Purification and characterization of a mucin-binding mycelial lectin from <Emphasis Type="Italic">Aspergillus nidulans</Emphasis> with potent mitogenic activity

Mucin-specific lectin from mycelium of Aspergillus nidulans was purified using anion exchange and gel filtration chromatographic techniques with an overall recovery of 32% and 21.97-fold purification. The purified lectin migrated as a single band in SDS–PAGE with an apparent molecular mass of 34 kDa. Carbohydrate analysis revealed that it is a glycoprotein with total sugar content of 2.54%. Optimal agglutination was observed when serially diluted lectin was incubated with human type O erythrocyte suspension at pH 7.0–8.0 and temperature 20–30°C. Lectin was found to be completely stable within pH 5.0–8.0 and temperature at or below 40°C. Demetallization by extensive dialysis against EDTA did not alter its haemagglutination activity. Lectin activity was reduced to half after 24 h incubation with urea and thiourea, with no such effect of guanidine HCl. The lectin showed potent mitogenic response towards mouse splenocytes, attaining a maximum at 200 μg/ml as compared to untreated control cells. Mitogenic lectins are invaluable tools to assess the functioning of immune cells. None of the microfungal lectin has yet been investigated for mitogenic activity. This is the first report on mitogenic activity of lectin from Aspergillus sp.     

<Emphasis Type="Italic">Narcissus tazetta</Emphasis> lectin shows strong inhibitory effects against respiratory syncytial virus,influenza A (H1N1, H3N2, H5N1) and B viruses

A mannose-binding lectin (Narcissus tazetta lectin [NTL]) with potent antiviral activity was isolated and purified from the bulbs of the Chinese daffodil Narcissus tazetta var. chinensis, using ion exchange chromatography on diethylaminoethyl (DEAE)-cellulose, affinity chromatography on mannose-agarose and fast protein liquid chromatography (FPLC)-gel filtration on Superose 12. The purified lectin was shown to have an apparent molecular mass of 26 kDa by gel filtration and 13 kDa by SDS-PAGE, indicating that it is probably a dimer with two identical subunits. The cDNA-derived amino acid sequence of NTL as determined by molecular cloning also reveals that NTL protein contains a mature polypeptide consisting of 105 amino acids and a C-terminal peptide extension. Three-dimensional modelling study demonstrated that the NTL primary polypeptide contains three subdomains, each with a conserved mannose-binding site. It shows a high homology of about 60%–80% similarity with the existing monocot mannose-binding lectins. NTL could significantly inhibit plaque formation by the human respiratory syncytial virus (RSV) with an IC₅₀ of 2.30 μg/ml and exhibit strong antiviral properties against influenza A (H1N1, H3N2, H5N1) and influenza B viruses with IC₅₀ values ranging from 0.20 μg/ml to 1.33 μg/ml in a dose-dependent manner. It is worth noting that the modes of antiviral action of NTL against RSV and influenza A virus are significantly different. NTL is effective in the inhibition of RSV during the whole viral infection cycle, but the antiviral activity of NTL is mainly expressed at the early stage of the viral cycle of influenza A (H1N1) virus. NTL with a high selective index (SI=CC₅₀/IC₅₀≥141) resulting from its potent antiviral activity and low cytotoxicity demonstrates a potential for biotechnological development as an antiviral agent.     

Isolation and characterization of a new phycoerythrin from the cyanobacterium <Emphasis Type="Italic">Synechococcus</Emphasis> sp. ECS-18

A new phycoerythrin, SCH-phycoerythrin, was purified from Synechococcus sp. ECS-18 by DEAE-Sephacel anion exchange chromatography and Sephacryl S-300 gel filtration. The protein pigment had an absorbance maximum at 542 nm and a fluorescence maximum at 565 nm. The native molecular mass was approximately 219 kDa as determined by gel filtration, and sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated the presence of two subunits, with molecular mass of 19 and 17.9 kDa. These observations are consistent with the (αβ)₆ subunit composition that is characteristic of phycoerythrins. The α- and β-subunits showed immunological identity by Ouchterlony double immunodiffusion with an anti-phycoerythrin antiserum. The DNA sequence of the SCH-phycoerythrin gene was determined by PCR amplification using primers based on the conserved N-terminal amino acid sequence of the α- and β-subunits of phycoerythrins.     

Purification and primary structure of a mannose/glucose‐binding lectin from Parkia biglobosa Jacq. seeds with antinociceptive and anti‐inflammatory properties

Parkia biglobosa (subfamily Mimosoideae), a typical tree from African savannas, possess a seed lectin that was purified by combination of ammonium sulfate precipitation and affinity chromatography on a Sephadex G‐100 column. The P. biglobosa lectin (PBL) strongly agglutinated rabbit erythrocytes, an effect that was inhibited by d ‐mannose and d ‐glucose‐derived sugars, especially α‐methyl‐d ‐mannopyranoside and N‐acetyl‐d ‐glucosamine. The hemagglutinating activity of PBL was maintained after incubation at a wide range of temperature and pH and also was independent of divalent cations. By sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis, PBL exhibited an electrophoretic profile consisting of a single band with apparent molecular mass of 45 kDa. An analysis using electrospray ionization–mass spectrometry indicated that purified lectin possesses a molecular average mass of 47 562 ± 4 Da, and the analysis by gel filtration showed that PBL is a dimer in solution. The complete amino acid sequence of PBL, as determined using tandem mass spectrometry, consists of 443 amino acid residues. PBL is composed of a single non‐glycosylated polypeptide chain of three tandemly arranged jacalin‐related domains. Sequence heterogeneity was found in six positions, indicating that the PBL preparations contain highly homologous isolectins. PBL showed important antinociceptive activity associated to the inhibition of inflammatory process. Copyright © 2013 John Wiley & Sons, Ltd.     

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, characterization, structural analysis and protein chemistry of a buffalo heart galectin-1

A soluble β-galactoside-binding lectin was purified by gel filtration chromatography from Bubalus bubalis heart. Its metal-independent nature, molecular weight of 14.5 kDa, preferential affinity for β-d-lactose, and 87–92% identity with carbohydrate recognition domain of previously reported galectin-1 confirmed its inclusion in galectin-1 subfamily. Stokes radii determination using gel filtration under reducing and non-reducing conditions revealed its homo-dimeric nature, further confirming its Gal-1 nomenclature. The purified lectin was found to be the most stable mammalian heart galectin purified till date, suggesting its preferential use in various recognition studies. Treatment of the purified lectin with oxidizing agent, thiol blocking reagents, denaturants, and detergents resulted in significant changes in UV–VIS, fluorescence, CD and FTIR spectra, which strongly emphasized the important aspect of regular secondary structure of galectins for the maintenance of their active conformation. Reduction of the activity of the purified lectin after oxidation by H₂O₂, with remarkable fluorescence quenching, may suggest potential role for galectin-1 in free radical-induced, oxidative stress-mediated cardiovascular disorders. The predictions of bioinformatics studies were found to be in accordance with the results obtained in wet lab.     

Isolation and Characterization of a Sialic Acid-specific Lectin from Hemolymph of the Southeast Asian Horseshoe Crab Tachypleus gigas

A lectin was isolated from hemolymph of the Southeast Asian horseshoe crab Tachypleus gigas by using glycophorin HA affinity chromatography and Sephacryl S-300 gel filtration. The purified lectin had a molecular mass of approximately 396 kDa and was composed of 13 identical subunits with molecular masses of 31 kDa. The serological specificity of the purified lectin was specifically inhibited by sialic acids sialoglycoproteins, but not by neutral sugars, hexosamines, N-acetylhexosamines, or asialoglycoproteins. Although the N-terminal amino acid sequence of the lectin from T. gigas was identical to that from American horseshoe crab (liphemin) by the same purification method and cross reacted with the anti-liphemin serum, the calcium concentration of hemagglutinating activity of the purified lectin showed a smaller optimal concentration than that of liphemin.     

Purification and characterization of a new mannose-specific lectin from Sternbergia lutea bulbs

A new mannose-binding lectin was isolated from Sternbergia lutea bulbs by affinity chromatography on an α(1-2)mannobiose-Synsorb column and purified further by gel filtration. This lectin (S. lutea agglutinin; SLA) appeared homogeneous by native-gel electrophoresis at pH 4.3, gel filtration chromatography on a Sephadex G-75 column, and SDS-polyacrylamide gel electrophoresis, These data indicate that SLA is a dimeric protein (20 kDa) composed of two identical subunits of 10 kDa which are linked by non-covalent interactions. The carbohydrate binding specificity of the lectin was investigated by quantitative precipitation and hapten inhibition assays. It is an α-D-mannose-specific lectin that interacts to form precipitates with various α-mannans, galactomannan and asialo-thyroglobulin, but not with α-glucans and thyroglobulin. Of the monosaccharides tested only D-mannose was a hapten inhibitor of the SLA-asialothryroglobulin precipitation system, whereas D-glucose, D-galactose and L-arabinose were not. The lectin appears to be highly specific for terminal α(1-3)-mannooligosaccharides. The primary structure of SLA appears to be quite similar to that of the snow drop (Galanthus nivalis) bulb lectin which is a mannose-binding lectin from the same plant family Amaryllidaceae. The N-terminal 46 amino acid sequence SLA showed 7% homology with that of GNA. Abbreviations: AAA, Allium ascalonicum agglutinin (shallot lectin); ASA, Allium sativum agglutinin (garlic lectin); AUA, Allium ursinum agglutinin (ramsons lectin); DAP, 1,3-diaminopropane; GNA, Galanthus nivalis agglutinin (snowdrop lectin); HHA, Hippeastrum hybr. agglutinin (amaryllis lectin); LOA, Listera ovata agglutinin (orchid twayblade lectin); NPA, Narcissus pseudonarcissus agglutinin (daffodil lectin); PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline, SLA, Sternbergia lutea agglutinin; SDS, sodium dodecyl sulfate; Me, methyl; Bn, benzyl; PNP, p-nitrophenyl. This revised version was published online in August 2006 with corrections to the Cover Date.     

Radical Scavenging Activity of Seela (<Emphasis Type="Italic">Sphyraena barracuda)</Emphasis> and Ribbon Fish (<Emphasis Type="Italic">Lepturacanthus savala)</Emphasis> Backbone Protein Hydrolysates

We have investigated the antioxidant activity of protein hydrolysates prepared from backbones of two commercially important fishes; seela (Sphyraena barracuda) and ribbon fish (Lepturacanthus savala). Pepsin and trypsin hydrolysates were found more potent to inhibit lipid peroxidation in case of ribbon and seela fish respectively and were further purified by using fast protein liquid chromatography on anion exchange and gel filtration chromatography. The active peaks after gel filtration chromatography of seela fish was able to scavenge 2,2-diphenyl-1-picryhydrazyl and hydroxyl radicals by 61 ± 2.3 and 58.7 ± 2.3% and ribbon fish hydrolysate by 60.0 ± 2.6 and 55.6 ± 1.8% as measured by ESR spectroscopy. And the active fractions showed presence of both essential and non-essential amino acids with high percentages of arginine (11.95 and 12.76%) and lysine (13.49 and 13.89%).     

Physicochemical Properties and Oxidative Inactivation of Soluble Lectin from Water Buffalo (<Emphasis Type="Italic">Bubalus bubalis</Emphasis>) Brain

Lectins are carbohydrate-binding proteins present in a wide variety of plants and animals, which serve various important physiological functions. A soluble β-galactoside binding lectin has been isolated and purified to homogeneity from buffalo brain using ammonium sulphate precipitation (40–70%) and gel permeation chromatography on Sephadex G_50–80 column. The molecular weight of buffalo brain lectin (BBL) as determined by SDS-PAGE under reducing and non-reducing conditions was 14.2 kDa, however, with gel filtration it was 28.5 kDa, revealing the dimeric form of protein. The neutral sugar content of the soluble lectin was estimated to be 3.3%. The BBL showed highest affinity for lactose and other sugar moieties in glycosidic form, suggesting it to be a β-galactoside binding lectin. The association constant for lactose binding as evidenced by Scatchard analysis was 6.6 × 10³ M⁻¹ showing two carbohydrate binding sites per lectin molecule. A total inhibition of lectin activity was observed by denaturants like guanidine HCl, thiourea and urea at 6 M concentration. The treatment of BBL with oxidizing agent destroyed its agglutination activity, abolished its fluorescence, and shifted its UV absorption maxima from 282 to 250 nm. The effect of H₂O₂ was greatly prevented by lactose indicating that BBL is more stable in the presence of its specific ligand. The purified lectin was investigated for its brain cell aggregation properties by testing its ability to agglutinate cells isolated from buffalo and goat brains. Rate of aggregation of buffalo brain cells by purified protein was more than the goat brain cells. The data from above study suggests that the isolated lectin may belong to the galectin-1 family but is glycosylated unlike those purified till date.     

Endogenous lectin from cultured soybean cells. Chemical characterization of the lectin of SB-1 cells

A lectin has been identified in the cell line, SB-1, originally derived from the roots of Glycine max. This lectin, which we shall refer to as SB-1 lectin, was isolated on the basis of its carbohydrate-binding activity (affinity chromatography on Sepharose column derivatized with N-caproyl-galactosamine) and its immunological cross-reactivity (immunoblotting with rabbit antibodies directed against seed soybean agglutinin (SBA]. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis of SB-1 lectin revealed a major polypeptide (Mr approximately equal to 30,000) which co-migrated with seed SBA. This form of the lectin was observed in fractions purified from culture medium of SB-1 cells or supernatant fraction of SB-1 cell suspension after enzymatic removal of cell wall. Extracts of SB-1 cells under some other conditions yielded a major band (Mr approximately equal to 60,000) as revealed by SDS-PAGE and immunoblotting with rabbit anti-seed SBA; prolonged incubation of these samples in the presence of SDS resulted in the appearance of the 30-kDa polypeptide. It appears that the 60-kDa band represented a highly stable, even under SDS-PAGE conditions, dimeric form of the 30-kDa subunit. The SB-1 lectin derived from the culture medium was compared with seed SBA by gel filtration and by peptide mapping after limited proteolysis; no difference between the lectins from the two sources was found. Extracts of soybean roots fractionated on N-caproyl-galactosamine-Sepharose affinity columns yielded, upon elution with galactose, polypeptides of Mr 30,000 and 60,000. These results suggest that soybean roots contain a lectin whose polypeptide composition corresponds to that of seed SBA and SB-1 lectin.     

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.     

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.     

High molecular weight precursors of glucans inSaccharomyces cerevisiae

Nascent -1,3 glucan synthesized by mixed membrane fractions fromSaccharomyces cerevisiae was solubilized by extraction with hot SDS or urea. Nature of the material was analyzed by electrophoresis and gel filtration. As determined by gel filtration, Mr of synthesized glucans exceeded 1,500 kDa, but was below 20,000 kDa. This nascent material served as an acceptor for further glucose transfer reactions, giving rise to glucan molecules over 20,000 kDa. It is suggested that the high Mr precursor components represent protein-bound glucan molecules in transit to the cell surface.     

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,characterization and induction of a C-type lectin in the freshwater planarian <Emphasis Type="Italic">Dugesia japonica</Emphasis>

Lectins are important components of the immune defense system of invertebrates. Given their important functions, numerous investigations have been carried out on the characterization and function of lectins in invertebrates. However, lectin studies with the freshwater planarian, an evolutionarily important animal, are rare. In this paper, we demonstrate agglutination of glutaraldehyde treated erythrocytes by a lectin with preference for rabbit erythrocytes. The result of hemagglutinating activity inhibition assays with several carbohydrates showed the most potent inhibitor was maltose. A natural lectin from the crude homogenates of freshwater planarian Dugesia japonica was purified by single step affinity chromatography using amylose-coupled agarose. The purified protein appeared as one band with a molecular mass of 350 kDa in PAGE, and as one band, approximately 56 kDa, in SDS-PAGE. The purified lectin showed dependence on calcium. The activity of the purified lectin was inhibited at temperatures greater than 50°C and showed a pH optimum between 5–8. The purified lectin also has binding activity to the Gram-negative bacteria E. coli, and the Gram-positive bacteria B. subtilis. Furthermore, the purified lectin obtained from injured and bacteria-induced planarians showed increased agglutinating activity against rabbit erythrocytes. These results suggest that the purified lectin may play an important role in the innate immunity of the freshwater planarian.     

Purification and characterization of an antimicrobial peptide produced by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. strain 4B

An antimicrobial peptide produced by a bacterium isolated from the effluent pond of a bovine abattoir was purified and characterized. The strain was characterized by biochemical profiling and 16S rDNA sequencing as Pseudomonas sp. The antimicrobial peptide was purified by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography. Direct activity on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was observed. A major band on SDS-PAGE suggested that the antimicrobial peptide has a molecular mass of about 30 kDa. The substance was inhibitory to a broad range of indicator strains, including pathogenic and food spoilage bacteria such as Listeria monocytogenes, Bacillus cereus, Staphylococcus aureus, among other. The partially purified antimicrobial substance remained active over a wide temperature range and was resistant to all proteases tested. This substance showed different properties than other antimicrobials from Pseudomonas species, suggesting a novel antimicrobial peptide was characterized.     

A unique post-translational processing of an exo-β-1,3-glucanase of Penicillium sp. KH10 expressed in Aspergillus oryzae

To characterize an exo-β-1,3-glucanase (ExgP) of an isolated fungal strain with high laminarin degradation activity, identified as Penicillium sp. KH10, heterologous secretory expression of the ExgP was performed in Aspergillus oryzae. Deduced amino acid sequence of the exgP gene possibly consisted of 989 amino acids which showed high sequence similarity to those of fungal exo-β-1,3-glucanases belonging to the glycoside hydrolase (GH) family 55. Notably, the purified recombinant ExgP showed a single protein peak in the native state (by gel-permeation chromatographic analysis), but showed two protein bands in the denatured state (by SDS–polyacrylamide gel electrophoresis). These two polypeptides exhibited activity in a coexisting state even under reducing conditions, suggesting that non-covalent association of both polypeptides took place. Taken together with the nucleotide sequence information, the ExgP precursor (104 kDa) would be proteolytically processed (cleaved) to generate two protein fragments (42 and 47 kDa) and the processed products (polypeptide fragments) would be assembled each other by a non-covalent interaction. Moreover, one of the matured ExgP polypeptides was N-glycosylated by the post-translational modification.