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ARS Home » Southeast Area » Fort Pierce, Florida » U.S. Horticultural Research Laboratory » Subtropical Insects and Horticulture Research » Research » Publications at this Location » Publication #386476
Research Project: IPM Method for Control of Insect Pests and Transmitted Diseases of Orchard Crops

Location: Subtropical Insects and Horticulture Research

Title: Cloning and Characterization of Aedes aegypti Trypsin Modulating Oostatic Factor (TMOF) Gut Receptor

Author
item BOROVSKY, DOV - University Of Colorado
item DECKERS, KATO - Zoological Institute
item VANHOVE, ANNE CATHERINE - Zoological Institute
item VERSTRAETE, MAUD - Zoological Institute
item ROUGE, PIERRE - Herbier De Guyane, Institut De Recherche Pour Le Développement (IRD)
item Shatters, Robert - Bob
item POWELL, CHARLES - Southwest Florida Research And Education Center

Submitted to: Biomolecules EISSN 2218-273X
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/20/2021
Publication Date: 7/23/2021
Citation: Borovsky, D., Deckers, K., Vanhove, A., Verstraete, M., Rouge, P., Shatters, R.G., Powell, C.A. 2021. Cloning and Characterization of Aedes aegypti Trypsin Modulating Oostatic Factor (TMOF) Gut Receptor. Biomolecules EISSN 2218-273X. https://doi.org/10.3390/biom11070934.
DOI: https://doi.org/10.3390/biom11070934

Interpretive Summary: Mosquitoes synthesize a peptide hormone called trypsin modulating Oostatic Factor (TMOF) that acts on cells of the insect gut to block the synthesis of enzymes that digest the blood meal taking in by the female. This peptide, when fed to immature mosquito larvae, induces mortality and thus has been used commercially as a natural insecticide against mosquitoes. Understanding how this enzyme works would allow further optimization of the mosquito control method and would also allow adapting this natural insecticidal strategy to other insects. Part of understanding how TMOF works in understanding how the peptide interacts with proteins on the surface of the gut cells (receptors) upon which this hormone acts. In this paper, we identify and characterize the gut cell receptor that binds TMOF, and as a result, present a new molecular target for pest insect control.

Technical Abstract: Trypsin Modulating Oostatic Factor (TMOF) receptor was solubilized from the guts of female Ae. Aegypti and cross linked to His6-TMOF and purified by Ni affinity chromatography. SDS PAGE identified two protein bands (45 and 61 kDa). The bands were cut digested and analyzed using MS/MS identifying a protein sequence (1306 amino acids) in the genome of Ae. aegypti. The mRNA of the receptor was extracted, the cDNA sequenced and cloned into pTAC-MAT-2. E. coli SbmA- was transformed with the recombinant plasmid and the receptor was expressed in the inner membrane of the bacterial cell. The binding kinetics of TMOF-FITC was then followed showing that the cloned receptor exhibits high affinity to TMOF (KD = 113.7 ± 18 nM ± SEM and Bmax = 28.7 ± 1.8 pmol ± SEM). Incubation of TMOF-FITC with E. coli cells that express the receptor show that the receptor binds TMOF and imports it into the bacterial cells, indicating that in mosquitoes the receptor imports TMOF into the gut epithelial cells. A 3D modeling of the receptor indicates that the receptor has ATP binding sites and TMOF transport into recombinant E. coli cells is inhibited with ATPase inhibitors Na Arsenate and Na Azide.