title: A hybrid, broad-spectrum inhibitor of Colorado potato beetle aspartate and cysteine digestive proteinases.
abstract: Protein engineering approaches are currently being devised to improve the inhibitory properties of plant proteinase inhibitors against digestive proteinases of herbivorous insects. Here we engineered a potent hybrid inhibitor of aspartate and cysteine digestive proteinases found in the Colorado potato beetle, Leptinotarsa 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(r) approximately 21 kDa), an approximately 33-kDa hybrid inhibitor was generated by fusing this inhibitor to the N terminus of corn 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 IC(50) values for the interaction of CDI-CCII with target proteinases similar to those measured for each inhibitor. These observations suggest the potential of plant CDIs and cystatins as functional inhibitory modules for the design of effective broad-spectrum, hybrid inhibitors of herbivorous insect cysteine and aspartate digestive proteinases.
authors.affiliation: Département de Phytologie, CRH/INAF, Université Laval, Québec, Canada.
authors.forename: France
authors.initials: F
authors.lastname: Brunelle
authors.forename: Cécile
authors.initials: C
authors.lastname: Girard
authors.forename: Conrad
authors.initials: C
authors.lastname: Cloutier
authors.forename: Dominique
authors.initials: D
authors.lastname: Michaud
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors*; D002402:Cathepsin D / Q000037:antagonists & inhibitors; D001517:Coleoptera / Q000187:drug effects / Q000201:enzymology*; D015891:Cystatins / Q000235:genetics / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism*; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D004063:Digestion / Q000187:drug effects; D004435:Eating / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel; D020128:Inhibitory Concentration 50; D007814:Larva / Q000187:drug effects / Q000254:growth & development; D018551:Solanum lycopersicum / Q000378:metabolism; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D011198:Solanum tuberosum / Q000378:metabolism
chemical_list: D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D017931:DNA Primers; D018076:DNA, Complementary; D010940:Plant Proteins; D011994:Recombinant Proteins; C077834:cystatin protein, Zea mays; D016282:Aspartic Acid Endopeptidases; D002402:Cathepsin D