Biochemical, Molecular, and Functional Characterization of PISCF-Allatostatin, a Regulator of Juvenile Hormone Biosynthesis in the Mosquito Aedes aegypti

• Published in: The Journal of biological chemistry 2006-11, Vol.281 (45), p.34048-34055
• Main Authors: Li, Yiping, Hernandez-Martinez, Salvador, Fernandez, Facundo, Mayoral, Jaime G., Topalis, Pantelis, Priestap, Horacio, Perez, Mario, Navare, Arti, Noriega, Fernando G.
• Format: Article
• Language: English
• Subjects: Aedes - drug effects; Aedes - growth & development; Aedes - metabolism; Aedes aegypti; Amino Acid Sequence; Animals; Chromatography, High Pressure Liquid; Cloning, Molecular; Corpora Allata - drug effects; Corpora Allata - metabolism; Enzyme-Linked Immunosorbent Assay; Female; Hormone Antagonists - chemical synthesis; Hormone Antagonists - chemistry; Hormone Antagonists - pharmacology; Insect Hormones - biosynthesis; Insect Hormones - genetics; Juvenile Hormones - biosynthesis; Juvenile Hormones - genetics; Molecular Sequence Data; Neuropeptides - chemical synthesis; Neuropeptides - chemistry; Neuropeptides - pharmacology; Peptide Fragments - immunology; Peptide Fragments - metabolism; Polymerase Chain Reaction; Rabbits; Sequence Homology, Amino Acid; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M606341200

Aedes aegypti PISCF-allatostatin or allatostatin-C (Ae-AS-C) was isolated using a combination of high performance liquid chromatography and enzyme-linked immunosorbent assay (ELISA). The matrix-assisted laser desorption/ionization time-of-flight (TOF) mass spectrum of positive ELISA fractions revealed a molecular mass of 1919.0 Da, in agreement with the sequence qIRYRQCYFNPISCF, with bridged cysteines. This sequence was confirmed by matrix-assisted laser desorption/ionization tandem TOF/TOF mass spectrometry analysis. The corresponding Ae-AS-C cDNA was amplified by PCR, and the sequence of the peptide was confirmed. An in vitro radiochemical assay was used to study the inhibitory effect of synthetic Ae-AS-C on juvenile hormone biosynthesis by the isolated corpora allata (CA) of adult female A. aegypti. The inhibitory action of synthetic Ae-AS-C was dose-dependent; with a maximum at 10–9m. Ae-AS-C showed no inhibitory activity in the presence of farnesoic acid, an immediate precursor of juvenile hormone, indicating that the Ae-AS-C target is located before the formation of farnesoic acid in the pathway. The sensitivity of the CA to inhibition by Ae-AS-C in the in vitro assay varied during the adult life; the CA was most sensitive during periods of low synthetic activity. In addition, the levels of Ae-AS-C in the brain were studied using ELISA and reached a maximum at 3 days after eclosion. These studies suggest that Ae-AS-C is an important regulator of CA activity in A. aegypti.