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Identification and characterization of juvenile hormone esterase gene from the yellow fever mosquito, Aedes aegypti.

ABSTRACT: Juvenile hormone esterase (JHE) plays an important role in regulating juvenile hormone titers. Recent sequencing and annotation of the Aedes aegypti genome identified ten putative jhe gene sequences. Analysis of these ten putative jhe gene sequences showed that only three of them, EAT43357, EAT43353 and EAT43354 contained GQSAG motif and showed high sequence similarity with the sequences of jhe genes identified from other insect species. To determine which putative jhe gene(s) code for functional JHE, the mRNA profiles of EAT43357, EAT43353 and EAT43354 were measured during the final instar larval and pupal stages by using quantitative real-time reverse transcriptase polymerase chain reaction (PCR). The mRNA for EAT43357 was detected during the late final instar larval stage. In contrast, EAT43354 mRNA was detected only during the pupal stage and EAT43353 mRNA was detected only during the larval stage. The mRNA of EAT43357 was detected in both fat body and midgut tissues. JHE enzyme levels gradually increased during the final instar larval stage reaching a peak at 42 h after ecdysis into the final instar larval stage. The mRNA expression profiles of EAT43357 correlate with the developmental expression profiles of JHE enzyme activity suggesting that this gene may encode for a functional JHE. The EAT43357 and EAT43354 cDNA were expressed in a baculovirus system. Proteins isolated from Sf9 cells infected with recombinant baculovirus expressing EAT43357 but not EAT43354 gene exhibited JHE activity confirming that EAT43357 gene codes for a functional JHE enzyme.

SUBMITTER: Bai H

PROVIDER: S-EPMC2020842 | biostudies-literature | 2007 Aug

REPOSITORIES: biostudies-literature

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Identification and characterization of juvenile hormone esterase gene from the yellow fever mosquito, Aedes aegypti.

Bai Hua H Ramaseshadri Parthasarathy P Palli Subba Reddy SR

Insect biochemistry and molecular biology 20070529 8

Juvenile hormone esterase (JHE) plays an important role in regulating juvenile hormone titers. Recent sequencing and annotation of the Aedes aegypti genome identified ten putative jhe gene sequences. Analysis of these ten putative jhe gene sequences showed that only three of them, EAT43357, EAT43353 and EAT43354 contained GQSAG motif and showed high sequence similarity with the sequences of jhe genes identified from other insect species. To determine which putative jhe gene(s) code for functiona ...[more]

PMID: 17628281

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Project description:Recent studies reported that JH-regulated phosphorylation status of the JH-receptor complex contributes to its transcription activity in Aedes aegypti. However, phosphorylation sites of these proteins have not yet been identified. In this study, we found that the fusion of an EGFP tag to Ae. aegypti Kr-h1 (AaKr-h1) and Met (AaMet) improved their stability in mosquito Aag-2 cells, which allowed their purification. The liquid chromatography and tandem mass spectrometry analysis of the purified AaKr-h1 showed that the phosphoserine residue at position 694, located in the evolutionarily conserved SVIQ motif, is dephosphorylated when the cells are exposed to JH. The AaKr-h1 dephosphorylation mutant (S694V) showed significantly higher activity in inducing the luciferase gene regulated by JH response elements. The phosphorylation profile of Met also changed after exposing Aag-2 cells to JH III. The Ser-77 and Ser-710 residues of Met were phosphorylated after JH III treatment. In contrast, the two phosphoserine residues at positions 73 and 747 were dephosphorylated after JH III treatment. JH exposure also induced transient and reversible phosphorylation of Thr-664 and Ser-723 residues. Overall, these data show that JH induces changes in post-translational modifications of AaMet and AaKr-h1. SIGNIFICANCE: Female Aedes aegypti mosquitoes are known to vector many disease agents, including Zika virus, dengue virus chikungunya virus, and Mayaro and yellow fever virus. In the present study, we developed an efficient method to prepare Ae. aegypti Met and Kr-h1, which are typically difficult to produce and purify, using a mosquito cell line expression system. A liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approaches were utilized to map the phosphorylation profiles of the isolated proteins. We then monitored the changes induced by JH activation in the phosphorylation profiles to check if the JH modulates post-translation modification of its key transcription factors. We found that the JH induced alterations in the phosphorylation profiles of the multiple residues of AaMet. In contrast, activation of the JH signaling pathway was accompanied by dephosphorylation of AaKr-h1 at phosphoserine-694, increasing its transcriptional activity. In addition, S694 of AaKr-h1 was located in the RMSSVIQYA motif highly conserved in orthologous proteins from other insect species. These results can help us further understand how JH modulates its key transcription factors and provide a basis for the development of novel insect control strategies.

Dataset Information

Identification and characterization of juvenile hormone esterase gene from the yellow fever mosquito, Aedes aegypti.

Publications

Identification and characterization of juvenile hormone esterase gene from the yellow fever mosquito, Aedes aegypti.

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