title: Recombinant expression, localization and in vitro inhibition of midgut cysteine peptidase (Sl-CathL) from sugarcane weevil, Sphenophorus levis.
abstract: A cDNA coding for a digestive cathepsin L, denominated Sl-CathL, was isolated from a cDNA library of Sphenophorus levis larvae, representing the most abundant EST (10.49%) responsible for proteolysis in the midgut. The open reading frame of 972 bp encodes a preproenzyme similar to midgut cathepsin L-like enzymes in other coleopterans. Recombinant Sl-CathL was expressed in Pichia pastoris, with molecular mass of about 42 kDa. The recombinant protein was catalytically activated at low pH and the mature enzyme of 39 kDa displayed thermal instability and maximal activity at 37°C and pH 6.0. Immunocytochemical analysis revealed Sl-CathL production in the midgut epithelium and secretion from vesicles containing the enzyme into the gut lumen, confirming an important role for this enzyme in the digestion of the insect larvae. The expression profile identified by RT-PCR through the biological cycle indicates that Sl-CathL is mainly produced in larval stages, with peak expression in 30-day-old larvae. At this stage, the enzyme is 1250-fold more expressed than in the pupal fase, in which the lowest expression level is detected. This enzyme is also produced in the adult stage, albeit in lesser abundance, assuming the presence of a different array of enzymes in the digestive system of adults. Tissue-specific analysis revealed that Sl-CathL mRNA synthesis occurs fundamentally in the larval midgut, thereby confirming its function as a digestive enzyme, as detected in immunolocalization assays. The catalytic efficiency of the purified recombinant enzyme was calculated using different substrates (Z-Leu-Arg-AMC, Z-Arg-Arg-AMC and Z-Phe-Arg-AMC) and rSl-CathL exhibited hydrolysis preference for Z-Leu-Arg-AMC (k(cat)/K(m)=37.53 mMS(-1)), which is similar to other insect cathepsin L-like enzymes. rSl-CathL activity inhibition assays were performed using four recombinant sugarcane cystatins. rSl-CathL was strongly inhibited by recombinant cystatin CaneCPI-4 (K(i)=0.196 nM), indicating that this protease is a potential target for pest control.
authors.affiliation: Department of Genetics and Evolution, Laboratory of Molecular Biology, Federal University of São Carlos, 13565-905 São Carlos, Brazil.
authors.forename: Fernando P P
authors.initials: FP
authors.lastname: Fonseca
authors.forename: Andrea
authors.initials: A
authors.lastname: Soares-Costa
authors.forename: Alberto F
authors.initials: AF
authors.lastname: Ribeiro
authors.forename: José César
authors.initials: JC
authors.lastname: Rosa
authors.forename: Walter R
authors.initials: WR
authors.lastname: Terra
authors.forename: Flávio
authors.initials: F
authors.lastname: Henrique-Silva
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D057056:Cysteine Proteases / Q000235:genetics / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors; D041981:Gastrointestinal Tract / Q000201:enzymology; D015870:Gene Expression; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D010843:Pichia; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D048889:Weevils / Q000201:enzymology* / Q000235:genetics
chemical_list: D015853:Cysteine Proteinase Inhibitors; D019476:Insect Proteins; D011994:Recombinant Proteins; D057056:Cysteine Proteases