Isolation of a glucosamine binding leguminous lectin with mitogenic activity towards splenocytes and anti-proliferative activity towards tumor cells.
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 IC(50) 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.
School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong.
Yau Sang
YS
Chan
Jack Ho
JH
Wong
Evandro Fei
EF
Fang
Wenliang
W
Pan
Tzi Bun
TB
Ng
D000818:Animals; D001483:Base Sequence; D049109:Cell Proliferation / Q000187:drug effects*; D016207:Cytokines / Q000096:biosynthesis; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D007887:Fabaceae / Q000378:metabolism*; D005944:Glucosamine / Q000378:metabolism*; D006386:Hemagglutination Tests; D006863:Hydrogen-Ion Concentration; D037102:Lectins / Q000378:metabolism* / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D008934:Mitogens / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D013154:Spleen / Q000166:cytology / Q000187:drug effects*; D013696:Temperature
D016207:Cytokines; D017931:DNA Primers; D037102:Lectins; D008934:Mitogens; D005944:Glucosamine