Termiticidal activity of lectins from Myracrodruon urundeuva against Nasutitermes corniger and its mechanisms

The isolation of lectins from Myracrodruon urundeuva bark (MuBL) and heartwood (MuHL) as well as the termiticidal activity of MuHL against Nasutitermes corniger has already been described. This work reports on the purification of a leaf lectin (MuLL) and the characterization of MuBL, MuHL, and MuLL; also described are the resistance of hemagglutinating activity of the three lectins to trypsin activity from N. corniger gut and the termiticidal activity on N. corniger of MuBL (LC₅₀ of 0.974 mg ml⁻¹ on workers and 0.787 mg ml⁻¹ on soldiers) and MuLL (LC₅₀ of 0.374 mg ml⁻¹ on workers and 0.432 mg ml⁻¹ on soldiers). The antibacterial effect of MuBL, MuHL, and MuLL on bacteria from gut of N. corniger was also investigated and lectins showed similar bacteriostatic activity (MIC of 62.5 ??g ml⁻¹ for workers and 125 ??g ml⁻¹ for soldiers). MuBL and MuHL were more efficient bactericidal agents on bacteria in the workers’ gut (MBC of 125 ??g ml⁻¹) than MuLL (MBC of 250 ??g ml⁻¹) and similar bactericidal activity was detected on bacteria in the gut of soldiers (MBC of 250 ??g ml⁻¹). The termiticidal activity of M. urundeuva lectins can be explained by the chitin-binding property, resistance to termite digestive enzyme, and the antibacterial effect on symbiotic bacteria of N. corniger gut.     

Antioxidant,Fusarium growth inhibition and Nasutitermes corniger repellent activities of secondary metabolites from Myracrodruon urundeuva heartwood

Myracrodruon urundeuva heartwood is resistant to biodeterioration and lectin purified from heartwood showed antifungal and termiticidal activities. This report deals with antioxidant, antifungal and termite repellent activities of secondary metabolites from M. urundeuva heartwood. Saline (SE, active hemagglutinin preparation) and methanolic (ME, without hemagglutinating activity) extracts contain phenolic compounds, gallic acid, flavonoids, luteolin, cinamic derivatives, proanthocyanidins, hydrolysable tannins, and leucoanthocyanidins. Both SE and ME showed antioxidant activity and were effective in Fusarium growth inhibition. SE was efficient against Fusarium decemcellulare, Fusarium moniliforme, Fusarium oxysporum, and Fusarium solani but it had little effect against Fusarium lateritium. ME practically had no effect on F. decemcellulare and was more active than SE against F. lateritium. SE induced mortality of Nasutitermes corniger (LC₅₀ of 1.81 mg ml^?1 for soldiers and 2.59 mg ml^?1 for workers) and had no repellent activity. ME had no termiticidal activity but was a good repellent. The detected bioactivities point out the possibility of participation of secondary metabolites in the resistance of M. urundeuva heartwood to biodeterioration. Additionally, the results indicate the use of wood residues, as extracts, a source of natural bioactive agents.     

A new chitin-binding lectin from rhizome of Setcreasea purpurea with antifungal,antiviral and apoptosis-inducing activities

A 48 kDa, chitin-binding lectin with antifungal, antiviral and apoptosis-inducing activities was isolated from the rhizomes of Setcreasea purpurea Boom, a member of family Commelinaceae. Setcreasea purpurea lectin (designated as SPL) is a homotetrameric protein consisting of 12031.9 Da subunits linked by non-covalent bonds as determined by SDS-PAGE, gel filtration and MS. The N-terminal 25 amino-acid sequence of SPL, NVLGRDAYCGSQNPGATCPGLCCSK was determined and homology analysis suggested that SPL belongs to the family of chitin-binding plant lectins composed of hevein domains. The lectin exhibited strong hemagglutinating activity towards rabbit erythrocytes at 0.95 μg/ml and the activity could be reversed exclusively by chitin hydrolysate (oligomers of GlcNAc). Its hemagglutinating activity was stable in pH range of 2.0–9.0 and it showed excellent thermal tolerance. SPL showed antifungal activity against Rhizoctonia solani, Sclerotinia sclerotiorum, Penicillium italicum and Helminthosporiun maydis. It also exhibited inhibitory effect on HIV-1 (IIIB) and HIV-2 (ROD), with an EC₅₀ of 13.8 ± 1.3 and 57.1 ± 15 μg/ml, respectively. Subsequently, MTT method, cell morphological analysis and LDH activity-based cytotoxicity assays demonstrated that SPL was highly cytotoxic to CNE-1 cells and induced apoptosis in a dose-dependent manner. Moreover, due to the caspase inhibitors analyses, caspase was also found to play an important role in the potential apoptotic mechanism of SPL.     

Purification and characterization of an N-acetyl-d-galactosamine-specific lectin from the edible mushroom Schizophyllum commune

An N-acetyl-d-galactosamine (GalNAc)-specific lectin was purified from the edible mushroom, Schizophyllum commune, using affinity chromatography on a porcine stomach mucin (PSM)-Sepharose 4B column. Under reducing and non-reducing conditions, SDS-polyacrylamide gel electrophoresis gave a major band of 31.5 kDa. The Schizophyllum commune lectin (SCL) showed high affinity toward rat erythrocytes and the sugar inhibition assay exhibited its sugar specificity highly toward lactose and N-acetyl-d-galactosamine. It was stable at 55 °C for 30 min and at pH 3–10 for 18-h test. The lectin was shown to be a glycoprotein with cytotoxic activity against human epidermoid carcinoma cells. The N-terminus of SCL was blocked but amino acid sequences of internal tryptic peptides showed moderately sequence similarities with some other fungal and plant lectins. Crystals of SCL were obtained by the sitting drop vapour-diffusion method using polyethylene glycol 8000 as the precipitant, and gave an X-ray diffraction pattern to approximately 3.8 Å resolution.     

Purification and characterization of a lectin from the banana shrimp Fenneropenaeus merguiensis hemolymph

A lectin from the hemolymph of the banana shrimp Fenneropenaeus merguiensis was purified by affinity chromatography on a fetuin-agarose column following by gel filtration on a Superose-12 column. The native molecular mass of purified F. merguiensis lectin (FmL) determined by gel filtration was 316.2 kDa and its carbohydrate content was estimated to be 4.4%. By SDS-PAGE analysis, purified FmL consisted of 32.3 kDa and 30.9 kDa subunits. These data suggest that this lectin is an oligomer. Two-dimensional electrophoresis showed that it had a pI value of 6.0 and was mainly composed of glycine, serine, histidine, glutamic acids and glutamine, with relatively lower amounts of methionine and tyrosine. Purified FmL expressed higher agglutination activity against rabbit and rat erythrocytes than with those from human, and its activity was Ca²⁺-dependent. The hemagglutinating activity of FmL was stable up to 55 °C and at pH 7.5–8. N-acetylated sugars, such as ManNAc, GlcNAc, GalNAc, and NeuNAc were strong inhibitors of the FmL induced hemagglutinating activity with NeuNAc being most effective. Porcine stomach mucin and fetuin were the most potent inhibitors of FmL. Purified FmL caused selective agglutination of Vibrio harveyi, and Vibrio parahemolyticus both pathogens of this Penaeus species and to a lesser extent Vibrio vulnificus but had no effect on the non-pathogenic strains; Vibrio cholerae, Salmonella typhi and Escherichia coli. Its bacterial agglutination was also completely inhibited by NeuNAc, mucin, fetuin and also anti-FmL antibody. This observation indicates that FmL may contribute to the defense response of this species of penaeid shrimps to potentially pathogenic bacteria.     

A lactose specific lectin from the sponge Cinachyrella apion: Purification,characterization, N-terminal sequences alignment and agglutinating activity on Leishmania promastigotes

Crude extract from the sponge Cinachyrella apion showed cross-reactivity with the polyclonal antibody IgG anti-CvL (Cliona varians lectin) and also a strong haemagglutinating activity towards human erythrocytes of all ABO groups. Thus, it was submitted to acetone fractionation, IgG anti-deglycosylated CvL Sepharose affinity chromatography, and Fast Protein Liquid Chromatography (FPLC-AKTA Purifier) gel filtration on a Superose 6 10/300 column to purify a novel lectin. C. apion lectin (CaL) agglutinated all types of human erythrocytes with preference for papainized type A erythrocytes. The haemagglutinating activity is independent of Ca²⁺, Mg²⁺ and Mn²⁺ ions, and it was strongly inhibited by the disaccharide lactose, up to a minimum concentration of 6.25 mM. CaL molecular mass, determined by FPLC-gel filtration on a Superose 12 10/300 column and SDS gel electrophoresis, was approximately 124 kDa, consisting of eight subunits of 15.5 kDa, assembled by hydrophobic interactions. The lectin was heat-stable between 0 and 60 °C and pH-stable. The N-terminal amino acid sequence of CaL was also determined and a blast search on amino acid sequences revealed that the protein showed similarity only with a silicatein. Leishmania chagasi promastigotes were agglutinated by CaL and this activity was abolished by lactose, indicating that lactose receptors could be presented in this parasite stage. These findings are indicative of the potential biotechnological application of CaL as diagnostic of pathogenic protozoa.     

Effect of lectins from Opuntia ficus indica cladodes and Moringa oleifera seeds on survival of Nasutitermes corniger

Biodegradation by termites is a serious problem for wood and crop industries worldwide, and new environmentally friendly alternatives for termite control have been developed. This work investigated the effects of crude and purified preparations containing lectins from Opuntia ficus indica cladodes (OfiL) and Moringa oleifera seeds (WSMoL and cMoL) on Nasutitermes corniger workers and soldiers. Purified OfiL was more active than cladode extracts, showing a stronger termiticidal activity against workers (LC₅₀ of 0.116 mg ml⁻¹) than against soldiers. OfiL was active against soldiers only at 1.5 mg ml⁻¹. All preparations containing WSMoL and cMoL were active only at concentrations of 1.0 and 1.5 mg ml⁻¹. The tested preparations did not exert repellent activity against N. corniger. OfiL was able to kill workers and therefore is potentially a new tool for N. corniger control; as a consequence, this lectin could disturb organization, structure, and maintenance of termite colonies.     

Isolation,enzymatic characterization and antiedematogenic activity of the first reported rattlesnake hyaluronidase from Crotalus durissus terrificus venom

A hyaluronidase (CdtHya1) from Crotalus durissus terrificus snake venom (CdtV) was isolated and showed to exhibit a high activity on hyaluronan cleavage. However, surveys on this enzyme are still limited. This study aimed at its isolation, functional/structural characterization and the evaluation of its effect on the spreading of crotoxin and phospholipase A₂ (PLA₂). The enzyme was purified through cation exchange, gel filtration and hydrophobic chromatography. After that, it was submitted to a reverse-phase fast protein liquid chromatography (RP-FPLC) and Edman degradation sequencing, which showed the first N-terminal 44 amino acid residues whose sequence evidenced identity with other snake venom hyaluronidases. CdtHya1 is a monomeric glycoprotein of 64.5 kDa estimated by SDS-PAGE under reducing conditions. It exhibited maximum activity in the presence of 0.2 M NaCl, at 37 °C, pH 5.5 and a specificity to hyaluronan higher than that to chondroitin-4-sulphate, chondroitin-6-sulphate or dermatan. Divalent cations (Ca²⁺ and Mg²⁺) and 1 M NaCl significantly reduced the enzyme activity. The specific activity of CdtHya1 was 5066 turbidity reducing units (TRU)/mg, against 145 TRU/mg for the soluble venom, representing a 34.9-fold purification. The pure enzyme increased the diffusion of crotoxin and PLA₂ through mice tissues. CdtHya1 (32 TRU/40 μL) potentiated crotoxin action, as evidenced by mice death, and it decreased the oedema caused by subplantar injections of buffer, crotoxin or PLA₂, thus evidencing the relevance of hyaluronidase in the crotalic envenoming. This work yielded a highly active antiedematogenic hyaluronidase from CdtV, the first one isolated from rattlesnake venoms.     

Isolation and antiproliferative activity of Lotus corniculatus lectin towards human tumour cell lines

The objective of the study was to investigate the anti cancer activity of a lectin isolated from Lotus corniculatus seeds. A tetrameric 70 kDa galactose specific lectin was purified using two step simple purification protocol which involved affinity chromatography on AF-BlueHC650M and gel filtration on Sephadex G-100. The lectin was adsorbed on AF-BlueHC650M and desorbed using 1 M NaCl in the starting buffer. Gel filtration on Sephadex G-100 yielded a major peak absorbance that gave two bands of 15 kDa and 20 kDa in SDS PAGE. Hemagglutination activity was completely preserved, when the temperature was in the range of 20–60 °C. However, drastic reduction in activity occurred at temperatures above 60 °C. Full hemagglutination activity was retained at ambient pH 4–12. Thereafter no activity was observed above pH 13. Hemaglutination of the lectin was inhibited by d-galactose. The lectin showed a strong antiproliferative activity towards human leukemic (THP-1) cancer cells followed by lung cancer (HOP62) cells and HCT116 with an IC₅₀ of 39 μg/ml and 50 μg/ml and 60 μg/ml respectively. Flow cytometry analysis showed an increase in the percentage of cells in sub G0G1 phase confirming that Lotus corniculatus lectin induced apoptosis. Morphological observations showed that Lotus corniculatus lectin (LCL) treated THP-1 cells displayed apparent apoptosis characteristics such as nuclear fragmentation, appearance of membrane enclosed apoptotic bodies and DNA fragmentation. Lotus corniculatus lectin (LCL) effectively inhibits the cell migration in a dose dependent manner as indicated by the wound healing assay.