title: Molt-inhibiting hormone from Chinese mitten crab (Eriocheir sinensis): Cloning, tissue expression and effects of recombinant peptide on ecdysteroid secretion of YOs.
abstract: Molt-inhibiting hormone (MIH), a member of the crustacean hyperglycemic hormone (CHH) family, inhibits the synthesis of ecdysteroid in Y-organ (YO) and plays a significant role in the regulation of molting and growth of crustaceans. A complete cDNA sequence encoding MIH (Ers-MIH, GenBank Accession No.: DQ341280) was cloned from eyestalk of Chinese mitten crab (Eriocheir sinensis) by 5' and 3' RACEs and PCR cloning. The full-length cDNA consists of 1457 bp with a 330 bp open reading frame, encoding 110 amino acids, containing a 75 amino acid mature peptide. The deduced amino acid sequence contains a typical CHH domain. Transcripts of Ers-MIH mRNA were detected in eyestalk by Northern blotting. The production of purified recombinant Ers-MIH (rErs-MIH) expressed in Escherichia coli was 0.3g/L. The LC-ESI-MS analysis showed that two peptide fragments of the recombinant protein were identical to the deduced amino acid sequence of Ers-MIH. By in vitro assay on E. sinensis YOs, a cGMP mediated suppression of rErs-MIH on ecdysteroidogenesis could be observed. Accumulation of cGMP in YOs showed a concentration-dependent manner within 0.01-1 nmol/mL of rErs-MIH; ecdysteroid secretion was inhibited significantly at the range of 0.01-100 nmol/mL rErs-MIH; furthermore, a significant inhibition effect on ecdysteroid releasing was shown when cGMP analog (8-Br-cGMP) concentration rose up to 100 nmol/mL. This study would facilitate to investigate the roles of MIH in molt cycle regulation.
authors.affiliation: College of Life Science/Tianjin Key Laboratory of Cyto-genetical and Molecular Regulation, Tianjin Normal University, Tianjin 300387, PR China.
authors.forename: Yichen
authors.initials: Y
authors.lastname: Zhang
authors.forename: Yan
authors.initials: Y
authors.lastname: Sun
authors.forename: Yichen
authors.initials: Y
authors.lastname: Liu
authors.forename: Xuyun
authors.initials: X
authors.lastname: Geng
authors.forename: Xuehui
authors.initials: X
authors.lastname: Wang
authors.forename: Yufan
authors.initials: Y
authors.lastname: Wang
authors.forename: Jinsheng
authors.initials: J
authors.lastname: Sun
authors.forename: Weijun
authors.initials: W
authors.lastname: Yang
mesh_terms: D000818:Animals; D001483:Base Sequence; D003386:Brachyura / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D003001:Cloning, Molecular; D026461:Ecdysteroids / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D007447:Invertebrate Hormones / Q000737:chemistry / Q000494:pharmacology / Q000502:physiology*; D008969:Molecular Sequence Data; D018978:Molting / Q000187:drug effects / Q000502:physiology*; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein
chemical_list: D026461:Ecdysteroids; D007447:Invertebrate Hormones; D012333:RNA, Messenger; D011994:Recombinant Proteins; C012445:molt-inhibiting hormone