Structural characterization and immunostimulatory activity of a novel protein-bound polysaccharide produced by Hirsutella sinensis Liu, Guo, Yu & Zeng

• A novel protein-bound polysaccharide HS002-II was isolated by Hirsutella sinensis.
• The backbone of HS002-II mainly contained glucopyranose units.
• Two branches were mannopyranose and galactopyranose units with arabinopyranose terminals.
• It consisted of 57.9% polysaccharide and 42.1% protein with N-type carbohydrate–protein linkage.
• HS002-II exhibited immunomodulatory activity by stimulating the IκB-NF-κB pathway.

HS002-II, a novel protein-bound polysaccharide with 44 kDa molecular weight, was fractionated from submerged cultures of Hirsutella sinensis Liu, Guo, Yu & Zeng by DEAE-Sepharose and Sephacryl S200 chromatography. Based on the results of infrared spectroscopy, high performance liquid chromatography, methylation, amino acid analysis, NMR spectroscopy and atomic force microscopy, the polysaccharide moieties of HS002-II mainly contained a long backbone of (1 → 3)-linked α-d-ribofuranosyl units (1 → 4)-linked α-d-xylopyranosyl units and (1 → 4)-linked β-d-glucopyranosyl units, which was substituted at C-6. The two branches were β-d-mannopyranosyl residues and β-d-galactopyranosyl residues terminated with α-l-arabinopyranosyl residues, respectively. HS002-II consisted of 57.9% polysaccharide and 42.1% protein with the existence of N-type carbohydrate–protein linkage. In terms of the pro-inflammatory cytokines assay (NO, TNF-α, IL-1β and NF-κB) using murine macrophages cell line (RAW264.7), HS002-II exhibited significant immunomodulatory activity by stimulating the IκB-NF-κB pathway.