title: Purification and characterization of a mandibular organ protein from the American lobster, Homarus americanus: a putative farnesoic acid O-methyltransferase.
abstract: Methyl farnesoate (MF) appears to have important roles in the development, morphogenesis, and reproduction of crustaceans. To better understand the regulation of MF synthesis, we studied farnesoic acid O-methyltransferase (FAOMeT, the final enzyme in the MF biosynthetic pathway) in the American lobster (Homarus americanus). FAOMeT purified from mandibular organ (MO) homogenates had a MW of approximately 38,000. The sequences of trypsin fragments of purified FAOMeT were used to design PCR primers to amplify a cDNA fragment, which was used to isolate a full-length cDNA containing a single open reading frame (ORF) of 828 bp encoding a protein of 276 amino acids. The deduced amino acid sequence of this putative FAOMeT protein contained two copies of a conserved approximately 135 amino acid domain we term the CF (CPAMD8/FAOMeT) domain; single copies of this domain also occur in the human CPAMD8 protein (a member of the alpha-2 macroglobulin family) and an uncharacterized Drosophila protein. The recombinant protein had no FAOMeT activity. However, its addition to MO homogenates from eyestalk ablated (ESA) lobsters increased enzyme activity by up to 75%, suggesting that FAOMeT may require an additional factor or modification (e.g., phosphorylation) for its activation. The mRNA for the putative FAOMeT was primarily found in the proximal region of the MO, the predominant site of MF synthesis. FAOMeT transcripts were found in muscle tissue from ESA animals, but not in green gland, hepatopancreas, or in muscle tissue from intact animals. FAOMeT mRNA was also detected in embryos and larval stages. This is the first comprehensive report of this protein in the lobster, and is an important step in elucidating the functions of MF in these animals.
authors.affiliation: Biology/Chemistry Division, Purdue University North Central, Westville, IN 46391, USA. cholford@pnc.edu
authors.forename: Kenneth C
authors.initials: KC
authors.lastname: Holford
authors.forename: Kevin A
authors.initials: KA
authors.lastname: Edwards
authors.forename: William G
authors.initials: WG
authors.lastname: Bendena
authors.forename: Stephen S
authors.initials: SS
authors.lastname: Tobe
authors.forename: Zhiwei
authors.initials: Z
authors.lastname: Wang
authors.forename: David W
authors.initials: DW
authors.lastname: Borst
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004625:Embryo, Nonmammalian / Q000201:enzymology / Q000378:metabolism; D004926:Escherichia coli; D007814:Larva / Q000201:enzymology / Q000235:genetics; D008334:Mandible / Q000201:enzymology* / Q000378:metabolism; D008780:Methyltransferases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D008121:Nephropidae / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA
chemical_list: D011994:Recombinant Proteins; D008780:Methyltransferases; C032433:farnesoic acid O-methyltransferase