Expression of crustacean (Callinectes sapidus) molt-inhibiting hormone in Escherichia coli: characterization of the recombinant peptide and assessment of its effects on cellular signaling pathways in Y-organs.

Academic Article

Abstract

  • A neuropeptide, 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 Escherichia coli. Bacteria were transformed with an expression plasmid containing a cDNA insert encoding MIH. After induction of protein synthesis, recombinant MIH (recMIH) was detected in the insoluble fraction of cell lysates. The insoluble recMIH was refolded and purified by reversed-phase high performance liquid chromatography (RP-HPLC). The refolded peptide was MIH-immunoreactive and comigrated with native MIH on RP-HPLC. Mass and CD spectral analyses showed the mass number and secondary structure of the recombinant peptide were as predicted for MIH. Bioassays showed recMIH dose-dependently suppresses ecdysteroid synthesis by Y-organs. The combined results suggest that recMIH is properly folded. In subsequent experiments, recMIH was used to assess cellular signaling pathways linked to MIH-mediated suppression of ecdysteroidogenesis. Incubation of Y-organs with recMIH produced an increase in intracellular cGMP content, but had no effect on intracellular cAMP. Further, a cGMP analog significantly suppressed ecdysteroid production, but neither cAMP analogs nor an activator of adenylyl cyclase had a detectable effect on ecdysteroidogenesis. The results are consistent with the hypothesis that MIH-induced suppression of ecdysteroidogenesis in Y-organs of C. sapidus is mediated by a cGMP second messenger. We anticipate recMIH will be a useful tool for additional studies of the cellular actions and physiological functions of MIH.
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    Keywords

  • Animals, Brachyura, Circular Dichroism, Colforsin, Endocrine Glands, Escherichia coli, Invertebrate Hormones, Mass Spectrometry, Molting, Nucleotides, Cyclic, Peptides, Protein Folding, Recombinant Proteins, Signal Transduction
  • Digital Object Identifier (doi)

    Author List

  • Nakatsuji T; Han D-W; Jablonsky MJ; Harville SR; Muccio DD; Watson RD
  • Start Page

  • 96
  • End Page

  • 104
  • Volume

  • 253
  • Issue

  • 1-2