Characterization and sequence elucidation of a novel peptide with molt-inhibiting activity from the South African spiny lobster, Jasus lalandii

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.     

Immunocytochemical demonstration of the neurosecretory systems containing putative moult-inhibiting hormone and hyperglycemic hormone in the eyestalk of brachyuran crustaceans

Summary By use of antisera raised against purified moultinhibiting (MIH) and crustacean hyperglycemic hormone (CHH) from Carcinus maenas, complete and distinct neurosecretory pathways for both hormones were demonstrated with the PAP and immunofluorescence technique. By double staining, employing a combination of silver-enhanced immunogold labelling and PAP, both antigens could be visualized in the same section. Immunoreactive structures were studied in Carcinus maenas, Liocarcinus puber, Cancer pagurus, Uca pugilator and Maja squinado. They were only observed in the X-organ sinus gland (SG) system of the eyestalks and consisted of MIH-positive perikarya, which were dispersed among the more numerous CHH-positive perikarya of the medulla terminalis X-organ (XO). The MIH-positive neurons form branching collateral plexuses adjacent to the XO and axons that are arranged around the CHH-positive central axon bundle of the principal XO-SG tract. In the SG, MIH-positive axon profiles and terminals, clustered around hemolymph lacunae, are distributed between the more abundant CHH-positive axon profiles and terminals. Colocalisation of MIH and CHH was never observed. The gross morphology of both neurosecretory systems was similar in all species examined, however, in U. pugilator and M. squinado immunostaining for MIH was relatively faint unless higher concentrations of antiserum were used. Possible reasons for this phenomenon as well as observed moult cycle-related differences in immunostaining are discussed.     

A comparative immunocytochemical investigation of the crustacean hyperglycemic hormone (CHH) in the eyestalks of some decapod crustacea

The eyestalk of the astacideans Orconects limosus, Nephrops norvegicus, and Homarus gammarus, and the palinuran Palinurus vulgaris, was examined with an antiserum raised against purified crustacean hyperglycemic hormone (CHH) of the astacidean species Astacus leptodactylus. A distinct immunopositive reaction occurs in a group of neurosecretory cells in the medulla terminalis ganglionic X-organ (MTGX), in the MTGX-sinus gland tractus, and in a considerable part of the sinus gland. The immunoreactive sites in the eyestalk of the investigated species correspond to the site of production, storage, and release of the CHH. Preliminary investigations with this antiserum also indicate that a positive immunoreaction can be obtained in the eyestalk of other decapod crustaceans, for example, of the brachyuran Macropipus puber and the caridean Palaemon serratus.     

The crustacean hyperglycemic hormone precursors a and b of the Norway lobster differ in the preprohormone but not in the mature peptide

The neuro-endocrine X-organ sinus-gland complex of crustaceans produces and releases the neuropeptides of the crustacean hyperglycemic hormone (cHH)/molt-inhibiting hormone (MIH)/gonad-inhibiting hormone (GIH) family that regulate important physiological processes, such as growth, reproduction and molting. We cloned two full-length cDNAs encoding the preprocHH-A and preprocHH-B of the Norway lobster Nephrops norvegicus of 132 and 131 amino acid residues. The two cHHs differ in the preprohormone but not in the mature peptide sequence. The mature cHH was expressed in bacteria as GST fusion protein that, in bioassay, shows a hyperglycemic activity similar to that of native cHH present in an eyestalk extract.     

Characterization of a molt-inhibiting hormone (MIH) of the crayfish, Orconectes limosus, by cDNA cloning and mass spectrometric analysis

The structure of the precursor of a molt-inhibiting hormone (MIH) of the American crayfish, Orconectes limosus was determined by cloning of a cDNA based on RNA from the neurosecretory perikarya of the X-organ in the eyestalk ganglia. The open reading frame includes the complete precursor sequence, consisting of a signal peptide of 29, and the MIH sequence of 77 amino acids. In addition, the mature peptide was isolated by HPLC from the neurohemal sinus gland and analyzed by ESI-MS and MALDI-TOF-MS peptide mapping. This showed that the mature peptide (Mass 8664.29 Da) consists of only 75 amino acids, having Ala75-NH2 as C-terminus. Thus, C-terminal Arg77 of the precursor is removed during processing, and Gly76 serves as an amide donor. Sequence comparison confirms this peptide as a novel member of the large family, which includes crustacean hyperglycaemic hormone (CHH), MIH and gonad (vitellogenesis)-inhibiting hormone (GIH/VIH). The lack of a CPRP (CHH-precursor related peptide) in the hormone precursor, the size and specific sequence characteristics show that Orl MIH belongs to the MIH/GIH(VIH) subgroup of this larger family. Comparison with the MIH of Procambarus clarkii, the only other MIH that has thus far been identified in freshwater crayfish, shows extremely high sequence conservation. Both MIHs differ in only one amino acid residue ( approximately 99% identity), whereas the sequence identity to several other known MIHs is between 40 and 46%.     

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.     

Functional analysis of crustacean Hyperglycemic Hormone by in vivo assay with wild-type and mutant recombinant proteins

The neuro-endocrine X-organ sinus-gland complex regulates important crustacean physiological processes, such as growth, reproduction and molting. Its major products are the neuropeptides of the cHH/MIH/GIH family. Until now the structure-function relationships of these neuropeptides were established by sequence comparison. To study the functional relevance of conserved amino acid residues or peptide motifs, we generated point and deletion mutants of the Norway lobster Nephrops norvegicus cHH. The wild type mature neuropeptide cHH and its mutant forms were expressed in bacteria as fusion proteins and assayed in vivo to assess their hyperglycemic activity. The wild type cHH had a hyperglycemic activity similar to that of cHH present in an eyestalk extract, and it was blocked by an anti-recombinant cHH antibody. Bioassays of cHHs, obtained by a progressive deletion of five highly conserved motifs, showed that the only deleted cHH, which conserves a hyperglycemic activity, is the one lacking the C-terminal motif, but still retaining all the motifs reported to be important for functional specificity and three-dimensional structure. All the cHH point mutants lacked a hyperglycemic activity. These results identify amino acid residues that are required for the hyperglycemic activity of cHH.     

Cloning of the crustacean hyperglycemic hormone and evidence for molt-inhibiting hormone within the central nervous system of the blue crab Portunus pelagicus

The crustacean X-organ–sinus gland (XO–SG) complex controls molt-inhibiting hormone (MIH) production, although extra expression sites for MIH have been postulated. Therefore, to explore the expression of MIH and distinguish between the crustacean hyperglycemic hormone (CHH) superfamily, and MIH immunoreactive sites (ir) in the central nervous system (CNS), we cloned a CHH gene sequence for the crab Portunus pelagicus (Ppel-CHH), and compared it with crab CHH-type I and II peptides. Employing multiple sequence alignments and phylogenic analysis, the mature Ppel-CHH peptide exhibited residues common to both CHH-type I and II peptides, and a high degree of identity to the type-I group, but little homology between Ppel-CHH and Ppel-MIH (a type II peptide). This sequence identification then allowed for the use of MIH antisera to further confirm the identity and existence of a MIH-ir 9 kDa protein in all neural organs tested by Western blotting, and through immunohistochemistry, MIH-ir in the XO, optic nerve, neuronal cluster 17 of the supraesophageal ganglion, the ventral nerve cord, and cell cluster 22 of the thoracic ganglion. The presence of MIH protein within such a diversity of sites in the CNS, and external to the XO–SG, raises new questions concerning the established mode of MIH action.     

Immunohistochemical Identification of Hyperglycemic Hormone- and Molt-Inhibiting Hormone-Producing Cells in the Eyestalk of the Kuruma Prawn, Penaeus japonicus

This study deals with the localization of crustacean hyperglycemic hormone (CHH, Pej-SGPIII) and molt-inhibiting hormone (MIH, Pej-SGP-IV) in the eyestalk of the kuruma prawn Penaeus japonicus using immunohistochemistry. High-titer and highly specific antisera were raised in rabbits against synthetic Pej-SGP-III C-terminal peptide (Glu-Glu-His-Met-Ala-Ala-Met-Gln-Thr-Val-NH2) and Pej-SGP-IV C-terminal peptide (Val-Trp-Ile-Ser-Ile-Leu-Asn-Ala-Gly-Gln-OH), both of which were conjugated with bovine serum albumin by a cross linker. Eyestalks were removed from mature male prawns at the intermolt stage of the molting cycle and fixed in Bouin's solution. Serial sections stained immunohistochemically showed that neurosecretory cells of Pej-SGP-III and Pej-SGP-IV were located in the same cluster of the medulla terminalis ganglionic X-organ (MTGX), and that three kinds of neurosecretory cells, which were stained with anti-PejSGP-III antiserum and/or anti-Pej-SGP-IV antiserum were present. The number of neurosecretory cells which stained with both antisera was much fewer than that of neurosecretory cells which stained with one of the antisera only. The axon and axon terminals in the sinus gland were also stained and the staining density of the sinus gland was always deeper than that of the neurosecretory cells.     

Crustacean Hyperglycemic Hormone Family: Old Paradigms and New Perspectives

I present an overview of recent research on the isolation andcharacterization of members of the crustacean hyperglycemichormone (CHH) neuropeptide family. Members of this arthropod-specificfamily include CHH, molt-inhibiting hormone (MIH), vitellogenesis-inhibitinghormone (VIH), and mandibular organ-inhibiting hormone (MOIH).There are two subfamilies of this neuropeptide group, basedupon the presence or absence of a C-terminal CHH precursor-relatedpeptide. There are also sequence motif differences between thesesubfamilies. Most of the peptides comprising this neuropeptidefamily are synthesized and released by the eyestalk X-organ/sinusgland complex. Recent experiments have demonstrated the presenceof extra-eyestalk cells that produce CHH and the assignmentof additional functions to this hormone family.     

Structural prediction and analysis of VIH-related peptides from selected crustacean species

The tentative elucidation of the 3D-structure of vitellogenesis inhibiting hormone (VIH) peptides is conversely underprivileged by difficulties in gaining enough peptide or protein, diffracting crystals, and numerous extra technical aspects. As a result, no structural information is available for VIH peptide sequences registered in the Genbank. In this situation, it is not surprising that predictive methods have achieved great interest. Here, in this study the molt-inhibiting hormone (MIH) of the kuruma prawn (Marsupenaeus japonicus) is used, to predict the structure of four VIHrelated peptides in the crustacean species. The high similarity of the 3D-structures and the calculated physiochemical characteristics of these peptides suggest a common fold for the entire family.     

Regulation of circulating levels of the crustacean hyperglycemic hormone: evidence for a dual feedback control system

The effects of glutamate, aspartate, glycine, proline, alanine, taurine, glycerol, glucose and lactate injections on the haemolymph levels of the crustancean hyperglycemic hormone and/or glucose and lactate in the shore crab, Carcinus maenas, were investigated. Only glucose and lactate caused significant changes of hyperglycaemic hormone levels. Glucose injections resulted in a drop of both hormone and lactate, while lactate had an opposite effect, i.e. it raised both crustacean hormone and glucose levels. The results suggest that during increases in glycolytic flux, lactate may cause a release of hormone by a positive feedback mechanism. The hormone would then stimulate glycogenolysis, thus increasing glucose availability. If more glucose is released than is metabolized, excess glucose may leak from the cells and suppress crustancean hyperglycemic hormone release from the X-organ/sinus gland complex by negative feedback.Abbreviations ABTS 2,2-azino-bis (3-ethylbenzthiazoline sulphonic acid) - ANOVA one-way analysis of variance - BSA bovine serum albumin - BW body weight - CHH crustacean hyperglycemic hormone - ELISA cnzyme-liked immunosorbent assay - GIH gonadinhibiting hormone - IgG immunoglobin G - MIH moult-inhibiting hormone - MTGXO medulla terminalis X-organ - PB sodium phosphate buffer - PBS phosphate buffered saline - Pi inorganic phosphate - XO-SG X-organ-sinus gland complex     

Expression of Biologically Active Crustacean Hyperglycemic Hormone (CHH) of <Emphasis Type="Italic">Penaeus monodon</Emphasis> in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

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 -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 µg/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.     

Do the neurohormones VIH (vitellogenesis inhibiting hormone) and CHH (crustacean hyperglycemic hormone) of crustaceans have a common precursor? Immunolocalization of VIH and CHH in the X-organ sinus gland complex of the lobster,Homarus americanus

Summary

The present study deals with the location of the vitellogenesis inhibiting hormone (VIH)-producing cells in the eyestalk of the lobster Homarus americanus. In the present study, the neurosecretory pathways of VIH in Homarus, have been described immunocytochemically by use of a mouse serum against Homarus VIH. The location of the VIH cells was compared with the location of the crustacean hyperglycemic hormone (CHH) cells visualized by a rabbit serum raised against CHH of the crayfish Astacus leptodactylus. Immunocytochemical detection procedures, both at the light and electron microscopic level, revealed frequent but not complete co-localization of VIH and CHH in a variable number of the same group of perikarya. In the sinus gland, both neuropeptides were mostly demonstrated in distinct axonal endings characterized by different granule types. Postulations on the biosynthesis of these factors and suggestions concerning the processing of both neurohormones have been made.     

Pigment-dispersing hormone-like immunoreactive neurons in the central nervous system of the gastropods, Helix pomatia and Lymnaea stagnalis

By using an antiserum raised against a crustacean #-pigment-dispersing hormone (PDH), the distribution and chemical neuroanatomy of PDH-like immunoreactive neurons was investigated in the central nervous system of the gastropod snails, Helix pomatia and Lymnaea stagnalis. The number of immunoreactive cells in the Helix central nervous system was found to be large (700-900), whereas in Lymnaea, only a limited number (50-60) of neurons showed immunoreactivity. The immunostained neurons in Helix were characterized by rich arborizations in all central ganglia and revealed massive innervation of all peripheral nerves and the neural (connective tissue) sheath around the ganglia and peripheral nerve trunks. A small number of Helix nerve cell bodies in the viscero-parietal ganglion complex were also found to be innervated by PDH-like immunoreactive processes. Hence, a complex central and peripheral regulatory role, including neurohormonal actions, is suggested for a PDH-like substance in Helix, whereas the sites of action may be more limited in Lymnaea.     

Hormonal Control of Sexual Differentiation and Reproduction in Crustacea

SYNOPSIS. Sexual differentiation in malacostracan Crustaceais controlled by the androgenic gland hormone (AGH). In males,the primordial androgenic glands (AG) develop and AGH inducesmale morphogenesis. In females, the primordial AG does not developand the ovaries differentiate spontaneously. Implantation ofthe AG into females yields various results, showing that thesensitivity to AGH differs with the species and the receptiveorgans. Purified AGH of the isopod Armadillidium vulgare consistsof at least two molecular forms, which exist as monomeric proteinswith molecular weights of 17,000 ± 800 and 18,300 ±1,000 Da and with isoelectric points of about 4.5 and 4.3, respectively.The antiserum raised against purified AGH makes it possibleto measure AGH activity by immunoassay. Neurohormones control male and female reproduction. In males,they are involved in the maintenance of the male germinativezone and the control of AG activity. In females, the secondaryvitellogenesis is controlled by the vitellogenesis-inhibitinghormone (VIH) and the vitellogenesis- stimulating hormone (VSH).VIH isolated from the lobster Homarus americanus is a peptidewith a molecular weight of 9,135 Da and shows homology to thecrustacean hyperglycemic hormone and moltinhibiting hormone.Involvement of the molting hormone and the juvenile hormone-likecompound in the secondary vitellogenesis have also been suggested.In the amphipod Orchestia gammarella, the vitellogenesis- stimulatingovarian hormone (VSOH) seems to control vitellogenin synthesis     

Bacterial expression of the shrimp molt-inhibiting hormone (MIH): antibody production, immunocytochemical study and biological assay

Molting in shrimp is controlled by the molt-inhibiting hormone (MIH) and ecdysone. MIH inhibits the synthesis of ecdysone in the Y-organ, resulting in molt suppression; it is a neuropeptide member belonging to the eyestalk CHH/MIH/GIH family. The cloning of MIH (formerly MIH-like) of the shrimp Metapenaeus ensis has been reported in a previous study. To obtain a large quantity of fusion protein for antibody production and biological assay, the cDNA encoding the shrimp MIH was inserted into the pRSET bacterial expression vector. His-tagged fusion protein was produced and purified by an Ni2+-charged affinity column. Polyclonal antibody to rMIH was subsequently obtained by immunizing rabbits with purified recombinant proteins. Results from Western blot analysis indicated that the antibody was specific. Furthermore, results from immunocytochemical analysis showed that specific cells in three different clusters of the X-organ, the sinus gland and the axonal tract of the eyestalk contain MIH. To test for the molt-inhibiting activity of rMIH, shrimp at intermolt stage were injected with rMIH and the molt cycle duration of the injected shrimp was monitored. A significant increase in molt cycle duration was recorded for the shrimp injected with the recombinant protein.