WCI, a novel wheat chymotrypsin inhibitor: purification, primary structure, inhibitory properties and heterologous expression

Abstract

A novel chymotrypsin inhibitor, detected in the endosperm of Triticum aestivum, was purified and characterized with respect to the main physical-chemical properties. On the basis of its specificity, this inhibitor was named WCI (wheat chymotrypsin inhibitor). WCI is a monomeric neutral protein made up of 119 residues and molecular mass value of 12,933.40 Da. Automated sequence and mass spectrometry analyses, carried out on several samples of purified inhibitor, evidenced an intrinsic molecular heterogeneity due to the presence of the isoform [des-(Thr)WCI], accounting for about 40% of the total sample. In vitro, WCI acted as a strong inhibitor of bovine pancreatic chymotrypsin as well as of chymotryptic-like activities isolated from the midgut of two phytophagous insects, Helicoverpa armigera (Hüb.) and Tenebrio molitor L., respectively. No inhibitory activities were detected against bacterial subtilisins, bovine pancreatic trypsin, porcine pancreatic elastase or human leukocyte elastase. The primary structure of WCI was significantly similar (45.7-89.1%) to those of several proteins belonging to the cereal trypsin/α-amylase inhibitor super-family and showed the typical sequence motif of this crowed protein group. The cDNA of the inhibitor (wci-cDNA) was isolated from wheat immature caryopses and employed to obtain a recombinant product in E. coli. Experimental evidences indicated that the recombinant inhibitor was localized in the inclusion bodies from which it was recovered as soluble and partially active protein by applying an appropriate refolding procedure. WCI reactive site localization, as well as its inhibitory specificity, was investigated by molecular modeling approach.

Extensive structural variation in the Bowman-Birk inhibitor family in common wheat (Triticum aestivum L.)

“…6), consistent with analyses of other monocot BBIs [36,37]. However, previous studies have detected chymotrypsin inhibition in protein extracts from the wheat endosperm, so it is likely that this activity is performed by a distinct family of protease inhibitors, potentially members of the cereal trypsin/a-amylase inhibitor family [66,67].…”

Thermo-reversible inhibition makes aqualysin 1 from Thermus aquaticus a potent tool for studying the contribution of the wheat gluten network to the crumb texture of fresh bread

“…Higher concentrations of WFE lowered the Aq1 activity even more as the losses in hydrolytic activity at 50 °C were about 87% or 100% in the presence of 1.5 µl or 5.0 µl WFE, respectively. Wheat flour contains different inhibitors of serine peptidases and, more specifically, of subtilisin (Di Maro, et al, 2011;Mundy, Hejgaard, & Svendsen, 1984;Odani, Koide, & Ono, 1986;Østergaard, Rasmussen, Roberts, & Hejgaard, 2000;Poerio, Di Gennaro, Di Maro, Farisei, Ferranti, & Parente, 2003). Some of the latter may inhibit Aq1 since, as noted above, it is a member of the subfamily of the subtilisin-like peptidases (Matsuzawa, et al, 1988).…”

“…Aq1 is optimally active at 70 °C (Matsuzawa, et al, 1988;Rawlings, Barrett, & Bateman, 2010) which would imply that it is active in the time frame during which starch gelatinization, dough / crumb transition and gluten polymerization occur. Also, as wheat flour contains serine peptidase inhibitors (Di Maro, Farisei, Panichi, Severino, Bruni, Ficca, et al, 2011;Mundy, Hejgaard, & Svendsen, 1984;Odani, Koide, & Ono, 1986;Østergaard, Rasmussen, Roberts, & Hejgaard, 2000;Poerio, Di Gennaro, Di Maro, Farisei, Ferranti, & Parente, 2003), we speculated that its activity during the dough and early baking stages would be very limited and that its inhibition, if any, would be thermo-reversible. In other words, we reasoned that, if the above proved to be true, use of Aql may help us to understand the importance of the thermoset gluten network for bread crumb and texture.…”

KP4 to control Ustilago tritici in wheat: Enhanced greenhouse resistance to loose smut and changes in transcript abundance of pathogen related genes in infected KP4 plants

“…Interestingly, other genes involved in pathogen defense, i.e. subtilisin-chymotripsin inhibitor 2 [29] , PR4 protein 4 [20] , and a multi antimicrobial extrusion (MATE) protein [30] correlated positively only in KP4 plants (with the high mRNA abundance of the polyubiquitin gene) and not in non-KP4 control plants. Apparently, in KP4 plants, the expression of certain pathogen defense related genes is induced with increasing amounts of kp4 .…”