Selenated polysaccharides - research into the biosynthesis and the relationship between the structure and immune activity

The aim of the presented project is to obtain by biosynthesis a series of new, hitherto undescribed macromolecular compounds - exopolysaccharides of fungal origin (β-glucans) containing selenium atoms in their structure, and then to study the influence of selenium incorporation into the polysaccharide chain on their structural features and immunomodulatory activity. The presented project is a continuation of the research carried out in 2010-2011 as part of the own research project MEiN N N405 613238. As a result of the research carried out in this period, the conditions for the cultivation of mycelium L. edodes on substrates enriched with selenium compounds were optimized, and speciation studies of organic selenium compounds biosynthesized were carried out by mycelial cultures, and one selenium-containing polysaccharide fraction was isolated. Preliminary studies of the effect of the isolated selenium polysaccharide fraction on the proliferation of human blood lymphocytes indicated its strong and selective immunosuppressive effect with very low toxicity. The research results were so promising that the possible use of biosynthesized selenium polysaccharides as immunosuppressants was covered by a patent application in 2012 (P. 402082). Both the mechanism of selenium incorporation into fungal exopolysaccharide molecules, as well as their structure and the mechanism of the immunosuppressive activity detected by us in preliminary studies are so far unknown. Therefore, in the presented project, research is planned to clarify this interesting problem. They also concern the hitherto unexplored issues of biosynthesis, structure and mechanism of action of selenopolysaccharides. We intend to use the methods in three areas of the research: In the biotechnological part of the research: - We will conduct the biosynthesis of selenium-enriched exoplisaccharides by deep mycelial culturing of the medicinal mushroom Lentinula edodes, belonging to the Basidiomycetes class, using culture media enriched with various selenium compounds. - We will use (we will develop) a method of fractionating these compounds that will enable the isolation of polysaccharides with different molar mass, monosaccharide composition and selenium content by using eluents of different polarity and chemical nature for the extraction. In the analytical part of the research: - We will apply the previously unknown method in which selenium is bound to the structure of polysaccharides by the method of X-ray absorption spectroscopy (XANES, XAFS) and nuclear magnetic resonance (selenium NMR spectra) - We will conduct a thorough analysis of the structure of selenopolysaccharides by using the following methods: to determine the monosaccharide composition, after complete hydrolysis, the method of high-performance liquid chromatography, • to determine the molar mass of isolated polysaccharides, we will use the gel chromatography technique (GPLC), • to determine the primary structure, we will use degradation methods, using enzymatic hydrolysis and a modified Hakomori method - by preliminary methylation and then hydrolysis of the analyzed fractions, • to determine the type of glycosidic bonds, we will use the IR and NMR spectral methods, • to determine the spatial structure, we will use (2D) NMR methods. e-polysaccharides with different molecular weight, selenium content and structure, and on the proliferation of human blood T and B lymphocytes, by conducting OKT3, PHA and SAC tests - We will investigate whether Se-polysaccharides have the ability to activate macrophages, NK cells, and whether they affect the dependent response from T lymphocytes. We assume that the results of the planned studies will allow us to formulate a hypothesis regarding the causes of atypical for polysaccharides of fungal origin, found in preliminary studies on the immunosuppressive activity of seleniated polysaccharide fractions. We also assume that we will be able to investigate to what extent selenium incorporation into the exopolysaccharide molecule changes their spatial structure, which in turn probably affects the binding of these compounds with CR-1 and CR-3 receptors. The research results should also broaden the scope of knowledge on the metabolism of selenium compounds by fungi and the possibility of their biosynthesis of selenopolysaccharides. The research should result in publications in journals from the JCR list and congress reports. We assume that the research will contribute to the development of young academic staff, constituting the subject of thesis / doctoral dissertations. It is also probable that their results will be used to design and produce an innovative drug with immunosuppressive activity, belonging to a new chemical group.