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

Nov 11, 2006General and comparative endocrinology

Molt-inhibiting hormone in the tropical land crab: gene cloning, where it’s made, and producing active hormone using yeast

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Abstract

A 1.4 kb cDNA encoding the molting-inhibiting hormone (MIH) from the land crab was successfully cloned and expressed.

MIH is associated with the inhibition of ecdysteroid production in the molting gland of crustaceans.
The cloned MIH prohormone contains a 35 amino acid signal peptide and a 78 amino acid mature peptide.
The mature peptide shares a highly conserved sequence with other crustacean MIHs, featuring six cysteines and other specific amino acids.
Expression of Gel-MIH was confirmed to occur exclusively in the eyestalk ganglia, with no presence in other examined tissues.
Recombinant MIH (rMIH) produced in yeast was shown to be biologically active by significantly inhibiting ecdysteroid secretion in cultured Y-organs.

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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.

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

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