To explore new anti-tumor constituents in the fungi, a water-soluble polysaccharide, WGLP was obtained from spores of Ganoderma lucidum (Fr.) Karst. The average molecular weight of this polysaccharide was estimated to be 15000 Da. Monosaccharide composition analysis indicated that it was composed solely of glucose. From methylation analysis, 1H and 13C NMR spectroscopy, and periodate oxidation and Smith degradation, it could be concluded that WGLP contained β-D-glucose residue exclusively, with an average repeating unit of hexasaccharide, having a backbone consisting of (1→3)-β-D-glucopyranosyl residues, to which the side chain consisting of terminal and (1→6)-β-D-glucopyranosyl residue is attached at position O-6 of the branching residues. Anti-tumor activity assays in vivo showed that WGLP could significantly inhibit the S180 tumor growth in the mice. Furthermore, no drug-related toxic reactions were observed. It was suggested that WGLP might be a potential anti-tumor agent used in the clinic.

structure, polysaccharide, β-glucan, Ganoderma lucidum, anti-tumor activity

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,3,3	bDGlcp	102.75	73.53	84.38	68.34	76.23	60.93
3,3	bDGlcp	102.75	73.53	84.38	68.34	76.23	60.93
3,6,6	bDGlcp	102.98	73.68	75.80	69.04	76.23	60.93
3,6	bDGlcp	102.75	73.53	75.13	68.34	76.08	69.80
3	bDGlcp	102.75	73.53	84.38	68.34	76.08	69.80
	bDGlcp	102.75	73.53	84.38	68.34	76.23	60.93


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,3,3	bDGlcp	4.78	3.56	3.77	3.52	3.50	3.68-3.90
3,3	bDGlcp	4.78	3.56	3.77	3.52	3.50	3.68-3.90
3,6,6	bDGlcp	4.57	3.28	3.38	3.36	3.50	3.68-3.90
3,6	bDGlcp	4.78	3.56	3.65	3.52	3.65	3.88-4.18
3	bDGlcp	4.78	3.56	3.77	3.52	3.65	3.88-4.18
	bDGlcp	4.78	3.56	3.77	3.52	3.50	3.68-3.90


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,3,3	bDGlcp	102.75/4.78	73.53/3.56	84.38/3.77	68.34/3.52	76.23/3.50	60.93/3.68-3.90
3,3	bDGlcp	102.75/4.78	73.53/3.56	84.38/3.77	68.34/3.52	76.23/3.50	60.93/3.68-3.90
3,6,6	bDGlcp	102.98/4.57	73.68/3.28	75.80/3.38	69.04/3.36	76.23/3.50	60.93/3.68-3.90
3,6	bDGlcp	102.75/4.78	73.53/3.56	75.13/3.65	68.34/3.52	76.08/3.65	69.80/3.88-4.18
3	bDGlcp	102.75/4.78	73.53/3.56	84.38/3.77	68.34/3.52	76.08/3.65	69.80/3.88-4.18
	bDGlcp	102.75/4.78	73.53/3.56	84.38/3.77	68.34/3.52	76.23/3.50	60.93/3.68-3.90

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

*O[C@H]1[C@H](O)[C@@H](CO)O[C@@H](O[C@@H]2[C@@H](O)[C@H](O[C@@H]3[C@@H](O)[C@H](O[C@@H]4[C@@H](O)[C@H](*)O[C@H](CO)[C@H]4O)O[C@H](CO[C@@H]4O[C@H](CO[C@@H]5O[C@H](CO)[C@@H](O)[C@H](O)[C@H]5O)[C@@H](O)[C@H](O)[C@H]4O)[C@H]3O)O[C@H](CO)[C@H]2O)[C@@H]1O

972.846 g/mol (C₃₆H₆₀R₂O₃₀, R = next and previous repeats, not counted in mol weight)

Pullulan is an important polysaccharide. Although its synthetic pathway in Aureobasidium melanogenum has been elucidated, the mechanism underlying its biosynthesis as regulated by signaling pathway and transcriptional regulator is still unknown. In this study, it was found that the expression of the UGP1 gene encoding UDPG-pyrophosphorylase (Ugp1) and other genes which were involved in pullulan biosynthesis was controlled by the transcriptional activator Msn2 in the nuclei of yeast-like fungal cells. The Ugp1 was a rate-limiting enzyme for pullulan biosynthesis. In addition, the activity and subcellular localization of the Msn2 were regulated only by the cAMP-PKA signaling pathway. When the cAMP-PKA activity was low, the Msn2 was localized in the nuclei, the UGP1 gene was highly expressed, and pullulan was actively synthesized. By contrast, when the cAMP-PKA activity was high, the Msn2 was localized in the cytoplasm and the UGP1 gene expression was disabled so that pullulan was stopped, but lipid biosynthesis was actively enhanced. This study was the first to report that pullulan and lipid biosynthesis in yeast-like fungal cells were regulated by the Msn2 and cAMP-PKA signaling pathway. Elucidating the regulation mechanisms was important to understand their functions and enhance pullulan and lipid biosynthesis.

pullulan biosynthesis, Msn2, UDPG-pyrophosphorylase, yeast-like fungi, cAMP-PKA

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

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

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

The structures and cytotoxic activities of water-soluble polysaccharides were investigated to search for biologically active polysaccharides from the fruit bodies of quinine conks (Fomitopsis officinalis). The decoctions of this medical fungus are actively used in folk medicine in many countries and traditional Chinese medicine. From the fungal extract we prepared, only branched β-glucan had cytotoxic activity among all the water-soluble polysaccharides. This glucan is characterized by a regular structure. Its backbone is formed by 1,3-linked β-D-Glcp residues, of which every third residue is substituted at O-6 by a single β-D-Glcp residue. It has a triple helix conformation according to the data obtained from a colorimetric assay with Congo red dye and is characterized by a high-weight average molar mass (Mw > 800 kDa). β-Glucan possessed cytotoxic activity against HeLa cells (IC50 = 318 ± 47 μg/mL) and induced the formation of apoptotic bodies around most cancer cells at a concentration of 200 μg/mL. It should be noted that extraction with boiling water, which is usually used to obtain extracts and decoctions, is unable to isolate active β-glucan. Active β-glucan can be obtained in an individual state by cold alkali extraction after dehydration of the fruit bodies and removal of the components extractable by boiling water.

NMR spectroscopy, β-glucan, Basidiomycota, HeLa cells, Fomitopsis officinalis, Fomitopsidaceae, fucosylated xylomannan, heteropolysaccharides

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,3	bDGlcp	102.83	72.37-72.47	85.87-86.57	68.37	75.88	60.61-60.74
3,6	bDGlcp	102.83	73.50	76.15	70.00	76.52	60.90
3	bDGlcp	102.76	72.62	86.18-86.57	68.49	74.54	68.27
	bDGlcp	102.83	72.37-72.47	85.87-86.57	68.37	75.88	60.61-60.74


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,3	bDGlcp	4.54	3.31	3.50	3.30	3.28	3.47-3.71
3,6	bDGlcp	4.23	3.02	3.19	3.09	3.12	3.47-3.69
3	bDGlcp	4.51	3.26	3.49	3.29	3.53	3.54-4.10
	bDGlcp	4.54	3.31	3.50	3.30	3.28	3.47-3.71


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,3	bDGlcp	102.83/4.54	72.37-72.47/3.31	85.87-86.57/3.50	68.37/3.30	75.88/3.28	60.61-60.74/3.47-3.71
3,6	bDGlcp	102.83/4.23	73.50/3.02	76.15/3.19	70.00/3.09	76.52/3.12	60.90/3.47-3.69
3	bDGlcp	102.76/4.51	72.62/3.26	86.18-86.57/3.49	68.49/3.29	74.54/3.53	68.27/3.54-4.10
	bDGlcp	102.83/4.54	72.37-72.47/3.31	85.87-86.57/3.50	68.37/3.30	75.88/3.28	60.61-60.74/3.47-3.71

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

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

648.564 g/mol (C₂₄H₄₀R₂O₂₀, R = next and previous repeats, not counted in mol weight)

The structures and cytotoxic activities of water-soluble polysaccharides were investigated to search for biologically active polysaccharides from the fruit bodies of quinine conks (Fomitopsis officinalis). The decoctions of this medical fungus are actively used in folk medicine in many countries and traditional Chinese medicine. From the fungal extract we prepared, only branched β-glucan had cytotoxic activity among all the water-soluble polysaccharides. This glucan is characterized by a regular structure. Its backbone is formed by 1,3-linked β-D-Glcp residues, of which every third residue is substituted at O-6 by a single β-D-Glcp residue. It has a triple helix conformation according to the data obtained from a colorimetric assay with Congo red dye and is characterized by a high-weight average molar mass (Mw > 800 kDa). β-Glucan possessed cytotoxic activity against HeLa cells (IC50 = 318 ± 47 μg/mL) and induced the formation of apoptotic bodies around most cancer cells at a concentration of 200 μg/mL. It should be noted that extraction with boiling water, which is usually used to obtain extracts and decoctions, is unable to isolate active β-glucan. Active β-glucan can be obtained in an individual state by cold alkali extraction after dehydration of the fruit bodies and removal of the components extractable by boiling water.

NMR spectroscopy, β-glucan, Basidiomycota, HeLa cells, Fomitopsis officinalis, Fomitopsidaceae, fucosylated xylomannan, heteropolysaccharides

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,3,3	aDManp	102.5	71.0	79.6	67.3-67.5	74.8	62.4
3,3	aDManp	102.5	71.0	79.6	67.3-67.5	74.8	62.4
3,4,2	aDFucp	100.6	69.6	71.2	73.1	68.2	16.8
3,4	bDXylp	102.7	79.5	77.8	70.7	66.3
3	aDManp	103.2	71.0	77.0	73.9	74.4	61.6
	aDManp	102.5	71.0	79.6	67.3-67.5	74.8	62.4


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,3,3	aDManp	5.17	4.20	4.03	3.82-3.86	3.81-3.86	3.78-3.91
3,3	aDManp	5.17	4.20	4.03	3.82-3.86	3.81-3.86	3.78-3.91
3,4,2	aDFucp	5.32	3.84	3.80	3.81	4.28	1.27
3,4	bDXylp	4.46	3.46	3.69	3.65	3.31-3.97
3	aDManp	5.14	4.23	4.13	3.90	4.05	3.85-3.93
	aDManp	5.17	4.20	4.03	3.82-3.86	3.81-3.86	3.78-3.91


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,3,3	aDManp	102.5/5.17	71.0/4.20	79.6/4.03	67.3-67.5/3.82-3.86	74.8/3.81-3.86	62.4/3.78-3.91
3,3	aDManp	102.5/5.17	71.0/4.20	79.6/4.03	67.3-67.5/3.82-3.86	74.8/3.81-3.86	62.4/3.78-3.91
3,4,2	aDFucp	100.6/5.32	69.6/3.84	71.2/3.80	73.1/3.81	68.2/4.28	16.8/1.27
3,4	bDXylp	102.7/4.46	79.5/3.46	77.8/3.69	70.7/3.65	66.3/3.31-3.97
3	aDManp	103.2/5.14	71.0/4.23	77.0/4.13	73.9/3.90	74.4/4.05	61.6/3.85-3.93
	aDManp	102.5/5.17	71.0/4.20	79.6/4.03	67.3-67.5/3.82-3.86	74.8/3.81-3.86	62.4/3.78-3.91

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

*O[C@H]1[C@H](O)[C@@H](CO)O[C@H](O[C@@H]2[C@H](O)[C@@H](O[C@@H]3[C@H](O)[C@@H](O[C@@H]4[C@H](O)[C@@H](*)O[C@H](CO)[C@H]4O)O[C@H](CO)[C@H]3O[C@@H]3OC[C@@H](O)[C@H](O)[C@H]3O[C@H]3O[C@H](C)[C@H](O)[C@H](O)[C@H]3O)O[C@H](CO)[C@H]2O)[C@H]1O

926.821 g/mol (C₃₅H₅₈R₂O₂₈, R = next and previous repeats, not counted in mol weight)