Biochemical, molecular, and functional characterization of PISCF-allatostatin, a regulator of juvenile hormone biosynthesis in the mosquito Aedes aegypti.
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(-9) m. 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.
Li
Yiping
Y
Department of Biological Sciences, Florida International University, Miami, Florida 33199, USA.
Hernandez-Martinez
Salvador
S
Fernandez
Facundo
F
Mayoral
Jaime G
JG
Topalis
Pantelis
P
Priestap
Horacio
H
Perez
Mario
M
Navare
Arti
A
Noriega
Fernando G
FG
D000330:Aedes / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003335:Corpora Allata / Q000187:drug effects* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006727:Hormone Antagonists / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D007301:Insect Hormones / Q000096:biosynthesis* / Q000235:genetics; D007605:Juvenile Hormones / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D006727:Hormone Antagonists; D007301:Insect Hormones; D007605:Juvenile Hormones; D009479:Neuropeptides; D010446:Peptide Fragments; C076523:allatostatin