitors but has 51% sequence homology with hirustasin. Guamerin has identical spacing of 10 cysteine residues as antistasin-type serine proteinase inhibitors, but the P1 reactive site residue is Met36 instead of Arg. The neighboring sequence of the reactive site consists primarily of hydrophobic amino acid residues. Based on examinations of the target proteinases and the reactive site specificity, guamerin is a new low molecular weight protein that inhibits elastases.

The period protein (PER) is a essential component of the circadian clock in Drosophila melanogaster. Although PER-containing pacemaker cells have been previously identified in the brain, the neuronal network that comprises the circadian clock remained unknown. Here it is shown that some PER neurons are also immunostained with an antiserum against the crustacean pigment-dispersing hormone (PDH). This antiserum reveals the entire arborization pattern of these pacemaker cells. The arborizations of these neurons are appropriate for modulation of the activity of many neurons and they might interact with PER-containing glial cells. A putative physiological role of PDH in the circadian system is discussed.

We described in a foregoing report findings on serpin, a serine protease inhibitor, newly identified in horseshoe crab (Tachypleus tridentatus) hemocytes and we name it limulus intracellular coagulation inhibitor, LICI (Miura, Y., Kawabata, S., and Iwanaga, S. (1994) J. Biol. Chem. 269, 542-547). This serpin specifically inhibits limulus lipopolysaccharide-sensitive serine protease, factor C. In ongoing studies on limulus serpin, we have found another inhibitor, LICI type-2 (LICI-2), which inhibits not only factor C (k1 = 7.1 x 10(4) M-1 S-1) but also limulus clotting enzyme (k1 = 4.3 x 10(5) M-1 S-1). LICI-2 inhibits mammalian serine proteases, including alpha-thrombin, salivary kallikrein, plasmin, and tissue plasminogen activator. The inactivation of plasmin is the most rapid (k1 = 1.2 x 10(6) M-1 S-1). The purified LICI-2 is a single chain glycoprotein with an apparent M(r) = 42,000. A cDNA for LICI-2 was isolated and the open reading frame coded for a mature protein of 386 amino acids, of which 160 residues were confirmed by peptide sequencing. Although LICI-2 shows significant sequence similarity to the previous limulus serpin, LICI-1 (42% identity), LICI-2 contains a unique putative reactive site, -Lys-Ser-, distinct from that of LICI-1 (-Arg-Ser-). Northern blotting revealed expression of LICI-2 mRNA only in hemocytes, and not in heart, brain, stomach, intestine, coxal gland, and skeletal muscle. The immunoblot of large and small granule components with antiserum against purified LICI-2 suggests that LICI-2 is stored specifically in large granules, as in the case of LICI-1, and is released in response to external stimuli. We propose that the LICIs be classified into a new subfamily of intracellular serpins, regulated secretory serpins.

Eight FMRFamide-like neuropeptides were isolated from an extract of heads and tails from the nematode Ascaris suum using seven steps of HPLC. The peptides ranged in size from 8 to 14 amino acid residues: AF3 (Ala-Val-Pro-Gly-Val-Leu-Arg-Phe-amide), AF4 (Gly-Asp-Val-Pro-Gly-Val-Leu-Arg-Phe-amide), AF5 (Ser-Gly-Lys-Pro-Thr-Phe-Ile-Arg-Phe-amide), AF7 (Ala-Gly-Pro-Arg-Phe-Ile-Arg-Phe-amide), AF9 (Gly-Leu-Gly-Pro-Arg-Pro-Leu-Arg-Phe-amide), AF10 (Gly-Phe-Gly-Asp-Glu-Met-Ser-Met-Pro-Gly-Val-Leu-Arg-Phe-amide), AF11 (Ser-Asp-Ile-Gly-Ile-Ser-Glu-Pro-Asn-Phe-Leu-Arg-Phe-amide), and AF12 (Phe-Gly-Asp-Glu-Met-Ser-Met-Pro-Gly-Val-Leu-Arg-Phe-amide). The effect of synthetic AF4 on muscle tension in a dorsal muscle strip preparation was a strong, long-lasting contraction. PF1, a peptide present in Panagrellus redivivus and Caenorhabditis elegans, relaxed the AF4-induced contraction.

So-called accessorial pulsatile circulatory organs in insects have developed during evolutionary reduction of the vascular system. As such a peripheral organ, the antenna-heart supplies the antennae with haemolymph. In the antenna-heart, the beat rhythm is generated by a myogenic automatism and is controlled neuronally. The electrical stimulation of the antenna-heart nerve produces an immediate heart beat inhibition brought about by octopamine release. Following this heart inhibition, an accelerating effect can be observed, which is based on a peptidergic mechanism. Proctolin-like material was found in both the dilator muscle and the antenna-heart nerve by means of immunocytochemistry. After HPLC-separation of antenna-heart tissue, proctolin was identified by sequencing the bioactive material and subsequent mass spectrometry. Proctolin proved to be extraordinarily effective producing stimulation of the antenna-heart beat rate up to 450%. The threshold concentration is 10(-10) mol.1(-1) and the value of the dissociation constant was fixed to KO = 1.3 x 10(-8) mol.1(-1). The chrono-trophic effect is caused by an increase in the steepness of the rising phase of pacemaker activity. The enhancement of the membrane resistance Rinput indicates a reduction of K(+)-conductance by proctolin. The PI-second messenger system is involved as well. Proctolin was identified and isolated from a real target organ. This fact and the physiological results furnished proof of peptidergic transmitter function of proctolin in the antenna-heart.

Tachypleus antilipopolysaccharide (LPS) factor (TALF) is a protein of 102 amino acids in the lysate of amebocytes of Tachypleus tridentatus that binds bacterial LPS with high affinity and blocks its biologic activity in numerous assays. To elucidate the minimal sequences that bind LPS, overlapping synthetic peptides based on the sequence of TALF were assessed for the ability to bind and neutralize LPS. TALF41-53 was the minimal sequence that bound LPS, as assessed by a slot blot capture assay. TALF29-59 bound LPS with the highest potency. TALF29-59 decreased LPS-induced coagulation of limulus amebocyte lysate, induction of cytokines from human monocytes, and LPS-induced lethality in sensitized mice. Synthetic peptides based on TALF or other LPS-binding proteins may be useful for the design of drugs for treatment of endotoxemia.

Several large peptidic neurohormones have been isolated in crustaceans. In lobster and other related species, each of these neurohormones, and particularly the crustacean hyperglycemic hormone, occurs as two isoforms having the same peptidic sequence and molecular mass. We report here that these isoforms differ by the configuration of a single amino acid residue. The third residue (Phe3) of the lobster hyperglycemic hormones is in either the L- or D-configuration. In addition, we have shown that the biological activity of the two isoforms differs when considering the kinetics of their hyperglycemic effect.

Antistasin is a Factor Xa inhibitor that is present in the salivary glands of the Mexican leech Haementeria officinalis. The antistasin protein consists of 119 amino acids, of which residues 1-55 (domain I) are 56% similar to residues 56-110 (domain II). Of the nine C-terminal amino acids (residues 111-119; domain III), four are positively charged. The reactive site for Factor Xa is located in domain I. In this study we assessed the role of separate domains and of individual amino acids in the reactive site for the inhibition of Factor Xa. A series of mutants was constructed and expressed in Chinese hamster ovary (CHO) cells. In vitro chromogenic assays for Factor Xa show that domain I is sufficient for inhibition of Factor Xa. Domains II and III neither contain any intrinsic Factor Xa inhibitory activity, nor contribute to the activity of domain I. Furthermore, domain II does not become a Factor Xa inhibitor by partially adaptating its sequence towards that of the reactive site in domain I. Mutation of the cysteine at position 33 is not crucial for Factor Xa inhibition, suggesting a relatively rigid reactive site loop structure.

Osmoregulatory capacity (OC) decreased by approximately 50% after eyestalk ablation in adult Homarus americanus when in dilute media. OC was used to assay sinus gland extracts. Injection of total extracts and of some HPLC-separated fractions of sinus glands into destalked lobsters increased OC. One of the described crustacean hyperglycemic hormone isoforms influences osmoregulation. Another fraction of the sinus gland extracts modifies osmoregulation but its nature remains unknown. Variations in OC were examined in response to ecdysterone, Phe-Met-Arg-Phe-NH2, and atrial natriuretic factor but effects were minimal.

The neuropeptides myomodulin, small cardioactive peptide (SCP), and buccalin are widely distributed in the phylum Mollusca and have important physiological functions. Here, we describe the detailed distribution of each class of peptide in the central nervous system (CNS) of the snail Lymnaea stagnalis by the use of immunocytochemical techniques combined with dye-marking of electrophysiologically identified neurons. We report the isolation and structural characterization of a Lymnaea myomodulin, PMSMLRLamide, identical to myomodulin A of Aplysia californica. Myomodulin immunoreactivity was localized in all 11 ganglia, in their connectives, and in peripheral nerves. In many cases, myomodulin immunoreactivity appeared localized in neuronal clusters expressing FMRFamide-like peptides, but also in a large number of additional neurons. Double-labelling experiments demonstrated myomodulin immunoreactivity in the visceral white interneuron, involved in regulation of cardiorespiration. SCP-like immunoreactivity also appeared in all ganglia, and double-labelling experiments revealed that in many locations it was specifically associated with clusters expressing distinct exons of the FMRFamide gene that are differentially expressed in the CNS. Characterization of the two types of SCP-antisera used in this study, however, suggested that they cross-reacted with both FMRFamide and N-terminally extended FMRFamide-like peptides. Selective preadsorption with these cross-reacting peptides resulted in elimination of the widespread staining and retention of bona fide SCP immunoreactivity in the buccal and pedal ganglia only. Buccalin immunoreactivity was limited to the buccal and pedal ganglia. It did not coincide with the distribution of either myomodulin or SCP. Most immunoreactive clusters were found in the pedal ganglia.

Antistasin, a potent inhibitor of the blood coagulation factor Xa, is the prototype of a novel family of serine-proteinase inhibitors. We have now isolated, sequenced and characterized an antistasin-type inhibitor from the medical leech Hirudo medicinalis. Hirustasin (Hirudo antistasin) was purified to apparent homogeneity by cation-exchange and affinity chromatography. Amino acid sequencing of the 55 amino acid protein (M(r) 5866) revealed that hirustasin is the only antistasin-type protein known to consist of one domain only; 27% and 32% sequence identity was found to the first and second domains of antistasin, respectively, and a nearly exact conservation of the spacing of the ten cysteine residues. Hirustasin is the first inhibitor of tissue kallikrein identified in leeches, and is also a tight-binding inhibitor of trypsin, chymotrypsin and neutrophil cathepsin G. However, despite the high similarity to antistasin, particularly in the vicinity of the putative reactive-site peptide bond, hirustasin neither inhibits blood coagulation in vitro nor amidolytic activity of isolated factor Xa. Thus, structural elements other than the reactive site sequence significantly influence the specificity of antistasin-type proteinase inhibitors.

The X-organ of the eyestalk in crustaceans is a source of neurosecretory peptides. Total RNA was isolated from X-organs of the white shrimp Penaeus vannamei and used to clone a cDNA encoding a molt-inhibiting hormone-like (MIH-like) neuropeptide by the 3' and 5' rapid amplification of cDNA ends (RACE) method. Sequence analysis of the 470-bp cDNA reveals a 306-bp open reading frame and a 164-bp 3' untranslated region. The deduced polypeptide consists of a 72-amino acid mature peptide and a 30-amino acid region of propeptide. The mature peptide shares 49 and 29% amino acid identity to the MIH from the lobster Homarus americanus and the crab Carcinus maenas, respectively. Northern hybridization shows that the MIH-like mRNA has a molecular size of 2.0 kb and is present in the eyestalk.

An oxytocin-vasopressin-related peptide, Cys-Phe-Val-Arg-Asn-Cys-Pro-Thr-Gly-NH2, was isolated from the lumbricid earthworm, Eisenia foetida and termed annectocin. Annetocin potentiated not only spontaneous contractions of the gut but also pulsatory contractions and bladder-shaking movement of the nephridia. Annetocin may be involved in osmoregulation of the animal through nephridial function.

Three FMRFamide-related peptides (FaRPs) were purified and characterized from the kidney of the snail, Helisoma trivolvis, by HPLC and detected using two radioimmunoassays (RIA) for FaRPs. Automated sequencing and mass spectrometry of the isolated peptides suggest the following sequences: Phe-Met-Arg-Phe-NH2 (FMRFamide), Phe-Leu-Arg-Phe-NH2 (FMRFamide), and Gly-Asp-Pro-Phe-Leu-Arg-Phe-NH2 (GDPFLRFamide). The FaRPs, predominantly the heptapeptides, were also detected by HPLC and RIA in other osmoregulatory tissues such as the skin, mantle, and the hemolymph. The level of FaRPs, detected by radioimmunoassay, appears to be lower in snails kept under hyposmotic stress than in snails kept under isosmotic stress. The FaRPs appear to be involved in osmoregulation in H. trivolvis.