Functional expression of an insect cathepsin B-like counter-defence protein

Insects are capable of readjusting their digestive regimes in response to dietary challenge. Cowpea bruchids (Callosobruchus maculatus) strongly induce C. maculatus cathepsin B-like cysteine protease 1 (CmCatB1) transcripts when fed diet containing a soybean cysteine protease inhibitor soyacystatin N (scN). CmCatB1 shares significant sequence similarity with cathepsin B-like cysteine proteases. In this study, we isolated another cDNA, namely CmCatB2 that encodes a protein sequence otherwise identical to CmCatB1, but lacking a 70-amino-acid internal section. CmCatB1 and CmCatB2 probably resulted from alternate splicing events. Only the CmCatB1 transcript, however, exhibited differential expression in response to dietary scN. Further, this expression was only detectable in larvae, which is the developmental stage associated with food ingestion. The scN-activated and developmentally regulated CmCatB1 expression pattern suggests it may have a unique function in insect counter-defence against antinutritional factors. Heterologously expressed recombinant CmCatB1 protein exhibited enzymatic activity in a pH-dependent manner. Activity of the protein was inhibited by both the cysteine protease inhibitor E-64 and the cathepsin B-specific inhibitor CA-074, verifying its cathepsin B-like cysteine protease nature. Interestingly, the enzymatic activity was unaffected by the presence of scN. Together, we have provided functional evidence suggesting that CmCatB1 confers inhibitor-insensitive enzymatic activity to cowpea bruchids, which is crucial for insect survival when challenged by dietary protease inhibitors.