TITLE:
Isolation, purification and characterization of an N-acetyl-D-lactosamine binding mitogenic and anti-proliferative lectin from tubers of a cobra lily Arisaema utile Schott
AUTHORS:
Vikram Dhuna, Kshitija Dhuna, Jatinder Singh, Ajit Kumar Saxena, Satyam Kumar Agrawal, Sukhdev Singh Kamboj
KEYWORDS:
N-acetyl-D-lactosamine; Arisaema Utile; Affinity Purification; Anti-Proliferative; Asialofetuin; Chemical Modification; Lectin; Mitogenicity
JOURNAL NAME:
Advances in Bioscience and Biotechnology,
Vol.1 No.2,
June
30,
2010
ABSTRACT: Lectins are the carbohydrate-binding proteins of non-immune origin which have been the subject of intense investigation over the last few decades owing to the variety of interesting biological properties. Most of the lectins which have been purified and characterized from plants have been obtained from dicotyledons. In the present study a lectin was purified from tubers of a monocot plant Arisaema utile (AUL) Schott by affinity chromatography on asialofetuin-linked amino activated silica beads. AUL gave a single band in SDS-PAGE at pH 8.3 corresponding to subunit Mr 13.5 kDa. The native molecular mass of AUL was 54 kDa suggesting a homotetrameric structure. AUL gave multiple bands in isoelectric focusing and in native PAGE at pH 8.3. AUL was inhibited by N-acetyl-D-lactosamine (Lac NAc), a disaccharide and asialofetuin, a complex desialylated serum glycoprotein. When treated with denaturing agents, the lectin was stable in the presence of urea (3 M), thiourea (4 M) and guanidine HCl (4 M). AUL was a glycoprotein with a carbohydrate content of 1.2%. Complete loss of activity was observed upon modification of tryptophan residues of the lectin. The activity was reduced to 25% after modification of tyrosine. Chemical modification of arginine, histidine, serine and cysteine residues of AUL did not affect its activity. Using Far UV CD spectra the estimated secondary structure was 37% α-helix, 25% β-sheet and 38% random contributions. The lectin showed potent mitogenic response towards human lymphocytes. In vitro anti-proliferative assay using 11 human cancer cell lines resulted in 50% inhibition of six cell lines viz. SW-620, HCT-15, SK-N-SH, IMR-32, Colo-205 and HT-29 at 38, 42, 43, 49, 50 and 89 µg/ml, respectively.