Effect of culture medium composition on the activity of extracellular lectins of Lentinus edodes

The time course of lectin production in culture liquid of the basidial fungus Lentinus edodes strain F-249 in different media under the conditions of submerged culture was studied. The activity of agglutinins depended on the ratio between carbon and nitrogen sources and pH of culture medium. The activity of lectin in culture medium was maximal when the fungus was grown in a medium containing L-arabinose as a source of carbon and L-asparagine as a source of nitrogen (C : N ratio, (9.5-12): 1)) on the day 15-18 of culturing at pH 8-9.     

Lectin activity of Lentinus edodes

The hemagglutinating activity of submerged mycelium and culture liquid for four strains of Lentinus edodes (Berk.) Sing [L. edodes (Berk.) Pegler] was studied in the search for lectins. The hemagglutinating activity of culture liquid was substantially higher, compared with mycelium. The carbohydrate-binding capacity of the agglutinins was established, and the lectin activity of extracts from mycelia grown on several agar media was elucidated in relation to fruiting. The lectin activity of L. edodes was examined at different morphogenetic steps: mycelium, brown mycelial film, primordium, and fruiting body. Hemagglutination titers at the brown film step were higher than in the mycelium, whereas activity at the primordial and fruiting bodies steps decreased. Lectins seem to be involved in the formation of hyphal aggregates of brown mycelial film.     

Optimization of the nutrient medium composition for directed biosynthesis of Bacillus mesentericus lectins

The medium has been optimized in order to increase the lectin-producing activity of Bacillus mesentericus. When a producer grew on the optimized medium, a 4-8-fold increase of the hemagglutination test titres and a 5.6-fold increase (as compared with the initial medium) of the specific lectin activity of the culture liquid was attained.     

Effect of culture medium composition on the activity of extracellular lectins of <Emphasis Type="Italic">Lentinus edodes</Emphasis>

The time course of lectin production in culture liquid of the basidial fungus Lentinus edodes strain F-249 in different media under submerged culture conditions was studied. The activity of agglutinins depended on the ratio between carbon and nitrogen sources and the pH of the culture medium. The lectin activity in culture medium was maximal when the fungus was grown in a medium containing L-arabinose as a source of carbon and L-asparagine as a source of nitrogen (C:N ratio, (9.5–12):1) on day 15–18 of culturing at pH 8.0–9.0.Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 2, 2005, pp. 200–203.Original Russian Text Copyright © 2005 by Tsivileva, Nikitina, Garibova.     

Erratum to: Extracellular protein production and morphogenesis of <Emphasis Type="Italic">Lentinula edodes</Emphasis> in submerged culture

Along with a brief review of Lentinula edodes (shiitake mushroom) submerged cultivation history within the framework of important extracellular proteins biosynthesis, this study contains the authors’ own results. The possibility of regulating the lectin activity of shiitake using the synthetic components is shown. The time course of lectin production in culture liquid of L. edodes in different media under submerged culture conditions was studied. The activity of agglutinins depended on the ratio between carbon and nitrogen sources and the pH of the culture medium. A relationship between the chemical composition of nutrient medium, the activity of extracellular lectins of L. edodes, and the formation of pigmented mycelial film in liquid culture has been found. The formulation of medium, on which the brown mycelial film appears in several days of submerged cultivation, is proposed. The results obtained make a contribution to the present notion of biochemical processes that give rise to the occurrence of the aforesaid morphological structure of shiitake. Finally, two extracellular lectins from the submerged culture of L. edodes have been isolated and purified to homogeneity. Their physicochemical properties and composition have been studied.     

Extracellular protein production and morphogenesis of <Emphasis Type="Italic">Lentinula edodes</Emphasis> in submerged culture

Along with a brief review of Lentinula edodes (shiitake mushroom) submerged cultivation history within the framework of important extracellular proteins biosynthesis, this study contains the authors’ own results. The possibility of regulating the lectin activity of shiitake using the synthetic components is shown. The time course of lectin production in culture liquid of L. edodes in different media under submerged culture conditions was studied. The activity of agglutinins depended on the ratio between carbon and nitrogen sources and the pH of the culture medium. A relationship between the chemical composition of nutrient medium, the activity of extracellular lectins of L. edodes, and the formation of pigmented mycelial film in liquid culture has been found. The formulation of medium, on which the brown mycelial film appears in several days of submerged cultivation, is proposed. The results obtained make a contribution to the present notion of biochemical processes that give rise to the occurrence of the aforesaid morphological structure of shiitake. Finally, two extracellular lectins from the submerged culture of L. edodes have been isolated and purified to homogeneity. Their physicochemical properties and composition have been studied.     

Change of the wheat lectin activity and degree of its interaction with different components of compositions of lectin nature

The wheat lectin hemagglutination activity and degree of its interaction with the bacterium Azotobacter chroococcum T79 and aminosaccharide N-acetyl-D-glucosamin hapten of wheat lectin was studied in laboratory experiments with the purpose of creation of biologic activity compositions of lectin nature for plant growing. It was shown that plant-bacterial compositions encloses the "bacteria+lectin" complex, free lectin and bacterial cells. The addition of aminosaccharide N-acetyl-D-glucosamin to wheat lectin, to the bacterial culture and plant-bacterial composition decreases its hemagglutination activity. The possibility of creation of new complexes in this compositions effected by hapten "lectin+hapten", "lectin+hapten+bacteria", "bacteria+hapten" is under discussion.     

Isolation and characterization of a novel lectin from the wild mushroom Xerocomus spadiceus

A lectin was isolated from extracts of fruiting bodies of the mushroom Xerocomus spadiceus using a procedure that involved ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on CM-cellulose, and gel filtration by fast protein liquid chromatography on Superdex 75. The lectin was unadsorbed on DEAE-cellulose and Affi-gel blue gel and adsorbed on CM-Sepharose. The lectin was capable of eliciting an approximately four-fold stimulation of mitogenic response in murine splenocytes. The hemagglutinating activity was stable up to 60 degrees C, halved at 70 degrees C, reduced to 25% at 75 degrees C and dwindled to an undetectable level at 80 degrees C. The activity remained unaltered in the presence of various divalent (Ca2+, Mg2+, Zn2+ and Mn2+) chlorides up to a salt concentration of 10 mM except in the case of ZnCl2. FeCl3 up to a concentration of 10 mM did not affect the hemagglutinating activity of the lectin. The hemagglutinating activity of the lectin was doubled in the presence of 5 mM AlCl3 or 10 mM ZnCl2 and quadrupled in the presence of 10 mM AlCl3. The activity was reduced in the presence of HCl and NaOH. Among the large number of carbohydrates tested, only inulin was able to inhibit the hemagglutinating activity of the lectin.     

Is the lectin binding pattern of human breast and colon cancer cells influenced by modulators of sialic acid metabolism?

Sialic acid residues are the most abundant terminal carbohydrate residues of mammalian cells. Modification of the sialic acid residues by exposure of cells in culture to sialic acid precursor analogues resulted in a modifed susceptibility to polyoma viruses. In the present study, human breast and colon cancer cell lines were exposed for 65 h to these acid precursor analogues at 5 mM and their lectin binding pattern was analysed. Use of a panel of several different lectins indicated that the pretreatment of these cell lines with the sialic acid analogues did not change their lectin binding profile. The incorporation of these precursors into membrane glycoproteins was assessed by reversed phase high-performance liquid chromatography, which clearly demonstrated that the precursors were incorporated. The results therefore indicate that these analogues are highly specific for sialic acid and do not interfere with other biosynthetic pathways of membrane glycoconjugates.     

Purification of Lectin from Micropropagated Roots Derived from Aseptic Seedling of <Emphasis Type="Italic">Canavalia ensiformis</Emphasis> L.

A well-organized protocol has been developed for high frequency root germination from the seed of Canavalia ensiformis on Murashige and Skoog (MS) medium. Surprisingly, the seeds that were grown on the MS medium having no growth hormone showed the best response. Roots of 30 days old aseptic seedling were homogenized and a lectin from them was purified on Sephadex G-50 affinity column. The finding that final product is a pure lectin was confirmed by specific hemagglutinating property. The final root lectin yield was 0.6% and eluted as a single peak. Root lectin specific activity was 50 times more than the seed lectin. Sugar specificity activity by hemagglutination-inhibition assay indicated that lectin belongs to glucose/mannose-specific group. Interestingly, the lectin was found to be 25 kDa, similar to molecular mass of Concanavalin A purified from seed of C. ensiformis, as revealed by SDS–PAGE. Thus, Concanavalin A from either source can be used for development of transgenic crops that are capable of expressing lectin gene and hence can efficiently perform biological nitrogen fixation by giving rise to nodules in their root. The advantage of this method is that purification of Concanavalin A in tissue culture conditions is easier, handy and is less time consuming.     

Isolation of a novel N-acetylglucosamine-specific lectin from fresh sclerotia of the edible mushroom Pleurotus tuber-regium

An N-acetylglucosamine-binding lectin with a molecular mass of 32kDa was isolated from fresh sclerotia of the edible mushroom Pleurotus tuber-regium. Its N-terminal sequence exhibited some similarity to that of Agaricus bisporus lectin. The isolation procedure was simple, involving (NH(4))(2)SO(4) precipitation, ion exchange chromatography on DEAE-cellulose, affinity chromatography on N-acetyl-D-glucosamine-agarose, and gel filtration by fast protein liquid chromatography on Superdex 75. The lectin exhibited hemagglutinating activity toward trypsinized rabbit erythrocytes but not toward untrypsinized rabbit erythrocytes.     

Purification and Characterization of Two Major Lectins from Araucaria brasiliensis syn. Araucaria angustifolia Seeds (Pinhão)

Two major lectins (lectin I and lectin II) were purified to homogeneity from the seeds of Araucaria brasiliensis (Gymnospermae). The purity of the lectins was confirmed by polyacrylamide gel electrophoresis, isoelectric focusing, and high performance liquid chromatography. They are glycoproteins in nature containing 6.3 and 2.9%, respectively, of neutral sugar and have absorption coefficients of 3.8 and 4.7, respectively, at 280 nanometers. The molecular weights of both lectins obtained by gel filtration on Sephacryl S-400 were equal: 200,000. After dissociation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, molecular weights were 20,000 and 34,000, respectively, for lectin I and lectin II, suggesting they are decameric and hexameric in nature. The amino acid composition of both lectins showed little difference, but both had high amounts of acidic amino acids and lacked methionine in their molecule. The carbohydrate binding specificity of lectins was directed towards mannose, glucose, and their oligomers. High inhibitory activity was also found with thyroglobulin. The erythroagglutinating activity of the lectins was enhanced in the presence of high-molecular-weight substances both at 37 and 4°C. Divalent cations do not appear to be essential for activity. They maintained their agglutinating activity over a broad but different range of pH: 5.5 to 7.5 and 6.5 to 7.5, respectively. Both lectins agglutinated erythrocytes of human ABO blood types equally well.     

Distribution of Lectins in Tissues, Derived Callus, and Roots of Psophocarpus tetragonolobus (Winged Bean)

The distribution of lectin in parental tissues, roots formed de novo from parental stem tissue, and derived callus cells of Psophocarpus tetragonolobus has been measured by hemagglutinating activity and radioimmunoassay. The antisera used for the radioimmunoassay was raised in rabbits to lectin isolated from seeds by affinity chromatography using insolubilized hog gastric mucin. The distribution of lectin in buffer extracts of the tissues (or cells) and the extracellular medium favors the tissues for in vitro grown roots, regardless of the culture conditions used. The lectin content of the extracellular medium is more significant for callus, regardless of its conditions of culture. The lectin activity of extracts of in vitro grown roots was higher than that of mature roots from whole plants. Differences in relative levels of lectin activity measured by hemagglutination and by radioimmunoassay, and differences in saccharide inhibition of hemagglutination, suggest the presence of multiple lectins in extracts of different tissues.     

Hormonal Regulation of the Lectin Biosynthesis in Callus Culture of the Phaseolus vulgaris Plant

Callus cultures established from Phaseolus vulgaris seedlings were used to investigate hormonal influence on lectin biosynthesis. The plant tissue cultures were initiated using defined levels of both a cytokinin (kinetin) and an auxin (2,4-dichlorophenoxyacetic acid) and were then transferred to media containing different amounts of these hormones. The lectin content of each callus culture was determined using an enzyme immunoassay specific for the seed lectin of the P. vulgaris plant. The lectin biosynthesis was directly affected by the levels of auxin and cytokinin in the culture media and no lectin was detected in hormone-free medium. This enabled us to compose culture media yielding a maximal or minimal lectin content of the callus cultures, illustrating the ability to induce an enhancement or suppression of the in vitro lectin biosynthesis. The lectin level of callus tissue during the growth cycle of a culture was, furthermore, related to the cellular growth rate which might indicate an involvement of the lectin in cellular events during rapid cell division.     

Site of hemolymph lectin production and its activation in vitro by 20-hydroxyecdysone

To identify the tissues which produce hemolymph lectin in larvae of Bombyx mori, ovary, testis, fat body, and hemocytes from 5th-instar larvae were cultured in vitro and the culture medium was partially purified and assayed for hemagglutinating activity. Among the tissues tested, hemocytes appeared to be a major source of the hemolymph lectins. Ovary produced lectins to about one-tenth of the amount observed for the hemocytes, whereas testis and fat body were not productive. To study the hormonal control of hemolymph lectin production by hemocytes, hemocytes from 4th-instar larvae were cultured in vitro. Hemagglutinating activity in the hemolymph of 4th-instar larvae was immunostainable with the monoclonal antibody raised against 350,000 dalton lectin found in the 5th-instar hemolymph, but their molecular sizes were larger than the 5th-instar hemolymph lectins. When 20-hydroxyecdysone was added into the medium, production of the lectin by the hemocytes was remarkably enhanced, depending upon the hormone concentration.