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.
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.
Nakatsuji
Teruaki
T
Department of Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
Han
Deug-Woo
DW
Jablonsky
Michael J
MJ
Harville
Steven R
SR
Muccio
Donald D
DD
Watson
R Douglas
RD
D000818:Animals; D003386:Brachyura / Q000378:metabolism*; D002942:Circular Dichroism; D005576:Colforsin / Q000032:analysis / Q000737:chemistry / Q000494:pharmacology; D004702:Endocrine Glands / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D007447:Invertebrate Hormones / Q000235:genetics* / Q000378:metabolism; D013058:Mass Spectrometry; D018978:Molting; D009712:Nucleotides, Cyclic / Q000032:analysis / Q000737:chemistry / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology
D007447:Invertebrate Hormones; D009712:Nucleotides, Cyclic; D010455:Peptides; D011994:Recombinant Proteins; C012445:molt-inhibiting hormone; D005576:Colforsin