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

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 Rosetta™ 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 L1 from larval gut of D. abbreviatus.

Molecular Identification of Cysteine and Trypsin Protease, Effect of Different Hosts on Protease Expression, and Rnai Mediated Silencing of Cysteine Protease Gene in the Sunn Pest

“…Since proteases play important roles in a wide range of physiological processes include diet digestion, it is important to study proteases to use them in insect integrated pest management. Majority of proteases are transcribed during larval feeding belong to the serine (chymotrypsins, trypsins, and serine carboxypeptidases) and cysteine (cathespsins B and L) protease families (Edwards et al., ; Ben‐Mahmoud et al., ).…”

“…Terra (1990) emphasized coleopterans and hemipterans utilize cathepsins to digest their food. The most extensively studied role of cysteine proteases is their functions as digestive enzymes because they are the major digestive enzymes in Coleoptera, Diptera, and Hemiptera (Zhu-Salzman et al, 2003;Cristofoletti et al, 2005;Ben-Mahmoud et al, 2015).…”

“…Since cysteine proteases play critical functions in the digestion and development of the different insects (Goptar et al., ; Zhang et al., ; Ben‐Mahmoud et al., ), they can be targets for developing new tools in the integrated pest management. So in this study, after sequence identification, cathepsin‐L cysteine was chosen as a target gene for RNAi gene silencing.…”

Cloning and characterization of Aedes aegypti blood downregulated chymotrypsin II

“…This pH range is lower than the pH optimum of 7.0–8.0 that is reported for chymotrypsin. This observation, however, is not unique, cathepsin L has an optimal activity in the acidic pH range (5–6.5), however, reports of basic cysteine proteases are known (Ben‐Mahmoud et al, 2015; Otto & Schirmeister, 1997). Our pH plots for adult chymotrypsin indicate a broad activity between pH 6 and 10 (Figure 7).…”

Peptidomics as a Tool to Assess the Cleavage of Wine Haze Proteins by Peptidases from Drosophila suzukii Larvae

“…Cathepsin L1 is a digestive peptidase in many organisms, transforming proteins into absorbable peptides [ 63 ]. Insects secrete cathepsins from epithelial cells into the gut [ 64 ], although they are only active in acidic regions [ 65 ]. For example, cathepsin L1 has been described as an acidic endopeptidase that is unstable at neutral pH [ 66 ].…”