A hybrid, broad-spectrum inhibitor of Colorado potato beetle aspartate and cysteine digestive proteinases

Protein engineering approaches are currently being devised to improve the inhibitory properties of plant proteinose inhibitors against digestive proteiflases of herbivorous insects. Here we engineered a potent hybrid inhibitor of aspartate and cysteine digestive proteinases found in the Colorado potato beetle, Leptinoturso decemlineata Say. Three cathepsin D inhibitors (CDIS) from stressed potato and tomato were first compared in their potency to inhibit digestive cathepsin D-like activity of the insect. After showing the high inhibitory potency of tomato CDI (M-i similar to 21 kDa), a similar to 33-kDa hybrid inhibitor was generated by fusing this inhibitor to the N terminus of (am cystatin II (CCII), a potent inhibitor of cysteine proteinases, Inhibitory assays with recombinant forms of CDI, CCII, and CDI-CCII expressed in Escherichia coli showed the CDI-CCII fusion to exhibit a dual inhibitory effect against cystatin-sensitive and cathepsin D-like enzymes of the potato beetle, resulting in detrimental effects against 3rd-instar larvae fed the hybrid inhibitor. The inhibitory potency of CDI and CCII was not altered after their fusion, as suggested by IC50 values for the interaction of CDI-CCII with target proteinoses similar to those measured for each inhibitor, These observations suggest the potential of plant CDls and cystatins as functional inhibitory modules for the design of effective broad-spectrum, hybrid inhibitors of herbivorous insect cysteine and aspartate digestive proteinases.