Protein hydrolysis by Colorado potato beetle, Leptinotarsa decemlineata, digestive proteases: the catalytic role of cathepsin D

摘要

Although several studies were carried out over the past 15 years to assess the nature and characteristics of digestive proteases in herbivorous insects, little is known about the relative importance of these enzymes in the hydrolysis of specific dietary proteins. In this study, the involvement of Colorado potato beetle (CPB; Leptinotarsa decemlineata) aspartate, cysteine, and serine digestive proteinases was assessed in the degradation of two model substrates: ribulose biphosphate carboxylase/oxygenase, the major protein in potato leaves, and the pro-region of papaya proteinase IV, a cysteine protease inhibitor (PI) susceptible to proteolysis by the insect "nontarget" proteases. As shown by the use of various combinations of diagnostic PIs specific to the different classes of CPB proteinases, the insect aspartate (cathepsin D-like) proteinase activity is important in initiating the hydrolysis of both proteins when the insect is feeding on potato, while cysteine (cathepsin B/ cathepsin H-like) andserine (chymotrypsin-like) proteinase activities would be involved in subsequent steps of the hydrolytic process. Similar observations were made with diet-induced variants of the insect protease system, suggesting the importance of digestive cathepsin Dand the sequential hydrolysis of dietary proteins in CPB, regardless of the diet ingested. Based on these observations, a preliminary model is proposed to explain dietary protein hydrolysis in CPB, also taking into account the information currently available about the distribution of digestive endo- and exopeptidases in the midgut of CPB. The potential of a wound-induced cathepsin D inhibitor from tomato in developing CPB-resistant transgenic potato lines is also discussed, after demonstrating the "pepstatin-like" effect of a recombinant form of this proteinaceous inhibitor against the insect cathepsin D.