Zbornik Matice srpske za prirodne nauke / Proc. Nat. Sci, Matica Srpska Novi Sad,
¥ 116, 225—233, 2009
Maja S. Kozarski
Anita S. Klaus
Miomir P. Nikšiã
Department for Chemistry and Biochemistry
Department for Industrial Microbiology, Institute for Food Technology
and Biochemistry, University of Belgrade — Faculty of Agriculture,
Nemanjina 6, 11081 Belgrade, Serbia
INFLUENCE OF STRUCTURAL FEATURES
ON IMMUNOSTIMULATING ACTIVITY OF GLUCANS
EXTRACTED FROM AGARICUS BLAZEI MUSHROOM
ABSTRACT: High molecular weight b-D-glucans derived from Basidiomycetes cell
walls are able to specifically activate cellular and humoral components of the host immune
system. The aim of this paper was to examine immunomodulating activity of native,
chemically and enzimatically modified glucans from Agaricus blazei mushroom and to de-
termine which structural features are of primary importance for their stimulation referring to
humane immune cells. The immunomodulating activities were tested in vitro, by stimula-
tion of peripheral blood mononuclear cells (PBMCs) and measuring of interferon-gamma
(IFN-g) production by enzyme linked immunosorbent assay (ELISA). Measurements of
immunomodulatory capacity of Agaricus blazei native glucans showed their expressive im
munostimulating effect on activated PBMCs and synthesis of IFN-g. The results obtained
after the stimulation of cells with 1M H
and 1M NaOH, the treated glucans showed
that primary structure is of more importance than the tertiary structure of the triple helix for
their immunostimulating activity and synthesis of IFN-g. Glucans of lower molecular weight
obtained after acid hydrolysis appeared as effective immunostimulators of PBMC's. The re
sults obtained after the incubation of cells with 1,6 b-glucanase modified glucans suggest
that b-(1,6) binding of glucose monomers probably has no importance for the production of
imunostimulating effects, in vitro. This confirmed that b-(1,3) bonds are the primary deter
minants of immunomodulatory activities and stimulation of IFN-g synthesis.
KEY WORDS: Agaricus blazei, b-D-glucans, IFN-g, immunostimulating activity,
b-D-glucans are important secondary metabolites isolated from microor
ganisms, mushrooms and plants. They exhibit prophylactic and therapeutic
properties and can function as biological response modifiers when administe
red to mammals. They have the ability to enhance or suppress both innate and
acquired immune response. The major immunopotentiation effects of these
active substances include mitogenicity and stimulation of hematopoietic stem
cells, such as T
cells, B cells, macrophages, DC
, and NK cells (L u l l
et al., 2005.).
The term “b-glucan" refers to the polymers which are generally com
posed of a linear backbone containing D-glucopyranosyl repeat units which are
linked together by b-(1,3) and b-(1,4)-linkages. Some, but not all, exhibit
b-(1,6)-side chains on the backbone. Glucans can assume a number of solution
conformations depending upon the solvent system. For water soluble glucans,
the two predominant conformations are single helix or triple stranded right
-winding helix. In the fungal cell wall, most glucans comprise a three-dimen
sional network of b-(1,3-1,6)-glucans that are connected to the other carbo
hydrates, proteins and lipids (Y oung and Castranova, 2005).
The mechanism of the imunomodulating effect of glucans is not yet fully
understood and probably depends on chemical characterisics, such as molecu
lar weight, branching patterns, solubility in water and conformational features
like the formation of helix (F r e i m u n d et al., 2003).
In this study we tried to determine which structural features of water so
luble glucans are of primary importance for their in vitro immunostimulatory
properties. Two types of glucan structure modification were applied, a che
mical with 1M NaOH and 1M H
and enzymatic with 1,6 b -glucanase.
NaOH changes the conformation of glucans from triple helix to single strand
( M a e d a et al., 1988). Glucans of lower molecular weight were obtained
after acid hydrolysis with 1M H
(D i a et al., 2003) and modification with
MATERIAL AND METHODS
Glucans were extracted by hot water and alcohol precipitation from pow-
der of fruit body of Agaricus blazei mushroom. Purification of extract was
done by dialysis. The immunomodulating activity of native and modified glu
cans was tested in vitro, by PBMCs and measurement of IFN-g production
was done by ELISA. Changes of molecular weight, after incubation with 1M
NaOH, 1M H
and 1,6 b-glucanase, were observed by exclusion chromato
graphy using Sephacryl S 200 (K o z a r s k i, 2006.).
Extraction of water soluble glucan fraction
Up to 10% of dried powdered tissue was suspended in water. Glucans
extraction was done by autoclaving 2 x at 121°C for 20 minutes. The extract
was cooled down and centrifuged at 12325 x g for 20 minutes. Supernatants
were collected and boiled to 10% of starting volume. Two volumes of 96%
ethanol were added and left at 4°C overnight. Supernatant was decanted,
washed 1 x with 70% ethanol and centrifuged at 12325 x g. Pellet was dried
at 42°C. Purification was done by dialysis, against 2 l of destilled water, ob
tained by Millipore purification system (MilliQ) for 24 hours at room tem
50 mg/ml of glucans were incubated in 1M NaOH and 1M H
at 37°C for 16 hours. Neutralization was done with 10M H
NaOH to pH 6.8—7.2. Glucans in 1M phosphat buffer saline (PBS) were used
as control, under the same conditions.
25 mg/ml of glucans in 5mM sodium acetate (NaAc) buffer at pH 5.4
were incubated with 6 mg/ml of 1,6 b -glucanase, Onuzuka R-10 (Yakult Hon
sha Co Ltd., Japan) for 1 hour at 55°C. Reaction was stopped by heating at
90°C for 30 minutes followed by precipitation of glucans by addition of 2 vo
lumes of 96% ethanol.
Size exclusion chromatography was done on a 1.5 x 90 cm column of
Sephacryl S 200. 25 mg/ml of each glucan sample in MilliQ applied on
column. Eluation was done using fast performance liquid chromatography
(FPLC) system (Pharmacia) with degassed MilliQ water at flow rate of 0.5
ml/min. The eluents were collected by a fraction collector (Pharmacia), each 5
ml in a tube. The void volume was determined to be 60 ml in each fraction,
and glucan content was semiquantified by the phenol-sulfuric acid method
with glucose as a reference (D ubois et al., 1956). Protein content was de-
termined using the Bradford method with bovine serum albumun (BSA) as a
standard (B r a d f o r d et al., 1976) Glucan and protein contents were mea-
sured in 5 times concentrated fractions.
Human PBMCs were prepared from buffy coats, obtained from various
healthy donors. Buffy coats were diluted 1 x with PBS and centrifuged for 15
minutes at 2500 x g over a layer of Histopaque 1077 (Sigma). PBMCs were
carefully collected at the interphase and washed with PBS. Cells were counted
and resuspended at 5—10 x 10
/ml in RPMI-1640 containing 10% fetal calf
serum (FCS), 1% penicilin and 1% streptomycin.
Glucan solutions were heated before application at 95°C for 20 minutes.
Immunomodulating activity of the various glucans was tested by exposing sti
mulated PBMCs. As transcription activators, 1 ng/ml phorbol 12-myristate
13-acetate (PMA) and 0,5 µl/ml Ca-ionophore were added. After incubation
for 48 hours at 37°C in 5% CO
atmosphere, medium was tested for IFN-g
concentration by sandwich ELISA.
ELISA was performed in Nunc Maxisorp high affinity 96 well plates.
Wells were coated overnight at 4°C wih 50 µl of mouse anti-human IFN-g in
PBS, pH 7. Blocking of non specific binding sites was carried out overnight at
4°C with 0,1% BSA and 1% skimmed milk protein in PBS. Plate was incu
bated for 2 hours at room temperature with samples diluted in reagent diluent
(0.1% BSA, 0.05% Tween 20 in Tris buffered saline, pH 7.2—7.4, 0.2 µm fil
tered) and duplicates of standard, human IFN-g (BioSource) reconstituted with
50% glycerol, that was serially diluted in reagent diluent. The concentration of
high standard was 1000 pg/ml. The 100 µl biotinylated goat anti-human IFN-g
in reagent diluent was added to each well, followed by 1 hour incubation at
room temperature. Streptavidin-HRP (BioSource) in 100 µl of reagent diluent
was added to the wells and incubated for 20 minutes at room temperature.
After each step, the plate was washed with wash buffer (0.05% Tween 20 in
PBS, pH 7.2—7.4). Then 100 µl of substrate solution (1:1 mixture of H
and tetramethylbenzidine) was added to each well and incubated at room tem
perature. After 20 minutes, 50 µl of stop solution (1M H
) was added.
IFN-g production was measured at 415 nm using Benchmark microplate spec-
RESULTS AND DISCUSSION
Size exclusion chromatography of A. blazei native glucans on Sephacryl
S 200 showed the presence of one highest peak eluting together with the void
volume of the column, indicating high molecular weight, over 80 kDa, and a
few small peaks containing molecules of much lower size (Figure 1). In each
fraction, the presence of protein was confirmed. This suggested that glucans
can bound to protein or peptide residues and form proteoglucans. Running the
glucanase digested glucans on the column showed a high fractionation of the
glucan extract (Figure 1). This confirmed that glucans of A. blazei fruiting bo
dies predominantly had a b-(1,6)-backbone structure with b-(1,3)-side branches
(K o z a r s k i, 2006).
Measurements of immunomodulatory capacity of Agaricus blazei native
glucans showed that A. blazei glucans express immunostimulating effect on ac
tivated PBMCs and synthesis of IFN-g (Figure 2). Titers of IFN-g measured
after stimulation of cells with acid-hydrolyzed fractions confirmed that glucans
of lower molecular weight are as effective as non-hydrolyzed glucans. ELISA
measurements of IFN-g titer obtained after the stimulation of PBMCs with 1M
NaOH treated glucan showed that the immunostimulating activity was not
changed (Table 1, Figure 2). This indicated that the primary structure of glu
cans is of more importance than the tertiary structure of the triple helix for
their immunostimulating activity and synthesis of IFN-g.
Tab. 1 — ELISA measurements of IFN-g titer obtained after the stimulation of PBMCs with A.
blazei native, in 1 M PBS glucans (Ab) and 1M NaOH (AbB) and 1M H
(AbA). Cell suspension in RPMI, with transcription activators, was used as control.
sample IFN-g titer (pg/mL)
56.09 ± 4.11
219.26 ± 29.45
215.95 ± 23.72
198.31 ± 25.09
The resulting b-(1,3)-glucan fragments of high molecular weight (MW >
80 kDa) and small b-(1,3)-glucan fragments (MW < 80 kDa), left after gluca
nase degradation, showed a strong enhancement of immunostimulatory activity
compared to the native glucans (Figure 3).
Fig. 1 — Separation of native and modified A. blazei (graphs A, B, C, D) fruiting body
glucans on Sephacryl S 200. Glucans in PBS, A; with 1,6 b-glucanase modified glucans B;
with 1M H
treated, C; and with 1M NaOH, D.
Fig. 2 — IFN-g response of stimulated PBMC's incubated with native, in 1M PBS,
A. blazei glucans (Ab) and glucans which have been exposed to 1M NaOH (AbB)
and 1M H
Fig. 3 — IFN-g response, after 48 hours, of stimulated PBMC's incubated with native,
in 1M PBS, A. blazei glucans (Ab) and with 1,6 b-glucanase digested glucans (AbE).
Tab. 2 — ELISA measurements of IFN-g titer obtained after the stimulation of PBMCs with A.
blazei native, in 1 M PBS glucans (Ab) and with 1,6 b-glucanase treated glucans (AbE). Cell
suspension in RPMI, with transcription activators, was used as control.
sample IFN-g titer (pg/mL)
135.23 ± 9.30
219.26 ± 29.45
198.31 ± 25.09
The obtained results suggest that b-(1,6) binding of glucose monomers
probably has no importance for the production of imunostimulating effects, in
Measurements of immunomodulatory capacity of Agaricus blazei native
glucans showed that A. blazei glucans express immunostimulating effect on the
activated PBMCs and synthesis of IFN-g. The results obtained after the stimu
lation of cells with chemical and enzimatically modified glucans showed that
primary structure is of more importance than the tertiary structure of the triple
helix for their immunostimulating activity and synthesis of IFN-g. The ob-
tained results confirmed that b-(1,6) binding of glucose monomers probably
has no importance for the production of imunostimulating effects, in vitro.
This suggests that b-(1,3) bonds are the primary determinants of immuno-
modulatory activities and stimulation of IFN-g synthesis. The results confirmed
that glucans of lower molecular weight are effective for stimulation of PBMCs
and production of IFN-g.
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UTICAJ STRUKTURNIH KARAKTERISTIKA
NA IMUNOSTIMULATIVNU AKTIVNOST GLUKANA
EKSTRAHOVANIH IZ GQIVE AGARICUS BLAZEI
Maja S. Kozarski
, Anita S. Klaus
, Miomir P. Nikšiã
Katedra za hemiju i biohemiju
Katedra za Tehnološku mikrobiologiju, Institut za
prehrambenu tehnologiju i biohemiju, Univerzitet u Beogradu —
Poqoprivredni fakultet, Nemawina 6, 11081 Beograd, Srbija
b-D-glukani velikih molekulskih masa izolovani iz ãelijskog zida gqiva
iz klase Basidiomycetes imaju sposobnost da specifiåno aktiviraju celularne i
humoralne komponente imunog sistema domaãina. Imunomodulatorska aktivnost
miko-D-glukana u funkciji je wihovih hemijskih karakteristika, kao što su
molekulska masa, stepen granawa, rastvorqivost u vodi i tercijarna struktura.
Ciq ovog rada je bio ispitivawe imunomodulatorske aktivnosti nativnih, he-
mijski i enzimski modifikovanih glukana gqive Agaricus blazei i da se utvrdi
koja je strukturna karakteristika od primarnog znaåaja za stimulaciju ãelija hu
manog imunog sistema. Imunomodulatorska aktivnost je testirana in vitro, sti
mulacijom mononuklearnih ãelija krvi iz perifernog krvotoka (PBMC) mole
kulima glukana i merewem koliåine sintetisanog interferona-gama (IFN-g)od
strane stimulisanih ãelija enzimoimunotestom (ELISA). Merewem imunomodu
latorskog kapaciteta Agaricus blazei nativnih glukana pokazano je da ovi mole
kuli imaju izraÿeno imunostimulativno dejstvo na aktivirane PBMC ãelije i
stimulaciju sinteze IFN-g. Stimulacijom ãelija glukanima koji su prethodno
bili parcijalno hidrolizovani 1M H
i 1M NaOH izmereni titar IFN-g se
nije znaåajno promenio u odnosu na nativne molekule. Glukani mawih molekul
skih masa, nastali nakon kisele hidrolize, pokazali su se kao efikasni stimu
latori PBMC ãelija. Merewem titra IFN-g nastalog nakon inkubacije aktivi
ranih ãelija sa 1,6 b-glukanazama modifikovanim glukanima potvrðeno je da su
fragmenti b-(1,3)-glukana velikih molekulskih masa (MM > 80 kDa) i mali
fragmenti b-(1,3)-glukana (MM <80 kDa), nastali nakon enzimske modifikaci
je, ispoqili znaåajno poveãawe imunostimulativne aktivnosti u odnosu na na
tivne molekule. Dobijeni rezultati su ukazali da b-(1,6)-glikozidne veze nemaju
znaåaja u ispoqavawu imunostimulativnog efekta, in vitro.
Ovim je potvrðeno da je za imunostimulativnu aktivnost i stimulaciju
sinteze IFN-g od primarnog znaåaja prisustvo b-(1,3)-glikozidnih veza. Zakqu
åeno je da je za imunomodulatorsku aktivnost ovih molekula bitna primarna
struktura, a ne konformacija trostrukog heliksa nativnih molekula, kao i da su
molekuli glukana mawih molekulskih masa efikasni stimulatori sinteze IFN-g.