Characterisation of cysteine proteinases responsible for digestive proteolysis in guts of larval Western corn rootworm (Diabrotica virgifera) by expression in the yeast Pichia pastoris

Cysteine proteinases are the major class of enzymes responsible for digestive proteolysis in western corn rootworm (Diabrotica virgifera), a serious pest of maize. A larval gut extract hydrolysed typical cathepsin substrates, such as Z-phe-arg-AMC and Z-arg-arg-AMC, and hydrolysis was inhibited by Z-phe-tyr-DMK, specific for cathepsin L. A cDNA library representing larval gut tissue mRNA contained cysteine proteinase-encoding clones at high frequency. Sequence analysis of 11 cysteine proteinase cDNAs showed that 9 encoded cathepsin L-like enzymes, and 2 encoded cathepsin B-like enzymes. Three enzymes (two cathepsin L-like, DvRS5 and DvRS30, and one cathepsin B-like, DvRS40) were expressed as recombinant proteins in culture supernatants of the yeast Pichia pastoris. The cathepsin L-like enzymes were active proteinases, whereas the cathepsin B-like enzyme was inactive until treated with bovine trypsin. The amino acid residue in the S2 binding pocket, the major determinant of substrate specificity in cathepsin cysteine proteinases, predicted that the two cathepsin L-like enzymes, DvRS5 and DvRS30, should differ in substrate specificity, with the latter resembling cathepsin B in hydrolysing substrates with a positively charged residue at P2. This prediction was confirmed; DvRS5 only hydrolysed Z-phe-arg-AMC and not Z-arg-arg-AMC, whereas DvRS30 hydrolysed both substrates. The enzymes showed similar proteolytic activity towards peptide substrate

Crystal structure of a novel Mid-gut procarboxypeptidase from the cotton pest Helicoverpa armigera

The cotton bollworm Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) is one of the most serious insect pests in Australia, India and China. The larva causes substantial economical losses to legume, fibre, cereal oilseed and vegetable crops. This pest has proven to be difficult to control by conventional means, mainly due to the development of pesticide resistance. We present here the 2.5 Å crystal structure from the novel procarboxypeptidase (PCPAHa) found in the gut extracts from H. armigera larvae, the first one reported for an insect. This metalloprotease is synthesized as a zymogen of 46.6 kDa which, upon in vitro activation with Lys-C endoproteinase, yields a pro-segment of 91 residues and an active carboxypeptidase moiety of 318 residues. Both regions show a three-dimensional structure quite similar to the corresponding structures in mammalian digestive carboxypeptidases, the most relevant structural differences being located in the loops between conserved secondary structure elements, including the primary activation site. This activation site contains the motif (Ala)5Lys at the C terminus of the helix connecting the pro- and the carboxypeptidase domains. A remarkable feature of PCPAHa is the occurrence of the same (Ala)6Lys near the C terminus of the active enzyme. The presence of Ser255 in PCPAHa instead of Ile and Asp found in the pancreatic A and B forms, respectively, enlarges the S1¿ specificity pocket and influences the substrate preferences of the enzyme. The C-terminal tail of the leech carboxypeptidase inhibitor has been modelled into the PCPAHa active site to explore the substrate preferences and the enzymatic mechanism of this enzym