title: Wheat Subtilisin/Chymotrypsin Inhibitor (WSCI) as a scaffold for novel serine protease inhibitors with a given specificity.
abstract: WSCI (Wheat Subtilisin/Chymotrypsin Inhibitor) is a small protein belonging to the Potato inhibitor I family exhibiting a high content of essential amino acid. In addition to bacterial subtilisins and mammalian chymotrypsins, WSCI inhibits chymotrypsin-like activities isolated from digestive traits of a number of insect larvae. In vivo, as suggested for many plant proteinase inhibitors, WSCI seems to play a role of natural defence against attacks of pests and pathogens. The functional region of WSCI, containing the inhibitor reactive site (Met48-Glu49), corresponds to an extended flexible loop (Val42-Asp53) whose architecture is somehow stabilized by a number of secondary interactions established with a small β-sheet located underneath. The aim of this study was to employ a WSCI molecule as a stable scaffold to obtain recombinant inhibitors with new acquired anti-proteinase activity or, alternatively, inactive WSCI variants. A gene sequence coding for the native WSCI, along with genes coding for muteins with different specficities, could be exploited to obtain transformed non-food use plants with improved insect resistance. On the other hand, the genetic transformation of cereal plants over-expressing inactive WSCI muteins could represent a possible strategy to improve the nutritional quality of cereal-based foods, without risk of interference with human or animal digestive enzymes. Here, we described the characterization of four muteins containing single/multiple amino acid substitutions at the WSCI reactive site and/or at its proximity. Modalities of interaction of these muteins with proteinases (subtilisin, trypsin and chymotrypsin) were investigated by time course hydrolysis and molecular simulations studies.
authors.affiliation: Dipartimento per la Innovazione nei sistemi Biologici, Agroalimentari e Forestali, Università degli Studi della Tuscia, I-01100 Viterbo, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Tedeschi
authors.forename: Antimo
authors.initials: A
authors.lastname: Di Maro
authors.forename: Angelo
authors.initials: A
authors.lastname: Facchiano
authors.forename: Susan
authors.initials: S
authors.lastname: Costantini
authors.forename: Angela
authors.initials: A
authors.lastname: Chambery
authors.forename: Natalia
authors.initials: N
authors.lastname: Bruni
authors.forename: Valeria
authors.initials: V
authors.lastname: Capuzzi
authors.forename: Anna Grazia
authors.initials: AG
authors.lastname: Ficca
authors.forename: Elia
authors.initials: E
authors.lastname: Poerio
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D020134:Catalytic Domain; D002918:Chymotrypsin / Q000037:antagonists & inhibitors; D006801:Humans; D019272:Leukocyte Elastase / Q000037:antagonists & inhibitors; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D015842:Serine Proteinase Inhibitors / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D014908:Triticum / Q000378:metabolism*; D014361:Trypsin Inhibitors / Q000032:analysis
chemical_list: D010940:Plant Proteins; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D014361:Trypsin Inhibitors; C504099:WSCI protein, Triticum; D002918:Chymotrypsin; D010196:Pancreatic Elastase; D019272:Leukocyte Elastase