Proregion of Acanthoscelides obtectus cysteine proteinase: A novel peptide with enhanced selectivity toward endogenous enzymes

Abstract

Acanthoscelides obtectus is a devastating storage insect pest capable of causing severe bean crop losses. In order to maintain their own development, insect pest larvae feed continuously, synthesizing efficient digestive enzymes. Among them, cysteine proteinases (CPs) are commonly produced as inactive precursors (procysteines), requiring a cleavage of the peptide proregion to become active. The proregion fits tightly into the active site of procysteines, efficiently preventing their activity. In this report, a CP cDNA (cpao) was isolated from A. obtectus midgut larvae. In silico studies indicated that the complete CP sequence contains a hydrophobic signal peptide, a prodomain and a conserved catalytic region. Moreover, the encoding cDNA contains 963bp translating into a 321 residue protein, CPAo, which was expressed in E. coli, fused with thioredoxin. Enzymatic assays using the recombinant protein revealed that the enzyme was catalytically active, being able to cleave the synthetic substrate Z-Phe-Arg-7-AMC. Additionally, this report also focuses the cpao propeptide (PCPAo) subcloning and expression. The expressed propeptide efficiently inhibited CPAo, as well as digestive CP of other bean bruchids. Little or no activity was found against proteolytic enzymes of two other coleopterans: Rhyzopertha dominica and Anthonomus grandis. The data reported here indicate the possibility of endogenous propeptides as a novel strategy on bruchids control, which could be applicable to bean improvement programs.

Inhibitory Properties of Cysteine Protease Pro-Peptides from Barley Confer Resistance to Spider Mite Feeding

“…In vitro assays and artificial diets using the recombinant pro-region of the papaya proteinase IV have demonstrated a selective inhibition of digestive CysProt from several phytophagous beetles [ 24 ]. Similarly, pro-regions from two plant pest species, the nematode Heterodera glycines and the bean bruchid Acanthoscelides obtectus , efficiently inhibited their own CysProt and proteases from other herbivory species [ 25 , 26 ]. Moreover, it has been demonstrated that the CysProt pro-region from H .…”

Structural Basis for Specificity of Propeptide-Enzyme Interaction in Barley C1A Cysteine Peptidases

“…Thus, C1A cysteine peptidases of Trypanosoma cruzi or Plasmodium falciparum (animal parasites) were inhibited by their cognate propeptides [17], [18]. Moreover, the cysteine peptidase activities of midgut crude soluble extracts from Colorado potato beetle or several bruchids (plant pests) were also inhibited by papaya proteinase IV propeptide or by the propeptide of a cysteine peptidase from the bean bruchid Acanthoscelides obtectus [19], [20]. Recently, it was demonstrated that genetically modified soybean expressing the propeptide of a C1A peptidase from Heterodera glycines (plant nematode pest) reduced soybean cyst nematode infection [21].…”

Protective effects of a cysteine proteinase propeptide expressed in transgenic soybean roots

“…Cysteine proteinase inhibitors affect both the growth rate and the development of several nematodes, such as pallida [60], H. glycines [62], H. schachtti, and M. incognita [61,64], suggesting the importance of these inhibitors in the parasite digestion process. Among the different classes of proteinase inhibitors, cysteine proteinases prodomains have been described as potent inhibitors with the ability to control nematodes [53,54]. Recombinant expressed papaya proteinase IV propeptide (a plant enzyme) inhibits Colorado potato beetle cysteine peptidases [65], and synthetic propeptides inhibit Manduca sexta midgut trypsin [56].…”

“…The cysteine proteinase of F. hepatica (animal parasite) also was inhibited by its propeptide [27,47]. Recently, it was demonstrated that the CP propeptide of A. obtectus inhibits midgut crude soluble extract of several bruchids, such as A. obtectus, Zabrotes subfasciatus, and Callosobruchus maculatus [54], showing a broad spectrum of inhibition of the propeptide against pests of similar species.…”

“…A similar mechanism of action also occurs for the propeptides present in precursors of cysteine proteinases, which are active as molecule inhibitors with the ability to inhibit proteolytic enzymes [24,67]. Different reports indicate that CPs propeptides are potent inhibitors of their cognate enzymes [6,22,34,46,[53][54][55].…”