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A glucuronic acid binding leguminous lectin with mitogenic activity toward mouse splenocytes
Yau Sang Chan1, Jack Ho Wong, Tzi Bun Ng
1School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China.
Insights
A French bean lectin was purified and characterized for its hemagglutinating and mitogenic activities. Glucuronic acid inhibited these activities, and the lectin showed no antiproliferative effects on cancer cells.
Area of Science:
- Biochemistry
- Molecular Biology
- Immunology
Background:
- Lectins are proteins known for their carbohydrate-binding properties.
- Plant lectins, such as those from Phaseolus vulgaris (French bean), are extensively studied for their biological activities.
Purpose of the Study:
- To purify and characterize a lectin from French bean seeds.
- To investigate the hemagglutinating, mitogenic, and antiproliferative activities of the purified lectin.
Main Methods:
- Purification using Q-Sepharose, Affi-gel blue gel, Mono S, and Superdex 75 chromatography.
- Hemagglutinating activity assays across a range of pH and temperatures.
- Mitogenic activity assays using murine splenocytes.
- Antiproliferative assays against cancer cell lines (HepG2, MCF7, CNE).
Main Results:
- A dimeric 64-kDa lectin was purified, showing a single 32-kDa band under SDS-PAGE.
- The lectin exhibited full hemagglutinating activity between pH 3-11 and temperatures of 20-60 °C.
- Glucuronic acid inhibited hemagglutinating and mitogenic activities.
- The lectin demonstrated maximum mitogenic activity at 0.488 µM and no antiproliferative activity against tested cancer cells.
Conclusions:
- The purified French bean lectin possesses significant hemagglutinating and mitogenic properties.
- Its activity is modulated by pH, temperature, and specifically inhibited by glucuronic acid.
- The lectin lacks antiproliferative effects on tested cancer cell lines and anti-HIV reverse transcriptase activity.
Abstract:
A dimeric 64-kDa lectin was purified from seeds of French bean (Phaseolus vulgaris) cultivar number 1. The purification protocol entailed Q-Sepharose, Affi-gel blue gel, Mono S and Superdex 75. The lectin-enriched fraction was adsorbed on Q-Sepharose and Affi-gel blue gel and desorbed using 1M NaCl in the starting buffer. Hemagglutinating activity was adsorbed on Mono S and eluted with a linear 0.3-1 M NaCl gradient. Gel filtration on Superdex 75 yielded a single absorbance peak which appeared as a single 32-kDa in sodium dodecyl sulfate poylacylamide gel electrophoresis. Full hemagglutinating activity was observed when the lectin was exposed to a pH ranging from 3 to 11. About 50% activity remained at pH 12, and about 25% at pH 0 to pH 2. Activity was totally abolished at pH 13-14. The activity was completely preserved when the ambient temperature was 20 °C-60 °C. However, only 50% and 12.5% of the activity remained at 65 °C and 70 °C, respectively. Activity was barely discernible at 75 °C and completely abrogated at and above 80 °C. Hemagglutinating activity of the lectin was inhibited by glucuronic acid. Maximum mitogenic activity of the lectin toward murine splenocytes occurred at a lectin concentration of 0.488 µM. The mitogenic activity was nearly eliminated in the presence of 250 mM glucuronic acid. The lectin did not exhibit antiproliferative activity toward hepatoma (HepG2) cells, breast cancer (MCF7) cells, and nasopharynegeal carcinoma CNE stage 1 and stage 2 cells. It was also devoid of significant anti-HIV reverse transcriptase activity.
