Purification, characterization and cloning of a ricin B-like lectin from mushroom Clitocybe nebularis with antiproliferative activity against human leukemic T cells

• Published in: Biochimica et biophysica acta 2009-03, Vol.1790 (3), p.173-181
• Main Authors: Pohleven, Jure, Obermajer, Nataša, Sabotič, Jerica, Anžlovar, Sabina, Sepčić, Kristina, Kos, Janko, Kralj, Bogdan, Štrukelj, Borut, Brzin, Jože
• Format: Article
• Language: English
• Subjects: Agaricales - chemistry; Amino Acid Sequence; Antiproliferative effect; Carbohydrate Sequence; Cell Line, Tumor; Cell Proliferation - drug effects; Chromatography, Gel; Clitocybe nebularis; DNA, Complementary; Electrophoresis, Polyacrylamide Gel; Humans; Hydrogen-Ion Concentration; Isoelectric Point; Lectins - chemistry; Lectins - genetics; Lectins - isolation & purification; Lectins - pharmacology; Leukemia, T-Cell - pathology; Leukemic T cells; Molecular Sequence Data; Molecular Weight; Mushroom; Ricin B-like lectin; Sequence Homology, Amino Acid; Spectrometry, Mass, Electrospray Ionization
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2008.11.006

Lectins are a diverse group of carbohydrate-binding proteins exhibiting numerous biological activities and functions. Two-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. A 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. The 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. CNL 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.