Roberto A. Sá

Larvicidal activity of lectins from Myracrodruon urundeuva on Aedes aegypti

Aedes aegypti transmits etiologic agents of yellow fever and dengue. Vaccine for dengue virus is not available and vector control is essential to minimize dengue incidence. This report deals with the larvicidal activity of lectins isolated from Myracrodruon urundeuva bark (MuBL) and heartwood (MuHL). The lectins were isolated by ammonium sulphate treatment of crude extracts followed by chromatography on chitin. MuBL and MuHL were evaluated by electrophoresis under native (PAGE) and denaturing conditions (SDS-PAGE). Carbohydrate specificity of lectins was evaluated by hemagglutinating activity (HA) inhibition assay using N-acetyl-d-glucosamine and by affinity chromatography on N-acetyl-D-glucosamine immobilized in agarose gel. Larvicidal activity against A. aegypti was investigated with the extracts, salt fractions and isolated lectins. MuBL and MuHL were characterized by PAGE as basic proteins of molecular masses of 14.0 and 14.4 kDa, respectively. The interaction of lectins with N-acetylglucosamine was detected by inhibition of HA by monosaccharide and lectin adsorptions on N-acetyl-D-glucosamine matrix. All M. urundeuva preparations promoted larvae mortality. LC16, LC50 and LC84 values of 0.077, 0.125, 0.173 for MuBL and 0.03, 0.04 and 0.05 mg/mL for MuHL were obtained. To our knowledge this is the first report of larvicidal activity of lectins against A. aegypti.

Antibacterial and antifungal activities of Myracrodruon urundeuva heartwood

The aim of this work was to isolate a lectin from Myracrodruon urundeuva heartwood and to evaluate its antimicrobial activity against bacteria and fungi that attack plants, including woods. The lectin was isolated from heartwood through affinity chromatography on a chitin column monitored by hemagglutination assay. The lectin inhibited Gram-negative and Gram-positive bacteria and was more effective than antifungal Cercobin in growth inhibition of phytopathogenic fungi. The detected antimicrobial activity reveals the possible role of the lectin in the resistance of M. urundeuva heartwood against deteriorative biological agents. The M. urundeuva lectin is the first bioactive peptide found in heartwood, probably stored as a chemical protection against biodegradation.

Digestive enzymes from workers and soldiers of termite Nasutitermes corniger

The digestive apparatus of termites may have several biotechnological applications, as well as being a target for pest control. This report discusses the detection of cellulases (endoglucanase, exoglucanase, and β-glucosidase), hemicellulases (β-xylosidase, α-l-arabinofuranosidase, and β-d-xylanase), α-amylase, and proteases (trypsin-like, chymotrypsin-like, and keratinase-type) in gut extracts from Nasutitermes corniger workers and soldiers. Additionally, the effects of pH (3.0-11.0) and temperature (30-100°C) on enzyme activities were evaluated. All enzymes investigated were detected in the gut extracts of worker and soldier termites. Endoglucanase and β-xylanase were the main cellulase and hemicellulase, respectively. Zymography for proteases of worker extracts revealed polypeptides of 22, 30, and 43kDa that hydrolyzed casein, and assays using protease inhibitors showed that serine proteases were the main proteases in worker and soldier guts. The determined enzyme activities and their response to different pH and temperature values revealed that workers and soldiers contained a distinct digestive apparatus. The ability of these termites to efficiently digest the main components of lignocellulosic materials stimulates the purification of gut enzymes. Further investigation into their biotechnological potential as well as whether the enzymes detected are produced by the termites or by their symbionts is needed.

Binding targets of termiticidal lectins from the bark and leaf of Myracrodruon urundeuva in the gut of Nasutitermes corniger workers: Binding targets of termiticidal lectins

BACKGROUND Lectins, carbohydrate-binding proteins, from the bark (MuBL) and leaf (MuLL) of Myracrodruon urundeuva are termiticidal agents against Nasutitermes corniger workers and have been shown to induce oxidative stress and cell death in the midgut of these insects. In this study, we investigated the binding targets of MuBL and MuLL in the gut of N. corniger workers by determining the effects of these lectins on the activity of digestive enzymes. In addition, we used mass spectrometry to identify peptides from gut proteins that adsorbed to MuBL-Sepharose and MuLL-Sepharose columns. RESULTS Exoglucanase activity was neutralized in the presence of MuBL and stimulated by MuLL. α-l-Arabinofuranosidase activity was not affected by MuBL but was inhibited by MuLL. Both lectins stimulated α-amylase activity and inhibited protease and trypsin-like activities. Peptides with homology to apolipophorin, trypsin-like enzyme, and ABC transporter substrate-binding protein were detected from proteins that adsorbed to MuBL-Sepharose, while peptides from proteins that bound to MuLL-Sepharose shared homology with apolipophorin. CONCLUSION This study revealed that digestive enzymes and transport proteins found in worker guts can be recognized by MuBL and MuLL. Thus, the mechanism of their termiticidal activity may involve changes in the digestion and absorption of nutrients.

Insecticide Activity of Lectins and Secondary Metabolites

Proteins are polymers of amino acids (molecules containing an amino group, a carboxylic group and a hydrophobic or hydrophilic side chain) present in all organisms. Apolar, polar uncharged and electrically charged amino acids are covalently linked through peptide bonds (amide bonds) and the sequence they form in the polypeptide chain (primary structure) determines the tertiary or quaternary structures ultimately presenting some biological activity. Proteins can be formed by one or multiple polypeptides (subunits) with or without a non-amino acid molecule (carbohydrate, ion, lipid, etc) linked to them. Lectins comprise a heterogeneous group of non-immune proteins that interact with carbohydrates. This interaction is behind a number of biological properties, including antimicrobial, antitumoral, hemagglutinating, mitogenic and insecticide activities. The specificity of the carbohydrate binding site is determined by the amino acids forming the lectin molecule, as well as shape and the spatial arrangement of neighboring amino acids; additionally, metal ions may contribute for correct positioning of the amino acid residues for binding to the carbohydrate (Sharon and Lis, 2001). Lectins can be divided into those that bind monosaccharides as well as oligosaccharides, and those that recognize only oligosaccharides (Sharon and Lis, 2007). Depending on carbohydrate specificity, they can be classified as: glucose/mannose, N-acetylglucosamine, galactose, N-acetylgalactosamine, fucose and sialic acid-binding lectins (Wu et al., 2001). The hemagglutinating activity assay (Figure 1A) in presence of free carbohydrates (Figure 1B) has been proved to be a useful tool to characterize lectin specificity. Plant lectins have been isolated from bark, cladodes, flowers, leaves, rhizomes, roots and seeds. They differ from each other with respect to their molecular structures, carbohydratebinding specificities, and biological activities. The compact globular structures, molecular aggregation and glycosylation of lectins in general result in high structural stability (Kawsar et al., 2008; Moreno et al., 2008). In general, lectin isolation procedures include protein extraction steps with aqueous solvent, the production of a lectin-rich fraction, and separation of lectin from protein or non-protein