Molecular and structural characterization of a trypsin highly expressed in larval stage of Zabrotes subfasciatus

Abstract

The Mexican bean weevil, Zabrotes subfasciatus, feeds on several seeds such as Vigna unguiculata, Phaseolus vulgaris, and Pisum sativum, causing severe crop losses. This ability to obtain essential compounds from different diets could possibly be explained due to a wide variability of digestive proteinases present in the weevil's midgut. These may improve digestion of many different dietary proteins. Coleopteran serine-like proteinases have not been thoroughly characterized at the molecular level. In this report, a full-length cDNA encoding a trypsin-like protein, named ZsTRYP, was isolated from Z. subfasciatus larvae using RT-PCR, 5' and 3' RACE techniques. The quantitative real-time PCR analysis strongly correlated the Zstryp transcript accumulation to the major feeding developmental larval stage. Zstryp cDNA was subcloned into pET101 vector and expressed in a Escherichia coli BL21(DE3) strain. Nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography was used to purify a 29.0-kDa recombinant enzyme. The purified ZsTRYP was then assayed with several synthetic peptide substrates and also challenged with different inhibitors. The biochemical data allowed us to classify ZsTRYP as a trypsin. Moreover, homology modeling analysis indicated a typical trypsin structural core and a conserved catalytic triad (His(41), Asp(86), and Ser(182)).

Identification of a trypsin gene from Scylla paramamosain and its expression profiling during larval development

“…FJ917598) encoding a 259-amino-acid protein (Figure 1). These characteristics were consistent with previous reports (Klein et al, 1996;Magalhaes et al, 2007;Rudenskaya et al, 2004) for other trypsins. Speaking with strict definition, the sequence cloned in this study was a trypsinogen gene.…”

Exclusive rewards in mutualisms: ant proteases and plant protease inhibitors create a lock–key system to protectAcaciafood bodies from exploitation

“…; Magalhães et al . ). Thus, both the dominance of trypsin‐like activity and the low sensitivity of chymotrypsin‐like activity to Kunitz‐type PIs appear to be common features of beetles.…”

“…In fact, in a recent study, trypsin and chymotrypsin were identified as the most active proteases in P. truncatus, and trypsin-like activity was more sensitive than chymotrypsin to plant PIs extracted from the seeds of tepary bean (Phaseolus acutifolius), soybean (G. max, both: Fabaceae) and chan (Hyptis suaveolens; Lamiaceae) (Castro-Guill en et al 2012). Similarly, chymotrypsin-like activities in Z. subfasciatus were not inhibited by natural PIs from soybean and common bean (Phaseolus vulgaris) (Silva et al 2001;Magalhães et al 2007). Thus, both the dominance of trypsin-like activity and the low sensitivity of chymotrypsin-like activity to Kunitz-type PIs appear to be common features of beetles.…”

Resistance of important bean genotypes to the Mexican bean beetle [Zabrotes subfasciatus (Bohemann)] during storage and its control with chemical synthetic and botanical insecticides

“…The Mexican bean beetle (MBB), Zabrotes subfasciatus (Bohemann) (Coleoptera: Chrysomelidae: Bruchinae), is a key pest of stored beans, attacking beans belong to the genera Phaseolus, Vicia and Vigna (Hill, 2002). In Brazil, MBB also infests other types of beans such as Vicia and Vigna and other legumes such as peas (Pisum sativum L. Fabaceae) probably because of the presence of a great number of digestive proteinases that exist in the insect's midgut (Magalhães et al, 2007). Infested seeds lose mass, quality and nutritional value, the latter two due to the presence of frass, eggs and dead insects in the product.…”