Crustacean hyperglycemic hormone from the tropical land crab, Gecarcinus lateralis: cloning, isoforms, and tissue expression.

Crustacean hyperglycemic hormone (CHH) regulates carbohydrate metabolism, molting, and ion and water transport. cDNAs encoding four CHH isoforms (designated EG-CHH-A, -B, -C, and -D) were cloned from eyestalk ganglia (EG) from land crab, Gecarcinus lateralis. The isoforms differed in the 3' region of the open reading frame and/or the length of the 3' untranslated region. All encoded essentially identical preprohormones containing a 28-amino acid (aa) signal peptide, a 42-aa precursor related peptide and a 72-aa mature CHH. All deduced aa sequences had the six cysteines, two arginines, one aspartate, one phenylalanine, and one arginine originally identified as characteristic of this neuropeptide family. There was a single aa difference between the EG-CHH-D mature hormone and the other three isoforms. The EG-CHH isoforms were expressed in EG, hindgut, and thoracic ganglion. A fifth CHH isoform, designated pericardial organ (PO)-CHH, was similar to the PO-CHH isoform described in green crab, Carcinus maenas. It was expressed in hindgut and testis, but not in eyestalk ganglia; its expression in PO was not determined. The deduced aa sequence of the PO-CHH was identical to that of the EG-CHH isoforms through aa #40 of the mature peptide. The divergent aa sequence between positions #41 and #73 was encoded by an insertion of a 111-bp sequence absent in EG-CHH cDNAs. The data suggest that EG-CHH and PO-CHH isoforms are generated by alternative splicing of at least two CHH genes.

Molt-inhibiting hormone from the tropical land crab, Gecarcinus lateralis: cloning, tissue expression, and expression of biologically active recombinant peptide in yeast.

Molt-inhibiting hormone (MIH), a member of the crustacean hyperglycemic neuropeptide hormone family, inhibits ecdysteroidogenesis in the molting gland or Y-organ (YO). A cDNA encoding MIH of the land crab (Gel-MIH) was cloned from eyestalk ganglia (EG) by a combination of reverse transcriptase polymerase chain reaction (RT-PCR) and 3'- and 5'-rapid amplification of cDNA ends (RACE). The cDNA (1.4 kb) encoded MIH prohormone containing a 35 amino acid signal peptide and a 78 amino acid mature peptide. The mature peptide had the six cysteines, one glycine, two arginines, one aspartate, one phenylalanine, and one asparagine in identical positions in the highly conserved sequence characteristic of other crustacean MIHs. Gel-MIH was expressed only in the EG, as determined by RT-PCR; it was not detected in Y-organ, heart, integument, gill, testis, ovary, hepatopancreas, thoracic ganglion, or skeletal muscle. A cDNA encoding the mature peptide was used to express recombinant MIH (rMIH) using a yeast (Pichia pastoris) expression system. Two constructs were designed to yield either a mature MIH fusion protein with a c-myc epitope and histidine (His) tag at the carboxyl terminus or an untagged mature protein without the c-myc and His sequences. Immunoreactive peptides were detected in Western blots of the cell culture media with both MIH constructs, indicating secretion of the processed rMIH into the medium. Culture media containing the untagged mature peptide significantly inhibited ecdysteroid secretion by YOs from land crab and green crab (Carcinus maenas) cultured in vitro, indicating that the Gel-rMIH was biologically active.

A crustacean nitric oxide synthase expressed in nerve ganglia, Y-organ, gill and gonad of the tropical land crab, Gecarcinus lateralis.

NO signaling is involved in many physiological processes in invertebrates. In crustaceans, it plays a role in the regulation of the nervous system and muscle contraction. Nested reverse transcription-polymerase chain reaction (RT-PCR) and 5' and 3' rapid amplification of cDNA ends (RACE) PCR generated a full-length cDNA sequence (3982 bp) of land crab NO synthase (Gl-NOS) from molting gland (Y-organ) and thoracic ganglion mRNA. The open reading frame encoded a protein of 1199 amino acids with an estimated mass of 135 624 Da. Gl-NOS had the highest sequence identity with insect NOS. The amino acid sequences for binding heme and tetrahydrobiopterin in the oxygenase domain, binding calmodulin and binding FMN, FAD and NADPH in the reductase domain were highly conserved. Gl-NOS had single amino acid differences in all three highly conserved FAD-binding sequences, which distinguished it from other NOS sequences. RT-PCR showed that the Gl-NOS mRNA was present in testis, ovary, gill, eyestalk neural ganglia, thoracic ganglion and Y-organ. NOS mRNA varied between preparations of Y-organ, thoracic ganglion and gill, while NOS mRNA was at consistently high levels in the ovary, testis and eyestalk ganglia. Immunohistochemistry confirmed that the Gl-NOS protein was expressed in Y-organ, ovary and gill. These results suggest that NOS has functions in addition to neuromodulation in adults, such as regulating or modulating ecdysteroid synthesis in the Y-organ.

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.

Expression of crustacean (Callinectes sapidus) molt-inhibiting hormone in insect cells using recombinant baculovirus.

Molt-inhibiting hormone (MIH) negatively regulates the synthesis of ecdysteroid molting hormones by crustacean Y-organs. We report here the expression of blue crab (Callinectes sapidus) MIH in insect cells using recombinant baculovirus. Insect Sf9 cells were transfected with recombinant baculovirus containing a DNA insert encoding the C. sapidus MIH prohormone (signal sequence plus mature hormone). The construct was designed to yield a mature, fully processed recombinant MIH (recMIH). Several baculovirus recombinants showing no contamination with wild-type viral DNA were subsequently analyzed for their ability to direct expression of recMIH. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins from infected cells revealed time-dependent expression of two proteins of approximately the predicted size for the C. sapidus MIH prohormone and mature hormone. Western blot results (using antiserum against MIH of Carcinus maenas) indicated that the proteins were MIH-immunoreactive. N-Terminal amino acid sequence data and mass spectral analysis indicated the expressed proteins were of the correct sequence and molecular mass. Cell lysates containing the recombinant protein dose-dependently suppressed the synthesis of ecdysteroids by Y-organs in vitro. We anticipate the recombinant peptide will prove useful for studies of the structure and function of MIH.