title: Tailoring the specificity of a plant cystatin toward herbivorous insect digestive cysteine proteases by single mutations at positively selected amino acid sites.
abstract: Plant cystatins, similar to other defense proteins, include hypervariable, positively selected amino acid sites presumably impacting their biological activity. Using 29 single mutants of the eighth domain of tomato (Solanum lycopersicum) multicystatin, SlCYS8, we assessed here the potential of site-directed mutagenesis at positively selected amino acid sites to generate cystatin variants with improved inhibitory potency and specificity toward herbivorous insect digestive cysteine (Cys) proteases. Compared to SlCYS8, several mutants (22 out of 29) exhibited either improved or lowered potency against different model Cys proteases, strongly suggesting the potential of positively selected amino acids as target sites to modulate the inhibitory specificity of the cystatin toward Cys proteases of agronomic significance. Accordingly, mutations at positively selected sites strongly influenced the inhibitory potency of SlCYS8 against digestive Cys proteases of the insect herbivore Colorado potato beetle (Leptinotarsa decemlineata). In particular, several variants exhibited improved potency against both cystatin-sensitive and cystatin-insensitive digestive Cys proteases of this insect. Of these, some variants also showed weaker activity against leaf Cys proteases of the host plant (potato [Solanum tuberosum]) and against a major digestive Cys protease of the two-spotted stinkbug Perillus bioculatus, an insect predator of Colorado potato beetle showing potential for biological control. Overall, these observations suggest the usefulness of site-directed mutagenesis at positively selected amino acid sites for the engineering of recombinant cystatins with both improved inhibitory potency toward the digestive proteases of target herbivores and weaker potency against nontarget Cys proteases in the host plant or the environment.
authors.affiliation: Département de Phytologie, Pavillon des Services, Centre de Recherche en Horticulture, Université Laval, Quebec, Canada.
authors.forename: Marie-Claire
authors.initials: MC
authors.lastname: Goulet
authors.forename: Cindy
authors.initials: C
authors.lastname: Dallaire
authors.forename: Louis-Philippe
authors.initials: LP
authors.lastname: Vaillancourt
authors.forename: Moustafa
authors.initials: M
authors.lastname: Khalf
authors.forename: Amine M
authors.initials: AM
authors.lastname: Badri
authors.forename: Andreja
authors.initials: A
authors.lastname: Preradov
authors.forename: Marc-Olivier
authors.initials: MO
authors.lastname: Duceppe
authors.forename: Charles
authors.initials: C
authors.lastname: Goulet
authors.forename: Conrad
authors.initials: C
authors.lastname: Cloutier
authors.forename: Dominique
authors.initials: D
authors.lastname: Michaud
mesh_terms: D000818:Animals; D001517:Coleoptera / Q000201:enzymology*; D015891:Cystatins / Q000235:genetics* / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism; D020387:Food Chain; D020062:Heteroptera / Q000201:enzymology; D006790:Host-Parasite Interactions / Q000235:genetics*; D018551:Solanum lycopersicum / Q000235:genetics* / Q000378:metabolism / Q000469:parasitology; D016297:Mutagenesis, Site-Directed
chemical_list: D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors