Demonstration of a cell-specific isomerization of invertebrate neuropeptides.

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.

Characterization of hyperglycemic and molt-inhibiting activity from sinus glands of the penaeid shrimp Penaeus vannamei.

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.

Evidence for a conformational polymorphism of invertebrate neurohormones. D-amino acid residue in crustacean hyperglycemic peptides.

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.

Detection of the mRNA encoding vitellogenesis inhibiting hormone in neurosecretory cells of the X-organ in Homarus americanus by in situ hybridization.

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.

Primary structure of two isoforms of the vitellogenesis inhibiting hormone from the lobster Homarus americanus.

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.

Immunological relationships between neuropeptides from the sinus gland of the lobster Homarus americanus, with special references to the vitellogenesis inhibiting hormone and crustacean hyperglycemic hormone.

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.

Comparative characterization of hyperglycemic neuropeptides from the lobster Homarus americanus.

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.

Neuropeptides from the sinus gland of the lobster Homarus americanus: Characterization of hyperglycemic peptides.

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.

Immunochemical and immunocytochemical studies of the crustacean vitellogenesis-inhibiting hormone (VIH).

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).