Structure of a lectin with antitumoral properties in king bolete (Boletus edulis) mushrooms

Michele Bovi 1 Maria E Carrizo 0 Stefano Capaldi 1 Massimiliano Perduca 1 Laurent R Chiarelli 2 Monica Galliano 2 Hugo L Monaco 1 0 Departamento de Qumica Biolgica , Facultad de Ciencias Qumicas, Universidad Nacional de Crdoba C.P. , 5016 Crdoba, Argentina 1 Biocrystallography Laboratory, Department of Biotechnology, University of Verona , Ca Vignal 1, strada Le Grazie 15, 37134 Verona, Italy 2 Department of Biochemistry A. Castellani, University of Pavia , via Taramelli 3b, 27100 Pavia, Italy A novel lectin has been isolated from the fruiting bodies of the common edible mushroom Boletus edulis (king bolete, penny bun, porcino or cep) by affinity chromatography on a chitin column. We propose for the lectin the name BEL (B. edulis lectin). BEL inhibits selectively the proliferation of several malignant cell lines and binds the neoplastic cell-specific T-antigen disaccharide, Gal1-3GalNAc. The lectin was structurally characterized: the molecule is a homotetramer and the 142-amino acid sequence of the chains was determined. The protein belongs to the salinesoluble family of mushroom fruiting body-specific lectins. BEL was also crystallized and its three-dimensional structure was determined by X-ray diffraction to 1.15 resolution. The structure is similar to that of Agaricus bisporus lectin. Using the appropriate co-crystals, the interactions of BEL with specific mono- and disaccharides were also studied by X-ray diffraction. The six structures of carbohydrate complexes reported here provide details of the interactions of the ligands with the lectin and shed light on the selectivity of the two distinct binding sites present in each protomer. Introduction Lectins are proteins of non-immune origin devoid of any catalytic activity that reversibly bind mono- and oligosaccharides with high specificity and are involved, through sugar binding, 1To whom correspondence should be addressed: Tel: +39-045-8027-903; Fax: +39-045-8027-929; e-mail: in many fundamental biological processes such as, among others, cell-to-cell interactions and innate immunity (Sharon and Lis 2004; Sharon 2007). They are present in all kinds of organisms from viruses to man, were initially identified in the plant kingdom for their hemagglutinating activity and are now being widely used in basic and clinical research to develop new drugs for cancer therapy (Gonzlez De Mejia and Prisecaru 2005), to treat microbial and viral infection (Sharon 2006; Wellens et al. 2008) and to fractionate hemopoietic stem cells for transplantation (Reisner et al. 1978). Fungi are heterotrophic organisms with a chitinous cell wall that depend on symbiosis for their source of energy and have to specifically recognize their host for adhesion, a process which is often achieved through the recognition of glycoconjugates by a fungal lectin (Imberty et al. 2005). The first fungal lectin was discovered in the toxic fungus Amanita phalloides as a result of research in the field of toxic substances present in higher fungi (reviewed by Khan and Khan 2011). Ectomycorrhizal symbiosis is the way of association formed between fungi and the roots of plants in which the fungus obtains mono- and disaccharides from the roots providing in exchange the use of the myceliums large surface area to absorb water and minerals from the soil, thus improving the mineral absorption capabilities of the plant root (Taylor and Alexander 2005). There are more than 140 mycorrhizal fungi with edible fruiting bodies (Hall et al. 1998), a group that includes some of the most expensive and globally diffused foods in the world (Hall et al. 2003). Among this group of mushrooms, the genus Boletus contains many members which are highly valued because they are edible and tasty, and several species that are closely related to each other and quite difficult to distinguish are often marketed together with the general name of Boletus edulis (Hall et al. 1998). The mushrooms from the genus Boletus have not yet been cultivated, whereas another common and widely diffused species, Agaricus bisporus, has, which explains why in most countries the volumes of Agaricus consumed are significantly higher (Hall et al. 2003). Agaricus bisporus contains a lectin (ABL) that has the remarkable property of binding selectively and with high affinity, the ThomsenFriedenreich antigen or T-antigen. The T-antigen is a disaccharide, Gal1-3GalNAc, linked to either serines or threonines on cell surface glycoproteins and hidden in healthy cells while exposed in a high percentage of human carcinomas and other neoplastic tissues (Springer 1984, 1997). ABL has the property of reversibly inhibiting the proliferation of malignant epithelial cell lines without any apparent cytotoxicity for normal cells (Yu et al. 1993). This effect is thought to be a consequence of the selective blocking by ABL of the nuclear localization sequencedependent protein import, essential for cell functioning (Yu et al. 1999, 2000; Yu 2007). Following our determination of the three-dimensional structure of ABL by X-ray crystallography, the characterization of the T-antigen-binding site and the discovery that the lectin has two distinct binding sites for N-acetylgalactosamine and N-acetylglucosamine (Carrizo et al. 2005), we decided to explore the possibility of the existence of a lectin with similar properties in the genus Boletus as well. We reasoned that the second binding site probably bound polymers of N-acetylglucosamine and the most common of these polymers is chitin, a constituent of the fungal cell wall and therefore present in high quantities in fungal cells (Wu et al. 2004). Therefore, we devised a protocol that uses as its major purification step the specific affinity for a chitin resin and, in this way, we isolated the lectin that we propose to call B. edulis lectin (BEL). In this paper, we report the isolation, amino acid sequencing, crystallization and three-dimensional structure determination of BEL. Similar to ABL, BEL is found to selectively inhibit the proliferation of several malignant cell lines. We also describe in detail the interactions of the lectin with specific mono- and disaccharides that can occupy the two distinct binding sites that are present in each protomer of the tetrameric protein. Results Amino acid sequence of BEL Information on the amino acid sequence of BEL was obtained from three sources: Edman degradation and sequence analysis of the intact protein, cDNA sequencing of the gene encoding the lectin obtained from RNA extracts of the mushrooms and the X-ray high-resolution electron density maps. The sequence was also checked by tandem mass spectrometric analysis of the peptides derived from tryptic, chymotryptic and S. Aureus V8 endopeptidase cleavage. Supplementary material, Figure S1 summarizes the results obtained. Several positions in the polypeptide chain showed significant variability. The amino acids present in those positions are labeled in red in the figure. This variability, (...truncated)