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Abstract

This study investigated the larvicidal activity on Culex quinquefasciatus of lectin purified from fresh fruiting bodies of woodland mushroom, Agaricus semotus. A. semotus lectin (ASL) was purified via ion-exchange chromatography on DEAE-cellulose A-25 and size exclusion chromatography on Sephadex G-100 matrix. Molecular weight (16.6 kDa) was estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The effects of temperature, pH, metal chelation- and larvicidal activity of ASL were also investigated. The ASL indifferently agglutinated the erythrocytes of the human ABO blood system and was stable at acidic pH and below 50 °C whereas 66% of its activity was lost at 60 °C with complete inactivation at 70 °C. ASL is a metalloprotein requiring barium ion as chelation of metals by 50 mM EDTA rendered the lectin inactive, while the addition of BaCl2, among other metal salts, restored the activity. ASL showed larvicidal activity against C. quinquefasciatus larvae after 24 h with a mortality of 5 and 95% at 5 and 25 mg/mL respectively, and LC50 of 13.80 mg/mL. This study concluded that purified A. semotus lectin showed impressive larvicidal activity, which could be exploited in its development as an insecticidal agent.
Introduction
Diseases transmitted by mosquitoes are one of the en-
demic health-related environmental menaces on the
rise worldwide, which contribute to a high loss of life,
especially in tropical countries with low income (Wilke
et al. 2017; Fonseca et al. 2019). Culex quinquefasciatus
Say, 1823 (Culicidae, southern house mosquito) is vastly
distributed in subtropical regions of the world (Lopes et
al. 2019). It is incursive, and belongs to the Culex pipiens
species complex, which feed on mammalian and avian
blood, C. quinquefasciatus is considered a medically sig-
nificant species, because of their implicated roles in the
transmission of zoonotic diseases including those caused
by Wuchereria bancrofti, West Nile virus, and arbovirus
(Fonseca et al. 2004; Lima-Camara et al. 2016).
Although majorly found in Africa, C. quinquefasciatus
usually spread beyond their domiciled environment,
posing a severe risk to public health (Farnesi et al. 2015;
Cuthbert et al. 2020). As part of control measures to curb
mosquito establishment and proliferation, the World
Health Organization (WHO) introduced a manual on
the management of these quintessential vectors (Takken
and van den Berg 2019). Due to the advent of mosquito
strains resistant to synthetic insecticides, together with
potential environmental and health threats associated
with these chemicals, alternative bio-control agents are
being sought (Bellinato et al. 2016; Camaroti et al. 2017;
Santos 2020).
The non-enzymatic lectins with unique sugar-binding
characteristics are ubiquitous proteins capable of revers-
ible discrete interaction with cell surface carbohydrate
associated structures and involved in diverse biological
and pathological functions (He et al. 2015; Muszyńska
et al. 2018; Nascimento et al. 2020; Perduca et al. 2020).
Aside from being a remarkable tool in blood group deter-
mination, lectins also perform defense-related functions
(Silva et al. 2020). Although originally identified in plants
and animals, exploration of fungal lectins is becoming
popular because of their peculiar carbohydrate specificity
coupled with potential application in biotechnology and
biomedicine (Singh et al. 2010; Varrot et al. 2013; Hassan
et al. 2015; Singh et al. 2015). Mushrooms have attracted
numerous research activities due their diverse inherent
bioactive constituents including lectins (Singh et al. 2010;
ARTICLE
A purifi ed lectin with larvicidal activity from a woodland
mushroom, Agaricus semotus Fr.
Isaiah O. Adedoyin1, Taiwo S. Adewole2, Titilayo O. Agunbiade3, Francis B. Adewoyin4,
Adenike Kuku1,*
1Department of Biochemistry and Molecular Biology, Obafemi Awolowo University, Ile-Ife, Osun State, Nigeria.
2Department of Chemical Sciences, Kings University, Ode-Omu, Osun State, Nigeria.
3Department of Chemical Sciences, Oduduwa University, Ipetumodu, Osun State, Nigeria.
4Drug Research and Production Unit, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Osun State, Nigeria.
This study investigated the larvicidal activity on Culex quinquefasciatus
of lectin puri ed from fresh fruiting bodies of woodland mushroom, Agaricus semotus.
A. semotus lectin (ASL) was puri ed via ion-exchange chromatography on DEAE-cellulose
A-25 and size exclusion chromatography on Sephadex G-100 matrix. Molecular weight
(16.6 kDa) was estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE). The eff ects of temperature, pH, metal chelation- and larvicidal activity of
ASL were also investigated. The ASL indiff erently agglutinated the erythrocytes of the
human ABO blood system and was stable at acidic pH and below 50 °C whereas 66% of
its activity was lost at 60 °C with complete inactivation at 70 °C. ASL is a metalloprotein
requiring barium ion as chelation of metals by 50 mM EDTA rendered the lectin inactive,
while the addition of BaCl2, among other metal salts, restored the activity. ASL showed
larvicidal activity against C. quinquefasciatus larvae after 24 h with a mortality of 5 and
95% at 5 and 25 mg/mL respectively, and LC
50
of 13.80 mg/mL. This study concluded that
puri ed A. semotus lectin showed impressive larvicidal activity, which could be exploited
in its development as an insecticidal agent. Acta Biol Szeged 65(1):65-73 (2021)
ABSTRACT
Agaricus semotus
Culex quinquefasciatus
larvicidal activity
lectin
KEY WORDS
Volume 65(1):65-73, 2021
Acta Biologica Szegediensis
http://abs.bibl.u-szeged.hu/index.php/abs
Submitted
21 December 2020.
Accepted
17 February 2021.
*Corresponding author
E-mail: adenikekuku@yahoo.com;
akuku@oauife.edu.ng
cta
iologica
zegediensis
DOI:10.14232/abs.2021.1.65-73
65
ARTICLE INFORMATION
Largeteau et al. 2011).
Worldwide, Agaricus genus has over 300 members,
which are distinguished by their unique spore coloration,
among other structural features (Zhang et al. 2019). In
this study a lectin was purified from the fresh fruiting
bodies of woodland mushroom and its larvicidal activity
was investigated towards C. quinquefasciatus to explore its
potential in insect control.
Materials and Methods
Materials and chemicals
Glutaraldehyde, Folin reagent, ethylenediaminetetraacetic
acid (EDTA), acrylamide, bovine serum albumin, sodium
dodecyl sulfate (SDS), sugars, Coomassie Brilliant Blue R
250 and molecular weight protein markers (10-170 kDa)
were purchased from Sigma-Aldrich (St Louis, Missouri,
USA). Purchase of Sephadex G-100 and DEAE cellulose
A-25 was from Pharmacia Fine Chemicals (Uppsala,
Sweden). The quality of all reagents was research-quality.
A, B, O human blood groups were collected with
informed consent from healthy subjects.
Collection of mushroom
Woodland mushroom (A. semotus Fries) was collected on
farmland near the Department of Electrical and Electron-
ics Engineering, Obafemi Awolowo University, Ile-Ife
(South-West Nigeria). Identification was done at the
Department of Microbiology (Mycology Laboratory),
Obafemi Awolowo University, Ile-Ife, Nigeria.
A. semotus crude extract preparation
Approximately 16 g mushroom sample was homogenized
and extracted using 0.025 M Phosphate Buffered Saline,
pH 7.2 (1:5 w/v) and was stirred at 4 °C overnight. Re-
sulting mixture was centrifuged at 4000 rpm for 15 min
and filtered through cheesecloth to get the crude extract,
which was stored at -20 °C until use.
Protein concentration assay
Assay method of Lowry et al. (1951) was employed in
estimating protein concentration. Standard protein used
was bovine serum albumin (1 mg/mL).
Hemagglutination and sugar specicity assays
Human A, B, and O blood groups were collected into
lithium-heparin bottles. Erythrocytes were recovered
from the centrifuged blood samples and fixed using
glutaraldehyde (Bing et al. 1967). Hemagglutination assay
and sugar specificity test was performed as described by
Kuku and Eretan (2004) in a U-shaped 96-well microtiter
plate.
Tested
sugars were D-mannitol, fructose, L-sorbose,
galactose, D-mannose, D-glucosamine hydrochloride,
N-acetyl-D-glucosamine, inulin, maltose, glycogen, D-
lactose monohydrate, D-glucose monohydrate, and starch.
Purication of A. semotus lectin (ASL)
Ion exchange chromatography on DEAE cellulose A-25
Chromatography column (1.5 x 20 cm, Bio-Rad) packed
with DEAE- cellulose A-25 matrix, and equilibrated with
10 mM Tris-HCl buffer, pH 7.3, was used to purify the
crude protein extract (3 mL, 33 mg protein). Fractions
(2 mL) were collected at 24 mL/h flow rate. Unadsorbed
fractions were eluted with the equilibration buffer (10
mM Tris-HCl buffer, pH 7.3), while adsorbed fractions
eluted with the same buffer containing 0.2 M NaCl. Frac-
tions were monitored at 280 nm, and hemagglutinating
activities were assayed. Active peak fractions (unadsorbed
peak) were pooled, dialyzed (against 10 mM Tris-HCl
buffer, pH 7.3, and distilled water) for 48 h, freeze-dried,
and kept at -20 °C.
Size exclusion chromatography on Sephadex G-100
Five milliliters ( 30 mg protein) of the pooled DEAE ac-
tive fractions (unadsorbed peak) was loaded on Sephadex
G-100 column (2.5 x 20 cm, Bio-Rad) equilibrated with
phosphate-buffered saline (0.02 M, pH 7.2), and fractions
(4 mL) collected at 30 mL/h flow rate. Fractions were
monitored at 280 nm, and hemagglutinating activity
assayed. Active peak fractions were collected, dialyzed,
freeze-dried, and kept frozen at -20 °C.
Physicochemical characterization of ASL
Denaturing gel electrophoresis (12.5% acrylamide gel,
Tris-HCl system) was employed in the determination of
ASL subunit molecular weight using standard marker
proteins of 10-170 kDa range, as described by Laemmli
Fractions Total protein (mg) Total activity (HU) Specic activity (HU/mg) Yield (%) Purication fold
Crude extract 5550 1024 0.184 100.0 1.00
Ion exchange chromatography (IEC-1) 585.90 512 0.873 50.0 4.74
Size exclusion chromatography (SEC-2) 101.8 128 1.257 12.5 6.83
Table 1. Purication summary.
Adedoyin et al.
66
and Favre (1970).
Assay method of Sampaio et al. (1998) was employed
in investigating temperature effect on ASL hemaggluti-
nating activity. Aliquot of ASL (500 µL) was subjected
to temperature variation (30-90 °C) for 60 min using a
water bath (Model DK-420, Movel Scientific Instruments,
Zhejiang, China). Incubated ASL was swiftly iced-cooled
and activity was assayed. Activity of ASL was served as
control at 25 °C.
The pH effect on ASL hemagglutinating activity was
investigated by incubating lectin aliquots (500 µL) at 25
°C for 60 min with buffer solutions of pH 2-11 range
(0.2 M citrate, pH 2-6; 0.2 M Tris-HCl, pH 7-8, and 0.2
M glycine-NaOH, pH 9-11). Control was ASL incubated
in PBS, pH 7.2 and results presented as described by
Nakagawa et al. (1996).
To investigate the divalent cation requirement of ASL,
assay experiment of Wang et al. (1996) was employed. Ali-
quot of ASL was dialyzed for 24 h against 50 mM EDTA
and hemagglutinating activity assayed. Thereafter, the
demetallized lectin aliquots were incubated differently
with BaCl2, HgCl2, MgCl2, SnCl2, and CaCl2 (10 mM, 50
µL) for 2 h, and each time, assayed for hemagglutinating
activity.
Larvicidal assay
C. quinquefasciatus larvae were cultured in an insectarium
under regulated conditions of relative humidity 70 ± 10%
and at 27 ± 2 °C with a 12-12 light-dark regime at the
Drug Research and Production Unit (DRPU), Faculty of
Pharmacy, Obafemi Awolowo University, Ile-Ife. Free
adult mosquitoes were allowed to lay eggs in the habitats
provided for them in the laboratory and egg rafts collected
for hatching in glass and plastic bowls. To promote larva
development and female fecundity, hatched mosquito
larvae were fed daily with fish food ad libitum.
Larvicidal activity of ASL was investigated as de-
scribed in our previous study ( Johnny et al. 2016). Labo-
ratory–reared fourth instar larvae of C. quinquefasciatus
were exposed to varying concentrations (5-25 mg/mL)
of ASL according to standard procedure (WHO 1981).
Comprehensively, 20 larvae were introduced into plastic
beakers (50 mL) for each concentration of ASL. Experi-
mentations were done in triplicates at 27 ± 2 °C, with one
control (distilled H2O) set for each. Motionless larvae or
those which settled at the bottom of test beakers with no
sensitivity to involuntary stimulus or light, or after mild
probing failed to move were considered dead. Percentage
mortalities were recorded for each concentration after 24
h of the exposure period and obtained data statistically
analyzed.
Statistical analysis
Experimental data were analyzed using StatPlus® 2006
(AnalystSoft, Canada) to find the lethal concentrations
(LC
50
) of larvae in 24 h by probit analysis with a reliability
interval of 95% (Finney 1971; Islam et al. 2013).
Results
Puri cation, hemagglutinating activity, and sugar speci-
city of Agaricus semotus lectin (ASL)
Ion exchange and size exclusion chromatography puri-
fied ASL from A. semotus to homogeneity with yield and
purification fold of 12.5% and 6.83, respectively (Table 1).
Two distinct peaks were obtained from the elution
profile of A. semotus crude extract on DEAE cellulose
A-25 column (Fig. 1), of which only the unadsorbed peak
(IEC-1) exhibited hemagglutinating activity. Size exclu-
sion chromatography of the DEAE-active peak (IEC-1)
on Sephadex G-100 matrix also resulted in two protein
peaks (Fig. 2), but only second peak (SEC-2) showed hem-
agglutinating activity, which constituted the homogeneous
ASL used for further studies.
Hemagglutination assay showed that ASL indifferently
agglutinated the erythrocytes of the human ABO blood
group, and this activity was strongly inhibited by inulin
among tested sugars (Table 2).
Physicochemical characterization of ASL
SDS-PAGE analysis of ASL revealed a distinct band with
a relative molecular weight of 16.6 kDa (Fig. 3). ASL lost
its activity at 70 °C (Fig. 4) and stable at acidic pH (Fig. 5).
Larvicidal study
The ASL treatment resulted the concentration-dependent
mortality of C. quinquefasciatus larvae with the lowest
mortality of 5% at 5 mg/mL and the highest mortality of
Figure 1. Ion exchange chromatography of A. semotus crude extract.
Lectin with larvicidal activity from Agaricus semotus
67
95% at 25 mg/mL concentration after 24 h (Table 3) with
LC50 of 13.767 mg/mL (Fig. 6).
Discussion
Puri cation, hemagglutinating activity and sugar speci-
city of Agaricus semotus lectin (ASL)
Diverse chromatographic techniques are being used in the
purification of mushroom lectins, however, recent studies
have reported the effectiveness of ion-exchange and size
exclusion chromatographic techniques as an alternative
to highly preferred single-step affinity chromatography,
which exploits sugar specificity of the lectin (Chandrasek-
aran et al. 2016; Wang et al. 2018; Panchak 2019). Elution
profile of ASL on DEAE cellulose A-25 matrix reported in
this study was similar to the chromatographic behavior
ofA. arvensislectin (AAL) (Zhao et al. 2011).
A. semotuslectin (ASL) exhibited a non-specific ag-
glutination towards the human ABO erythrocytes,
characteristics mainly associated with an unique group
of promiscuous lectins (panalectins), which have been
identified mainly in plants, but rarely in mushrooms
(Kuku et al. 2000; Akinyoola et al. 2016; Oladokun et
al. 2019). However, lectins have been already identified
and isolated within the genusAgaricus, there is not any
previous report onthe lectins of A. semotus (Kawagishi
et al. 1988; Mikiashvili et al. 2006; Nakamura-Tsuruta
et al. 2006; Zhang et al. 2019).
The hapten inhibition test to determine the sugar
specificity of ASL showed that D-mannose, L-sorbose,
galactose, and fructose slightly decreased the hemaggluti-
nating activity, while inulin (a fructan) strongly inhibited
the activity, although starch enhanced activity among the
sugars tested (Table 2). Inhibition of the hemagglutinating
activity of ASL by more than one simple and/or complex
sugar reported in this study is not uncommon among
mushroom lectins, especially within the genusAgaricus,
as similar characteristics have been reported for lec-
Figure 2. Size exclusion chromatography of the DEAE-Unadsorbed
peak (IEC-1).
Sugar Hemagglutination titre
Control 29
Fructose 27
L-Sorbose 27
Galactose 27
D- Mannose 27
D- Mannitol 29
D- Glucosamine-hydrochloride 29
D- Glucose monohydrate 28
N-acetyl-D-glucosamine 28
Inulin 22
Maltose 28
Glycogen 28
D-Lactose monohydrate 29
Starch 212
Table 2. Hapten inhibition test of ASL puri ed from A. semotus.
Experiments comprised ASL (100 µL) diluted serially in a 96-well microtitre
plate. Equal volumes (50 µL) of respective sugar solutions (0.2 M) and 2%
human blood group A erythrocytes suspension were introduced to the wells.
Positive control contained no sugars, and negative control contained neither
ASL nor sugars. Experiments were done in triplicates.
S/N Concentration (mg/mL) Number of larvae Recorded death Mortality %
1Control (distilled water) 20.00 0.00 ± 0.00 0.00 ± 0.00
25.00 20.00 1.00 ± 0.05 5.00 ± 0.12
3 10.00 20.00 5.00 ± 0.25 25.00 ± 0.31
415.00 20.00 8.00 ± 0.40 40.00 ± 0.11
520.00 20.00 15.00 ± 0.75 75.00 ± 0.25
6 25.00 20.00 19.00 ± 0.95 95.00 ± 0.11
Table 3. Larvicidal activity of ASL on C. quinquefasciatus.
Experiments were done in triplicates. Data were presented as mean ± SEM (standard error of mean).
Adedoyin et al.
68
tins isolated fromA. blazei,A. bisporus,andA. pilatianus
(Kawagishi et al. 1988; Nakamura-Tsuruta et al. 2006;
Mikiashvili et al. 2006).
Inulin specificity obtained for ASL is similar to that
ofA. arvensislectin (Zhao et al. 2011). Zhanget al.(2019)
also reported inulin inhibition ofA.bitorquislectin sug-
gesting a conserved inulin-binding specificity regarding
isolated lectins from the genusAgaricus, which might be
exploited in a structure-function relationship for prac-
ticable biotechnological application, especially through
molecular docking. Other mushroom lectins reportedly
inhibited by inulin includeArmillaria luteo-virenslec-
tin,Pholiota adiposelectin, andHericium erinaceumlectin
(Feng et al. 2006; Zhang et al. 2009; Li et al. 2010).
Physicochemical characterization of ASL
Fungal lectins, especially those isolated from mushrooms
exhibit profound variation in their physicochemical
properties (Hassan et al. 2015; Singh et al. 2020). The
molecular weight of ASL (16.6 kDa) is similar to the
weight reported forA. blazeilectin (16 kDa) (Kawagishi
et al. 1988). ASL maintained 100% activity up to 50 ºC,
however, lost 66% of the activity at 60 ºC and completely
inactivated at 70 ºC (Figure 4). ASL is moderately thermo-
stable, as mushroom lectins are reportedly usually stable
at a moderate temperature with gradual loss of activity
as the temperature increases (Alborés 2014; Wang et al.
Figure 3. Electrophoretogram of SDS-PAGE of A. semotus lectin (ASL)
and molecular weight markers (10-170 kDa). Lane A (Standard marker
proteins), lane B (puri ed ASL).
Figure 4. Eff ect of temperature on the hemagglutinating activity of ASL.
Figure 5. Eff ect of pH on the hemagglutinating activity of ASL.
Figure 6. Probit graph for determination of LC50.
Lectin with larvicidal activity from Agaricus semotus
69
2019). However, this result contradicts with the study
of Zhaoet al.(2011), who reportedA. arvensislectin was
thermostable up to 90 °C. Several reports have shown that
the thermostability of lectins differs, as lectins usually
undergo unprecedented conformational changes under
harsh temperatures, which might culminate in inactiva-
tion mostly owing to the destabilization of interactions
necessary for their native conformation (Wang et al.
1996; Singh and Saxena 2013). ASL was stable at acidic
pH with maximum activity within pH 2–5, with a 41%
loss of activity within pH 7-9 and 50% loss at pH 11 (Fig.
5). Stability of ASL at acidic pH shown was similar to
that reported forGymnopilus spectabilis lectin (Alborés
et al. 2014).
ASL was completely inactivated by 50 mM EDTA,
however, when different metal salts were added to the
assay medium, BaCl2 restored 33% of the lectin activity
suggesting ASL might be a metalloprotein. The result
obtained for ASL contradicts the report on A. arven-
sislectin (AAL), which was unaffected by divalent ions
and enhanced by trivalent ions (Zhao et al. 2011).
Larvicidal study
Chemical method of insect control is one of the major
mechanisms of mitigating diseases propagated by these
organisms with long-term effects linked with insect resis-
tance, together with detrimental effects on the ecosystem,
and human health, paving way for exploration of safer and
eco-friendly natural alternatives such as lectins (Gautam
et al. 2013; Shaurub et al. 2015; Demok et al. 2019; Satoto
et al. 2019; Kumar et al. 2020).
As a distinctive component of the innate defense system
of their host, mushroom lectins have a unique ability to
recognize diverse carbohydrate-associated structures that
mediate most of their varying biological activities (Singh
and Saxena 2013; Sabotič et al. 2016). Previous studies on
entomotoxic lectins have suggested the fatality induced
by these proteins usually involves multiple complemen-
tary mechanisms including induction of apoptosis and
interaction with specific carbohydrates/glycan structures
of vital enzymes, especially those engaged in metabolism
and detoxification (de Oliveira et al. 2016; Napoleão et
al. 2018). In fact, because of their multivalent assembly,
fungal lectins can cross-link cell surface glycoconju-
gates or trigger distinct oligomerization of glycosylated
signaling receptors, which may affect their turnover
(Hamshou et al. 2012). Morphological damages of the gut
initiated by excessive proteolysis due to specific binding
to peritrophic matrixes or epithelial cells of target insects
have also been connected to their insecticidal/larvicidal
activity (Coelho et al. 2009). As reported by Coelho and
co-workers,Moringa oleiferalectin increased lumen volume
and disrupted the midgut epithelium ofAedes aegyptilarvae
and peritrophic membrane ofAnagasta kuehniellalarvae,
ultimately resulting in gut cell death (Coelho et al. 2009;
de Oliviera et al. 2017).
Entomotoxic lectins have also been reported to alter
critical target insects’ biological functions such as pupa-
tion, survival, larval development, and adult emergence
(Kaur et al. 2009).Dioscorea batataslectin was reported
to alter the development ofHelicoverpa armigeralarvae by
interacting strongly with vital intracellular structures
(Ohizumi et al. 2009).
The entomotoxic lectins vary in physicochemical
characteristics, and there are many potential targets be-
cause of their multivalent highly stereospecific binding
with diverse arrays of insect glycan structures, however,
differences in their spatial arrangement together with
the proportion of carbohydrate recognition domain may
expound the disparity in toxicity mechanisms elicited
by these quintessential bioactive proteins (Fitches et al.
2012; Yang et al. 2014; Rani et al. 2017; Singh et al. 2020).
Conclusion
This study concluded thata lectin purified from A. semotus
showed potent larvicidal activity againstC. quinquefas-
ciatus,which suggests its application as an alternative
and eco-friendly larvicide/insecticide in the control of
mosquitoes to mitigate mosquitoes-borne diseases and
mortalities. However, a better comprehension of the
action mechanism used by the lectin might help further
research to investigate the possibilities of biotechnological
applications of mushroom lectins in agriculture against
phytophagous insects.
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