Digestive physiology and characterization of digestive cathepsin L-like proteinase from the sugarcane weevil Sphenophorus levis

Abstract

Sugarcane is an important crop that has recently become subject to attacks from the weevil Sphenophorus levis, which is not efficiently controlled with chemical insecticides. This demands the development of new control devices for which digestive physiology data are needed. In the present study, ion-exchange chromatography of S. levis whole midgut homogenates, together with enzyme assays with natural and synthetic substrates and specific inhibitors, demonstrated that a cysteine proteinase is a major proteinase, trypsin is a minor one and chymotrypsin is probably negligible. Amylase, maltase and the cysteine proteinase occur in the gut contents and decrease throughout the midgut; trypsin is constant in the entire midgut, whereas a membrane-bound aminopeptidase predominates in the posterior midgut. The cysteine proteinase was purified to homogeneity through ion-exchange chromatography. The purified enzyme had a mass of 37 kDa and was able to hydrolyze Z-Phe-Arg-MCA and Z-Leu-Arg-MCA with k(cat)/K(m) values of 20.0±1.1 μM(-1)s(-1) and 30.0±0.5 μM(-1)s(-1), respectively, but not Z-Arg-Arg-MCA. The combined results suggest that protein digestion starts in the anterior midgut under the action of a cathepsin L-like proteinase and ends on the surface of posterior midgut cells. All starch digestion takes place in anterior midgut. These data will be instrumental to developing S. levis-resistant sugarcane.

Recombinant expression, localization and in vitro inhibition of midgut cysteine peptidase (Sl-CathL) from sugarcane weevil, Sphenophorus levis

“…SDS-PAGE analysis revealed that purification of the cysteine peptidase from S. levis larvae was efficient, as demonstrated by the presence of a single band corresponding to a molecular mass of approximately 37 kDa (Soares-Costa et al, 2011). This information is in agreement with the expected molecular mass calculated from the EST clone (gene bank accession number FJ467290) coding for this cysteine peptidase.…”

“…Both amino acid residues have hydrophobic side chains and therefore share similar Amino acid sequences were deduced from product ions and identified (bold) in the cysteine peptidase sequence deduced from S. levis ESTs obtained from the larval cDNA library, shown below. These data prove that the SlCathL clone (FJ467290) chosen for recombinant expression and characterization corresponds to the previous purified digestive enzyme (Soares-Costa et al, 2011). The peptide "VPESIDWR" (A) is a non-tryptic peptide, considering the preceding serine residue in the Sl-CathL protein sequence.…”

“…The K m value obtained for hydrolysis of the ZLeu-Arg-AMC substrate by rSl-CathL was 1.79 mM, which indicates a very high affinity for this type of substrate when compared with other insect cathepsin Ls, as displayed in Table 1. The slight differences between the recombinant and purified enzyme (Soares-Costa et al, 2011) can be assigned to the fact that purified enzyme from the gut may constitute a pool of Sl-CathL isoforms with distinct features, as expected for an insect digestive arsenal against plant inhibitors.…”

Molecular characterization of cathepsin L cDNA and its expression during oogenesis and embryogenesis in the oriental river prawn Macrobrachium nipponense (Palaemonidae)

“…Similar functions of CatL in vitellogenesis were also found in rainbow trout Oncorhynchus mykiss (Kwon et al, 2001) and Danio rerio (Carnevali et al, 2006). Most studies on CatL in arthropods have focused on the function of CatL in immunity (Ren et al, 2010;Zhang et al, 2010b) and digestion (Hu and Leung, 2007;Soares-Costa et al, 2011), but little is known of its role in VTG utilization. Current evidence in Metapenaeus ensis (Crustacean) suggests that the ovary is the only non-alimentary organ showing CatL expression (Hu and Leung, 2004).…”

Plant Proteinaceous α-Amylase and Proteinase Inhibitors and Their Use in Insect Pest Control

“…It has been revealed that cathepsin L-like enzymes are the only quantitatively important member of cysteine proteinases presented in midgut of insects. Digestive cathepsin L-like enzymes have been purified from Diabrotica virgifera (Coleoptera: Cucujiformia) (Koiwa et al, 2000), Acyrthosiphon pisum (Hemiptera: Sternorrhyncha) (Cristofoletti et al, 2003), T. molitor (Coleoptera: Cucujiformia) (Cristofoletti et al , 2005), Sphenophorus levis (Coleoptera: Curculionidae) (Soares-Costa et al, 2011;Fonseca et al, 2012), and Triatoma brasiliensis (Reduviidae, Triatominae) (Waniek et al, 2012).…”