Purification and characterization of a thermostable mycelial lectin from basidiomycete Lentinus squarrosulus

Lectins are non-immune carbohydrate-binding proteins or glycoproteins with specific binding sites for certain glycoconjugates. Fungal lectins have been documented for their antitumour, antiproliferative, immunomodulatory, hypotensive and insecticidal effects. In the present study, a mycelial lectin having molecular mass 55 kDa has been purified and characterized from Lentinus squarrosulus. Biological action spectrum of the lectin revealed agglutination of all human blood types (A, B, O, AB), goat, sheep, rabbit and pig erythrocytes. Neuraminidase treatment of blood type O erythrocytes considerably augmented hemagglutination titre. Carbohydrate inhibition studies showed its high affinity to mucin and asialofetuin. Lectin was purified by a combination of ammonium sulphate precipitation, dialysis, ion exchange chromatography and gel filtration chromatography. Optimum pH for lectin activity was observed to be 6.5–8.0 and optimum temperature was 25–30°C. Lectin showed poor pH stability and was stable within pH 7.0–7.5. It was highly thermostable and could withstand temperature upto 70°C. Lectin activity was sensitive to ethylenediaminetetraacetic acid and denaturants.     

Isolation and characterization of a lectin from the cephalochordate Branchiostoma lanceolatum (Pallas).

1. A lectin was isolated from an extract of Branchiostoma lanceolatum by affinity chromatography using an asialo-A-peptone-cellulose column. 2 The lectin is a glycoprotein with a carbohydrate content of 2.7%. The mol. wt is 392,000 +/- 28,000. Two subunits of identical size (183,000 +/- 3000) are linked by non-covalent bonds. 3. The lectin agglutinates a variety of erythrocytes including human A, B, O red blood cells as well as human lymphocytes. 4. Hemagglutination activity is inhibited best by N,N',N"-triacetylchitotriose, followed by N,N'-diacetylchitobiose, which is half as inhibitory. 5. Lectin activity is constant between pH 5 and 10. Divalent cations are not required for binding reactions. Activity is totally destroyed by heating to 60 degrees C for 30 min. 6. The lectin is precipitated from the extract by 30-40% ammonium sulfate saturation.     

Lectins in Immature Pods of Winged Bean,Psophocarpus tetragonolobus (L.) DC.

Lectin activity in immature pods from 30 strains of winged bean was investigated. Most of the lectin activity occurred in green shells and a small portion in immature seeds. Hemagglutinating activity of green shells was classified into four groups, according to the agglutination of trypsinized human type A, B, and O erythrocytes. Extracts from green shells of four representative strains which showed different hemagglutination patterns gave different elution profiles of lectin activity from ion exchange columns. Four types of lectin activity with different blood group specificities were apparently found in green shells.     

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.     

Production and characterization of lectin from <Emphasis Type="Italic">Bauhinia holophylla</Emphasis> (Fabaceae:Cercideae) calli

Bauhinia holophylla is a woody plant found in the Brazilian Cerrado used in folk medicine to treat diabetes. Lectins are glycoproteins with several biotechnological applications and play important roles in plant defenses and in medicine. Lectins have been produced in vitro using plant tissue culture techniques. This study shows the production and characterization of lectin from B. holophylla by callus culture. Calli were obtained from leaf segments placed on woody plant and Murashige and Skoog media supplemented with 2,4-dichlorophenoxyacetic acid (0, 4.52, 9.05, 18.10 µM) or 6-benzylaminopurine (0, 4.44, 8.88, 17.75 µM), in the presence or absence of light. The highest concentrations of lectin expressed by hemagglutination activity were observed in green and compact callus induced in woody plant medium supplemented with 6-benzylaminopurine in the presence of light, which showed specificity by all types of erythrocytes tested. The highest concentrations of lectin (128 HU mg^?1) and fresh weight of callus were observed in the stationary phase (41st–60th day). Lectin was not detected in subcultured calli. d-Galactose promoted the highest inhibitory effect on the lectin activity in human erythrocytes ABO system, especially B-type. Lectin has been shown to be a stable protein, maintaining its hemagglutination activity after incubation at a wide range of temperatures, pH values and ethylenediaminetetraacetic acid concentrations.     

THE NATURE OF SULPHATION OF URONIC ACID-CONTAINING GLYCOSAMINOGLYCANS CATALYSED BY BRAIN SULPHOTRANSFERASE

—A sulphotransferase system of rat brain catalyses the transfer of sulphate from 3′-phosphoadenosine 5′-phosphosulphate to the low-sulphated glycosaminoglycans isolated from normal adult human brain. These were shown to be precursors of higher-sulphated glycosaminoglycans by DEAE-Sephadex column chromatography and paper electrophoresis. Nitrous acid degradation and mild acid hydrolysis of enzymically-sulphated fractions further confirmed the presence of heparan sulphate in human brain. A partially purified sulphotransferase preparation was obtained from neonatal human brain using chondroitin-4-sulphate as sulphate acceptor. This sulphotransferase catalyses the transfer of sulphate to the various uronic acid containing glycosaminoglycans. Heparan sulphate was the best sulphate acceptor followed by dermatan sulphate, N-desulphoheparin, chondroitin-4-sulphate and chondroitin-6-sulphate in decreasing order. Sulphotransferase obtained from 1-day-old rat, rabbit and guinea pig brain also had the same pattern of specificity towards various sulphate acceptors. This sulphotransferase catalyses both N-sulphation and O-sulphation. Studies on the sulphotransferase obtained from both rat and human brain of various age groups indicate that the ratio of N-sulphation: O-sulphation decreases as the brain matures.     

Human erythrocyte specific lectin from the seeds of Indian coral tree,<Emphasis Type="Italic">Erythrina variegata</Emphasis> Linn,var.<Emphasis Type="Italic">orientali</Emphasis> Linn,Merrill

Human erythrocyte specific lectin was isolated from the seeds ofErythrina variegata Linn. var.orientalis Linn. Merrill. The lectin preferentially agglutinated erythrocytes in the sequence of O>B>A = AB. The lectin was purified 19-fold by affinity chromatography on acid treated sepharose 4B with an yield of 81%. The purified lectin was found homogeneous on polyacrylamide gel electrophoresis. The erythroagglutination reaction was inhibited by N-acetyl-D-galactosamine, D-galactose and lactose at very low concentration. The haemagglutination by the purified lectin was not inhibited by different hexose and pentose sugars even at high concentration. The purified lectin was a glycoprotein and agglutinated leucocytes at 3 μg protein concentration. The lectin induced transformation of peripheral blood lymphocytes in cultures.     

Haemolysis of rabbit erythrocytes by a lectin from the seeds of<Emphasis Type="Italic">Croton tiglium</Emphasis>

The kinetics of haemolysis of rabbit erythrocytes byCroton tiglium lectin was studied as a function of concentration of the lectin and erythrocytes. The length of the prelytic period decreased with increasing lectin concentrations, indicating that the secondary events at the membrane which follow the binding of the lectin to cell surface carbohydrate receptors are accelerated at higher surface concentrations of the lectin. The rate or extent of haemolysis was not affected by the inclusion of ions like K⁺, Ca²⁺ and Mg²⁺ in the medium or by the substitution of ionic medium by a non-ionic medium. The inhibition of haemagglutination and haemolysis of rabbit red cells byCroton tiglium lectin by antilectin rabbit serum was observed. A possible mechanism of haemolysis by the lectin is discussed.     

Multiple molecular forms of peanut lectin: Classification of isolectins and isolectin distribution among genotypes of the genus Arachis

Peanut lectin (or an immunologically indistinguishable material) is present in seeds of 4556 genotypes of the peanut, Arachis hypogaea, and in 65 genotypes of related species of Arachis. Seeds of one line of A. villosa and three lines of unclassified Arachis spp. are devoid of the lectin. Peanut lectin from 116 A. hypogaea genotypes is resolved by isoelectric focusing into three related isolectin profiles, which are designated the V, S, and V₂ types. Each is composed of from six to eight separate isolectins. Peanut lectin from A. monticola, A. pusilla, and one genotype of Arachis spp. is of the V type; isolectin profiles from other wild Arachis genotypes are variable, but comprise several distinct groups. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate resolves peanut lectin preparations from 37 genotypes into the lectin subunit of M_r 30,000 and a second polypeptide of M_r 60,000. Lectin preparations from five genotypes lack the M_r 60,000 polypeptide band and have a subunit that migrates slightly faster (and therefore probably is of lower molecular weight) than the subunits of all other tested lines. Peanut lectin preparations from 62 lines have specific hemagglutinating activities ranging from 1024 to 4196 with desialyzed human Type O erythrocytes. The lectin from one genotype exhibits substantially less hemagglutinating activity and is hemolytic.     

Blood group B-specific lectin of Plecoglossus altivelis (Ayu fish) eggs

A lectin that agglutinates human blood group B erythrocytes but not blood group A and O erythrocytes was isolated from eggs of Ayu sweet fish (Plecoglossus altivelis). The lectin also agglutinates Ehrlich ascites carcinoma cells but not rat ascites hepatoma AH109 or rat sarcoma 150 cells tested. The lectin agglutination was most effectively inhibited by monosaccharides with the first type of configuration, i.e., L-rhamnose, L-mannose and L-lyxose at a concentration of 0.03 mM. The lectin agglutination was moderately inhibited by monosaccharides with the second type of configuration, i.e., D-galactose, D-fucose and D-galacturonic acid at a concentration of 0.4 mM. However, the agglutination was not inhibited by various other monosaccharides and oligosaccharides that have other types of configuration. The basis for an apparent B-specific hemagglutination may be due to the steric similarity of the C₂ and C₄ of the galactosyl series, the B-specific determinant, and the L-rhamnosyl series, which are the best inhibitors of the lectin activity. The lectin was affinity purified on an L-rhamnosyl-Sepharose column and was characterized as a homogeneous low molecular weight protein (M_r 14 000) with an abundance of hydrophobic amino acids and dicarboxylic amino acid.     

Properties of the human erythrocyte membrane receptors for peanut and Dolichos biflorus lectins.

Neuraminidase treatment of blood type A and B human erythrocytes, which is required for the agglutination of these cells by peanut (Arachis hypogaea) lectin, increased the number of receptor sites for the lectin from about 5 × 10⁴ to 1.8 × 10⁶ sites/ cell for both blood types. The same treatment also increased the agglutinability of type A cells by the blood group A-specific Dolichos biflorus lectin, but the number of receptor sites for this lectin (~6 × 10⁵ sites/cell) did not change. D. biflorus lectin binding and agglutination of blood type B cells were negligible both before and after neuraminidase treatment. To isolate the peanut agglutinin receptor from the membrane of these cells, washed type A erythrocytes were incubated with neuraminidase and galactose oxidase and then treated with NaB³H₄, thus labeling the galactose residues on the membrane. For measuring peanut agglutinin receptor activity, a radioaffinity assay was developed based on the displacement of [¹⁴C]asialofetuin from peanut agglutinin by receptor and precipitation of the complex in the presence of polyethyleneglycol. Membranes were isolated by hypotonic lysis and were solubilized in 0.5% Empigen BB, a zwitterionic detergent, which was found to be superior to Triton X-100 for this purpose. The cell extract, after centrifugation, was subjected to affinity chromatography on peanut agglutinin-polyacrylhydrazido-Sepharose. Elution with lactose afforded a peak of radioactivity (32% yield) containing 70% of the applied receptor activity. The eluting sugar and the receptor were separated by chromatography on Bio-Gel P-2 with subsequent dialysis against 80% acetone to remove the detergent. The bulk of the isolated receptor radioactivity (91%) precipitated with peanut agglutinin. The amino acid composition, the glucosamine and galactosamine content and the electrophoretic mobility, on polyacrylamide gel electrophoresis in sodium dodecyl sulfate of the peanut receptor were similar to those of asialoglycophorin. In addition, the peanut receptor coprecipitated with asialoglycophorin and with isolated erythrocyte T antigen on Ouchterlony double-diffusion plates against peanut agglutinin and the Ricinus communis lectin, but not with D. biflorus lectin, suggesting that the receptor for the latter lectin is distinct from the peanut agglutinin receptor.     

Purification of an N-acetyl-d-glucosamine specific lectin (P. B. A.) from epidermal cell membranes of Pieris brassicae L

We report the isolation and the purification of an N-acetyl-d-glucosamine specific lectin capable of agglutinating either fixed trypsinized rabbit erythrocytes or chitin particles. An agglutinin assay based on the affinity of this lectin for the chitin was devised with fluorescent particles of scorpion cuticle to measure lectin activity during purification steps.Lectin was isolated from epidermal cell membranes; its molecular weight was determined by gel filtration and polyacrylamide electrophoresis in sodium dodecyl sulfate. Mr was estimated to be 43,000. Lectin could be constituted by two subunits, Mr of which was estimated to be 23,000. The specificity of this lectin against N-acetyl-d-glucosamine and its oligomers suggests a possible role in the dynamics of these saccharides during the cuticle cycle.     

Purification and characterization of an N-acetyl-D-galactosamine-specific lectin from seeds of chickpea (Cicer arietinum L.)

A novel lectin (CAA-II) was isolated and purified from the seeds of Cicer arietinum by ammonium sulphate fractionation and affinity chromatography on an N-acetyl-D-galactosamine-linked agarose column. The lectin is composed of four identical subunits of 30 kDa and the molecular mass of the native lectin was estimated to be 120 kDa by gel filtration chromatography and confirmed by mass spectrometry. The lectin showed agglutination activity against rabbit erythrocytes (trypsin-treated and untreated) as well as against human erythrocytes. Haemagglutination inhibition assays showed that the lectin is a galactose-specific protein having a high affinity for N-acetyl-D-galactosamine. The molecular weight, haemagglutination pattern, carbohydrate specificity and N-terminal amino acid sequence indicated that the lectin is clearly distinct from the previously reported chickpea lectin CAA-I.     

Expression of c-Fos in subcutaneous adipose tissue of the fetal pig

Calcium oxalate crystal growth and aggregation leads to the formation of renal calculi. It is known to be inhibited by several compounds both in vitro and in vivo conditions. The present study highlights the inhibitory potential of sodium pentosan polysulphate (SPP), a semi-synthetic glycosaminoglycan (GAG) on calcium oxalate crystal growth in vitro. Its efficacy was compared with those of known inhibitors like pyrophosphate, heparin and chondroitin-4-sulphate. Of the above compounds pyrophosphate was found to be the most potent inhibitor. Among the GAGs, SPP exhibited 80% inhibitory activity as compared to heparin. A lesser degree of inhibition was observed with chondroitin-4-sulphate.     

A phytohaemagglutinin from the Azolla-anabaena symbiosis

Proteins recovered from cell-free extracts of the Azolla-Anabaena azollae symbiosis exhibited haemagglutination activity; galactose was the most effective carbohydrate tested in preventing haemagglutination. Extracts of cyanobacteria-free Azolla also caused haemagglutination but extracts of free-living or symbiotic Anabaena azollae did not. Symbiotic Azolla plants grown on NO₃^? showed lower haemagglutination activity than did those grown on N₂; activity increased on removal of NO₃^?. The lower activity of the NO₃^?-grown material may be due to NO₃su? exerting a direct effect on lectin activity/synthesis, or it may act indirectly by inhibiting the development of Anabaena which in turn affects the Azolla lectin. The purified lectin was shown to be composed of 6 sub-units, each of M.W. 21000.     

One-step partial purification of Solanum tuberosum tuber lectin using magnetic chitosan particles

A one-step procedure for a partial purification of Solanum tuberosum tuber lectin has been developed. Lectin from tuber extract or from potato wastewater was adsorbed to magnetic chitosan particles and eluted with low pH buffer. The specific activity of separated lectin increased 13 times during the purification process and the recovery was 50%.     

Hemolysis and clearance of erythrocytes in Scylla serrata are related to the agglutination by the native sialic acid-specific lectin

The hemolymph of the crab Scylla serrata contains a lectin specific for N-glycolylneuraminic acid. The role of the sialic acid-specific lectin on natural immunity of the crab is studied by using several kinds of mammalian erythrocytes as a pathogen model. A significant correlation is observed between in vivo clearance of exogenous erythrocytes with the extent of erythrocyte agglutination by the lectin. Similarly, another correlation is noticed between the susceptibility of erythrocytes to lectin-dependent hemocytc-mediated hemolysis and the extent of lectin-mediated erythrocyte agglutination. Two hours after administration of the erythrocytes into the hemocoel, induced augmentation of hemagglutinating activity was observed against all erythrocytes, whether agglutinated highly or least by the lectin, suggesting an increase in the circulating lectin. This study documents that “opsonization” of foreign pathogen by the native lectin is an important step in hemocyte recognition, hemolysis and clearance of the pathogen.     

Purification of a lectin from Eugenia uniflora L. seeds and its potential antibacterial activity

Aims: The aim of this work was to analyse the antimicrobial properties of a purified lectin from Eugenia uniflora L. seeds. Methods and Results: The E. uniflora lectin (EuniSL) was isolated from the seed extract and purified by ion‐exchange chromatography in DEAE‐Sephadex with a purification factor of 11·68. The purified lectin showed a single band on denaturing electrophoresis, with a molecular mass of 67 kDa. EuniSL agglutinated rabbit and human erythrocytes with a higher specificity for rabbit erythrocytes. The haemagglutination was not inhibited by the tested carbohydrates but glycoproteins exerted a strong inhibitory action. The lectin proved to be thermo resistant with the highest stability at pH 6·5 and divalent ions did not affect its activity. EuniSL demonstrated a remarkable nonselective antibacterial activity. EuniSL strongly inhibited the growth of Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella sp. with a minimum inhibitory concentration (MIC) of 1·5 μg ml^?1, and moderately inhibited the growth of Bacillus subtilis, Streptococcus sp. and Escherichia coli with a MIC of 16·5 μg ml^?1. Conclusions: EuniSL was found to be effective against bacteria. Significance and Impact of the Study: The strong antibacterial activity of the studied lectin indicates a high potential for clinical microbiology and therapeutic applications.     

Effect of intestinal erythrocyte agglutination on the feeding performance of Triatoma brasiliensis (Hemiptera: Reduviidae)

Triatoma brasiliensis is an important vector of Trypanosoma cruzi in Brazil. The feeding efficiency on its hosts depends on several parameters including the maintenance of the ingested blood at low viscosity, which could be modulated by the anterior midgut (crop) anticoagulant and haemagglutinant activities. In the present study, we characterized T. brasiliensis crop haemagglutination activity and evaluated its importance in the feeding process. Soluble crop contents (SCC) of T. brasiliensis were able to agglutinate rat, mouse and rabbit eryhtrocytes, but had no activity on cattle and Thrichomys apereoides, a rodent species commonly associated with T. brasiliensis in the wild. The haemagglutination was characterized by the immediate formation of several clusters of erythrocytes connected by flexible elastic-like fibers. The feeding efficiency of T. brasiliensis on rat (agglutinated by SCC) was almost double that from T. apereoides (not agglutinated by SCC). The influence of haemagglutination on feeding was confirmed by artificially feeding bugs on a diet composed of cattle or rat erythrocytes. The bugs fed on cattle erythrocytes had lower ingestion rates in comparison to those fed on rats. The results indicate that, in addition to other parameters, haemagglutination brought about by SCC has an important role in the feeding efficiency of T. brasiliensis.     

Purification and Characterization of a Mucin Specific Mycelial Lectin from Aspergillus gorakhpurensis: Application for Mitogenic and Antimicrobial Activity

Background

Lectins are carbohydrate binding proteins or glycoproteins that bind reversibly to specific carbohydrates present on the apposing cells, which are responsible for their ability to agglutinate red blood cells, lymphocytes, fibroblasts, etc. Interest in lectins has been intensified due to their carbohydrate specificity as they can be valuable reagents for the investigation of cell surface sugars, purification and characterization of glycoproteins. The present study reports the purification, characterization and evaluation of mitogenic and antimicrobial potential of a mycelial lectin from Aspergillus gorakhpurensis.

Methods

Affinity chromatography on mucin-sepharose column was carried out for purification of Aspergillus gorakhpurensis lectin. The lectin was characterized for physico-chemical parameters. Mitogenic potential of the lectin was evaluated against splenocytes of Swiss albino mice by MTT assay. Antimicrobial activity of the purified lectin has also been evaluated by disc diffusion assay.

Results

Single-step affinity purification resulted in 18.6-fold purification of the mycelial lectin. The molecular mass of the lectin was found to be 70 kDa and it was composed of two subunits of 34.8 kDa as determined by gel filtration chromatography, SDS-PAGE and MALDI-TOF analysis. pH optima of the lectin was found to be 6.5–9.5, while optimum temperature for lectin activity was 20–30°C. Lectin was stable within a pH range of 7.0–10.5 and showed fair thermostability. EDTA did not affect lectin activity whereas it was found susceptible to the denaturants tested. MTT assay revealed strong mitogenic potential of A. gorakhpurensis lectin at a concentration upto 150 µg/mL. Antimicrobial activity assay showed its potent antibacterial activity against Bacillus cereus, Staphylococcous aureus and Escherichia coli and marginal antifungal activity against Saccharomyces cerevisiae.

Conclusion

This is the first report on the mitogenic and antimicrobial potential of Aspergillus gorakhpurensis lectin. The results will provide useful guidelines for further research in clinical applications of this lectin.