Mimicking The Biosynthesis Of Native Cordyceps Polysaccharides: Structure-based Functional Studies

Cordyceps is a rare and exotic mushroom that grows out of the head of amummified caterpillar. Many companies are cultivating Cordyceps to meet theincreased demand for its medicinal applications. The major functional ingredients inCordyceps are polysaccharides, which are the most complex bio-molecules made bynature. Due to the complexities, the structure-based functional studies of thepolysaccharides remain to be the most challenging area in Cordyceps research. Thebiosynthesis of polysaccharide is a non-template driven process, where many factorsincluding genes, proteins, metabolic products‘and salts regulate the quality, quantity,structure, and functions of the polysaccharides. Therefore, how to establish thestructural and functional relationships of the Cordyceps polysaccharides and how toreproduce the native Cordyceps polysacchairde structures in culture are importantscientific issues.To establish the structural and functional relationships of Cordycepspolysaccharides, we first isolated28polysaccharides from3native species ofCordyceps. Eighteen out of the28polysaccharides were further purified into72different fractions based on different chemical properties of the polysaccharides. Wealso puirifed46different kinds of polysacchairdes from different Cordyceps cultures.The physical, chemical, and biological properties of the purified polysaccharides weresystematically compared. We found that the polysacchairdes from the native andcultured Cordyceps had different molecular weight, carbohydrate, and proteincontents. Cellulose acetate electrophoresis analysis indicated that the Cordycepspolysaccharides were negatively charged. According to the element analysis.Cordyceps polysaccharide contained sulfur. Based on composition analysis, threemajor monosaccharides in the polysaccharide were found to be mannose. glucose, andgalatose. However，the relative proportion of each monosacchairde was different in each puirifed polysaccharide. When incubated with cancer cells at100P g/ml. only7out of164purified polysaccharides not from the wild Cordvceps inhibited the HI299cancer cell growth by30-40%. In contrast, only the polysaccharides purified from themushroom part of wild Cordvceps stimulated FGF/FGFR signals in a cell-basedmodel system, a property requires the presence of sulfated polysaccharides. Ouroverall results indicated that the structures and functions of the polysaccharides fromwild Cordvceps were different from that of cultured ones.To make cultured Cordvceps polysaccharides resemble the wild ones, wehypothesized that mimicking the salty environment inside caterpillar bodies mightmake the cultured fungus synthesize polysaccharides with similar structures andfunctions to that of wild Cordvceps. By adding either sodium sulfate or sodiumchloride into growth media, we observed the salinity-induced anti-angiogenesisactivities of the polysaccharides purified from the cultured C Milharis. To correlatethe activities with the polysaccharide structures, we performed the,3C-NMR analysisand observed profound structural changes including different proportions of a and pglycosidic bonds and appearances of uronic acid signals in the polysaccharidespuirfied from the culture after the salts were added. By coupling the techniques ofstable34S-sulfate isotope labeling, aniline-and Ds-aniline tagging, and stable isotopefacilitated uronic acid-reduction with LC-MS analysis, our data revealed for the firsttime the existence of covalentlv linked sulfate and the presence of polvgalacuronicacids in the polysaccharides purified from the salt added C. Militaris culture. Our datashowed that cultuirng C Militaris with salts added changed the biosynthetic schemeand resulted in novel polysaccharide structures and functions. These ifndings might beinsightful in terms of how to make C. Militaris cultures to reach or to exceed thepotency of wild Cordvceps in future.