Purification,characterization and cloning of a ricin B-like lectin from mushroom Clitocybe nebularis with antiproliferative activity against human leukemic T cells |
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Authors: | Jure Pohleven,Nata&scaron a Obermajer,Jerica Sabotič,Sabina Anžlovar,Kristina Sepčić,Janko Kos,Bogdan Kralj,Borut &Scaron trukelj,Jože Brzin |
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Affiliation: | 1. Department of Biotechnology, Jo?ef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia;2. Faculty of Pharmacy, University of Ljubljana, Ašker?eva 7, SI-1000 Ljubljana, Slovenia;3. Department of Biology, Biotechnical Faculty, University of Ljubljana, Ve?na pot 111, SI-1000 Ljubljana, Slovenia;4. Department of Environmental Sciences, Jo?ef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia |
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Abstract: | BackgroundLectins are a diverse group of carbohydrate-binding proteins exhibiting numerous biological activities and functions.MethodsTwo-step serial carbohydrate affinity chromatography was used to isolate a lectin from the edible mushroom clouded agaric (Clitocybe nebularis). It was characterized biochemically, its gene and cDNA cloned and the deduced amino acid sequence analyzed. Its activity was tested by hemagglutination assay and carbohydrate-binding specificity determined by glycan microarray analysis. Its effect on proliferation of several human cell lines was determined by MTS assay.ResultsA homodimeric lectin with 15.9-kDa subunits agglutinates human group A, followed by B, O, and bovine erythrocytes. Hemagglutination was inhibited by glycoprotein asialofetuin and lactose. Glycan microarray analysis revealed that the lectin recognizes human blood group A determinant GalNAcα1–3(Fucα1–2)Galβ-containing carbohydrates, and GalNAcβ1–4GlcNAc (N,N'-diacetyllactosediamine). The lectin exerts antiproliferative activity specific to human leukemic T cells.ConclusionsThe protein belongs to the ricin B-like lectin superfamily, and has been designated as C. nebularis lectin (CNL). Its antiproliferative effect appears to be elicited by binding to carbohydrate receptors on human leukemic T cells.General significanceCNL is one of the few mushroom ricin B-like lectins that have been identified and the only one so far shown to possess immunomodulatory properties. |
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Keywords: | CNL, Clitocybe nebularis lectin RP-HPLC, reversed-phase high-performance liquid chromatography TFA, trifluoroacetic acid SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis FPLC, fast protein liquid chromatography PCR, polymerase chain reaction ESI, electrospray ionization 3&prime RACE, 3&prime rapid amplification of cDNA ends RicB1, ricin B chain lectin domain 1 Abra1, abrin-a B chain lectin domain 1 RicB2, ricin B chain lectin domain 2 Abra2, abrin-a B chain lectin domain 2 Flav, flavastacin MOA, Marasmius oreades agglutinin LSL, Laetiporus sulphureus lectin |
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