Cloning and characterization of a basic Cysteine-like protease (Cathespsin L1) expressed in the gut of larval Diaprepes abbreviatus L. (Coleoptera: Curculionidae)

Authors: BEN-MAHMOUD, S - University Of Florida; Ramos, John; Shatters, Robert - Bob; ROUGE', PIERRE - University Of Toulouse; POWELL, CHARLES - University Of Florida; SMAGGHE, GUY - Ghent University; BOROVSKY, DOV - Dov Borovsky

Submitted to: Journal of Insect Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/5/2014
Publication Date: 11/13/2014
Citation: Ben-Mahmoud, S., Ramos, J.E., Shatters, R.G., Rouge', P., Powell, C.A., Smagghe, G., Borovsky, D. 2014. Cloning and characterization of a basic cysteine-like protease (cathespsin L1) expressed in the gut of larval Diaprepes abbreviatus L. (Coleoptera: Curculionidae). Journal of Insect Physiology. (72):1-13.

Interpretive Summary: The Diaprepes root weevil is an invasive insect that has causes millions of dollars of damage to the Florida citrus industry annually. Control of this weevil is problematic because the larvae are in the soil in close association with citrus roots and therefore difficult to reach with effective pesticides. Furthermore, both the adult and larvae of this insect can feed on hundreds of different plant hosts resulting in a reservoir insects that can migrate into citrus fields at a time after a treatment has been applied and is no longer affective. This paper presents work on the analysis of an important digestive process, protein digestion, in the insect gut and specifically on the characterization of a predominant protease the insect uses to digest ingested proteins. This proteolytic enzyme has unique enzymatic characteristics for this class of proteins (cathepsins). These findings have led us to speculate that the unique characteristics of this enzyme may explain one mechanism this insect uses to avoid many plant defense strategies that typically block insect feeding and therefore explain how this insect can have such a broad range of plant hosts. This enzymatic characterization research has led us to hypothesize that developing inhibitors that block this enzymes function may provide a robust control strategy for this insect. Research is now continuing to develop inhibitors that could be deployed commercially.

Technical Abstract: Diaprepes abbreviatus is an important pest that causes extensive damage to citrus in the USA. Analysis of an expressed sequence tag (EST) library from the digestive tract of larvae and adult D. abbreviatus identified cathepsins as major putative digestive enzymes. One class, sharing amino acid sequence identity with cathepsin L’s, was the most abundant in the EST dataset representing 14.4% and 3.6% of the total sequences in feeding larvae and adults, respectively. The predominant cathepsin (Da-CTSL1) among this class was further studied. Three dimensional modeling of the protein sequence showed that the mature Da-CTSL1 protein folds into an expected Cathepsin L structure producing a substrate binding pocket with appropriate positioning of conserved amino acid residues. A full-length cDNA was obtained and the proCTSL1 encoding sequence was expressed in RosettaTM Escherichia coli cells engineered to express tRNAs specific for eukaryotic codon usage. The Da-CTSL1 was expressed as a fusion protein with GST and His6 tags and purified in the presence of 1% Triton X-100 by Ni-NTA affinity and size exclusion chromatography. Recombinant mature Da-CTSL1 (23 KDa) exhibits optimal activity at pH 8, rather than at acidic pH that was shown of all previously characterized cathepsins L. Substrate specificity supports the hypothesis that Da-CTSL1 is a unique basic cathepsin L and protease inhibitor studies also suggest unique activity, unlike other characterized acidic cathepsin Ls. This paper describes for the first time a prokaryotic expression system for the production of a functional eukaryotic cathepsin L 1 from larval gut of D. abbreviatus.