An Inulin-Specific Lectin with Anti-HIV-1 Reverse ...downloads.hindawi.com/journals/bmri/2019/1341370.pdf · ResearchArticle An Inulin-Specific Lectin with Anti-HIV-1 Reverse Transcriptase,

Research ArticleAn Inulin-Specific Lectin with Anti-HIV-1 ReverseTranscriptase Antiproliferative and Mitogenic Activities fromthe Edible Mushroom Agaricus bitorquis

Guo-Qing Zhang 1 Qing-Jun Chen2 Jing Hua1 Zi-Lu Liu1 Yue Sun1 Xin Xu1

Peng Han1 and He-XiangWang 3

1Key Laboratory of Urban Agriculture (North China) of Ministry of Agriculture College of Biological Science and EngineeringBeijing University of Agriculture Beijing 102206 China2Beijing Key Laboratory for Agricultural Application and New Technique College of Plant Science and TechnologyBeijing University of Agriculture Beijing 102206 China3State Key Laboratory for Agrobiotechnology and Department of Microbiology China Agricultural University Beijing 100193 China

Correspondence should be addressed to He-Xiang Wang hxwangcaueducn

Received 20 December 2018 Accepted 27 February 2019 Published 19 March 2019

Academic Editor Gail B Mahady

Copyright copy 2019 Guo-Qing Zhang et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

A novel lectin (ABL) was purified from the dried fruiting bodies of Agaricus bitorquis An efficient 3-step purification protocolinvolved two consecutive steps of ion exchange chromatography on Q-Sepharose and SP-Sepharose and gel filtration by FPLC onSuperdex 75 ABL is a monomeric protein with the molecular mass of 276 kDa which is different from other lectins from genusAgaricus Its N-terminal amino acid sequence is EYTISIRVYQTNPKGFNRPV which is unique and sharing considerably highsimilarity of othermushroom lectinsThehemagglutinating activity of the lectinwas inhibited by inulin Based on hemagglutinationtests ABL prefers rabbit human type A and AB erythrocytes to human type B and O erythrocytes The lectin inhibits the activityof HIV-1 reverse transcriptase and the proliferation of leukemia cell (L1210) with an IC

50value of 469 and 497 120583M respectively

Furthermore ABL demonstrates the highest mitogenic activity with a response of 241777 plusmn 9406 [3H-methyl] thymidine countsper minute (CPM) at a concentration of 091 120583M

1 Introduction

Lectins are carbohydrate-binding proteins of nonimmuneorigin that specifically recognize and bind to carbohydratemoieties of glycoproteins or glycolipids They play crucialrole in various biological and pathological processes suchas cellular signaling cell-cell and host-pathogen interactionsdifferentiation innate immune responses serum glycopro-tein turnover tissue metastasis etc [1 2] Lectins are widelydistributed in plants fungi animals bacteria and virusesAmong all the sources plant lectins have been most exten-sively investigated including classification structure andfunctions They act as the defensive system against animalsand phytopathogenic microorganisms and also mediate theinteractions of plants and their mycorrhizal fungi [3 4]

Remarkably numerous fungal lectins have been reportedsince their great therapeutic potentials as antitumor antipro-liferative antivirus immunomodulatory activities etc [5]Mushrooms have been recognized for their nutritious andmedicinal properties for thousands of years More than 80of reported fungal lectins come from mushroom speciesand play crucial roles in dormancy growth morphogenesismorphological changes and molecular recognition duringtheir life cycles [1 6] Nowadays mushroom lectins haveattracted considerable attention due to their treatment orprevention of cancer viral diseases atherosclerosis hyper-tension hypercholesterolemia etc [4 7]

Agaricus is a genus of mushrooms from phylum Basid-iomycota class Agaricomycetes order Agaricales and familyAgaricaceae with possibly over 300 members worldwide

HindawiBioMed Research InternationalVolume 2019 Article ID 1341370 9 pageshttpsdoiorg10115520191341370

2 BioMed Research International

Members of the genus Agaricus are characterized by havinga fleshy cap and stipe and chocolate-brown spores Somespecies are commercially manufactured including A bis-porus A blazei A bitorquis etc In recent years lectinsfrom Agaricus were widely reported including purificationproperties and therapeutic applications A bisporus lectinscan inhibit the growth of colon cancer cell breast cancer celland human retinal pigment epithelial cells [8]A blazei lectinclaimed to exert an in vitro antitumor activity in leukemiccells with no significant effects on normal lymphatic cells [9]

A bitorquis is an edible mushroom species and can becommercially produced similar to the commonbuttonmush-room A bisporus It is an important supplement cultivationspecies of the button mushroom production in China Uptill now only hemagglutination activity of A bitorquis fromAustralia was reported [10] In the present study we aimedto purify a novel lectin from the artificially cultivated Abisporus and find out its properties and potential therapeuticapplications

2 Materials and Methods

21 Assay for Hemagglutinating Activity Hemagglutinatingactivity was determined in the 96-well microtiter plates witha final volume of 50 120583l A serial twofold dilution of the lectinsolution (25 120583l) was mixed with a 2 rabbit erythrocytesuspension (25 120583l) in phosphate-buffered saline (pH 72) andincubated at room temperature for 1 hThe hemagglutinationtiter one hemagglutination unit was defined as the reciprocalof the highest dilution exhibiting hemagglutination Specificactivity is the number of hemagglutination unitsmg protein[11] The hemagglutination profiles of the lectin towardserythrocytes from other origins were similarly determinedHuman blood (A B O and AB types) was donated by healthyvolunteers and the blood typewas confirmed by clinical tests

22 Purification of Lectin The mushroom A bitorquis wasartificially cultivated in woodland of Beijing mountain areasusing a commercial strain from Hebei Province in Chinaand followed the cultivation method of Chen et al described[14] Dried fruiting bodies (100 g) were homogenized andextracted in 015MNaCl (1 L) at 4∘C overnight Subsequentlythe homogenate was centrifuged at 9500 rpm for 20 min at4∘C The supernatant was collected and ammonium sulphatewas added to the supernatant to 80 saturation Themixturewas left at 4∘C for 8 hours before another centrifugationat 9500 rpm for 20 min at 4∘C Then the precipitate wasdissolved and dialyzed to remove ammonium sulphate beforeapplying to a Q- Sepharose (GE Healthcare USA) column(25times20 cm) which had previously been equilibrated withand was then eluted with 10 mM Tris-HCl buffer (pH 76)After removal of the unadsorbed fraction (Q1) containingslight hemagglutinating activity two adsorbed fractions (Q2and D3) were eluted with 200 mM NaCl and 1000 mMNaCl in the starting buffer respectively Fractions Q2 demon-strated strong hemagglutinating activity and were dialyzedfor further purification on cation exchange chromatographyof SP-Sepharose (GE Healthcare USA) column (25times20 cm)

with 10 mM NH4OAc buffer (pH 46) After removal of an

unadsorbed fraction (SP1) two adsorbed fractions (SP2 andSP3) were eluted by using a linear concentration gradient of0-1000 mM NaCl in the same buffer (pH 46) Lectin activefraction SP3 was finally applied to to gel filtration by fastprotein liquid chromatography (FPLC GE Healthcare USA)on a Superdex 75 gel filtration column (02 M NH

4HCO3

buffer pH 94) using an AKTA Purifier (GE HealthcareUSA) The second fraction (SU2) was the purified lectin

23 Determination of Molecular Mass Molecular mass (Mr)of the purified lectin was determined based on FPLC-gelfiltration and sodium dodecyl sulphate-polyacrylamide gelelectrophoresis (SDS-PAGE) During the FPLC chromatog-raphy Mr of natural proteins were calculated using thestandard curve of Log Mr versus elution volume made bymolecular mass standards (GEHealthcare USA) SDS-PAGEwas performed using the standard procedure with a 12resolving gel and a 5 stacking gelMr of denatured proteinswas obtained using another standard curve of LogMr versusrelative mobilities of molecular mass standards (GenviewUSA)Mr of the present purified laccase was evaluated basedon the two curves [13]

24 N-Terminal Amino Acid Sequencing After SDS-PAGEthe purified enzymeon the gel was transferred to a polyvinyli-dene difluoride (PVDF Bio-RadUSA)membrane by electro-blotting and stained with CBB R-250 The stained band wasthen excised and analyzed by the automated Edman degrada-tion method using an HP G1000A Edman degradation unit(Hewlett Packard Company USA) and an HP1000 HPLCsystem (Hewlett Packard Company USA) [11]

25 Assay of Hemagglutinating Inhibition by CarbohydratesThe hemagglutinating inhibition tests to investigate inhibi-tion of lectin induced hemagglutination by various carbo-hydrates [11] Serial twofold dilutions of sugar samples (200mM to 156mM)were prepared in phosphate-buffered salineAll of the dilutions were mixed with an equal volume (25120583L) of the purified lectin solution with 16 hemagglutinationunits The mixture was allowed to stand for 60 minutes at4∘C and then mixed with 50 120583L of a 2 rabbit erythrocytesuspension The assay for lectin activity was conducted asdescribed above The minimum concentration of the sugarin the final reaction mixture which completely inhibited16 hemagglutination units of the lectin preparation wascalculated The carbohydrates tested included D-fructose D-galactose D-maltose D-mannose D-melibiose D-xylose L-rhamnose L-sorbose cellobiose inulin and raffinose

26 Assay of HIV-1 Reverse Transcriptase (HIV-1 RT)Inhibitory Activity Inhibitory activity towards humanimmunodeficiency virus type 1 (HIV-1) reverse transcriptase(RT) of the purified lectin was assayed using an enzyme-linked immunosorbent assay (ELISA) kit (BoehringerMannheim Germany) by determining the ability of HIV-1RT to synthesize DNA [13] A fixed amount (4-6 ng) ofrecombinant HIV-1 RT was used The absorbance of the

BioMed Research International 3

Table 1 Yields and hemagglutinating activities of various chromatographic fractions derived from 100g dried fruiting bodies

Purification step Yield (mg) Total activity(U)

Specific activity(Umg)

Recovery ofactivity () Purification fold

Crude extract 5230 39 times 106 745 1000 10Q-Sepharose (Q2) 814 26 times 106 3218 672 43SP-Sepharose (SP3) 57 51 times 105 9026 132 121FPLC (SU2) 29 35 times 105 12064 90 162

samples at 405 nm can bemeasured with an ELISAmicrotiterplate reader and was directly correlated to the level of RTactivity The inhibitory activity of the lectin was calculatedas percentage inhibition compared with the control withoutany protein added Lectin form the mushroom H erinaceumwas used as a positive control [15] All treatments wereperformed in triplicate

27 Assay of Antiproliferative Activity towards Tumor CellLines Antiproliferative activity of the purified lectin wasdetermined using the MTT (3-[45-dimethylthiazol-2-yl]-25-diphenyltetrazolium bromide) method towards thehuman liver cancer cell line Hep G2 and mouse lymphocyticleukemia cell line L1210 (AmericanTissueCulture CollectionUSA) [11] The cell lines (2times104 cells100 120583l) were seededinto 96-well culture plates in RPMI 1640 culture mediumsupplemented with 10 FBS 100 mgL streptomycin and100 IUml penicillin in a humidified atmosphere of 5 CO

2

at 37∘C for 24 h Serials concentrations of the purified lectin(100 120583l per well) were added before further incubation for48 h Subsequently the medium was removed and the platewas washed with phosphate-buffered saline (PBS) MTTsolution (100 120583l per well) was then added followed by furtherincubation for 1 h under the same conditions Absorbanceat 540 nm was measured after incubation with dimethylsulfoxide (100 120583l per well) for 30 min Reagent and controlwere included with the absence of cells or respectively Alltreatments were performed in triplicate

Viability () of tumor cells = [OD540 nm (sample)OD540 nm(control)] times 100

28 Assay of Mitogenic Activity In this assay four BALBCmice (20-25 g in body weight) were purchased from BeijingVital River Experimental Animal Technical Co Ltd (China)and sacrificed by cervical dislocation Spleens were removedaseptically and splenocytes were collected using a sterilizedstainless steel sieve (100-mesh) The obtained splenocyteswere subsequently resuspended to 5times106 cellsml in RPMI1640 culture medium supplemented with 10 fetal bovineserum (FBS) 100 mgL streptomycin and 100 IUml peni-cillin The splenocytes were seeded into a 96-well cultureplate (Nunc Denmark) with the final concentration of 7times105cells100 120583lwell Serials concentrations of the purified lectin(625-200 120583gml) in 100 120583l medium were added followed byincubation at 37∘C in a humidified atmosphere of 5 CO

2

for 24 h Each well was then pulsed with 10 120583l [methyl-3H]-thymidine (025 120583Ci Amersham Biosciences Sweden)and incubated for another 6 h under the same conditions

Finally the splenocyteswere harvestedwith an automated cellharvester onto a glass fiber filterThe radioactivity of eachwellwasmeasuredwith a Beckmanmodel LS 6000SC scintillationcounter Con A was used as a positive control because ofits high potency [15] All treatments were performed intriplicate

3 Results

31 Lectin Purification The lectin from fruiting bodies ofthe edible mushroom A bisporus (abbreviated as ABL) waspurified following an isolation protocol that entailed twoconsecutive steps of ion exchange chromatography of Q-Sepharose and SP-Sepharose and a final gel filtration stepof FPLC The results of lectin purification at different stepswere summarized in Table 1 The purification factor andspecific activity of the purified lectin ABL was increased43- 121- and 162-fold respectively after Q-Sepharose SP-Sepharose and FPLC ABL possessed a hemagglutinatingactivity towards rabbit erythrocytes of 12064 Umg and a90 recovery of activity

32 Properties of the Purified Lectin ABL ABL demonstrateda molecular mass (Mr) of 276 kDa when it was chro-matographed on FPLC (Figure 1(a)) and exhibited one bandwith the sameMr in SDS-PAGE (Figure 1(b)) The first 20 N-terminal amino acids of ABL were sequenced to be EYTISIRVYQTNPKGFNRPV A comparison of N-terminal aminoacid sequence of ABL with that of other fungal lectins wasshown in Table 2 Among the variety of sugars tested inulin(with the assay concentration up to 625 mM) was able toinhibit the hemagglutinating activity of the purified lectin(Table 3) The purified lectin can agglutinate both rabbitand human erythrocytes with different capacity (Table 4)ABL preferred rabbit human type A and AB erythrocytes tohuman type B and O erythrocytes ABL possessed significantHIV reverse transcriptase (RT) inhibitory activity with anIC50value of 469 120583M (1294 120583gml) (Figure 2) and inhibited

the proliferation ofmouse lymphocytic leukemia cells (L1210)with an IC

50value of 497 120583M (1372 120583gml) (Figure 3)

However the inhibitory activity towards the proliferationof human HepG2 cells was very weak Furthermore ABLdemonstrated the highest mitogenic activity with a responseof 241777 plusmn 9406 [3H-methyl] thymidine counts per minute(CPM) at a concentration of 091 120583M (25 120583gml) while ConA induced maximal mitogenic response with a response of271440 plusmn 11938 CPM at a concentration of 012 120583M (125120583gml) (Figure 4)


Table2Com

paris

onof

theN

-term

inalsequ

ence

ofABL

with

otherfun

gallectin

s

Species

N-te

rminalsequ

ence

Max

ident

Accessionnu

mber

Ref

Agaricu

sbito

rquis

1EY

TISIRVYQ

TNPK

GFN

RPV

20100

Presentstudy

Agaricu

sbisp

orus

2TY

TISIRVYQ

TTPK

GFF

RPV

2185

AAA858131

Agaricu

sbisp

orus

var

bisporus

H97

2SY

TITIRVYQ

TDPN

AFF

RRV

2170

XP00

64555551

Agaricu

sarvensis

1TY

AVLNFV

YG10

30

[12]

Pleurotuscornu

copiae

2SY

TIKVRV

YQTN

PNAFF

RIV

2165

BAB6

39231

Sisto

tremastru

mniveocremeum

2AYT

I TIH

VYQ

TNPK

IFFK

IV21

65

KZS904

471

Boletopsis

grise

a2

SYKI

CVRV

YQTN

PNAFF

RIV

2160

ANW379211

Scler

otium

rolfsii

2TY

KITVRV

YQTN

PNAFF

HPV

2160

2O

FCA

Metarhiziu

man

isopliae

2SY

TITVQ

VYQ

TNPN

AFF

HLV

2155

KFG856971

Punctularia

strigo

sozonata

2SY

TITVSV

YQTN

PNCFF

NIV

2155

XP00

73839861

Hygrophorus

russu

la1

TIGNAKP

VLV

QQEIVGGRR

I20

10

[13]

Stropharia

rugosoan

nulata

1IK

SGVYR

IVSW

QGALGPE

AR

205

[11]

Aminoacid

resid

uesidenticalto

correspo

ndingresid

ueso

fABL

areu

nderlin

ed


Table 3 Effect of various carbohydrates on hemagglutination induced by ABL (16 hemagglutinating units)

Sugar (mM) 100 50 25 125 625 3125 156 078D-Fructose + + + + + + + +D-Galactose + + + + + + + +D-Maltose + + + + + + + +D-Mannose + + + + + + + +D-Melibiose + + + + + + + +D-Xylose + + + + + + + +L-Rhamnose + + + + + + + +L-Sorbose + + + + + + + +N-acetyl-D-galactosamine + + + + + + + +

Cellobiose + + + + + + + +Inulin minus minus minus minus minus + + +Raffinose + + + + + + + ++ hemagglutination activity minus no hemagglutination activity the experiment was repeated twice and the results were reproducible

A28

0nm

Elution volume (ml)

SU2

SU1

035

03

025

02

015

01

005

0

0 4 8 12 16 20 24 28

(a)

9467

43

30

kDa Marker ABL

20

144

(b)

Figure 1 (a) FPLC-gel filtration on Superdex 75 HR 1030 column Eluent 02 MNH4HCO

3buffer (pH 94) Fraction size 08 ml Flow rate

05 mlmin Fraction SU2 represents purified lectin ABL (b) SDS-PAGE of ABL (right lane) and molecular weight markers (left lane) Themarkers were phosphorylase b (94kDa) bovine serum albumin (67kDa) ovalbumin (43kDa) carbonic anhydrase (30 kDa) soybean trypsininhibitor (20kDa) and 120572-lactalbumin (144kDa) all from GE Healthcare

4 Discussion

Lectins are widely distributed among Basidiomycetes in notonly fruiting bodies but also vegetative mycelia About 61reported lectins among Basidiomycetes come from orderAgaricales followed by Russulales and Boletales of 15 and10 respectively [16] Up till now lectins from genus Agar-icus have been reported from A abruptibulbus A bisporusA arvensis A blazeiA campestris A pilatianusA silvicolaand A sylvaticus some of which were purified [4 12 17 18]Mushroom species from genus Agaricus such as A bisporusand A blazei are delicious and nutritious and possess healthprotection and clinical applications Many of them can beartificially cultivated among which A bisporus is one of themost popular and widely cultivated all over the world In

the present study the strain A bitorquis was isolated fromHebei Province inChina and artificially domesticated by localhorticulturists The fruiting bodies we used were cultivatedin mountain areas of Beijing China It is more and morepopular in China since it can be artificially cultured usingthe production equipment for A bisporus or just in the wildHowever few reports on its lectins can be found

Owing to differential binding strength on the Q-Sepharose and SP-Sepharose exchange columns the lectinABL could be purified from the crude protein solutionswith an acceptable recovery (harvest) rate of 90 whichis quite higher than that of other lectins from mushroomStropharia rugosoannulata (79) [11] Hygrophorus russula(12) [13] etc Many reported lectins can be adsorbed onboth anion exchange chromatography as DEAE-Cellulose


Table 4 Specific hemagglutinating activity of ABL towards erythro-cytes from different origins

Species Strainline Hemagglutinating activity (U)Human

A Chinese 16B Chinese 12O Chinese 8AB Chinese 16

Rabbit Cuniculus 16The experiment was repeated twice and results were reproducible

Inhi

bito

ry ra

te (

)

100

80

60

40

20

0

0 100 200 300 400

Protein concentration (gml)

Figure 2 HIV-1 reverse transcriptase inhibitory activity of ABL(data represent means plusmn SD n = 3)

and Q-Sepharose and cation exchange chromatography asCM-Cellulose and SP-Sepharose The less chromatographyprocesses were used the higher recovery rate would beobtained A lectin from A blazei was purified using chro-matography of DEAE-Toyopearl and Sepharose 4B withthe recovery rate of 92-16 [18] A lectin from a wildtoxic mushroom Inocybe umbrinella which is from thesame order as A bitorquis was purified with a recoveryrate of 11 following a protocol comprised ion exchangechromatography onDEAE-Cellulose andCM-Cellulose andgel filtration on Superdex 75 [19] However H russula lectinwas isolated with a very low recovery rate of 12 and along purification procedure encompassing five successiveion exchange chromatography on CM-Cellulose DEAE-Cellulose Q-Sepharose SP-Sepharose and Mono S [13]Therefore simple and efficient purification processes arethe important guarantees for protein purification and therecovery rate

Based on both FPCL and SDS-PAGE the present lectinwas determined to be a monomeric protein with a molecularmass of 276 kDa which is different from many other lectinsreported Since the first lectin was reported from a famoustoxic mushroom Amanita muscaria (Fly agaric) in the year1910 more than 300 mushroom lectins have been reportedwhich demonstrate different molecular masses and subunitstructure [4 16]The common mushroom A bisporus whichis very close to A bitorquis genetically was reported to havebetween two and four hemagglutinin proteins of 58 kDa

Inhi

bito

ry ra

te (

)

100

80

60

40

20

0

0 100 200 300 400


HepG2L1210

Figure 3 Inhibitory effect of ABL on proliferation of HepG2 andL1210 cancer cells Cell proliferation was determined by MTT assay(data represent means plusmn SD n = 3)


Inco

rpor

atio

n of

[3H

-met

hyl]t

hym

idin

e

0 50 100 150 200

30000

25000

20000

15000

10000

5000

0

ABLConA

Figure 4 Mitogenic response of murine splenocytes induced byABL and ConA (data represent means plusmn SD n = 3)

to 64 kDa composed of identical subunits of 16 kDa [20]Molecular mass of the lectin from A blazei was determinedto be 60-70 kDa using gel filtration and SDS-PAGE with fouridentical subunits of sim16 kDa [18] A thermostable 304-kDalectin from dried fruiting bodies of Agaricus arvensis wasreported to be a dimermade up of two 152 kDahomogeneoussubunits [12] Lectins from Polyporus squamosus (28 kDa)and Xylaria hypoxylon (288 kDa) possess a very closemolecular masswith ABL but both of them are homodimericlectins To date a lectin from Pholiota squarrosa possesses thelowest molecular mass of 45 kDa [4]

Although ABL demonstrates a unique molecular massand subunit structure comparing with other lectins fromthe genus Agaricus N-terminal sequence of ABL sharesconsiderably high homology with that of A bisporus (85AAA858131) and A bisporus var bisporus H97 (70XP 0064555551) but low homology with that of A arvensis(30) (Table 2) [12] It also shows high sequence similarity


of about 55-65 with fungal lectins from other genera suchas Pleurotus cornucopiae (65 BAB639231) Sistotremastrumniveocremeum (65 KZS904471) Boletopsis grisea (60ANW379211) Sclerotium rolfsii (60 2OFC A) Metarhiz-ium anisopliae (55 KFG856971) and Punctularia strigoso-zonata (55 XP 0073839861) On the other hand less than10 homology of N-terminal amino acid sequence has beenfound between ABL and lectins from H russula (10) [13]and Stropharia rugosoannulata (5) [11]

The hemagglutinating activity of the purified lectin can-not be inhibited by a variety of monosaccharides tested butcan be inhibited by the polysaccharides inulin which is aheterogeneous collection of fructose polymers Many mush-room lectins can be inhibited by monosaccharides or theirderivatives The hemagglutinating activity of X hypoxylonlectin is inhibited by xylose [4]Thehemagglutinating activityof the lectin from I umbrinella is inhibited by both D-galactose and D-melibiose [19] Lectin from A bisporus isinhibited by N-acetyl-D-galactosamine while lectins fromother Agaricus species including A blazei A campestrisand A edulis are devoid of inhibitory activity by commonmonosaccharides [4] It suggests that the present lectinfrom artificially cultivated A bitorquis manifests a distinc-tive carbohydrate-binding specificity with other Agaricuslectins as indicated by the ability of only inulin to inhibitits hemagglutinating activity In addition inulin can alsoinhibits the hemagglutinating activity of other lectins fromAgaricomycetes as Pholiota adiposa and R lepida [4]

Most lectins are nonspecific and can agglutinate erythro-cytes of all human blood groupswithout any noticeable speci-ficity [16]The present lectin ABL can agglutinate human typeA B O AB and rabbit erythrocytes while hemagglutinationin human type A AB and rabbit erythrocytes will be strongerthan that in human type B and O erythrocytes Just likeABL lectins fromA bisporus and A campestris also manifesthemagglutinating activity towards human type A B O ABand rabbit erythrocytes A abruptibulbus lectin possessesagglutination activity towards human type O erythrocytesdouble than human A B and rabbit erythrocytes [16]Moreover a homodimeric lectin from Clitocybe nebularisagglutinates human type A erythrocytes with highest affinityfollowed by human type B O and bovine erythrocytes [21]

ABL demonstrate potential therapeutic applications withconsiderably high anti-HIV antitumor and mitogenic activ-ities The lectin inhibits the HIV-1 RT activity with an IC

50

value of 469 120583M which is more efficient than a lectin fromA bisporus with an IC

50value of 8 120583M [16] I umbrinella

lectin exhibits HIV-1 RT inhibitory activity with an IC50

value of 47 120583M which is just the same as ABL [19] Othermushroom lectins show extremely high inhibitory activitytowards HIV-1 RT with very low IC

50values such as lectins

from P adiposa (IC50value of 19 120583M) [22] Moreover lectins

from S rugosoannulata and Hericium erinaceus inhibit HIV-1 RT with a higher IC


[11 15] On the other hand many mushroom lectins do notexhibit HIV-1 RT inhibitory activity

ABL displays inhibitory activity against mouse lympho-cytic leukemia cells L1210 with an IC

50value of 497 120583M

but has no significant effect on human HepG2 cells Many

lectins isolated from order Agaricales exhibit significantinhibitory activity against tumor cells A bisporus lectin(50 mgml) can inhibit the proliferation in human coloncancer HT29 cells without cytotoxicity [16] Lectin from themushroom Armillaria luteo-virens inhibit MBL2 HeLa andL1210 cellswith an IC

50value of 25 5 and 10120583M respectively

However proliferation ofHepG2 cells was not affected whichis similar with that of ABL in the present study [23] Itis worth mentioning that the lectin from Armillaria luteo-virens was also inulin-specific just like the present one Whileanother inulin-specific lectin from P adiposa demonstratedantiproliferative activity towards both Hep G2 and MCF7cells with an IC

50value of 21 and 32 120583M respectively [22]

That is to say the sugar specificity is very important factor forantiproliferative activity but not the only one

Mushroom lectins share great diversity in differentspecies and even one species in different growth conditionsThat is why the lectins are unique in the sugar specificityand antiproliferative potential Tumor cell surfaces vary incomposition of glycoconjugates and their terminal saccharideunits which is believed to be correlated to tumor metas-tasis [4] Lectins may display antiproliferative potential bycross-linking the glycoconjugates of tumor cell surfaces orthrough immunomodulatory effects [7] Lectin from Tri-choloma mongolicum inhibits mouse mastocytoma cells invitro and Sarcoma S 180 cells in Bal bc mice by modulationof the immune system rather than direct cytotoxicity [24]Lectin and fermentation extract from European mistletoe(Viscum album) have been investigated in clinical treatmentsof malignant melanoma which is shown to be safe andwithout any further tumor enhancement [25] The potentantiproliferative activity of ABL is remarkable and hopefullyit can be developed into an agent for cancer therapy andalso diagnostic and experimental tools to study the variousfeatures of cell growth and differentiation

Many mushroom lectins possess the remarkable propertyof stimulating the transformation of lymphocytes from smallresting cells to large blast-like cells that may undergo mitosissuch as A luteo-virens [23] Agrocybe cylindracea [26] andB edulis [27] The present lectin is capable of eliciting amitogenic response from mouse splenocytes with a responseof 241777 CPM at a concentration of 091 120583M (25 120583gml)which is similar to that ofA luteo-virens lectin of 25000CPMat a concentration of 1 120583M [23] Although the magnitude ofthe maximal mitogenic response to ABL (241777 CPMof 091120583M ) is less than that to Con A (271440 CPM of 012 120583M)it manifests higher mitogenic activity than lectins from Acylindracea (6000 CPM of 2 120583M) [26] and B edulis (14000CPM of 1 120583M) [27] Moreover S commune lectin exhibits amitogenic response of 23000 CMP at lectin concentration of4 120583M [28]

5 Conclusion

In summary a novel lectin was purified for the first timefrom the artificially cultivated mushroom A bitorquis Itpossesses unique properties of different molecular mass andsingle subunit structure comparing with other lectins from


the genus Agaricus It is an inulin-specific lectin and prefersrabbit human type A and AB erythrocytes It manifests sig-nificant HIV-1 RT inhibitory activity antiproliferative activitytowards cancer cells and mitogenic activity towards murinesplenocytes In this regard it is noteworthy that the presentlectin demonstrates potential therapeutic applications

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare no conflicts of interest

Acknowledgments

Thisworkwas financially supported byNatural Science Foun-dation of Beijing Municipality (5162006) National Grant ofChina (31200070)Opening Foundation of Key Laboratory ofUrban Agriculture (North China) of Ministry of Agriculture(kf2017012) and Natural Science Foundation of ShandongProvince (ZR2015CL041)

References

[1] A Varrot S M Basheer and A Imberty ldquoFungal lectinsstructure function and potential applicationsrdquoCurrent Opinionin Structural Biology vol 23 no 5 pp 678ndash685 2013

[2] B Muszynska A Grzywacz-Kisielewska K Kała and J Gdula-Argasinska ldquoAnti-inflammatory properties of edible mush-rooms A reviewrdquo Food Chemistry vol 243 pp 373ndash381 2018

[3] S K Lam and T B Ng ldquoLectins production and practicalapplicationsrdquo Applied Microbiology and Biotechnology vol 89no 1 pp 45ndash55 2011

[4] S S Singh H Wang Y S Chan et al ldquoLectins from ediblemushroomsrdquoMolecules vol 20 no 1 pp 446ndash469 2015

[5] N A Amirullah N Zainal Abidin and N Abdullah ldquoThepotential applications of mushrooms against some facets ofatherosclerosis A reviewrdquo Food Research International vol 105pp 517ndash536 2018

[6] T B Ng ldquoPeptides and proteins from fungirdquo Peptides vol 25no 6 pp 1055ndash1073 2004

[7] M A A Hassan R Rouf E Tiralongo T W May and J Tira-longo ldquoMushroom lectins specificity structure and bioactivityrelevant to human diseaserdquo International Journal of MolecularSciences vol 16 no 4 pp 7802ndash7838 2015

[8] W T Ismaya Yunita A Efthyani et al ldquoA novel immune-tolerable and permeable lectin-like protein from mushroomAgaricus bisporusrdquo Biochemical and Biophysical Research Com-munications vol 473 no 4 pp 1090ndash1093 2016

[9] HKawagishi T Kanao R Inagaki et al ldquoFormolysis of a potentantitumor (1997888rarr 6)-120573-d-glucan-protein complex fromAgaricusblazei fruiting bodies and antitumor activity of the resultingproductsrdquo Carbohydrate Polymers vol 12 no 4 pp 393ndash4031990

[10] R Rouf E Tiralongo A Krahl et al ldquoComparative study ofhemagglutination and lectin activity in Australian medicinal

mushrooms (higher Basidiomycetes)rdquo International Journal ofMedicinal Mushrooms vol 13 no 6 pp 493ndash504 2011

[11] W Zhang G Tian X Geng et al ldquoIsolation and characteri-zation of a novel lectin from the edible mushroom Strophariarugosoannulatardquo Molecules vol 19 no 12 pp 19880ndash198912014

[12] J K Zhao Y C Zhao S H Li H X Wang and T B NgldquoIsolation and characterization of a novel thermostable lectinfrom the wild edible mushroom Agaricus arvensisrdquo Journal ofBasic Microbiology vol 51 no 3 pp 304ndash311 2011

[13] Q Liu M Zhu F Du H Wang and T B Ng ldquoIsolationand characterization of a novel mannose- and fructose-bindinglectin from the edible wild mushroom Hygrophorus russula(Fr) Quelrdquo Food Science and Technology Research vol 20 no6 pp 1101ndash1108 2014

[14] Q J Chen J H Cheng H Y Dong Z X Li Z H Ren andC Liu ldquoArtificial cultivation techniques of Agarics bitorquis onwoodland in mountain areasrdquo Journal of Beijing University ofAgriculture vol 23 no 3 pp 19ndash21 2008

[15] Y Li G Zhang T B Ng and H Wang ldquoA novel lectin withantiproliferative and HIV-1 reverse transcriptase inhibitoryactivities from dried fruiting bodies of the monkey headmushroom Hericium erinaceumrdquo Journal of Biomedicine andBiotechnology vol 2010 Article ID 716515 9 pages 2010

[16] R S Singh R Bhari and H P Kaur ldquoMushroom lectinscurrent status and future perspectivesrdquo Critical Reviews inBiotechnology vol 30 no 2 pp 99ndash126 2010

[17] N Mikiashvili V Elisashvili S P Wasser and E NevoldquoComparative study of lectin activity of higher basidiomycetesrdquoInternational Journal of Medicinal Mushrooms vol 8 no 1 pp31ndash38 2006

[18] H Kawagishi A Nomura T Yumen T Mizuno T Hagiwaraand T Nakamura ldquoIsolation and properties of a lectin from thefruiting bodies of Agaricus blazeirdquo Carbohydrate Research vol183 no 1 pp 150ndash154 1988

[19] J K Zhao H X Wang and T B Ng ldquoPurification andcharacterizationof a novel lectin from the toxic wildmushroomInocybe umbrinellardquo Toxicon vol 53 no 3 pp 360ndash366 2009

[20] R W Crenshaw S N Harper M Moyer and L S PrivalleldquoIsolation and characterization of a cDNA clone encoding alectin gene from Agaricus bisporusrdquo Plant Physiology vol 107no 4 pp 1465-1466 1995

[21] J Pohleven N Obermajer J Sabotic et al ldquoPurificationcharacterization and cloning of a ricin B-like lectin frommushroom Clitocybe nebularis with antiproliferative activityagainst human leukemic T cellsrdquo Biochimica et Biophysica Acta(BBA) - General Subjects vol 1790 no 3 pp 173ndash181 2009

[22] G Q Zhang J Sun H X Wang and T B Ng ldquoA novel lectinwith antiproliferative activity from the medicinal mushroomPholiota adiposardquo Acta Biochimica Polonica vol 56 no 3 pp415ndash421 2009

[23] K FengQH Liu T BNg et al ldquoIsolation and characterizationof a novel lectin from the mushroom Armillaria luteo-virensrdquoBiochemical and Biophysical ResearchCommunications vol 345no 4 pp 1573ndash1578 2006

[24] H X Wang T B Ng V E C Ooi W K Liu and S TChang ldquoActions of lectins from the mushroom Tricholomamongolicum on macrophages splenocytes and life-span insarcoma-bearing micerdquo Anticancer Reseach vol 17 no 1 A pp419ndash424 1997


[25] M Augustin P R Bock J Hanisch M Karasmann andB Schneider ldquoSafety and efficacy of the long-term adju-vant treatment of primary intermediate- to high-risk malig-nant melanoma (UICCAJCC stage II and III) with a stan-dardized fermented European mistletoe (Viscum album L)extract Results from a multicenter comparative epidemiolog-ical cohort study in Germany and Switzerlandrdquo Arzneimittel-ForschungDrug Research vol 55 no 1 pp 38ndash49 2005

[26] HWang T B Ng andQ Liu ldquoIsolation of a newheterodimericlectin with mitogenic activity from fruiting bodies of themushroom Agrocybe cylindraceardquo Life Sciences vol 70 no 8pp 877ndash885 2002

[27] S Zheng C Li T B Ng and H X Wang ldquoA lectin withmitogenic activity from the edible wild mushroom Boletusedulisrdquo Process Biochemistry vol 42 no 12 pp 1620ndash1624 2007

[28] C H Han Q H Liu T B Ng and H X Wang ldquoA novelhomodimeric lactose-binding lectin from the edible split gillmedicinal mushroom Schizophyllum communerdquo Biochemicaland Biophysical Research Communications vol 336 no 1 pp252ndash257 2005

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of



Journal of Parasitology Research

GenomicsInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018


BioinformaticsAdvances in

Marine BiologyJournal of



Neuroscience Journal


BioMed Research International

Cell BiologyInternational Journal of



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ArchaeaHindawiwwwhindawicom Volume 2018


Genetics Research International


Advances in

Virolog y Stem Cells International



Enzyme Research



MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom


Members of the genus Agaricus are characterized by havinga fleshy cap and stipe and chocolate-brown spores Somespecies are commercially manufactured including A bis-porus A blazei A bitorquis etc In recent years lectinsfrom Agaricus were widely reported including purificationproperties and therapeutic applications A bisporus lectinscan inhibit the growth of colon cancer cell breast cancer celland human retinal pigment epithelial cells [8]A blazei lectinclaimed to exert an in vitro antitumor activity in leukemiccells with no significant effects on normal lymphatic cells [9]

A bitorquis is an edible mushroom species and can becommercially produced similar to the commonbuttonmush-room A bisporus It is an important supplement cultivationspecies of the button mushroom production in China Uptill now only hemagglutination activity of A bitorquis fromAustralia was reported [10] In the present study we aimedto purify a novel lectin from the artificially cultivated Abisporus and find out its properties and potential therapeuticapplications

2 Materials and Methods

21 Assay for Hemagglutinating Activity Hemagglutinatingactivity was determined in the 96-well microtiter plates witha final volume of 50 120583l A serial twofold dilution of the lectinsolution (25 120583l) was mixed with a 2 rabbit erythrocytesuspension (25 120583l) in phosphate-buffered saline (pH 72) andincubated at room temperature for 1 hThe hemagglutinationtiter one hemagglutination unit was defined as the reciprocalof the highest dilution exhibiting hemagglutination Specificactivity is the number of hemagglutination unitsmg protein[11] The hemagglutination profiles of the lectin towardserythrocytes from other origins were similarly determinedHuman blood (A B O and AB types) was donated by healthyvolunteers and the blood typewas confirmed by clinical tests

22 Purification of Lectin The mushroom A bitorquis wasartificially cultivated in woodland of Beijing mountain areasusing a commercial strain from Hebei Province in Chinaand followed the cultivation method of Chen et al described[14] Dried fruiting bodies (100 g) were homogenized andextracted in 015MNaCl (1 L) at 4∘C overnight Subsequentlythe homogenate was centrifuged at 9500 rpm for 20 min at4∘C The supernatant was collected and ammonium sulphatewas added to the supernatant to 80 saturation Themixturewas left at 4∘C for 8 hours before another centrifugationat 9500 rpm for 20 min at 4∘C Then the precipitate wasdissolved and dialyzed to remove ammonium sulphate beforeapplying to a Q- Sepharose (GE Healthcare USA) column(25times20 cm) which had previously been equilibrated withand was then eluted with 10 mM Tris-HCl buffer (pH 76)After removal of the unadsorbed fraction (Q1) containingslight hemagglutinating activity two adsorbed fractions (Q2and D3) were eluted with 200 mM NaCl and 1000 mMNaCl in the starting buffer respectively Fractions Q2 demon-strated strong hemagglutinating activity and were dialyzedfor further purification on cation exchange chromatographyof SP-Sepharose (GE Healthcare USA) column (25times20 cm)

with 10 mM NH4OAc buffer (pH 46) After removal of an

unadsorbed fraction (SP1) two adsorbed fractions (SP2 andSP3) were eluted by using a linear concentration gradient of0-1000 mM NaCl in the same buffer (pH 46) Lectin activefraction SP3 was finally applied to to gel filtration by fastprotein liquid chromatography (FPLC GE Healthcare USA)on a Superdex 75 gel filtration column (02 M NH

4HCO3

buffer pH 94) using an AKTA Purifier (GE HealthcareUSA) The second fraction (SU2) was the purified lectin

23 Determination of Molecular Mass Molecular mass (Mr)of the purified lectin was determined based on FPLC-gelfiltration and sodium dodecyl sulphate-polyacrylamide gelelectrophoresis (SDS-PAGE) During the FPLC chromatog-raphy Mr of natural proteins were calculated using thestandard curve of Log Mr versus elution volume made bymolecular mass standards (GEHealthcare USA) SDS-PAGEwas performed using the standard procedure with a 12resolving gel and a 5 stacking gelMr of denatured proteinswas obtained using another standard curve of LogMr versusrelative mobilities of molecular mass standards (GenviewUSA)Mr of the present purified laccase was evaluated basedon the two curves [13]

24 N-Terminal Amino Acid Sequencing After SDS-PAGEthe purified enzymeon the gel was transferred to a polyvinyli-dene difluoride (PVDF Bio-RadUSA)membrane by electro-blotting and stained with CBB R-250 The stained band wasthen excised and analyzed by the automated Edman degrada-tion method using an HP G1000A Edman degradation unit(Hewlett Packard Company USA) and an HP1000 HPLCsystem (Hewlett Packard Company USA) [11]

25 Assay of Hemagglutinating Inhibition by CarbohydratesThe hemagglutinating inhibition tests to investigate inhibi-tion of lectin induced hemagglutination by various carbo-hydrates [11] Serial twofold dilutions of sugar samples (200mM to 156mM)were prepared in phosphate-buffered salineAll of the dilutions were mixed with an equal volume (25120583L) of the purified lectin solution with 16 hemagglutinationunits The mixture was allowed to stand for 60 minutes at4∘C and then mixed with 50 120583L of a 2 rabbit erythrocytesuspension The assay for lectin activity was conducted asdescribed above The minimum concentration of the sugarin the final reaction mixture which completely inhibited16 hemagglutination units of the lectin preparation wascalculated The carbohydrates tested included D-fructose D-galactose D-maltose D-mannose D-melibiose D-xylose L-rhamnose L-sorbose cellobiose inulin and raffinose

26 Assay of HIV-1 Reverse Transcriptase (HIV-1 RT)Inhibitory Activity Inhibitory activity towards humanimmunodeficiency virus type 1 (HIV-1) reverse transcriptase(RT) of the purified lectin was assayed using an enzyme-linked immunosorbent assay (ELISA) kit (BoehringerMannheim Germany) by determining the ability of HIV-1RT to synthesize DNA [13] A fixed amount (4-6 ng) ofrecombinant HIV-1 RT was used The absorbance of the









2




2



3 Results







Table2Com

paris

onof

theN

-term

inalsequ

ence

ofABL

with

otherfun

gallectin

s

Species

N-te

rminalsequ

ence

Max

ident

Accessionnu

mber

Ref

Agaricu

sbito

rquis

1EY

TISIRVYQ

TNPK

GFN

RPV

20100

Presentstudy

Agaricu

sbisp

orus

2TY

TISIRVYQ

TTPK

GFF

RPV

2185

AAA858131

Agaricu

sbisp

orus

var

bisporus

H97

2SY

TITIRVYQ

TDPN

AFF

RRV

2170

XP00

64555551

Agaricu

sarvensis

1TY

AVLNFV

YG10

30

[12]

Pleurotuscornu

copiae

2SY

TIKVRV

YQTN

PNAFF

RIV

2165

BAB6

39231

Sisto

tremastru

mniveocremeum

2AYT

I TIH

VYQ

TNPK

IFFK

IV21

65

KZS904

471

Boletopsis

grise

a2

SYKI

CVRV

YQTN

PNAFF

RIV

2160

ANW379211

Scler

otium

rolfsii

2TY

KITVRV

YQTN

PNAFF

HPV

2160

2O

FCA

Metarhiziu

man

isopliae

2SY

TITVQ

VYQ

TNPN

AFF

HLV

2155

KFG856971

Punctularia

strigo

sozonata

2SY

TITVSV

YQTN

PNCFF

NIV

2155

XP00

73839861

Hygrophorus

russu

la1

TIGNAKP

VLV

QQEIVGGRR

I20

10

[13]

Stropharia

rugosoan

nulata

1IK

SGVYR

IVSW

QGALGPE

AR

205

[11]

Aminoacid

resid

uesidenticalto

correspo

ndingresid

ueso

fABL

areu

nderlin

ed





A28

0nm

Elution volume (ml)

SU2

SU1

035

03

025

02

015

01

005

0

0 4 8 12 16 20 24 28

(a)

9467

43

30

kDa Marker ABL

20

144

(b)




4 Discussion









Inhi

bito

ry ra

te (

)

100

80

60

40

20

0

0 100 200 300 400





Inhi

bito

ry ra

te (

)

100

80

60

40

20

0

0 100 200 300 400


HepG2L1210



Inco

rpor

atio

n of

[3H

-met

hyl]t

hym

idin

e

0 50 100 150 200

30000

25000

20000

15000

10000

5000

0

ABLConA









50











50value of 497 120583M









5 Conclusion




Data Availability




Acknowledgments


References

































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018










2




2



3 Results







Table2Com

paris

onof

theN

-term

inalsequ

ence

ofABL

with

otherfun

gallectin

s

Species

N-te

rminalsequ

ence

Max

ident

Accessionnu

mber

Ref

Agaricu

sbito

rquis

1EY

TISIRVYQ

TNPK

GFN

RPV

20100

Presentstudy

Agaricu

sbisp

orus

2TY

TISIRVYQ

TTPK

GFF

RPV

2185

AAA858131

Agaricu

sbisp

orus

var

bisporus

H97

2SY

TITIRVYQ

TDPN

AFF

RRV

2170

XP00

64555551

Agaricu

sarvensis

1TY

AVLNFV

YG10

30

[12]

Pleurotuscornu

copiae

2SY

TIKVRV

YQTN

PNAFF

RIV

2165

BAB6

39231

Sisto

tremastru

mniveocremeum

2AYT

I TIH

VYQ

TNPK

IFFK

IV21

65

KZS904

471

Boletopsis

grise

a2

SYKI

CVRV

YQTN

PNAFF

RIV

2160

ANW379211

Scler

otium

rolfsii

2TY

KITVRV

YQTN

PNAFF

HPV

2160

2O

FCA

Metarhiziu

man

isopliae

2SY

TITVQ

VYQ

TNPN

AFF

HLV

2155

KFG856971

Punctularia

strigo

sozonata

2SY

TITVSV

YQTN

PNCFF

NIV

2155

XP00

73839861

Hygrophorus

russu

la1

TIGNAKP

VLV

QQEIVGGRR

I20

10

[13]

Stropharia

rugosoan

nulata

1IK

SGVYR

IVSW

QGALGPE

AR

205

[11]

Aminoacid

resid

uesidenticalto

correspo

ndingresid

ueso

fABL

areu

nderlin

ed





A28

0nm

Elution volume (ml)

SU2

SU1

035

03

025

02

015

01

005

0

0 4 8 12 16 20 24 28

(a)

9467

43

30

kDa Marker ABL

20

144

(b)




4 Discussion









Inhi

bito

ry ra

te (

)

100

80

60

40

20

0

0 100 200 300 400





Inhi

bito

ry ra

te (

)

100

80

60

40

20

0

0 100 200 300 400


HepG2L1210



Inco

rpor

atio

n of

[3H

-met

hyl]t

hym

idin

e

0 50 100 150 200

30000

25000

20000

15000

10000

5000

0

ABLConA









50











50value of 497 120583M









5 Conclusion




Data Availability




Acknowledgments


References

































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



Table2Com

paris

onof

theN

-term

inalsequ

ence

ofABL

with

otherfun

gallectin

s

Species

N-te

rminalsequ

ence

Max

ident

Accessionnu

mber

Ref

Agaricu

sbito

rquis

1EY

TISIRVYQ

TNPK

GFN

RPV

20100

Presentstudy

Agaricu

sbisp

orus

2TY

TISIRVYQ

TTPK

GFF

RPV

2185

AAA858131

Agaricu

sbisp

orus

var

bisporus

H97

2SY

TITIRVYQ

TDPN

AFF

RRV

2170

XP00

64555551

Agaricu

sarvensis

1TY

AVLNFV

YG10

30

[12]

Pleurotuscornu

copiae

2SY

TIKVRV

YQTN

PNAFF

RIV

2165

BAB6

39231

Sisto

tremastru

mniveocremeum

2AYT

I TIH

VYQ

TNPK

IFFK

IV21

65

KZS904

471

Boletopsis

grise

a2

SYKI

CVRV

YQTN

PNAFF

RIV

2160

ANW379211

Scler

otium

rolfsii

2TY

KITVRV

YQTN

PNAFF

HPV

2160

2O

FCA

Metarhiziu

man

isopliae

2SY

TITVQ

VYQ

TNPN

AFF

HLV

2155

KFG856971

Punctularia

strigo

sozonata

2SY

TITVSV

YQTN

PNCFF

NIV

2155

XP00

73839861

Hygrophorus

russu

la1

TIGNAKP

VLV

QQEIVGGRR

I20

10

[13]

Stropharia

rugosoan

nulata

1IK

SGVYR

IVSW

QGALGPE

AR

205

[11]

Aminoacid

resid

uesidenticalto

correspo

ndingresid

ueso

fABL

areu

nderlin

ed





A28

0nm

Elution volume (ml)

SU2

SU1

035

03

025

02

015

01

005

0

0 4 8 12 16 20 24 28

(a)

9467

43

30

kDa Marker ABL

20

144

(b)




4 Discussion









Inhi

bito

ry ra

te (

)

100

80

60

40

20

0

0 100 200 300 400





Inhi

bito

ry ra

te (

)

100

80

60

40

20

0

0 100 200 300 400


HepG2L1210



Inco

rpor

atio

n of

[3H

-met

hyl]t

hym

idin

e

0 50 100 150 200

30000

25000

20000

15000

10000

5000

0

ABLConA









50











50value of 497 120583M









5 Conclusion




Data Availability




Acknowledgments


References

































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018






A28

0nm

Elution volume (ml)

SU2

SU1

035

03

025

02

015

01

005

0

0 4 8 12 16 20 24 28

(a)

9467

43

30

kDa Marker ABL

20

144

(b)




4 Discussion









Inhi

bito

ry ra

te (

)

100

80

60

40

20

0

0 100 200 300 400





Inhi

bito

ry ra

te (

)

100

80

60

40

20

0

0 100 200 300 400


HepG2L1210



Inco

rpor

atio

n of

[3H

-met

hyl]t

hym

idin

e

0 50 100 150 200

30000

25000

20000

15000

10000

5000

0

ABLConA









50











50value of 497 120583M









5 Conclusion




Data Availability




Acknowledgments


References

































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018







Inhi

bito

ry ra

te (

)

100

80

60

40

20

0

0 100 200 300 400





Inhi

bito

ry ra

te (

)

100

80

60

40

20

0

0 100 200 300 400


HepG2L1210



Inco

rpor

atio

n of

[3H

-met

hyl]t

hym

idin

e

0 50 100 150 200

30000

25000

20000

15000

10000

5000

0

ABLConA









50











50value of 497 120583M









5 Conclusion




Data Availability




Acknowledgments


References

































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018







50











50value of 497 120583M









5 Conclusion




Data Availability




Acknowledgments


References

































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




Data Availability




Acknowledgments


References

































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018









Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


Documents

An Inulin-Specific Lectin with Anti-HIV-1 Reverse ...downloads.hindawi.com/journals/bmri/2019/1341370.pdf · ResearchArticle An Inulin-Specific Lectin with Anti-HIV-1 Reverse Transcriptase,