Volume 114, Issue 2 e22042 pp. 1-24
RESEARCH ARTICLE

Heliothis virescens chymotrypsin is translationally controlled by AeaTMOF binding ABC putative receptor

Dov Borovsky

Corresponding Author

Dov Borovsky

Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz School of Medicine, Aurora, Colorado, USA

Correspondence Dov Borovsky, Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz School of Medicine, Aurora, CO 80045, USA.

Email: [email protected]

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Pierre Rougé

Pierre Rougé

UMR 152 Pharma-Dev, Faculté des Sciences Pharmaceutiques, Institut de Recherche et Développement, Université Toulouse 3, Toulouse, France

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First published: 01 August 2023

Abstract

Heliothis virescens larval chymotrypsin (GenBank accession number AF43709) was cloned, sequenced and its three dimensional (3D) conformation modeled. The enzyme's transcript was first detected 6 days after larval emergence and the transcript level was shown to fall between larval ecdysis periods. Comparisons between the activities of larval gut chymotrypsin and trypsin shows that chymotrypsin activity is only 16% of the total trypsin activity and the pH optimum of the larval chymotrypsin is between pH 9−10, however the enzyme also exhibited a broad activity between pH 4−6. Injections of AeaTMOF and several shorter analogues into 3rd instar larvae followed by Northern blot analyses showed that although the chymotrypsins activities were inhibited by 60%−80% the transcript level of the sequenced chymotrypsin was not reduced and was similar to controls in which the chymotrypsin activity was not inhibited, indicating that AeaTMOF and its analogues exert a translational control. Based on these observations a putative AeaTMOF receptor (ABCC4) homologous to the Ae. aegypti ABC receptor sequence was found in the H. virescens genome. 3D molecular modeling and docking of the AeaTMOF and several of its analogues to the ABCC4 receptor showed that it can bind AeaTMOF and its analogues as was shown before for the Ae. aegypti receptor.

Research Highlights

Heliothis virescens chymotrypsin was cloned from 3rd instar larval gut, characterized, three dimensional (3D) modeled and compared with gut trypsin. Chymotrypsin activity accounts for only 16% of the trypsin activity in the larval gut. The larval chymotrypsins exhibit a pH peak at basic pH, and broad activity at acidic pHs. AeaTMOF and its analogues inhibited the activity of the larval cloned chymotrypsin using translational control. Homology studies of the H. virescens genome using the AeaTMOF ABC receptor found a homologous ABCC4 putative AeaTMOF receptor. 3D molecular modeling and docking showed that it can bind AeaTMOF and several of its analogues in a similar fashion that was shown for the Ae. aegypti ABC receptor. A control binding experiment of AeaTMOF to H. virescens ABC transporter that transports lipids HvABCA2 and has low homology to the HvABCC4 transporter showed that the binding affinity of AeaTMOF to HvABCA2 is 1.6-fold lower than the binding of AeaTMOF to HvABCC4 and the binding is not specific using only hydrophobic and not hydrogen bonding that is commonly used by ligands when they bind their specific receptors.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are openly available in GenBank at https://www.ncbi.nih.gov, reference number AF237417.

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