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Biomolecules
Volume 11
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10.3390/biom11070934
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Article

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

by
Dov Borovsky
1,*,
Kato Deckers
2,
Anne Catherine Vanhove
2,
Maud Verstraete
2,
Pierre Rougé
3,
Robert G. Shatters, Jr.
4 and
Charles A. Powell
5
1
Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz School of Medicine, Aurora, CO 80045, USA
2
Zoological Institute, Katholieke Universiteit Leuven, 3000 Leuven, Belgium
3
UMR 152 Pharma-Dev, Faculté des Sciences Pharmaceutiques, Institut de Recherche et Développement, Université Toulouse 3, F-31062 Toulouse, France
4
USDA ARS, Subtropical Horticultural Laboratory, 2001 Rock Road, Ft. Pierce, FL 34945, USA
5
UF-IFAS Indian River Research and Education Center, Fort Pierce, FL 34945, USA
*
Author to whom correspondence should be addressed.
Biomolecules 2021, 11(7), 934; https://doi.org/10.3390/biom11070934
Submission received: 8 June 2021 / Revised: 18 June 2021 / Accepted: 20 June 2021 / Published: 23 June 2021
(This article belongs to the Special Issue Insect Receptors: Biochemical, Physiological and Molecular Studies)
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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.
Keywords: mosquito; ABC-TMOF receptor; molecular modeling; sequencing; kinetic characterization; fluorescence microscopy; E. coli mosquito; ABC-TMOF receptor; molecular modeling; sequencing; kinetic characterization; fluorescence microscopy; E. coli
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MDPI and ACS Style

Borovsky, D.; Deckers, K.; Vanhove, A.C.; Verstraete, M.; Rougé, P.; Shatters, R.G., Jr.; Powell, C.A. Cloning and Characterization of Aedes aegypti Trypsin Modulating Oostatic Factor (TMOF) Gut Receptor. Biomolecules 2021, 11, 934. https://doi.org/10.3390/biom11070934

AMA Style

Borovsky D, Deckers K, Vanhove AC, Verstraete M, Rougé P, Shatters RG Jr., Powell CA. Cloning and Characterization of Aedes aegypti Trypsin Modulating Oostatic Factor (TMOF) Gut Receptor. Biomolecules. 2021; 11(7):934. https://doi.org/10.3390/biom11070934

Chicago/Turabian Style

Borovsky, Dov, Kato Deckers, Anne Catherine Vanhove, Maud Verstraete, Pierre Rougé, Robert G. Shatters, Jr., and Charles A. Powell. 2021. "Cloning and Characterization of Aedes aegypti Trypsin Modulating Oostatic Factor (TMOF) Gut Receptor" Biomolecules 11, no. 7: 934. https://doi.org/10.3390/biom11070934

APA Style

Borovsky, D., Deckers, K., Vanhove, A. C., Verstraete, M., Rougé, P., Shatters, R. G., Jr., & Powell, C. A. (2021). Cloning and Characterization of Aedes aegypti Trypsin Modulating Oostatic Factor (TMOF) Gut Receptor. Biomolecules, 11(7), 934. https://doi.org/10.3390/biom11070934

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