Cathepsin L-like cysteine proteinase (DcCathL) from Delia coarctata (wheat bulb fly): Basis of insecticidal activity q Prashant S. Pyati a , Howard A. Bell b , Elaine Fitches b , Daniel R.G. Price a , Angharad M.R. Gatehouse c , John A. Gatehouse a, * a School of Biological and Biomedical Sciences, Durham University, South Road, Durham DH1 3LE, UK b Central Science Laboratory, Sand Hutton, York YO411LZ, UK c Institute for Research on the Environment and Sustainability, Newcastle University, Newcastle upon Tyne NE1 7RU, UK article info Article history: Received 6 March 2009 Received in revised form 5 May 2009 Accepted 16 May 2009 Keywords: Wheat bulb fly (Delia coarctata) Cathepsin L-like proteinase Functional characterisation Cabbage moth (Mamestra brassicae) Haemolymph Phenoloxidase activation cascade Serpin Negative regulation Melanisation abstract 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. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction Cysteine proteinases are peptidases having a cysteine residue at the active site, which provides the catalytic nucleophile for peptide bond hydrolysis. Cathepsin L (EC 3.4.22.15) is a cysteine proteinase with a broad specificity for cleavage of peptide bonds, which is normally located in lysosomes, and is ubiquitous in eukaryotic cells. It is synthesized as a pre–proenzyme by ribosomes bound to the ER, and may be modified by glycosylation in the Golgi network prior to transport. The propeptide acts as an intramolecular inhibitor, and is necessary for proper folding, stability and transport to Golgi apparatus (Tao et al., 1994). In contrast to higher animals, cysteine proteinases play a major role in extracellular protein digestion in some insects. The biochem- istry of protein digestion in insect guts is complex, and variable between species, but in Coleoptera and Hemiptera, cysteine proteinases are important digestive enzymes (Terra et al., 1996). Although cysteine proteinases have been identified in gut tissue in a number of dipteran insect species, their role in digestion of dietary proteins remains to be established. A cathepsin L-like cysteine proteinase from Drosophila melanogaster Cp-1 (CG6692), was repor- ted as present in alimentary organs (Matsumoto et al., 1995), and was suggested to play a role in digestion, but the encoding gene is also expressed throughout the insect (FlyAtlas; http://www.flyatlas.org). A cathepsin B-like cysteine proteinase whose expression was upre- gulated after feeding was cloned from gut tissue of tsetse fly (Glossina morsitans morsitans; Yan et al., 2002), although a role in digestion was not established. Cysteine proteinase activity, and a specific cathepsin L-like proteinase designated DrCP1 was present in gut tissue of crucifer root fly (Delia radicum), but like D. melanogaster CP-1, DrCP1 was also present in other tissues, and was not considered to act as a digestive enzyme (Hegedus et al., 2002). Several studies have concluded that the major role for cathepsin L-like cysteine proteinases in insects is that of tissue remodelling, based on a seminal study of the enzyme in flesh fly, Sarcophaga peregrina (Homma et al., 1994). The similar enzyme in D. radicum, q The sequences of DcCathL, M. brassicae MbSpn1A and MbSpn1B/C have been deposited in the EMBL sequence database with following accession numbers: FJ763762, FJ763763 and FJ763764 respectively. * Corresponding author. Tel.: þ44 191 334 1264; fax: þ44 191 334 1201. E-mail address: j.a.gatehouse@durham.ac.uk (J.A. Gatehouse). Contents lists available at ScienceDirect Insect Biochemistry and Molecular Biology journal homepage: www.elsevier.com/locate/ibmb 0965-1748/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibmb.2009.05.003 Insect Biochemistry and Molecular Biology 39 (2009) 535–546