Cathepsin L-like cysteine proteinase (DcCathL) from Delia coarctata (wheat bulb fly): Basis of insecticidal activity

A cDNA encoding a cathepsin L-like cysteine proteinase (DcCathL) was prepared from gut tissue of larvae of wheat bulb fly (Delia coarctata: Diptera). The predicted protein is a homologue of the product of Drosophila melanogaster gene Cp-1 (CG6692), and is similar to a sub-family of cysteine proteinases found in other insects which have roles in tissue remodelling during development, and moulting. Recombinant DcCathL was produced using the yeast Pichia pastoris as expression host, and showed hydrolytic activity in vitro towards the synthetic substrate Z-Phe-Arg-AMC with a pH optimum of 4.5. DcCathL was insecticidal to lepidopteran larvae when injected into haemolymph, causing mortality that was accompanied by systemic melanisation, suggesting that DcCathL was affecting the immune-related proteolytic activation cascade leading to production of active phenoloxidase. This process is normally negatively regulated by serpins in the haemolymph. Recombinant serpins from cabbage moth (Mamestra brassicae) did not inhibit DcCathL, and were susceptible to degradation by the enzyme in vitro in buffer and extracted haemolymph. When M. brassicae larvae were co-injected with a lethal dose of DcCathL and exogenous recombinant serpins, no mortality or systemic melanisation was observed, suggesting that the insecticidal effects of DcCathL in vivo result from degradation of endogenous serpins.