Abstract
A dimeric 64-kDa glucosamine-specific lectin was purified from seeds of Phaseolus vulgaris cv. “brown kidney bean.” The simple 2-step purification protocol involved affinity chromatography on Affi-gel blue gel and gel filtration by FPLC on Superdex 75. The lectin was absorbed on Affi-gel blue gel and desorbed using 1M NaCl in the starting buffer. Gel filtration on Superdex 75 yielded a major absorbance peak that gave a single 32-kDa band in SDS-PAGE. Hemagglutinating activity was completely preserved when the ambient temperature was in the range of 20°C–60°C. However, drastic reduction of the activity occurred at temperatures above 65°C. Full hemagglutinating activity of the lectin was observed at an ambient pH of 3 to 12. About 50% activity remained at pH 0–2, and only residual activity was observed at pH 13–14. Hemagglutinating activity of the lectin was inhibited by glucosamine. The brown kidney bean lectin elicited maximum mitogenic activity toward murine splenocytes at 2.5 µM. The mitogenic activity was nearly completely eliminated in the presence of 250 mM glucosamine. The lectin also increased mRNA expression of the cytokines IL-2, TNF-α and IFN-γ. The lectin exhibited antiproliferative activity toward human breast cancer (MCF7) cells, hepatoma (HepG2) cells and nasopharyngeal carcinoma (CNE1 and CNE2) cells with IC50 of 5.12 µM, 32.85 µM, 3.12 µM and 40.12 µM respectively after treatment for 24 hours. Flow cytometry with Annexin V and propidum iodide staining indicated apoptosis of MCF7 cells. Hoechst 33342 staining also indicated formation of apoptotic bodies in MCF7 cells after exposure to brown kidney bean lectin. Western blotting revealed that the lectin-induced apoptosis involved ER stress and unfolded protein response.
Highlights
Lectins are sugar binding proteins or glycoproteins that consist of one or more binding sites for interaction with their specific carbohydrates
Plant lectins exhibit a variety of biological activities like anti-bacterial [4], anti-viral [5], anti-fungal [2] and anti-insect [3] activities
The first chromatographic step on Affi-gel blue gel helped to remove most of the impurities in the crude extract, allowing brown kidney bean lectin (BKBL) to remain as the major protein in the adsorbed fraction (Fig. 2B)
Summary
Lectins are sugar binding proteins or glycoproteins that consist of one or more binding sites for interaction with their specific carbohydrates. The anti-pathogenic activities of plant lectins enable themselves to act as defense proteins to protect the plants from invasion of harmful organisms. Other than for the plants’ own uses, the plant lectins exhibit other biological activities, such as inducing mitogenic response in mammalian splenocytes [1,2,6] and anti-proliferative effects toward tumor cells [7,8]. These activities provide insights for plant lectins to be applied to humans for therapeutic uses, such as development of immunomodulatory drugs or anti-tumor drugs. Mistletoe lectin from Viscum album exhibited immunostimulatory and cytotoxic effects while having a low toxicity [9,10], allowing it to be used for treatment of cancers [11]
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