Purification and characterization of lectin from fruiting body of Ganoderma lucidum: Lectin from Ganoderma lucidum

A novel 114 kDa hexameric lectin was purified from the fruiting bodies of the mushroom Ganoderma lucidum. Biochemical characterization revealed it to be a glycoprotein having 9.3% neutral sugar and it showed hemagglutinating activity on pronase treated human erythrocytes. The lectin was stable in the pH range of 5–9 and temperature up to 50 °C. The hemagglutinating activity was inhibited by glycoproteins that possessed N-as well as O-linked glycans. Chemical modification of the G. lucidum lectin revealed contribution of tryptophan and lysine to binding activity. The thermodynamics of binding of bi- and triantennary N-glycans to G. lucidum lectin was studied by spectrofluorimetry. The lectin showed very high affinity for asialo N-linked triantenary glycan and a preference for asialo glycans over sialylated glycans. The binding was accompanied with a large negative change in enthalpy as well as entropy, indicating primarily involvement of polar hydrogen, van der Waals and hydrophobic interactions in the binding.     

A mannose-specific tetrameric lectin with mitogenic and antibacterial activities from the ovary of a teleost,the cobia (<Emphasis Type="Italic">Rachycentron canadum</Emphasis>)

A tetrameric lectin, with hemagglutinating activity toward rabbit erythrocytes and with specificity toward d-mannosamine and d(+)-mannose, was isolated from the ovaries of a teleost, the cobia Rachycentron canadum. The isolation protocol comprised ion exchange chromatography on CM-cellulose and Q-Sepharose, ion exchange chromatography by fast protein liquid chromatography (FPLC) on Mono Q, and finally gel filtration by FPLC on Superose 12. The lectin was adsorbed on all ion exchangers used. It exhibited a molecular mass of 180 kDa in gel filtration on Superose 12 and a single 45-kDa band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that it is a tetrameric protein. The hemagglutinating activity of the lectin was stable up to 40°C and between pH 4 and pH 10. All hemagglutinating activity disappeared at 60°C and at pH 1 and pH 13. The hemagglutinating activity was doubled in the presence of 0.1 μM FeCl₃. The lectin exerted antibacterial activity against Escherichia coli with 50% inhibition at 250 μg. There was no antifungal activity toward Coprinus comatus, Fusarium oxysporum, Mycosphaerella arachidicola, and Rhizoctonia solani at a dose of 300 μg. The lectin exhibited maximal mitogenic response from mouse splenocytes at a concentration of 14 μM.     

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.     

A mushroom lectin from ascomycete Cordyceps militaris

A mushroom lectin has been purified from ascomycete Cordyceps militaris, which is one of the most popular mushrooms in eastern Asia used as a nutraceutical and in traditional Chinese medicine. This lectin, designated CML, exhibited hemagglutination activity in mouse and rat erythrocytes, but not in human ABO erythrocytes. SDS-PAGE of CML revealed a single band with a molecular mass of 31.0 kDa under both nonreducing and reducing conditions that was stained by silver nitrate, and a 31.4 kDa peak in a Superdex-200 HR gel-filtration column. The hemagglutination activity was inhibited by sialoglycoproteins, but not in by mono- or disaccharides, asialoglycoproteins, or de-O-acetylated glycoprotein. The activity was maximal at pH 6.0–9.1 and at temperatures below 50 °C. Circular dichroism spectrum analysis revealed that CML comprises 27% α-helix, 12% β-sheets, 29% β-turns, and 32% random coils. Its binding specificity and secondary structure are similar to those of a fungal lectin from Arthrobotrys oligospora. However, the N-terminal amino acid sequence of CML differs greatly from those of other lectins. CML exhibits mitogenic activity against mouse splenocytes.     

Isolation of a new heterodimeric lectin with mitogenic activity from fruiting bodies of the mushroom Agrocybe cylindracea

From the dried fruiting bodies of the mushroom Agrocybe cylindracea a heterodimeric lectin with a molecular weight of 31.5 kDa and displaying high hemagglutinating activity was isolated. The molecular weights of its subunits were 16.1 kDa and 15.3 kDa respectively. The larger and the smaller subunits resembled Agaricus bisporus lectin and fungal immunomodulatory protein from Volvariella volvacea respectively in N-terminal sequence. The lectin was adsorbed on DEAE-cellulose in 10 mM Tris-HCl buffer (pH 7.4) and was eluted by the same buffer containing 150 mM NaCl. It was adsorbed on SP-Sepharose in 10 mM NH4OAc (pH 4.5) and eluted by approximately 0.19 M NaCl in the same buffer. The lectin was obtained in a purified form after the mushroom extract had been subjected to (NH4)2SO4 precipitation and the two aforementioned ion exchange chromatographic steps. The lectin exhibited potent mitogenic activity toward mouse splenocytes. The hemagglutinating activity of the lectin was inhibited by lactose, sialic acid and inulin.     

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.     

Purification and characterization of a galactose-specific lectin from corn (Zea mays) coleoptyle

We purified and characterized a lectin from the corn coleoptyle (Zea mays). The lectin (CCL) was purified by affinity chromatography on a Lactosyl–Sepharose 4B column. It is a glycoprotein of 88.7 kDa, composed mainly by glutamic, aspartic, glycine, and Ser residues; in a minor proportion, it contained methionine and cysteine residues. Carbohydrates that constituted 12% of the total weight comprised galactose, mannose, and N-acetyl-D-glucosamine. The lectin contained the blocked amino-terminus. Analysis of the lectin, determined from peptides obtained after trypsin digestion by MALDI-TOF (matrix-assisted laser desorption ionization-time of flight), indicated that CCL has 18% homology with a putative calcium-dependent Ser/Thr protein kinase, from Arabidopsis thaliana, and 39% homology with a NADPH-dependent reductase from Z. mays. The lectin showed hemagglutinating activity toward several erythrocytes, including human A, B, and O. Hapten inhibition assays indicated that the lectin interacts specifically with the OH on C4 from galactose residues. OH- on C1 plays a relevant role in the interaction with CCL, since β-galactose residues are better recognized than those from the anomeric α-galactose. Lack of lectin activity was observed in corn extracts; the highest specific activity was obtained from coleoptyle obtained at the 7th day after seeding.     

A novel lectin from the wild mushroom Polyporus adusta

A lectin with antiproliferative activity toward tumor cell lines and mitogenic activity toward splenocytes was isolated from the mushroom Polyporus adusta. The lectin was composed of two identical subunits each with a molecular weight of 12 kDa. It was adsorbed on both DEAE-cellulose and Q-Sepharose and unadsorbed on CM-Sepharose. The hemagglutinating activity of the lectin was inhibited by turanose and by a large variety of other carbohydrates. It was adversely affected in the presence of NaOH or HCl at a concentration of 7.5mM and above, and when the ambient temperature was raised above 70 degrees C. All divalent and trivalent metallic chlorides tested at 1.25-10mM including CaCl(2), MgCl(2), ZnCl(2), MnCl(2), and AlCl(3), did not alter the hemagglutinating activity of the lectin. FeCl(3) at 10mM caused the hemagglutinating activity to increase by 100%, but it did not change the lectin activity when tested at lower concentrations up to 5mM.     

A Calcium Ion-Dependent Dimeric Bean Lectin with Antiproliferative Activity Toward Human Breast Cancer MCF-7 Cells

In this study, a 60.8-kDa dimeric lectin was isolated from the Phaseolus vulgaris cv. jade bean and characterized. The lectin was bound on Blue Sepharose 6 and Q Sepharose and was finally purified by size exclusion chromatography on Superdex 200. Its hemagglutinating activity toward rabbit erythrocytes was dependent on divalent cations, especially calcium ions. Various carbohydrates tested were devoid of any effect on the hemagglutinating activity. The lectin was stable at pH between 4.5 and 9.4 and temperatures between 30 and 70 °C. It did not exert antifungal activity toward Valsa mali, Setosphaeria turcica, Mycosphaerella arachidicola, Fusarium oxysporum and Bipolaris maydis. The IC₅₀ of the antiproliferative activity of the lectin toward MCF-7 human breast cancer cells was 174 μM. It did not inhibit proliferation of WRL-68 human normal embryonic hepatocytes. The lectin was dependent on calcium ions for hemagglutinating activity and possessed a blocked N-terminus. These two characteristics make the lectin unique among Phaseolus lectins.     

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.     

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.     

First report of an arabinose-specific fungal lectin

To date, arabinose-binding lectins have been reported only from the human opportunistic pathogen Pseudomonas aeruginosa, the plant aggressive pathogen Ralstonia solanacearum, and the sponge Pellina semitubulosa. An arabinose-binding lectin with mitogenic activity toward splenocytes and a high specific hemagglutinating activity was isolated in the present study from a wild discomycete mushroom, Peziza sylvestris. The maximal mitogenic activity was induced by a lectin concentration of 8 microM. The lectin was a single-chained protein with a molecular mass of 20 kDa. Its N-terminal sequence showed only slight resemblance to other mushroom lectins. It was adsorbed on both diethylaminoethyl-cellulose and carboxymethyl-cellulose. Unlike previously reported mushroom lectins, the hemagglutinating activity of the lectin was inhibited by arabinose, but not by a large variety of other carbohydrates. The lectin activity was adversely affected in the presence of 0.05 M NaOH or 0.025 M HCl, and when the ambient temperature was elevated above 35 degrees C.     

First report of a xylose-specific lectin with potent hemagglutinating, antiproliferative and anti-mitogenic activities from a wild ascomycete mushroom

From fresh fruiting bodies of the wild ascomycete mushroom (Xylaria hypoxylon) a lectin with N-terminal sequence resemblance to a part of Aspergillus oryzae genome and only slight similarity to fungal immunomodulatory protein from the mushroom Flammulina velutipes was isolated. The protocol comprised extraction with water, precipitation from the aqueous extract using 80% saturated (NH(4))(2)SO(4), ion exchange chromatography on DEAE-cellulose and CM-cellulose, and then gel filtration by fast protein liquid chromatography on Superdex 75. Lectin activity was adsorbed on DEAE-cellulose and unadsorbed on CM-cellulose. The lectin appeared as a single band with a molecular mass of 14.4 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a single 28.8-kDa peak in gel filtration on Superdex 75. The lectin exhibited highly potent antiproliferative activity toward tumor cell lines, and exerted a potent anti-mitogenic action on mouse splenocytes. The hemagglutinating activity of the lectin was inhibited by inulin and xylose. It was stable up to 35 degrees C. At 40 degrees C its hemagglutinating activity was reduced by 50%, and it dwindled to 12.5% of the original activity at 50 degrees C. The hemagglutinating activity was also sensitive to NaOH and HCl solutions. The hemagglutinating activity was unaffected by CaCl(2) and ZnCl(2), and was potentiated substantially in the presence of AlCl(3) and FeCl(3). The distinctive features of this lectin comprise a unique sugar specificity, and highly potent hemagglutinating, antiproliferative and anti-mitogenic activities. X. hypoxylon lectin differs in molecular mass, N-terminal sequence and sugar specificity from previously reported ascomycete mushroom lectins.     

Isolation of a seed coagulant Moringa oleifera lectin

In this work hemagglutinating activity (HA) was investigated in distinct Moringa oleifera tissue extracts. A new lectin from seeds (cMoL) was purified and characterized; hemagglutinating and coagulating activities were evaluated. HA was detected in 0.15 M NaCl extracts from flowers and rachis inflorescence (5%, w/v), seeds, leaves, fundamental tissue of stem and steam bark (10%, w/v). cMoL isolated after saline extraction and guar gel column chromatography was active at pH range 4.0–9.0 agglutinating erythrocytes from rabbit and human blood types. Extracts of tissues and cMoL activities were carbohydrate inhibited; azocasein and asialofetuin abolished cMoL HA. The lectin was thermostable at 100 °C during 7 h. Polyacrylamide gel electrophoresis under reduced conditions revealed a main polypeptide band of 26.5 kDa; native basic cMoL was detected as a unique band. Seed lectin preparations and cMoL showed coagulant activity, similar to aluminium sulphate, the coagulant most widely used in water treatment.     

A new Bauhinia monandra galactose-specific lectin purified in milligram quantities from secondary roots with antifungal and termiticidal activities

This work describes the purification in milligram quantities of a lectin from Bauhinia monandra secondary roots (BmoRoL) and its antifungal and termiticidal activities. The BmoRoL (6.2 mg) was isolated through ammonium sulfate fractionation and affinity chromatography on guar gel. Native lectin was resolved as a single band on polyacrylamide gel electrophoresis for basic proteins. Under denaturing and reducing conditions it appeared as a unique glycosylated polypeptide of 26 kDa. The highest agglutination activity of BmoRoL was found with glutaraldehyde-treated rabbit erythrocytes. BmoRoL showed antifungal activity against phytopathogenic species of Fusarium and was more active on Fusarium solani. The lectin also showed termiticidal activity on Nasutitermes corniger workers and soldiers with LC₅₀ of 0.09 and 0.395 mg ml⁻¹ for 12 days. In conclusion, BmoRoL is a new antifungal and termiticidal lectin that can be purified in milligram quantities and has potential biotechnological application for control of agricultural pests.