A novel linear 3-O-methylated galactan isolated from Cantharellus cibarius activates macrophages

Article abstract of DOI:10.1016/j.carbpol.2019.03.002

A novel polysaccharide (WCCP-N-b) with a molecular weight of 18 kDa was isolated and purified from the fruiting bodies of Cantharellus cibarius. Monosaccharide composition, methylation analysis and NMR spectra indicated that WCCP-N-b was a linear α-1,6-galactan, partially methylated at O-3 of galactose. The molar ratio of Gal, 3-methylated-Gal, Glc and Man was 14.4:4.6:1.0:1.2. WCCP-N-b could significantly increase macrophage phagocytosis, release of NO and secretion of TNF-α, IL-6 and IL-1β. On a cellular mechanistic level, WCCP-N-b activated MAPKs and NF-κB signaling pathway via Toll-like receptor 2 (TLR2). To further elucidate the structure-function relationship, WCCP-N-b was hydrolyzed by acid. Four degraded fragments were obtained, with molecular weights of 16.1 kDa, 11.2 kDa, 5 kDa and 3.5 kDa, respectively. Their macrophage activation effects were significantly decreased along with the molecular weight decrease. Collectively, WCCP-N-b could activate RAW264.7 cells, and the activation effect was related to its molecular weight.

Full text of DOI:10.1016/j.carbpol.2019.03.002

Accepted Manuscript
Title: A novel linear 3-O-methylated galactan isolated from
Cantharellus cibarius activates macrophages
Authors: Guang Yang, Yunhe Qu, Yue Meng, Yushi Wang,
Chengcheng Song, Hairong Cheng, Xiaomeng Li, Lin Sun,
Yifa Zhou
PII:
S0144-8617(19)30268-1
DOI:
Reference:
https://doi.org/10.1016/j.carbpol.2019.03.002
CARP 14671
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Received date:
Revised date:
Accepted date:
11 December 2018
18 February 2019
3 March 2019
Please cite this article as: Yang G, Qu Y, Meng Y, Wang Y, Song C, Cheng
H, Li X, Sun L, Zhou Y, A novel linear 3-O-methylated galactan isolated
from Cantharellus cibarius activates macrophages, Carbohydrate Polymers (2019),
https://doi.org/10.1016/j.carbpol.2019.03.002
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A novel linear 3-O-methylated galactan isolated from Cantharellus
cibarius activates macrophages
Guang Yang, Yunhe Qu, Yue Meng, Yushi Wang, Chengcheng Song, Hairong Cheng,
Xiaomeng Li, Lin Sun*, Yifa Zhou*
Jilin Province Key Laboratory on Chemistry and Biology of Changbai Mountain Natural Drugs,
School of Life Sciences, Northeast Normal University, Changchun 130024, China
*Correspondence:
Lin Sun, email: sunl925@nenu.edu.cn
Yifa Zhou, email: zhouyf383@nenu.edu.cn;
Highlights

A novel linear 3-O-methylated galactan (WCCP-N-b) from C. cibarius was
identified.


WCCP-N-b increased phagocytosis and secretion of NO, TNF-α, IL-6 and IL-1β.
WCCP-N-b activated macrophages through Akt/NF-κB, MAPKs via TLR2
receptor.

The molecular weight of WCCP-N-b impacted the activation.
Abstract
A novel polysaccharide (WCCP-N-b) with a molecular weight of 18 kDa was
isolated and purified from the fruiting bodies of Cantharellus cibarius.
Monosaccharide composition, methylation analysis and NMR spectra indicated that
WCCP-N-b was a linear α-1,6-galactan, partially methylated at O-3 of galactose. The
1

molar ratio of Gal, 3-methylated-Gal, Glc and Man was 14.4:4.6:1.0:1.2. WCCP-N-b
could significantly increase macrophage phagocytosis, release of NO and secretion of
TNF-α, IL-6 and IL-1β. On a cellular mechanistic level, WCCP-N-b activated
MAPKs and NF-κB signaling pathway via Toll-like receptor 2 (TLR2). To further
elucidate the structure-function relationship, WCCP-N-b was hydrolyzed by acid.
Four degraded fragments were obtained, with molecular weights of 16.1 kDa, 11.2
kDa, 5 kDa and 3.5 kDa, respectively. Their macrophage activation effects were
significantly decreased along with the molecular weight decrease. Collectively,
WCCP-N-b could activate RAW264.7 cells, and the activation effect was related to its
molecular weight.
Key words: Cantharellus cibarius, Polysaccharide, 3-O-methylated galactan,
Macrophage, TLR2
2

1
. Introduction
Macrophages are important components of the innate immune system, playing a
crucial role in host defense against infection trough immune-inflammatory responses,
recognition of pathogens and phagocytosis. In addition, macrophages also exert an
important role as an interface between innate and adaptive immunity (Van den
Bossche, O'Neill & Menon, 2017). Hence, macrophages are thought to be the
important target cells of some antitumor and immunomodulatory drugs. Many
mushroom sugars that can bind to pattern recognition receptors (PRRs) such as TLR2,
TLR4, Dectin-1 (Gordon, 2002) and stimulate immune system, which present on the
surface of macrophage cells, and then activate MAPKs and NF-κB signaling pathway,
induce the activation of immune response (Takeuchi & Akira, 2010).
Mushrooms are used as healthy food in the world as they are rich in a great
variety of bioactive components, such as proteins (Zhang et al., 2014),
lipopolysaccharides (Wasser, 2011), glycoproteins (Cui et al., 2013) and
polysaccharides (Meng, Cheng, Han, Chen, & Wang, 2017). Among the bioactive
compounds, polysaccharides are the most abundant and extensively studied.
Polysaccharides from mushrooms possess antioxidant activity (Nowacka et al., 2014),
antitumor activity (Wasser, 2002) and immunoregulatory activity (Meng, et al. 2018).
These biologically active mushroom polysaccharides are mostly glucans (Liana et al.,
2
018) and galactans (Rosado et al., 2003) The main chains of the galactans are
composed of (1→6)-linked α-D-Galp units, which are partially methylated at O-3 or
substituted at C-2 by L-fucopyranosyl, D-mannopyranosyl, D-galactopyranosyl or
3
-O-α-D-mannopyranosyl-α-L-fucopyranosyl residues as side chains (Rosado et al.,
3

2
003; Ruthes, Smiderle & Iacomini, 2016) have been reported, possess
immunoregulatory activity.
Cantharellus cibarius (C. cibarius, the golden chanterelle) is an edible mushroom
which widely distributes in Asia, America and some European countries. It belongs to
the phylum Basidiomycota, family Cantharel-laceae. It is not only consumed as
health-supplementary food, but also considered as adjuvant in improving eyesight and
strengthening both spleen and stomach functions in traditional Chinese medicines
(
Wang, Ngai, & Ng, 2003; Han, et al., 2013). Polysaccharides, such as mannans
Nyman, Aachmann, Rise, Ballance, &Samuelsen, 2016) and glucans (Villares, et al.,
(
2
013; Zhao, et al., 2018) have been purified from C. cibarius fruit bodies. However,
few studies about galactans from C. cibarius have been reported. In this paper, a
novel galactan was isolated and purified from the fruit bodies of C. cibarius. The
present study is aimed to study the structure of this galactan, evaluate its
immunostimulatory activity on RAW264.7 cells, investigate the molecular
mechanism responsible for its immunostimulatory activity, and illuminate its
structure-activity relationship.
2
2
. Materials and methods
.1 Materials
Fruiting bodies of C. cibarius were purchased at the local market in Jilin Province,
PR China and were identified by using rDNA-ITS sequencing analysis.
DEAE-cellulose was purchased from Shanghai Chemical Reagent Research Institute
(Shanghai, China). Sepharose CL-6B and Sephadex G-10 were purchased from GE
4

Healthcare (Little Chalfont, Buckinghamshire, UK). LPS was obtained from Sigma
Sigma Aldrich, St Louis, MO, USA). ELISA kits were obtained from Boster
(
Biological Technology (Wuhan, China). LY294002, BAY11-7082, SP600125, U0126
and SB203580 were acquired from Selleck (Shanghai, China). TLR2 siRNA was
obtained from JTSBIO (Wuhan, China). All of the other reagents were of analytical
grade or better.
2
.2 Extraction and purification of the polysaccharide
Fruiting bodies of C. cibarius were defatted with 95% ethanol (material/ethanol,
w/v, 1:10), and the residues were first extracted with distilled water at 100 °C for 4 h
1:25w/v) followed by extracting again at 100 °C for 2 h (1:20 w/v). The extracts
(
were concentrated under vacuum at 60 °C, and 95% ethanol was added at a final
concentration of 65% to precipitate the polysaccharides. The precipitate was collected
by centrifugation at 4000 rpm for 15 min and vacuum dried. A water-soluble
polysaccharide named as WCCP with a yield of 5.5% (yield in relation to dry weight
of initial material) was obtained. WCCP was dissolved in distilled water and
centrifuged at 4000 rpm for 15 min. The supernatant was applied to a
-
DEAE-Cellulose column (Cl , 1.5×14 cm) pre-equilibrated with distilled water. The
column was first eluted with distilled water, yielding a neutral polysaccharide fraction
(WCCP-N), and then eluted with 0.3 M NaCl to give an acidic fraction (WCCP-A).
WCCP-N was further purified using gel-permeation chromatography with Sepharose
Cl-6B to give a homogeneous fraction WCCP-N-b.
5

2
.3 Analysis of chemical properties
Total carbohydrate content was determined by using the phenol-sulfuric acid
protocol with glucose as the standard (Dubois, Gilles, Hamilton, Rebers, & Smith,
951). Protein content was determined by using the Bradford assay with bovine serum
1
albumin as the standard (Sedmak & Grossberg, 1977).
Monosaccharide composition was determined by using high performance liquid
chromatography (HPLC) as described by Zhang (Zhang et al., 2009).
Methyl-galactose (Me-Gal) was determined by UPLC-MS (Yan, et al., 2019).
Molecular weight distributions were determined by using high performance
gel-permeation chromatography (HPGPC) on a TSK-gel G-3000PWXL column (7.8
×
300 mm, TOSOH, Japan) coupled to a Shimadzu HPLC system as described by
Zhang et al. (2009). The column was pre-calibrated by using standard dextrans (50
kDa, 25 kDa, 12 kDa, 5 kDa and 1 kDa) using linear regression.
2
.4 Acid hydrolysis of WCCP-N-b
WCCP-N-b (2 mg) was hydrolyzed with 1 ml TFA (1 M, 0.5 M, 0.25 M and 0.1 M)
at 80 °C for 1 h, respectively, and then the aliquot was adjusted to pH 7 by adding 0.3
M NaOH. The hydrolysates were desalted by using Sephadex G-10 column and freeze
dried.
2
.5 Methylation analysis
Methylation analysis was carried out according to the method of Needs and
6

Selvendran (Needs & Selvendran, 1993). The partially methylated sugars in the
hydrolysate were reduced by using NaBH and acetylated (Sweet, Albersheim &
Shapiro, 1975). The resulting alditol acetates were analyzed by GC-MS
7890B-5977B, Agilent, USA) with a HP-5ms capillary column (30 m × 0.32 mm ×
4
(
0
2
.25 mm). The oven temperature was programed from 120 °C (hold for 1 min) to
10 °C (hold for 2 min) at 3 °C/min, then up to 260 °C (hold for 4 min) at 10 °C/min.
Both temperature of inlet and detector were 300 °C. Helium was used as carrier gas.
The mass scan range was 50-500 m/z.
2
.6 NMR analyses
Samples (20 mg) were dissolved in D O (0.5 ml) and centrifuged to remove
2
1
13
undissolved polysaccharide. H, C, HSQC, and HMBC NMR spectra were recorded
at 20 °C on a Bruker Avance 600 MHz spectrometer (Germany) with a Bruker 5 mm
1
13
broadband probe operating at 600 MHz for H NMR and 150 MHz for C NMR,
respectively. Data were analyzed using standard Bruker software.
2
.7 Cell culture
RAW264.7 cells were purchased from the American Type Culture Collection.
RAW264.7 cells were maintained in DMEM high glucose medium supplemented with
00 U/ml penicillin, 100 μg/ml streptomycin and 10% heat-inactivated FBS. Cells
were cultured at 37 °C in a 5% CO incubator.
1
2
2
.8 Cell viability assay
5
RAW264.7 cells were seeded at a density of 2×10 cells/ml in a 96-well plate
7

overnight and then treated with various concentrations of WCCP-N-b or 1 μg/ml of
LPS for 24 h. Cell viability was determined by MTT assay (Meng, et al., 2018).
2
.9 Measurement of NO
RAW264.7 cells at a density of 8×10 /ml were incubated with different
5
concentrations of WCCP-N-b or LPS (1 μg/ml) for 24 h. After incubation,
supernatants were collected and reacted with Griess reagent (Meng, et al., 2018).
2
.10 Determination of phagocytic uptake capacity of macrophage
5
RAW264.7 cells were seeded at a density of 4×10 /well in 6-well plate overnight
and incubated with different concentrations of WCCP-N-b or LPS (1 μg/ml) for 24 h.
The phagocytic activity of macrophages was analyzed using flow cytometry (Yan, et
al., 2018). The phagocytosis index was calculated as the number of phagocytosed
beads/total number of macrophages.
2
.11 Measurement of TNF-α, IL-6 and IL-1β
RAW264.7 cells were pretreated with or without specific inhibitors (25 μM of
SB203580, 25 μM of U0126, 20 μM of SP600125, 25 μM of LY294002, 10 μM of
BAY11-7082) for 2 h, followed by incubation with WCCP-N-b (200 μg/ml) for 24 h.
The concentrations of TNF-α, IL-6 and IL-1β were assessed using ELISA kits
according to the manufacturer’s instructions.
2
.12 Antibody inhibition experiments
Cells were pretreated with function-blocking antibodies (20 μg/ml each) to TLR2,
TLR4, Dectin-1 for 2 h, followed by incubation with WCCP-N-b for 24 h. Cytokine
8

concentration was examined using ELISA kits according to the manufacturer’s
instruction.
2
.13 Immunofluorescence assay
Macrophages were fixed in 4% paraformaldehyde for 30 min and incubated with
anti-NF-κB p65 antibody (1:200) for 1 h at room temperature. Then cells were
washed three times with PBS and incubated with FITC secondary antibody (1:200)
for 1 h at room temperature. Nucleus was stained with Hoechst reagent for 10 min at
room temperature. The location of NF-κB p65 was assessed using
immunofluorescence microscopy.
2
.14 Western blotting
WCCP-N-b (200 μg/ml) was added to each well (8×10 cells) and maintain at 37 ℃
for 30 min. The Western blotting assay was performed as previously described
Meng, et al. 2018).
5
(
2
.15 Transfection
Transfection of RAW264.7 cells was conducted with Lipofectamine 3000 (Thermo
Fisher Scientific, USA) following the manufacturer’s instructions. High purity
controls (scrambled RNA), along with TLR2 siRNA oligos, were obtained from
JTSBIO (Wuhan, China). The targeting sequences of the siRNA constructs are: TLR2
siRNA-1,
5'-GCCUUGACCUGUCUUUCAATT-3';
TLR2
siRNA-2,
5
'-CCAGCAGAAUCAAUACAAUTT-3'. After 48 h of transfection, RAW264.7 cells
were treated with WCCP-N-b (200 μg/ml) for 30 min or 24 h, and then analyzed by
western blotting or ELISA assay, respectively.
9

2
.16 Statistical analysis
Data are provided as the mean ± SD from at least three independent experiments.
Differences between the groups were analyzed through a one-way analysis of
variance (ANOVA). Significance was defined as P-values < 0.05, 0.01 or 0.001,
respectively.
3
3
. Results
.1 Extraction and purification of polysaccharide from Cantharellus cibarius
The water-soluble polysaccharide (WCCP) was extracted from the fruiting bodies
of C. cibarius (Fig. 1A), with a yield of 5.5% in relation to dry weight of initial
material. WCCP was fractionated by ion-exchange chromatography into a neutral
polysaccharide fraction WCCP-N (yield 50.2%) and an acid polysaccharide fraction
WCCP-A (yield 29.2%) (Fig. 1B). WCCP-N was further purified with gel permeation
chromatography and a major homogeneous fraction WCCP-N-b was obtained (Fig.
1
C). The molecular weight of WCCP-N-b was around 18.0 kDa determined by
HPGPC (Fig. 1D). Monosaccharide composition analysis showed that WCCP-N-b
contained galactose (Gal) and 3-methyl-Gal as the main component, followed by
minor of glucose (Glc) and mannose (Man), with the molar ratio of 14.4:4.6:1.0:1.2
(Table 4), respectively.
10

Fig. 1. Extraction and purification of polysaccharides. (A) Extraction scheme of
water-soluble polysaccharide (WCCP) from C. cibarius. (B) WCCP was separated by
DEAE-cellulose anion-exchange chromatography to give WCCP-N and WCCP-A. (C)
WCCP-N was purified by Sepharose Cl-6B column to give WCCP-N-b. (D) Elution
profile of WCCP-N-b on HPGPC.
3
.2 Methylation analysis
In order to determine the glycosyl linkage types of WCCP-N-b, it was methylated,
hydrolyzed and acetylated, and the partially methylated alditol acetates were analyzed
by GC-MS. The methylation analysis result (Table 1) indicated that major glycosyl
linkage type in WCCP-N-b was 1,6-linked-Galp (including 1,6-linked-Me-Galp), with
the molar ratio of 91.1%. Small amounts of 1,2,6-linked-Galp were also detected
(3.2%). Therefore, the backbone of WCCP-N-b should be 1,6-linked-Galp. The
11

degree of branching (DB) was only 3.4%. Trace amounts of 1,6-linked-Glcp (2.9%)
and non-reducing terminals Manp (2.7%) were found. According to these results, the
backbone of WCCP-N-b was composed of around one hundred Gal residues., among
which around three Gal residues in the backbone were substituted at C-2 by very short
branches.
Table.1. Linkage type analysis of WCCP-N-b by GC-MS
Metylated sugars
Linkages
1,6-
Molar ratio (mol %)
Mass fragments (m/z)
2
3
2
2
,3,4-Me
3
-Galp
91.1
3.2
87,101,117,129,161,173,189,233
87,99,117,129,159, 189,233
87,101,117,129,161,173,189,233
87,101,117,129,145,161,205
,4-Me
2
-Galp
1,2,6-
1,6-
,3,4-Me
3
-Glcp
2.9
,3,4,6-Me
4
-Manp
1-
2.7
3
.3 NMR analysis.
The structure of WCCP-N-b was further characterized by 1D/2D NMR spectra. In
1
the H-NMR spectrum, the signal at 4.93 ppm was assigned to H-1 of α-1,6-linked
D-Galp and 3-O-Me-D-Galp residues (Zhang, Xu, Fu & Sun, 2013). The signal at 3.37
ppm was assigned to proton of O-CH group (Ruthes et al., 2013) (Fig. 4A). No
3
obvious signals for Glcp and Manp residues were detected since their quite lower
content. In the 13C-NMR spectrum, signals at 97.61 ppm and 97.55 ppm were
assigned to anomeric carbons of 1,6-linked Galp and 3-O-Me-Galp, respectively.
Weak signal peak at 98.19 ppm was attributed to 1,2,6-linked Galp. The signal at
7
8.56 ppm was corresponded to C-3 of α-1,6-linked 3-O-Me-D-Galp (Cho, Yun, Yoo
Koshino, 2011). The signal at 55.88 ppm was assigned to carbon of O-CH group
Fig. 4B). No obvious signal of C-2 for 1,2,6-linked α-D-Galp was detected in the
&
3
(
12

13C-NMR spectrum, which was consistent with methylation analysis result that quite
lower content (3.2%) of 1,2,6-linked Galp was determined. The other chemical shifts
were assigned mainly according to carbon and hydrogen correlations in the HSQC
(Fig. 4C) and HMBC spectra (Fig. 4D). On the basis of the proton assignments, the
chemical shifts of H-1/C-1~H-6/C-6 for Galp and 3-O-Me-Galp were readily obtained
from the HSQC spectrum (Carbonero et al., 2008). The sequence of the major
residues in the repeating unit was identified from the HMBC spectrum, which showed
clear correlations: AC-1/BH-6a/6b; AH-1/AC5; BC-1/AH-6a/6b; BH-1/BC-5;
O-CH
3
H-BC3; O-CH C-BH3. In conclusion, the possible structural feature of
3
WCCP-N-b was proposed as a linear methylated galactan which was composed of
α-1,6-linked D-Gal and 3-O-Me-D-Gal residues. This structure has never been
reported in C. cibarius.
13

1
13
Fig. 2. NMR spectra of WCCP-N-b. (A) H NMR spectrum, (B) C NMR spectrum,
C) HSQC spectrum, (D) HMBC spectrum.
Table 2. HSQC spectral assignments for WCCP-N-b.
(
Linkage type
1
2
3
4
5
6
O-CH
3
(
(
(
A)α-1,6-D-3-Me-Galp
B) α-1,6-D-Galp
H
C
H
C
H
C
4.93
3.80
3.50
4.24
4.15
3.85;3.62
66.24
3.37
55.88
--
97.55
4.93
67.06
3.78
78.56
3.81
64.84 68.67
3.96
4.15
3.85;3.62
66.24
97.61
4.87
68.02
3.80
69.31
3.78
69.21 68.60
--
C) α-1,2,6-D-Galp
3.96
4.15
3.86;3.62
64.83
--
98.19
78.60
69.38
69.21 68.58
--
Table 3. HMBC spectral assignments for WCCP-N-b
14

Glycosidic linkage
H-3/C-3
H-1/C-1
4.93
Coupling relationship
δ
H
/δ
C
Residue
Atom
(C)α-1,6-D-3-Me-Galp
66.24
D
C
C
C6
6
7
8.54
8.56
C5
C3
3
7
.50
55.80
3.37
3.86
O-CH
3
3
Me(C)
Me(H)
H6a
H6b
C6
8.56
O-CH
D
9
7.55
3
.62
D
(D)α-1,6-D-Galp
4.93
66.24
C
6
8.58
D
C5
9
7.71
3.85
C
H6a
H6b
3
.62
C
3
.4 WCCP-N-b promotes activation of macrophages.
The cytotoxicity effect of WCCP-N-b on RAW264.7 macrophage cells was
investigated by the MTT assay. Results showed that WCCP-N-b minimally affect cell
viability (Fig. 3A), indicating the absence of cytotoxic effects. Macrophage activation
was assessed by assessing release of NO, phagocytic uptake and secretion of the
cytokines (TNF-α, IL-6, IL-1β). LPS was applied as a positive control. Compared to
15

the control group, WCCP-N-b significantly induced production of NO (Fig. 3B),
obviously increased phagocytic uptake (Fig. 3C), as well as robustly provoked the
secretion of TNF-α, IL-6 and IL-1β (Fig. 3D).
Fig. 3. WCCP-N-b promoted the activation of RAW264.7 cells. RAW264.7 cells
were treated with the indicated doses of WCCP-N-b for 24 h. (A) Cell viability was
determined by MTT assay. (B) Releases of NO was analyzed with Griess reagents. (C)
Phagocytic uptake was assessed by determining the uptake of fluorescent
microspheres using flow cytometry. (D) Secretion of the cytokines TNF-α, IL-6 and
IL-1β was examined using the ELISA assay. Error bars in (A), (B), (C), and (D)
represent the S.D. (N = 3 independent experiments). *P< 0.05, ** P< 0.01 and ***P<
0
.001, compared to control group.
3
.5 MAPK pathway is related to RAW264.7 activation.
Mitogen-activated protein kinases (MAPKs), including the extracellular
signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs), and p38-MAPKs,
have been reported to be involved in macrophage activation (Meng et al., 2018). In
the present study, whether MAPK pathway was involved in the WCCP-N-b-induced
16

macrophage activation was investigated. The results showed that WCCP-N-b
increased phosphorylation of ERK, JNK and p38 (Fig. 4A). Pre-treatment cells with
ERK inhibitor U0126, JNK inhibitor SP600125 or p38 inhibitor SB203580
significantly reduced the phosphorylation of ERK, JNK and p38, respectively.
Furthermore, secretion of NO, as well as TNF-α and IL-6 production, were also
significantly reduced (Fig. 4C). Taken together, we concluded that MAPK pathway
accounted for, at least partially, the macrophage activation by WCCP-N-b.
17

Fig. 4. Effect of WCCP-N-b on MAPKs signaling pathways. RAW264.7 cells were
treated with 200 μg/ml of WCCP-N-b for the indicated concentrations for 30 min (A).
RAW 264.7 cells were pretreated with or without the ERK inhibitor U0126, the JNK
inhibitor SP600126 and the p38 inhibitor SB203580 for 2 h, followed by incubation
18

with WCCP-N-b (200 μg/ml) or LPS (1 μg/ml) for 30 min (B). Whole-cell extracts
were analyzed by western bolt. (C) RAW264.7 cells were treated with 200 μg/ml of
WCCP-N-b for 24 h, NO production, TNF-α and IL-6 secretion were detected by
using Griess reagents and ELISA assay, respectively. Error bars in (C) represent the
S.D. (N= 3 independent experiments). **P< 0.01 and ***P < 0.001.
3
.6 WCCP-N-b induces AKT/NF-κB activation in RAW264.7 cells.
The nuclear factor κB (NF-κB) signaling pathway is essential for regulation of a
wide variety of cellular genes, especially those involved in immune and inflammatory
responses (Cao, Li, Chen, Xue, & Liu, 2016). In resting cells, inactive NF-κB forms a
complex with IκB-α and exists as a latent form in the cytosol. However, upon
stimulation, IκB-α in the cytosolic complex is phosphorylated and degraded, which
results in release of NF-κB and translocation to the nucleus allowing NF-κB to
interact with its promoter elements and mediate transcription of a variety of target
genes (Yan, et al., 2018). It has been reported that Akt activation is the upstream of
NF-κB activity (Cianciulli et al., 2016). In the present study, immunofluorescence
assay results showed that most cytoplasmic NF-κB p65 translocated from the
cytoplasm to the nucleus when cells were treated with WCCP-N-b (Fig. 5A). Western
blot analysis indicated that WCCP-N-b increased phosphorylation of AKT,
degradation of IκB-α as well as phosphorylation of NF-κB (Fig. 5B). To validate the
roles of AKT and NF-κB, inhibitor LY294002 to AKT and inhibitor BAY11-7082 to
NF-κB were applied. The results showed that NF-κB inhibitor BAY11-7082
significantly reduced NF-κB phosphorylation (Fig. 5C). AKT inhibitor significantly
19

reduced phosphorylation of AKT, and subsequent IκB-α degradation as well as NF-κB
phosphorylation (Fig. 5C). In addition, the secretion of NO, TNF-α and IL-6 were
also robustly inhibited upon pretreatment of LY294002 or BAY11-7082 (Fig. 5D).
Overall, these results indicated that the AKT/NF-κB signaling pathway is also
involved in WCCP-N-b-induced activation of macrophages.
20

Fig. 5. Effect of WCCP-N-b on NF-κB signaling pathways. (A) The localization of
NF-κB subunit (green) and nucleus (blue) were analyzed by double
immunofluorescence staining following treatment with WCCP-N-b for 30 min. (B)
RAW264.7 cells were treated with 200 μg/ml of WCCP-N-b for the indicated
concentrations for 30 min, whole-cell extracts were prepared and analyzed by western
21

blotting. RAW 264.7 cells were pretreated with or without the AKT inhibitor
LY294002) or the NF-κB inhibitor (BAY11-7082) for 2 h, (C) followed by
(
incubation with WCCP-N-b (200 μg/ml) for 30 min. Whole-cell extracts were
analyzed by western bolt. (D) RAW 264.7 cells were pretreated with or without the
AKT inhibitor (LY294002) or the NF-κB inhibitor (BAY11-7082) for 2 h, then treated
with 200 μg/ml of WCCP-N-b for 24 h. NO production, TNF-α and IL-6 secretion
were detected by using Griess reagents and ELISA assay, respectively. Error bars in
(D) represent the S.D. (N= 3 independent experiments). ***P< 0.001.
3
.7 Knockdown of TLR2 in RAW264.7 cells suppresses WCCP-N-b-induced
macrophage activation
Polysaccharides cannot penetrate cells directly, because of their relatively large
molecular mass. Therefore, polysaccharides need to bind to cell membrane receptors
in order to mediate intracellular events and thereby trigger an immune response.
Several pattern recognition receptors (PRRs), including TLR2, TLR4 and Dectin-1,
have been reported to participate in signal transmission and thereby macrophage
activation (Gordon, 2002)
.
To determine which PRR participate in
WCCP-N-b-induced macrophage activation, function-blocking antibodies of Dectin-1,
TLR2 or TLR4 were applied. We found that only the anti-TLR2 antibody could
abrogate WCCP-N-b-induced TNF-α and IL-6 cytokine release (Fig. 6A). To further
confirm the role of TLR2, we transfected RAW264.7 cells with TLR2 siRNA.
Compared with scrambled RNA group, TLR2-siRNA1 and 2 significantly decreased
TLR2 expression (Fig. 6B). Moreover, WCCP-N-b-induced production of NO, TNF-α
22

and IL-6 were significantly inhibited in TLR2 siRNA transfected cells (Fig. 6C).
These data indicated that TLR2 plays a vital regulatory role in the macrophage
activation induced by WCCP-N-b.
Fig. 6. WCCP-N-b activated RAW264.7 cell via TLR2 receptor. (A) RAW 264.7
cells were pretreated with or without function-blocking antibodies (20 μg/ml each) to
Dectin-1, TLR2, TLR4 for 2 h, followed by incubation with WCCP-N-b (200 μg/ml)
for 24 h. The production of TNF-α and IL-6 in the supernatants were determined by
ELISA assay. RAW264.7 cells were transfected with scrambled RNA or TLR2 siRNA,
and then treated with WCCP-N-b (200 μg/ml) for 30 min or 24 h. Protein expressions
were detected by western blot (B). NO production or secretion of TNF-α and IL-6 in
the culture supernatant were measured by the Griess assay (B) or ELISA assay (C),
respectively. Error bars in represent the S.D. (N =3 independent experiments). *P<
0
.05, **P < 0.01 and ***P < 0.001, ns, no significance.
23

3
.8 Molecular weight of WCCP-N-b plays critical role on its macrophage
activation effect
To further elucidate the structure-function relationship, WCCP-N-b was treated
with 0.1 M, 0.25 M, 0.5 M and 1.0 M TFA solutions, respectively. A series of
degraded fragments were obtained, named as WCCP-N-b1 (yield 60%), WCCP-N-b2
(
yield 54%), WCCP-N-b3 (yield 37.8%) and WCCP-N-b4 (yield 33.8%). Their
molecular weights were 16.1 kDa, 11.2 kDa, 5 kDa and 3.5 kDa, respectively (Fig.
A). Compared with WCCP-N-b (molecular weight of 18 kDa), their molecular
7
weights were all decreased. The monosaccharide composition of these degraded
fragments (WCCP-N-b1~WCCP-N-b4) were similar to that of WCCP-N-b (Fig. 7B,
Table 4.). Immunological activity analyses showed that the macrophage activation
effects of these degraded polysaccharide fragments were significantly weaker than
that of WCCP-N-b. The lower the molecular weight, the weaker the macrophage
activation effect (Fig. 7C, D and E). Furthermore, we noted that when the molecular
weight was decreased to 3.5 kDa, the polysaccharide fragment could not induce the
secretion of NO, TNF-α and IL-6 (Fig. 7C, D and E). Therefore, we speculated that
molecular weight of WCCP-N-b plays a crucial role inactivation of macrophages.
24

Fig. 7. Molecular weight of WCCP-N-b affected the activation of macrophages.
A) Molecular weight distribution of WCCP-N-b, WCCP-N-b1, WCCP-N-b2,
(
WCCP-N-b3, WCCP-N-b4. (B) Monosaccharide composition analysis of WCCP-N-b,
WCCP-N-b1, WCCP-N-b2, WCCP-N-b3, WCCP-N-b4 by HPLC, and the peak of
Me-Gal appeared in 36min was confirmed by LC-MS analysis (Yan, et al., 2019).
RAW264.7 cells were treated with polysaccharides (200 μg/ml) for 24 h, release of
NO was analyzed by using Griess reagents (C), secretion of TNF-α and IL-6 were
examined using the ELISA assay (D & E). Error bars represent the S.D. (N = 3
independent experiments). *P< 0.05, ** P < 0.01and***P < 0.001 compared to
control group.
25

Table 4. Yield and monosaccharide composition of collected fractions
Fraction
Yield (g%) a
Monosaccharide composition (%)
Gal
67.9
67.8
66.6
67.5
66.5
Me-Gal
21.8
Man
5.5
6.2
6.8
5.4
6.0
Glc
4.7
3.0
4.5
5.6
5.5
WCCP-N-b
WCCP-N-b1
WCCP-N-b2
WCCP-N-b3
WCCP-N-b4
--
60
23.0
54
22.1
37.8
33.8
21.5
22.0
aYield in relation to fraction WCCP-N-b
4
. Discussion
Cantharellus cibarius is an edible mushroom widely used as healthy food in the
world. Polysaccharide is one of the major components in C. cibarius. Lemieszek,
Nunes, Cardoso, Marques, & Rzeski, (2018) isolated four polysaccharide fractions
from C. cibarius fruit bodies, and these fractions were all made up of Man in the form
of 1,6- and 1,4-linkages, with Glu and Fuc branches. Nyman, et al. (2016) purified
three polysaccharide fractions from C. cibarius which were branched (1 →
6
)-α-mannan and β-glucans. Two D-Glcp polysaccharides were isolated from C.
cibarius by Villares, et al. (2013). Zhao, et al., (2018) purified a
,4-linked-β-D-glucose which branched by α-D-xylopyranose residue at O-6 from C.
1
cibarius fruit bodies. In the present study, we obtained a novel linear 3-O-methylated
galactan (WCCP-N-b) from C. cibarius, which was composed of 67% of
α-1,6-D-Galp and 22% of α-1,6-D-Me-Galp. The structure of WCCP-N-b was
completely different from those polysaccharides above-mentioned. To our knowledge,
this polysaccharide structure has not been reported in C. cibarius.
Mushrooms have been used both as food and medicine for last few decades.
26

Polysaccharide, as the main active ingredient in mushroom, has attracted much
attention. Polysaccharides from mushrooms possess immunoregulatory activity. In
most cases, polysaccharides cannot enter cells directly. A lot of evidences showed that
the first step for polysaccharides to exert any function on cells was the recognition by
PRRs (Yan, et al., 2018). A heterogalactan which consisted of a (1,4)-Galp and
(1,4)-GalAp backbone activated NF-κB signaling pathway, induced macrophages to
release TNF-α via bingding to TLR4（Wang, H. T., et al., 2018）. Meng, et al. (2018)
isolated an α-(1,6)-D-galactan which was branched at the O-2 of Galp residues by
α-D-(1,6)-linked Manp attached to t-β-D-Glcp or t-α-D-Fucp side chains from
Flammulina velutipes, activated macrophage via TLR4 receptor. Yan, et al., (2019)
extracted a partially 3-O-methylated galactans (WPEP-N-b) from Pleurotus eryngii
which was composed of α-1,6-D-Galp and α-1,6-D-3-O-Me-Galp as main chains,
branched at O-2 with single t-β-D-Manp as major side chain, could bind to TLR2 to
activate macrophage. In the present study, the linear 3-O-methylated galactan
WCCP-N-b could also activate MAPKs and NF-κB signaling pathway via TLR2 to
promote immune activity. We speculated the galactan structure lead to its ability to
bind to Toll-like receptor and heterogalactan partially substituted at O-3 by methyl
groups might play a crucial role on its specific recogonization to TLR2. TLR2 could
mediate activation of macrophage, B cells (Han et al., 2003), human monocytes
(Balachandran, Pugh, Ma, & Pasco, 2006), NK cells (Ke, et al., 2014). Hence, we
speculated that our 3-O-methylated galactan might be a promising and broad
immunopotentiator via binding to TLR-2 receptor. Studies on the role of O-3 methyl
27

groups in the binding to TLR2 are ongoing in our laboratory.
In the present research, we found that the macrophage activation effect of galactan
was decreased following with the decrease of the molecular weights of the galactan
during acid hydrolysis. When the molecular weight was decreased to 3.5 kDa, the
polysaccharide fragment could not induce the secretion of NO, TNF-α and IL-6. We
speculated that the binding of the galactan to TLR2 was correlation with its spatial
structure. However, further studies are needed to substantiate this hypothesis.
5
. Conclusion
WCCP-N-b, the polysaccharide purified from the fruiting bodies of Cantharellus
cibarius, was a linear galactans composed of 67% of α-1,6-D-Galp and 22% of
α-1,6-D-Me-Galp. Immunomodulatory activity analysis showed that WCCP-N-b
significantly activated RAW264.7 macrophages through increasing phagocytic uptake
and inducing secretion of NO, TNF-α, IL-6 and IL-1β. On a cellular mechanistic level,
WCCP-N-b activated MAPK/NF-kB signaling pathways via TLR2 receptor. The
molecular weight of WCCP-N-b played a critical role on its macrophage activation
effect.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (No:
3
1500274, 31770852), the University S & T Innovation Platform of Jilin Province for
Economic Fungi (#2014B-1) and Natural Science Foundation of Changchun City of
China (17DY026).
28

Author Contributions
Y. Zhou conceived the study and revised the manuscript. G. Yang performed the
activities and mechanisms research of the polysaccharide and drafted the manuscript.
Y. Qu performed the polysaccharide extraction, isolation and purification. Y. Meng
and Y. Wang repeated the macrophage activation results. L. Sun and H. Cheng helped
to draft the manuscript. All authors have read and approved the final manuscript.
Conflict of interest
The authors declare that they have no conflicts of interest.
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