Elsevier

Phytomedicine

Volume 17, Issue 10, August 2010, Pages 775-781
Phytomedicine

First isolation and characterization of a novel lectin with potent antitumor activity from a Russula mushroom

https://doi.org/10.1016/j.phymed.2010.02.001Get rights and content

Abstract

To date only a ribonuclease and a protein with anti-HIV-1 reverse transcriptase activity have been isolated from mushrooms of the genus Russula. In this study a novel lectin, with a molecular weight of 32 kDa, and a unique N-terminal sequence different from other lectins, was isolated from the mushroom Russula lepida. It represents the first lectin isolated from Russula mushrooms. The purification scheme involved (NH4)2SO4 precipitation, ion exchange chromatography on diethylaminoethyl DEAE-cellulose and SP-Sepharose, and fast protein liquid chromatography-gel filtration on Superdex 75. The hemagglutinating activity of the lectin (RLL) was inhibited by inulin and O-nitrophenyl-β-D-galacto-pyranoside. The lectin was stable at temperatures up to 70 °C (half of the activity was preserved at 80 °C), and in the presence of NaOH or HCl solutions up to a concentration of 12.5 mM. Its hemagglutinating activity was reduced in the presence of Mn2+, Co2+, and Hg2+ ions, and enhanced by Cu2+ ions. It exhibited antiproliferative activity toward hepatoma Hep G2 cells and human breast cancer MCF-7 cells with an IC50 of 1.6 μM and 0.9 μM, respectively. Daily intraperitoneal injections of RLL (5.0 mg/kg body weight/day for 20 days) brought about 67.6% reduction in the weight of S-180 tumor. RLL was devoid of antifungal, ribonuclease, and HIV-1 reverse transcriptase inhibitory activities.

Introduction

Lectins are carbohydrate-binding proteins or glycoproteins of nonimmune origin that can agglutinate cells or precipitate glycoconjugates (Goldstein et al., 1980). Although they were first identified in plants, they are now known to distribute widely throughout the nature, including plants, animals, fungi, bacteria and viruses. In mushrooms, lectins have been localized on the caps, stipes and the mycelia (Ng, 2004). Most plant lectins are storage proteins and defence proteins when the plant or the seed is assaulted by insects or fungi (Janzen et al., 1976; Mirelman et al., 1975). In mammals, lectins play a part in the migration of lymphocytes from the bloodstream into the lymphoid organs and in metastasis of cancer cells (Wang et al., 1998). In mushrooms, lectins may play crucial roles in dormancy, growth, morphogenesis, morphological changes and molecular recognition during the early stages of mycorrhization (Guillot and Konska, 1997; Ng, 2004). Mushroom lectins manifest exploitable biological activities such as antiproliferative (Wang et al., 1995), antitumor (Wang et al., 2000), immunomodulatory (She et al., 1998), and HIV-1 reverse transcriptase inhibiting (Wang and Ng, 2001) activities. Hence they have captured the attention of many investigators.

Russula lepida, which is also called red mushroom by people in northeast China, is a wild mushroom. It belongs to Phylum Basidiomycota, Order Agaricales, Family Russulaceae. A prolyl endopeptidase and a seco-ring-A cucurbitane triterpenoid were reported from R. lepida (Jian-Wen et al., 2002; Yoshimoto et al., 1988). Recently, some new terpenoids and ceramides have been reported from Russula sp. (Gao et al., 2000; Gao et al., 2001; Tan et al., 2001). However, there are few reports on its proteinaceous constituents. The objective of the present study was to isolate a lectin from the mushroom R. lepida and find out if it has any distinctive characteristics.

Section snippets

Isolation of lectin

Dried fruiting bodies of the mushroom Russula lepida (100 g) were homogenized in 0.15 M NaCl at 4 °C and extracted overnight at 4 °C. Then the homogenate was centrifuged at 8000g for 15 min. (NH4)2SO4 was added to the supernatant to 80% saturation and the mixture was stirred. Subsequently, the mixture was centrifuged at 8000g for 15 min. Then the precipitate was dissolved, and dialyzed to remove (NH4)2SO4 before applying to a DEAE-cellulose (Sigma) column (2.5×20 cm) which had previously been

Purification of RLL

The fraction of the fruiting body extract unadsorbed on DEAE-cellulose (D1) but not the adsorbed fractions (D2,3,4) contained the hemagglutinating activity (Table 1). Fraction D1 was divided into two fractions, SP1 and SP2, upon ion exchange chromatography on a SP-Sepharose column (Fig. 1). Hemagglutinating activity resided in the adsorbed fraction SP2 (Table 1). Subsequently, fraction SP2 was resolved into a larger fraction SU1 and a tiny fraction SU2 upon gel filtration on a Superdex 75 HR

Acknowledgements

This work was financially supported by National Grants of China (nyhyzx07-008, 2007BAD89B00 and 2010CB732202).

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