Purification, Characterization and Studies of Biological Activity of a Novel Lectin from Nymphaea nouchali Tuber

Abstract

A novel lectin (named as NNTL) have been isolated from the crude extracts of Nymphaea nouchali (White water-lily) tubers and purified by using anion-exchange chromatography on DEAE cellulose column followed by hydrophobic chromatography on HiTrap Phenyl HP column and then re-applying to anion-exchange chromatography on a HiTrap Q FF column. The purified NNTL moved as a single band and monomer in nature as judged by SDS-PAGE both in presence and absence of 2-mercaptoethanol and its molecular weight was estimated to be 27±1 kDa. The NNTL was glycoprotein in nature with neutral sugar content of 8%, which agglutinated rat, chicken and different groups of human blood cells. In the sugar inhibition study, O-nitrophenyl-ß-D-galactopyranoside was found to be most potent inhibitor and the minimum inhibitory concentration needed for visible agglutination was 25 mM. The lectin showed toxicity against brine shrimp nauplii with the LC50 value of 120±29 µg/ml and strong agglutination activity against four pathogenic bacteria (Bacillus subtilis, Sarcina lutea, Shigella shiga and Shigella sonnei). The NNTL was more thermostable and showed full activity at the temperature range 30 to 600C, and did not lose its activity between pH 5.0 to 9.0. The lectin was a divalent ion-dependent glycoprotein, which lost its activity markedly by treating with denaturants such as DTT and EDTA, but the activity was restored sequentially by the addition of Ca2+, Ba2+ and Mg2+. The N-terminal sequences of the lectin upto 10-residues were identified except the first position and the sequence homology showed that NNTL was not identical with the sequence of any other reported lectins so far available. The analysis of amino acid composition revealed that NNTL was rich in leucine, methionine, and glycine residues as well as significant amounts of acidic amino acids. The study of the conformational changes by fluorescence spectroscopy indicates that the tryptophan environment of NNTL was greatly affected upon binding to different concentrations of metal ions, sugars and denaturants and the stability of NNTL was markedly dependent on Ca2+ requirement. The antifungal activity of both the lectin and non-lectin protein of Nymphaea nouchali tuber were assayed against five pathogenic fungi and it was found that the NNTL was devoid of any antifungal activity, whereas the purified protein (named NNTP-I) had notable effect only against Candida albicans. The study of antitumor activity of NNTL showed that the lectin inhibits 56% and 76% of proliferation of EAC (Ehrlich’s Ascites Carcinoma) cells in vivo in mice at the dose of 1.5 and 3.0 mg/Kg/day respectively. Furthermore, we also studied the antitumor effect of NNTL to assess the apoptotic nuclear morphology in fluorescence microscopy by staining with Hoechst 33342, but there was no nuclear damage markedly found in the EAC cells treated with NNTL. Subsequently, from the assessment of the expression of apoptosis regulating gene bcl-1, bcl-X, and bax by RT-PCR, it was observed that the apoptosis causing gene bax was not expressed in the experimental cell, suggesting that the lectin does not induce apoptosis.