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.
Subject(s)
Coleoptera/enzymology , Cysteine Endopeptidases/metabolism , Cysteine Proteinase Inhibitors/metabolism , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Cloning, Molecular , Conserved Sequence , Cysteine Endopeptidases/chemistry , Cysteine Endopeptidases/genetics , Cysteine Endopeptidases/isolation & purification , Cysteine Proteinase Inhibitors/chemistry , Cysteine Proteinase Inhibitors/genetics , DNA, Complementary/genetics , Escherichia coli/genetics , Molecular Sequence Data , Protein Binding , Protein Sorting Signals , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Substrate Specificity , Thioredoxins/metabolismABSTRACT
Cysteine proteinases (CPs) are synthesized as zymogens and converted to mature proteinase forms by proteolytic cleavage and release of their pro domain peptides. A cDNA encoding a papain-like CP, called hgcp-Iv, was isolated from a Heterodera glycines J2 cDNA library, expressed and utilized to assess the ability of its propeptide to inhibit proteinase in its active form. The hgcp-Iv cDNA sequence encodes a polypeptide of 374 amino acids with the same domain organization as other cathepsin L-like CPs, including a hydrophobic signal sequence and a pro domain region. HGCP-Iv, produced in Escherichia coli as a fusion protein with thioredoxin, degrades the synthetic peptide benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin and is inhibited by E-64, a substrate and inhibitor commonly used for functional characterization of CPs. Recombinant propeptides of HGCP-Iv, expressed in E. coli, presented high inhibitory activity in vitro towards its cognate enzyme and proteinase activity of Meloidogyne incognita females, suggesting its usefulness in inhibiting nematode CPs in biological systems. Cysteine proteinases from other species produced no noticeable activity.
