An Immunological Polysaccharide from Tremella fuciformis: Essential Role of Acetylation in Immunomodulation

Abstract

The edible fungus Tremella fuciformis was shown to have a high molecular weight (1.87 × 10³ kDa) bioactive polysaccharide, denoted as TFP-F1. Monosaccharide composition and NMR analysis of the polysaccharide and its derivatives indicated it contained fucose (Fucp), xylose (Xylp), mannose (Manp), and glucuronic acid (GlcAp) in a ratio of 0.9:1.0:3.2:1.2. Using IR, NMR, and GC-MS spectroscopic data, the structure of TFP-F1 was elucidated as {→3)-[β-D-GlcAp-(1→2)]-α-D-Manp-(1→3)-α-D-Manp-(1→3)-[α-L-Fucp-(1→2)-β-D-Xylp-(1→2)]-α-D-Manp-(1→}_n, with partial acetylation of C6-OH in mannoses. Furthermore, at a concentration of 1 μg/mL, TFP-F1 was found to stimulate the secretion of TNF-α and IL-6 in J774A.1 macrophage cells in vitro via interaction with toll-like receptor 4 (TLR4). The removal of O-acetyl groups led to the loss of immunomodulatory activities, demonstrating that O-acetyl groups play an essential role in enhancing the production of pro-inflammatory cytokines.

Keywords: Tremella fuciformis; acetylation; immunomodulatory activities; polysaccharide; structure elucidation; toll-like receptor 4.

Figure 1

(a) A flow chart showing the procedures used to purify polysaccharides from T. fuciformis and evaluate their immunomodulatory activity (b) Size-exclusion chromatography HW-65F profile of TFP-F1. Fractions containing carbohydrates were determined using the phenol–sulfuric acid method. TFP-F1 was collected from tubes 26 to 33. (c) Standard curves of the dextran samples in HPLC, including 2000 kDa, 500 kDa, 70 kDa, and 10 kDa, were run in a BioBasic SEC-1000 column. (d) MW analysis of TFP-F1 was followed by (c).

Figure 2

The chemical structure and symbolic nomenclature of TFP-F1.

Figure 3

Immunomodulatory activity of TFP-F1. J774A.1 macrophage cells were incubated for 24 h with or without different concentrations of TFP-F1 or LPS (1 μg/mL). (a) Induction of TNF-α secreted by TFP-F1 (5, 10, and 20 μg/mL) in J774A.1 macrophage cells. (b,c) Wild-type RAW-Dual^TM cells and RAW-Dual^TM KO-TLR4 cells were incubated with or without different concentrations of TFP-F1 (1, 3, and 5 μg/mL) or LPS (1 μg/mL). The levels of (b) TNF-α and (c) IL-6 in the RAW-Dual^TM KO-TLR4 cells. (d,e) Wild-type HEK293 cells and TLR4-expressing HEK 293 cells were incubated for 24 h with or without different concentrations of TFP-F1 (1, 3, and 5 μg/mL) or LPS (1 μg/mL), and the level of (d) TNF-α and (e) IL-6 were measured by ELISA.

Figure 4

(a) Preparation of different TFP-F1 derivatives. TFP-F1R was prepared by treating the TFP-F1 with the CMC and NaBH₄, while deOAc TFP-F1 was afforded by treating TFP-F1R with NaOH. TFP-F1P was the product of the periodate cleavage of TFP-F1. (b) The level of TNF-α was stimulated in the J774A.1 macrophage cells induced by the treatment of TFP-F1 of TFP-F1P. (c) The level of TNF-α secreted in the J774A.1 macrophage cells by the treatment of TFP-F1, TFP-F1R, and deOAc TFP-F1R.

Figure 5

The activation levels of NF-κB were measured by an NF-κB reporter assay.

Figure 6

Purification of a water-soluble bioactive polysaccharide TFP-F1 from T. fuciformis induced the production of pro-inflammatory cytokines through interaction with TLR4. The O-acetyl groups at C-6 in mannose residues were essential for immunomodulation.