Stage-specific gut proteinases of the cotton stainer bug Dysdercus peruvianus: Role in the release of entomotoxic peptides from Canavalia ensiformis urease Angela R. Piovesan a,1 , Fernanda Stanisçuaski b, * , 1 , Juliana Marco-Salvadori a , Rafael Real-Guerra a , Marina S. Defferrari a , Ce ´ lia R. Carlini a, b a Graduate Program in Cellular and Molecular Biology, Center of Biotechnology, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves, 9500, Building 43421, CEP 91501-970 Porto Alegre, Rio Grande do Sul, Brazil b Department of Biophysics and Center of Biotechnology, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves, 9500, Building 43422, CEP 91501-970 Porto Alegre, Rio Grande do Sul, Brazil article info Article history: Received 12 May 2008 Received in revised form 24 August 2008 Accepted 11 September 2008 Keywords: Dysdercus peruvianus Urease Proteolytic enzyme Insecticidal peptide Enzyme inhibitor abstract Canavalia ensiformis ureases are toxic to insects of different orders. The entomotoxicity of urease is due to a 10 kDa internal peptide released by proteinases in the insect digestive tract. We previously observed that, given orally, urease is toxic to nymphs of Dysdercus peruvianus, but does not affect adults. Here we characterized the major proteolytic activities of D. peruvianus midgut homogenates and investigated their in vitro-catalyzed release of the 10 kDa entomotoxic peptide from urease. Cysteine, aspartic and metalloproteinases are present in both homogenates. Variations in optimal pH and susceptibility to inhibitors indicated differences in the enzyme profiles in the two developmental stages. Only nymph homogenates released w10 kDa fragment(s) from urease, recognized by antibodies against the ento- motoxic peptide. Fluorogenic substrates containing urease partial sequences flanking the N-terminal or the C-terminal portion of the entomotoxic peptide were efficiently cleaved by homogenates from nymphs, but much more slowly by the adult homogenate. Different classes of enzymes in the homog- enates cleaved both substrates suggesting that in vivo the release of the entomotoxic peptide results from the concerted action of at least two different proteinases. Our findings support the view that a differential processing of ingested urease by the insects explains at least in part the lack of toxicity in adults. Ó 2008 Elsevier Ltd. All rights reserved. 1. Introduction Ureases (urea amidohydrolase; EC 3.5.1.5), nickel-dependent enzymes that catalyze the hydrolysis of urea to ammonia and carbon dioxide, are synthesized by plants, fungi and bacteria (Mobley et al., 1995; Follmer, 2008). In plants, ureases are homo- trimers or hexamers of a w90 kDa subunit and participate in the use of urea as nitrogen source (Polacco and Holland, 1993; Sirko and Brodzik, 2000; Follmer, 2008). Canatoxin, a toxic protein isolated from Canavalia ensiformis seeds (Carlini and Guimara ˜es, 1981) and more recently identified as a minor isoform of urease (Follmer et al., 2001), displays insecticidal properties (Carlini et al., 1997). Cana- toxin and the major isoform of urease of C. ensiformis seeds (herein designated ‘‘urease’’) are toxic to Dysdercus peruvianus (Hemiptera: Pyrrhocoridae), and this toxicity is independent of their ureolytic activity (Follmer et al., 2004a; Stanisçuaski et al., 2005; Follmer et al., 2004b). Insects with cathepsin-like digestive enzymes are susceptible to the toxic effects of urease, while insects with trypsin-like digestive enzymes are not (Carlini et al., 1997), possibly due to differences in the proteolytic processing of ureases in the insects. The entomo- toxic activity is due to a 10 kDa internal peptide released from ureases by insect digestive enzymes. This peptide, called pepca- natox, was isolated and characterized (Ferreira-DaSilva et al., 2000). A recombinant peptide, Jaburetox-2Ec equivalent to pepcanatox, obtained by heterologous expression in Escherichia coli (Mulinari et al., 2004, 2007), is highly insecticidal and did not affect mice or neonate rats when administered by oral or intraperitoneal routes (Mulinari et al., 2007). Interestingly, Jaburetox-2Ec kills insects that are resistant to intact ureases, such as Spodoptera frugiperda (Mulinari et al., 2007) or adults of Triatoma infestans (Tomazzeto et al., 2007). The cotton stainer bug D. peruvianus feeds on cotton seeds (Gossypium hirsutum), staining the cotton fibers, damaging the seeds, besides being a vector for phytopathogenic microorganisms, and thus can cause heavy losses in cotton production (Gallo, 1988). * Corresponding author. Tel.: þ55 51 3308 7606; fax: þ55 51 3308 7003. E-mail address: fernanda.staniscuaski@ufrgs.br (F. Stanisçuaski). URL: http://www.ufrgs.br/laprotox 1 These authors have contributed equally to this work. Contents lists available at ScienceDirect Insect Biochemistry and Molecular Biology journal homepage: www.elsevier.com/locate/ibmb 0965-1748/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibmb.2008.09.004 Insect Biochemistry and Molecular Biology 38 (2008) 1023–1032