Harnessing the functional diversity of plant cystatins to design inhibitor variants highly active against herbivorous arthropod digestive proteases

Protein engineering approaches have been proposed to improve the inhibitory properties of plant cystatins against herbivorous arthropod digestive proteases. These approaches typically involve the site-directed mutagenesis of functionally relevant amino acids, the production and selection of improved inhibitory variants by molecular phage display procedures, or the design of bi/multifunctional translational fusions integrating one or several cystatin inhibitory domains. Here, we propose a new approach where the function-related structural elements of a cystatin are substituted by the corresponding elements of an alternative cystatin. Cys protease inhibitory assays were first performed with 20 representative plant cystatins and model Cys proteases, including herbivorous arthropod digestive proteases, to appreciate the extent of functional variability among plant cystatin protein family members. The most, and less, potent of these cystatins were then used as donors of structural elements to create hybrids of tomato cystatin SlCYS8 used as a model recipient inhibitor. Our data confirm the wide variety of cystatin protease inhibitory profiles among plant taxa. They also demonstrate the usefulness of these proteins as a pool of discrete structural elements for the design of cystatin variants with improved potency against herbivorous pest digestive Cys proteases.

Cystatins: a versatile family

Cystatins are small proteins, typically composed of 100–120 amino acids, which together with similar proteins devoid of inhibitory properties, belong to a cystatin ‘superfamily’. Cystatins can do more than just inhibit proteases: two important aspects described here are aggregation properties linked to misfolding diseases and the unique ability of monellin, a plant cystatin, to elicit sweet taste. The explanation of the puzzling phenomenon of ‘sweet proteins’ required an in-depth structural study of monellin, also regarding the causes of the high thermal stability of its single chain structure. The detailed mechanisms by which cystatins aggregate could be relevant in the study of misfolding diseases involving cystatins. They are reviewed here with emphasis on 3D domain swapping, typical of aggregating cystatins. While studying monellin, we noticed that it aggregates in a conventional way, probably through the cross-β spine mechanism. However, several cystatins derived from oryzacystatin_I to emulate the taste behavior of monellin aggregate via different mechanisms.

Antifungal Activity of a Plant Cystatin

Purified chestnut cystatin inhibited the growth of the phytopathogenic fungi Botrytis cinerea, Colletotrichum graminicola, and Septoria nodorum, but not that of the saprophyte Trichoderma viride. Furthermore, the cystatin strongly affected the protease activity of B. cinerea but had no effect on the protease activity of T. viride. These results suggest that chestnut cystatin contributes to plant defense against phytopathogenic fungi.