ABSTRACT
PC2 prohormone convertases are enzymes involved in the proteolytic maturation of neuropeptide precursors. In the present work, a cDNA encoding a PC2-like enzyme (OrlPC2) was cloned from crayfish eyestalk ganglia (medulla terminalis) containing the X-organ, a major neuroendocrine center. The predicted 634 amino acid preproprotein exhibits highest sequence identity, especially in the catalytic domain, with PC2s from arthropods and nematodes, and less with mollusc and vertebrate enzymes. It was demonstrated by in situ hybridization on crayfish medulla terminalis sections that OrlPC2 is expressed in a large number of neuron perikarya, including those producing the well known crustacean hyperglycemic hormone.
Subject(s)
Astacoidea/genetics , Subtilisins/genetics , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , DNA Primers , DNA, Complementary/biosynthesis , DNA, Complementary/chemistry , In Situ Hybridization , Molecular Sequence Data , Proprotein Convertase 2 , Sequence Alignment , Subtilisins/biosynthesis , Subtilisins/chemistryABSTRACT
Total extracts of sinus glands (SG) of the euryhaline grapsid crab Pachygrapsus marmoratus contain peptidic factor(s) that stimulate osmoregulatory processes in isolated and perfused posterior gills from crabs acclimated to dilute seawater. This study investigated the nature of the active factor(s). Separation of P. marmoratus SG peptides by reverse-phase HPLC, followed by a direct enzyme-linked immunosorbent assay using an anti-Carcinus maenas crustacean hyperglycemic hormone (CHH) antiserum, identified a major immunoreactive chromatographic peak. A glucose quantification bioassay demonstrated a strong and specific hyperglycemic activity following injection of the immunoreactive peak, therefore defined as the CHH of P. marmoratus. Isolated posterior gills were then perfused with HPLC fractions using a dose of 4 SG equivalents/assay. The CHH fraction consistently and significantly increased the transepithelial potential difference and Na(+) influx by about 50%. The effect was rapid and reversible. Another substance of unknown nature (eluted earlier than CHH in the HPLC gradient) caused a small increase in Na(+) influx (14%) but had no effect on the transepithelial potential difference. No other peptidic product from the SG had significant effect on the measured osmoregulatory parameters. These results indicate that CHH, in addition to its hyperglycemic activity, is also implicated in the control of branchial ionic transport. This neuropeptide may thus constitute a major factor involved in the control of osmoregulation in decapod crustaceans.
Subject(s)
Crustacea/metabolism , Gills/metabolism , Ion Transport , Nerve Tissue Proteins/physiology , Animals , Arthropod Proteins , Chromatography, High Pressure Liquid , Enzyme-Linked Immunosorbent Assay , Epithelium/metabolism , Female , Invertebrate Hormones , Male , Membrane Potentials , Sodium/metabolism , Water-Electrolyte BalanceABSTRACT
Modification of the chirality of a single amino acid residue within a peptide chain appears to be novel additional mechanism leading to structural and functional diversification of eukaryotic bioactive peptides. This phenomenon has been studied at the cellular level in a neuroendocrine organ which elaborates a mixture of diastereoisomers of a 72-residue neuropeptide, crustacean hyperglycemic hormone. For the first time, amino acid isomerization has been shown to occur in the perikarya of fully specialized neurosecretory cells, as a late step of the maturation of the hyperglycemic hormone precursor and after propeptide cleavage. The specificity and efficiency of this phenomenon indicates the existence of a new enzyme family involved in the biogenesis of peptide hormones.
Subject(s)
Nerve Tissue Proteins/metabolism , Protein Processing, Post-Translational , Amino Acid Sequence , Animals , Arthropod Proteins , Astacoidea , Female , Invertebrate Hormones , Isomerism , Male , Nerve Tissue Proteins/chemistry , Neurosecretory Systems/cytologyABSTRACT
The crustacean hyperglycemic hormone (CHH) is the major neuropeptide produced by the X-organ-sinus gland neurosecretory system of the crayfish, Orconectes limosus. This hormone is synthesized by two different cell types, as two isomers (CHH and D-Phe3-CHH) which display different activities The aim of this report is to analyze and compare the synthetic and secretory activities of these specialized cells. In vitro pulse-chase incubations and time-course experiments were conducted on isolated X-organ-sinus gland (XO-SG) complexes, followed by analysis of the labeled peptides. The different steps of the post-translational processing of the CHH precursor, including proteolytic cleavage of the propeptide, C-terminal amidation and N-terminal pyroglutamylation were characterized and the kinetics of CHHs maturation were estimated in the different parts of the neuroendocrine complex. Furthermore, synthesis of CHHs in XO-SG complexes and release in incubation media were investigated using combined HPLC/immunoassay. Under basal conditions, i.e. without stimulation, similar dynamics for both isomers were found and results indicate that newly synthesized CHHs are preferentially released.
Subject(s)
Astacoidea/metabolism , Invertebrate Hormones/metabolism , Nerve Tissue Proteins/metabolism , Amino Acid Sequence , Animals , Arthropod Proteins , Exocrine Glands/metabolism , Eye , Invertebrate Hormones/biosynthesis , Kinetics , Leucine/metabolism , Nerve Tissue Proteins/biosynthesis , Nerve Tissue Proteins/chemistry , Protein Isoforms/biosynthesis , Protein Isoforms/chemistry , Protein Isoforms/metabolism , Protein Processing, Post-TranslationalABSTRACT
The combination of two sensitive and powerful analytical techniques on the same biological sample was examined: (i) matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), which gives informative peptide profiling on complex samples such as organs or cells; (ii) immunological tools such as enzyme-linked immunosorbent assay (ELISA) and immunocytochemistry to probe for specific peptides in biological extracts or cells. The cellular expression of the two precursors of the hyperglycemic hormone (cHH) was analyzed in neurosecretory cells (30-micron diameter) from the crayfish Orconectes limosus. Neurohemal organs were used to optimize the sample preparation and to demonstrate that, after peptide fingerprinting by MALDI-TOF MS, the sample can be recovered from the MALDI plate for further immunological analysis by ELISA. It was also established that, after immunocytochemistry following 4% paraformaldehyde fixation of the organ, the stained tissue could be recovered for further MALDI-TOF MS analysis. This dual characterization was successfully scaled down to the level of a single crayfish neurosecretory cell. Direct peptide profiling by MALDI-TOF MS on a single cHH-producing cell previously identified by immunocytochemistry demonstrated that both procHH isoforms were expressed in each cell analyzed.
Subject(s)
Astacoidea/chemistry , Cells/chemistry , Endocrine Glands/chemistry , Peptides/analysis , Animals , Enzyme-Linked Immunosorbent Assay , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Neurohemal organs of the lobster Homarus americanus contain isoforms of the crustacean hyperglycemic hormone, which differ by the third amino acid (phenylalanyl) residue that is either in the L- or in the D-configuration. Polyclonal antisera have been raised in rabbit against synthetic octapeptides with the sequence corresponding to the N-terminal part of the L- or D-phenylalanine-containing isoforms. Their specificity was shown by immunoassays, indicating that they discriminate the isoforms of the lobster hyperglycemic neuropeptides. It was demonstrated that the two major forms of the crayfish Orconectes limosus hyperglycemic hormone also correspond to peptide isomers containing the L- or D-phenylalanyl residue. The cellular distribution of the isoforms among the neurosecreting cells of the major neuroendocrine complex in lobster and crayfish has been studied by immunohistochemistry. Every hyperglycemic hormone-containing cell was labelled with the anti-L antisera while only some of them were visualized with the anti-D antisera. These results constitute the first observation of peptide isomerization at the cellular level and suggest that the isomerization process occurs in specialized neuroendocrine cells.
Subject(s)
Astacoidea/metabolism , Nephropidae/metabolism , Neuropeptides/metabolism , Amino Acids/metabolism , Animals , Chromatography, High Pressure Liquid , Enzyme-Linked Immunosorbent Assay , Exocrine Glands/metabolism , Immunohistochemistry , Invertebrate Hormones/metabolism , Isomerism , Neuropeptides/chemistrySubject(s)
Crustacea/genetics , Invertebrate Hormones/genetics , Nerve Tissue Proteins/genetics , Neuropeptides/genetics , Amino Acid Sequence , Animals , Arthropod Proteins , Arthropods/genetics , Genetic Variation , Molecular Sequence Data , Polymorphism, Genetic , Sequence Homology, Amino Acid , Species SpecificityABSTRACT
To design a homologous bioassay for the molt-inhibiting hormone and the crustacean hyperglycemic hormone of the shrimp Penaeus vannamei, the effect of sinus gland homogenate (SGh), in vitro, on ecdysteroid production by Y-organs (YOs), and the effect of the injection of SGh, in vivo, on the glycemia of shrimps have been investigated. Addition of SGh to incubation medium of shrimp YOs dose dependently reduced, within a few hours, ecdysteroid release into the medium. Moreover, inhibition by SGh decreases drastically in YOs from animals in late premolt stages, when there is maximal ecdysteroid production. Injection of SGh into shrimps evokes a hyperglycemic response maximal after 2 hr. Immunoadsorption of SGh with an anti-Homarus americanus cHHA antiserum inhibited both biological activities of the homogenate. After fractionation of acidic sinus gland extract by RP-HPLC, the maximal response in both bioassays was associated with the major UV absorbent peak, which was also the major immunoreactive peak when tested by ELISA with the anti-lobster cHHA. After a further purification step, the molecular mass of the bioactive and immunoreactive peptide was found to be 8627 +/- 0.3 Da by electrospray ionization mass spectrometry. The amino acid sequence of the first 38 residues of this peptide was established by gas-phase microsequencing. This sequence shows 55% homology with the first 38 residues of the lobster cHHA.
Subject(s)
Hyperglycemia/metabolism , Invertebrate Hormones/physiology , Neurosecretory Systems/physiology , Penaeidae/physiology , Amino Acid Sequence , Animals , Biological Assay , Chromatography, High Pressure Liquid , Ecdysone/analogs & derivatives , Ecdysone/metabolism , Ecdysterone/metabolism , Enzyme-Linked Immunosorbent Assay , Invertebrate Hormones/chemistry , Mass Spectrometry , Molecular Sequence Data , Nephropidae/physiology , Neurosecretory Systems/chemistry , Tissue Extracts/pharmacologyABSTRACT
The primary structure of the neurohormone crustacean hyperglycemic hormone (CHH-II) was determined by means of enzymatic digestions, manual Edman degradation, and mass spectrometry. CHH-II is a 72 residue peptide (molecular mass 8388 Da), with six cysteines forming three disulfide bridges that connect residues 7-43, 23-39, and 26-52. The peptide has blocked N- and C-termini, and lacks tryptophan, histidine, and methionine. The CHH-I and CHH-II of Procambarus bouvieri have identical sequences and elicit levels of hyperglycemia that are not distinguishable. The difference between the two isomorphs consists in a posttranslational modification of a L-Phe in CHH-I to a D-Phe in CHH-II at the third position from the N-terminus.
Subject(s)
Astacoidea/chemistry , Invertebrate Hormones/chemistry , Nerve Tissue Proteins/chemistry , Amino Acid Sequence , Amino Acids/chemistry , Animals , Arthropod Proteins , Astacoidea/genetics , Astacoidea/metabolism , Chromatography, High Pressure Liquid , Cysteine/chemistry , Enzyme-Linked Immunosorbent Assay , Invertebrate Hormones/genetics , Invertebrate Hormones/metabolism , Mass Spectrometry , Molecular Sequence Data , Molecular Structure , Molecular Weight , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Peptide Fragments/chemistry , Protein Processing, Post-Translational , StereoisomerismABSTRACT
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.
Subject(s)
Invertebrate Hormones/chemistry , Nephropidae/chemistry , Nerve Tissue Proteins/chemistry , Polymorphism, Genetic , Protein Conformation , Amino Acid Sequence , Animals , Arthropod Proteins , Invertebrate Hormones/isolation & purification , Mass Spectrometry , Molecular Sequence Data , Nerve Tissue Proteins/isolation & purification , Oligopeptides/chemical synthesis , Oligopeptides/chemistry , Peptide Fragments/chemistry , Peptide Fragments/isolation & purification , Sebaceous Glands/chemistry , Sequence Homology, Amino AcidABSTRACT
Vitellogenesis inhibiting hormone (VIH)-mRNA in secretory cells of the eyestalk of Homarus americanus was detected by nonradioactive in situ hybridization (ISH) using two digoxigenin-tailed oligonucleotide probes deduced from the peptide sequence. Two distinct clusters of positive cells were observed in the medulla terminalis ganglionic X-organ (MGTX). Only one of them gave a strong immunoreaction after incubation with a specific polyclonal anti-VIH serum and corresponded to the conventionally described VIH producing cells. The significance of the cells reacting positively in ISH but not in immunocytochemistry (ICC) is discussed. Northern blot analysis using 32P-labeling confirms the specificity of the probes and indicates an approximate size of 2.5 kb for VIH mRNA.
Subject(s)
Carrier Proteins/genetics , Invertebrate Hormones , Nephropidae/genetics , RNA, Messenger/analysis , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Eye/chemistry , Immunohistochemistry , Molecular Sequence Data , Neurosecretory Systems/chemistry , Nucleic Acid Hybridization , Oligonucleotide Probes/chemistry , RNA, Messenger/chemistryABSTRACT
From a medium in which Daudi cells had been grown, we isolated by HPLC a protein that caused ovarian abnormalities in adult females of Drosophila melanogaster when injected into preblastoderm embryos. This protein, whose apparent M(r) is between 30,000 and 50,000, was found to be a moderately polar compound which is heat stable and whose activity is destroyed by acidification. The protein is characteristic of medium conditioned from Daudi cells.
Subject(s)
Drosophila melanogaster/anatomy & histology , Neoplasm Proteins/physiology , Animals , Cell Count , Chromatography, High Pressure Liquid , Culture Media, Conditioned/chemistry , Female , Humans , Hydrogen-Ion Concentration , Male , Neoplasm Proteins/isolation & purification , Ovary/abnormalities , Temperature , Tumor Cells, Cultured , beta 2-Microglobulin/physiologyABSTRACT
The amino acid sequence of two isoforms of the Vitellogenesis Inhibiting Hormone from the lobster Homarus americanus (one biologically active and one inactive in a heterologous bioassay) has been established by gas-phase microsequencing and fast-atom bombardment mass spectrometry. These two isoforms, isolated from sinus glands display the same sequence of 77 amino acid residues (m.w.: 9135 Da) and have a free N-terminus. Structurally related to Crustacean Hyperglycemic Hormone and Molt Inhibiting Hormone, the Vitellogenesis Inhibiting Hormone of the lobster clearly appears as an original member of the newly described family of neuropeptides, so far proper to crustaceans, which are involved in the control of major physiological functions.
Subject(s)
Carrier Proteins/chemistry , Nephropidae/chemistry , Amino Acid Sequence , Animals , Chromatography, High Pressure Liquid , Crustacea/chemistry , Invertebrate Hormones/chemistry , Isoelectric Point , Molecular Sequence Data , Molecular Weight , Spectrometry, Mass, Fast Atom BombardmentABSTRACT
With the use of a two-step HPLC purification procedure, two sets of two isoforms of the crustacean hyperglycemic hormone (CHH) were isolated from sinus glands of the lobster Homarus americanus. Structural differences between the two groups of isoforms were found in their amino acid sequences, amino acid compositions and precise molecular weights. Using peptide mapping, the difference between the isoforms in each group was located within the first eight amino acids at the N-termini. The nature of this difference remained unclear as all four peptides had the same N-terminal amino acid sequence unto residue 19.
Subject(s)
Nephropidae/metabolism , Neuropeptides/isolation & purification , Amino Acid Sequence , Amino Acids/analysis , Animals , Chromatography, High Pressure Liquid , Hyperglycemia/metabolism , Molecular Sequence Data , Molecular Weight , Neuropeptides/chemistry , Peptide Mapping , Sequence Homology, Nucleic Acid , Spectrometry, Mass, Fast Atom BombardmentABSTRACT
Antisera raised in guinea pigs against four major neuropeptides purified from sinus glands of the lobster, Homarus americanus, were used to study the immunological relationships between several sinus gland peptides. On the basis of their behavior in ELISA and in absorption procedures, three groups of peptides are defined. Two groups may be related to the crustacean hyperglycemic hormone (CHH groups); the third one is composed of three immunologically identical peptides and, since one of these peptides was characterized in previous studies as a vitellogenesis inhibitor, is referred to as VIH group. This closely meets our present knowledge about the physiological effects and biochemical characteristics of these neuropeptides and gives immunological insights on the question of molecular polymorphism of lobster neurohormones.
Subject(s)
Nephropidae/metabolism , Nerve Tissue Proteins/immunology , Neuropeptides/immunology , Neurosecretory Systems/chemistry , Animals , Antibodies/immunology , Antibody Specificity , Arthropod Proteins , Enzyme-Linked Immunosorbent Assay , Epitopes/immunology , Immunosorbent Techniques , Invertebrate Hormones , Neuropeptides/isolation & purificationABSTRACT
In order to characterize hyperglycemic peptides from the sinus gland of the lobster, Homarus americanus, a bioassay was developed with juvenile H. gammarus. This assay was used for determining the hyperglycemic activity of peptides perified by reversed-phase high-performance liquid-chromatography, from acidic extracts of sinus gland. The major peptides are eluted in three sets of two peptides. Among them, two pairs show hyperglycemic activity when assayed on lobster; when assayed on crayfish, three peptides are active. The less hydrophobic pair consists of basic peptides (pI: 8.7), with a MW of 8633 Da., determined by fast-atom bombardment mass spectrometry. The most hydrophobic pair consists of acid peptides (pI: 5.0), with a MW of 8577 Da. Amino acid composition of the hyperglycemic peptides shows strong homologies within each pair.
Subject(s)
Nephropidae/metabolism , Nerve Tissue Proteins/pharmacology , Neuropeptides/pharmacology , Amino Acids/analysis , Animals , Arthropod Proteins , Astacoidea/drug effects , Astacoidea/metabolism , Biological Assay , Blood Glucose/metabolism , Chromatography, High Pressure Liquid , Eye , Invertebrate Hormones , Isoelectric Point , Mass Spectrometry , Molecular Weight , Nephropidae/drug effects , Nerve Tissue Proteins/isolation & purification , Neuropeptides/isolation & purificationSubject(s)
Birds/metabolism , Enzymes/blood , Liver/metabolism , Polychlorinated Biphenyls/toxicity , Testosterone/blood , Xenobiotics/metabolism , Alanine Transaminase/metabolism , Animals , Chromatography, High Pressure Liquid , Cytochrome P-450 Enzyme System/metabolism , Diet , Liver/drug effects , Liver/enzymology , Microsomes, Liver/enzymology , Oxidation-Reduction , gamma-Glutamyltransferase/metabolismABSTRACT
Immunochemical investigations, using dot immunobinding assay (DIA) and enzyme-linked immunosorbent assay (ELISA), and immunocytochemical studies reveal the following new information about crustacean vitellogenesis-inhibiting hormone (VIH): (1) The structure of VIH is sufficiently different from that of the other sinus gland neuropeptides to allow a selective recognition of VIH by polyclonal antibodies. (2) From immunochemical criteria, VIH does not seem strictly species specific. The antisera raised against VIH of Homarus americanus cross-react with sinus gland extracts of Palaemonetes varians, Palaemon serratus, Macrobrachium rosenbergii, Carcinus maenas, and Porcellio dilatatus. (3) In the sinus gland of H. americanus, VIH immunoreactivity is localized mainly in electron-dense granules of medium size (110-185 nm in diameter) while, in P. dilatatus, the labeling is mostly on the largest granules (200-270 nm in diameter).