Structural studies of a Tremella fuciformis mushroom lectin: a novel protein with antitumoral properties

Lectins are carbohydrate-binding proteins of non-immune origin, widely distributed in all the living organisms. They play a role in different biological processes and, due to their carbohydrate specific binding, some lectins have been found to possess antitumoral properties. A particular case is the recognition of the T-antigenic determinant (Galβ1-3GalNAcα) on the malignant cell surfaces, which is present in 90% of human carcinomas. Upon binding to the carbohydrate, some lectins can cause apoptosis, cytotoxicity, inhibition of tumour growth, thus preventing the proliferation of tumoral cells. Considering the fact that these carbohydrates are masked on healthy cells, the highly specific carbohydrate-lectin interaction can be exploited to target only malignant cells. Although the function and the biological properties of several lectins have been determined, there are still many lectins that remain to be structurally and functionally characterized. As reported in the literature, some Tremella fuciformis proteins have been investigated for their potential therapeutical properties and in the light of this, the crude extract of this fungus was examined to assess the presence of lectins. A lectin of 11 KDa, named TFL, was isolated and purified from the dried fruiting bodies and used for testing several crystal screening conditions. The best crystals were grown in 0.1 M TRIS pH 8.5, 1.5 potassium phosphate dibasic, 1 % DMSO and the final data sets were collected at the ESRF of Grenoble, revealing the three-dimensional structure of the protein. TFL belongs to the space group P1211 and the cell parameters of the crystal are the following: a = 61.62 Å, b = 61.83 Å, c = 67.84 Å, with β = 106.87 °. The protein is a monomer, composed of six β-sheets (from A to F) that arrange to form the so called “β-barrel”. In addition, two α-helixes, named H1 and H2 can be recognized in the structure, which is also stabilized by the presence of two disulphide bridges that connect Cys 54 to Cys 70 and Cys 97 to Cys 100, respectively. Thermal protein stability was analyzed by means of differential scanning calorimetry, revealing that TFL does not undergo a two-state unfolding process and its denaturation is reversible, a feature that has been rarely observed for lectins before. In addition, chemical and pH-induced unfolding were investigated using fluorescence spectroscopy, highlighting the high stability of TFL in a wide range of conditions. Exploiting isothermal titration calorimetry, more information about sugar-binding were acquired, identifying N-acetylgalactosamine as the best candidate, which has micromolar affinity for TFL. It has also been observed that Tremella fuciformis lectin shows no cytotoxicity on malignant and healthy cells and interestingly, it seems to reduce skin malignant cells migration and to have a positive effect in the upregulation of certain genes involved in cancer arresting (SBPP1). In addition, immunomodulatory activity of TFL was analyzed by means of ELISA essay, resulting in the up-regulation of the most important inflammatory markers (IL-6, TNFα), that could be usefully exploited in order to kill intracellular microorganisms and to help the human body to develop a stronger tumoral resistance. In conclusion, this work provides a new and interesting insight into cancer treatment, being an essential prerequisite for future in vitro and in vivo experiments. The encouraging results obtained so far, alongside with the fact that TFL has been produced also heterologously in E.coli, pave the way for further studies, including the feasibility to perform mutagenesis and to explore the possibility to further encapsulate the protein inside tailor-made nanoparticles, with the aim of reducing the amount of protein used, increasing its therapeutical efficiency, and preventing side effects.