We isolated and purified a new polysaccharide (WFVP-N-b1) with a molecular weight of 20 kDa from Flammulina velutipes. Results showed that WFVP-N-b1 is composed of an α (1→6)-linked D-galactan backbone and branched at the O-2 of its Galp residues by an α-D-(1→6)-linked Manp attached to t-β-D-Glcp or t-α-D-Fucp side chains. WFVP-N-b1 can significantly induce cytokines secretion and release of toxic molecules. On a cellular level, WFVP-N-b1 is recognized by Toll-like receptor 4 (TLR4). Thereby, the hetero-galactan increased the phosphorylation of mitogen-activated protein kinases (MAPKs) and Akt, promoted degradation of IκB-α and the nuclear translocation of the NF-κB p65 subunit. Importantly, our results indicate that WFVP-N-b1 activated macrophage is mediated by autophagy, as blockade of WFVP-N-b1-induced autophagy by Baf-A1 significantly decreases macrophage activation. This is the first report that hetero-galactan-induced macrophage activation is mediated by autophagy. Collectively, WFVP-N-b1 activated RAW264.7 cells through MAPKs, autophagy, and Akt/NF-κB signaling pathways via TLR4 receptor.

macrophage, Flammulina velutipes, MAPKs, Akt/NF-κB, autophagy, hetero-galactan

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
2	34%aDManp	101.34	68.84	69.69	69.52	77.45	69.36
	aDGalp	97.67	77.29	69.26	69.69	68.90	66.94
2,6	27%aLFucp	101.36	71.50	71.26	77.25	73.17	15.37
2,6	73%bDGlcp	102.80	72.86	75.42	69.30	74.70	60.80


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
2	34%aDManp	5.06	4.10	4.02	3.85	3.79	3.83-3.97
	aDGalp	5.00	3.94	3.83	4.02	4.09	3.93-4.12
2,6	27%aLFucp	5.03	3.79	3.93	3.96	3.73	1.17
2,6	73%bDGlcp	4.48	3.28	3.44	3.43	3.58	3.71-3.85


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
2	34%aDManp	101.34/5.06	68.84/4.10	69.69/4.02	69.52/3.85	77.45/3.79	69.36/3.83-3.97
	aDGalp	97.67/5.00	77.29/3.94	69.26/3.83	69.69/4.02	68.90/4.09	66.94/3.93-4.12
2,6	27%aLFucp	101.36/5.03	71.50/3.79	71.26/3.93	77.25/3.96	73.17/3.73	15.37/1.17
2,6	73%bDGlcp	102.80/4.48	72.86/3.28	75.42/3.44	69.30/3.43	74.70/3.58	60.80/3.71-3.85

There are 2 chemically distinct structures. Please, select:
-6)[bDGlcp(1-6)34%aDManp(1-2)]aDGalp(1- -6)[aLFucp(1-6)34%aDManp(1-2)]aDGalp(1-

SVGMWSMILES3D molIonize

 

[*]OC[C@H]1O[C@H]([*])[C@H](O[C@H]2O[C@H](CO[C@@H]3O[C@H](CO)[C@@H](O)[C@H](O)[C@H]3O)[C@@H](O)[C@H](O)[C@@H]2O)[C@@H](O)[C@H]1O

486.423 g/mol (C₁₈H₃₀R₂O₁₅, R = next and previous repeats, not counted in mol weight)