New lectins from aspergilli and their carbohydrate specificity

Biologia 69/1: 15—23, 2014Section Cellular and Molecular BiologyDOI: 10.2478/s11756-013-0293-0

New lectins from aspergilli and their carbohydrate specificity

Ram S. Singh1*, Hemant P. Kaur1 & Jatinder Singh2

1 Carbohydrate and Protein Biotechnology Laboratory, Department of Biotechnology, Punjabi University, Patiala 147 002,Punjab, India; e-mail: [email protected] of Molecular Biology and Biochemistry, Guru Nanak Dev University, Amritsar 143 005, Punjab, India

Abstract: Lectin activity was assessed in sixteen Aspergillius species using human A, B, O, AB, rabbit, goat, pig andsheep erythrocytes. Neuraminidase and protease treated blood group O erythrocytes were also used to evaluate lectinactivity from all the cultures unable to agglutinate native red blood cells. Lectin activity was revealed from Aspergillusacristatus, A. gorakhpurensis, A. panamensis and A. carbonarius extracts, while undiluted extract of A. fischeri showed weakhaemagglutination. Lectin activity was expressed after 5 days of growth by A. acristatus, A. gorakhpurensis, A. panamensisand A. carbonarius and after 8 days of cultivation a sharp decline in lectin activity was observed. Higher titres were observedfrom these species with enzymatically modified blood type O erythrocytes. A variety of carbohydrates were used to studytheir minimum inhibitory concentration capable of inhibiting haemagglutination. Porcine stomach mucin was found tobe the most potent inhibitor of all the lectins. A. gorakhpurensis lectin showed high specificity for chondroitin-6-sulphateand N-acetyl-D-galactosamine. Significant specificity for L-fucose, D-arabinose and 2-deoxy-D-ribose was identified withA. panamensis lectin. Low concentrations of 0.625 mM of D-galactosamine HCl and 0.12 mg/mL of chondroitin-6-sulphatewere found optimal to prevent haemagglutination of A. carbonarius extract. A. carbonarius lectin was partially purified2.75-fold using ammonium sulphate precipitation, dialysis and ultrafiltration. It was found to be stable upto 40◦C and withinthe pH range of 7.0–8.0. Lectin activity was not affected by guanidine-HCl, while it was reduced to half after incubationwith urea and thiourea after 24 h.

Key words: Aspergillus; lectin; haemagglutination; carbohydrate specificity; partial purification; lectin characterization.

Abbreviations: PBS, phosphate buffered saline; MIC, minimum inhibitory concentration; NI, non-inhibitory; IU, inter-national units of enzyme.

Introduction

Lectins are ubiquitous proteins or glycoproteins ofplant/animal or microbial origin possessing an ability tospecifically bind carbohydrate moieties including cells(Pajic et al. 2002). The wide distribution of lectins inviruses, protozoa, bacteria, algae, yeasts, fungi, animalsand plants attribute their important physiological func-tions. Many biological functions of these lectins havebeen elucidated and they have been used in biologicaland biomedical applications (Shimokawa et al. 2012).The carbohydrate binding properties of lectins are crit-ically important not only to clarify their biological roles,but also to develop them as carbohydrate probes ormedicines. Carbohydrate binding properties of lectinshave been applied in the fields of immunology, cell bi-ology, cancer research and genetic engineering (Han etal. 2012). Recently, new technology such as lectin arrayis becoming more popular, therefore lectins with spe-cific sugar-binding properties are required (Suzuki etal. 2012).Fungal lectins especially from either mushroom

or filamentous fungi have been the focus of research

for the past few years. Mushroom lectins are endowedwith antiproliferative, antitumor, mitogenic, hypoten-sive, vasorelaxing, haemolytic, anti-HIV1 reverse tran-scriptase and immunepotentiating activities (Singh etal. 2010a). They have also been studied for biochemicalreagents with valuable carbohydrate binding specificity(Horibe et al. 2010). Owing to unique carbohydrate-binding specificities of mushroom lectins, isolation ofnew lectins has been carried out by various workers(Mikiashvili et al. 2006; Devitashvili et al. 2008; Roufet al. 2011; Albores et al. 2013). There are many re-ports on lectins from micromycetes and pathogenicfungi but their physiological role still remains uncer-tain (Tronchin et al. 2002). The roles of microfungallectins include specific recognition molecules, effectorsof fungal pathogenesis, role as storage proteins and in-volvement in growth and morphogenesis (Singh et al.2011a). The fungal lectins reported so far constitute aminority among carbohydrate interacting proteins andhaemagglutinins (Khan & Khan 2011).Aspergilli are filamentous, cosmopolitan and ubiq-

uitous fungi belonging to the class Eurotiomycetes.These are commonly isolated from soil, plant debris and

* Corresponding author

c©2013 Institute of Molecular Biology, Slovak Academy of Sciences

16 R.S. Singh et al.

indoor air environment. Aspergillus sp. is well-knownto play a role in opportunistic infections, allergic statesand toxicoses in man. Immunosuppression is the majorfactor predisposing to develop opportunistic infections.This genus exhibits lectins possessing unique carbohy-drate specificity and few lectins from this genus haveparticular value as specific probes for investigating thedistribution, structure and biological function of car-bohydrate chains on the cell surface of animals, plantsand microorganisms (Matsumara et al. 2007). A lectin(32 kDa) has been reported from Aspergillus fumigatusthat showed specificity for sialic acid containing gly-coconjugates (Tronchin et al. 2002). Aspergillus terri-cola has been reported to possess a novel thermostablemycelial lectin having approximately similar molecu-lar mass (Singh et al. 2010d). A fucose-specific lectinhas been reported from Aspergillus oryzae (Matsumaraet al. 2007). The mitogenic ability, immunomodulatoryand therapeutic potential have been reported from As-pergillus nidulans lectin (Singh et al. 2011b,c). In earlierstudies, our group has reported high incidence of lectinsfrom aspergilli (Singh et al. 2008, 2010b) and presentinvestigations were carried out as an extension of ourwork to screen sixteen species of fungi for catalogu-ing them for lectin activity. Keeping in view the aboveproperties of aspergilli and possible therapeutic poten-tial of fungal lectins, characteristics of these haemagglu-tinins from Aspergillus species have also been studied.Amongst the species of Aspergillus in this investiga-tion, Aspergillus carbonarius is a primary producer ofochratoxin and possesses teratogenic, immunosuppres-sive and carcinogenic properties (Cabanesa et al. 2002).Therefore, lectin from A. carbonarius has been partiallypurified and characterized.

Material and methods

Fungal cultures, media and growth conditionsSixteen fungal strains of Aspergillus were obtained from Mi-crobial Type Culture Collection (MTCC), Institute of Mi-crobial Technology, Chandigarh, India. Each culture wasmaintained on specific medium prescribed by MTCC andstored at 4 ± 1◦C, until further use. Aspergillus acristatus(MTCC 1257), Aspergillus carbonarius (MTCC 4872), As-pergillus caespitosus (MTCC 6326), Aspergillus asperences(MTCC 1380), Aspergillus awamori (MTCC 2879) and As-pergillus foetidus (MTCC 2737) were grown on Czapek agarslants containing (in g/L): sodium nitrate 3.0, potassiumchloride 0.5, magnesium sulphate 0.5, ferrous sulphate 0.01,dipotassium hydrogen orthophosphate 1.0, yeast extract 5.0,sucrose 30.0 and agar 30.0. Aspergillus columnaris (MTCC4883), Aspergillus janus (MTCC 3563), Aspergillus fischeri(MTCC 4883) and Aspergillus stromatoides (MTCC 1282)were grown on malt extract agar slants containing (in g/L):malt extract 20.0, agar 30.0 and adjusted to pH 6.5. As-pergillus brevipes (MTCC 1988) and Aspergillus panamensis(MTCC 949) were grown on malt extract agar (Blakeslee’sformula) slants containing (in g/L): malt extract 20.0, glu-cose 20.0, peptone 10.0 and agar 30.0. Aspergillus tonophilus(MTCC 1385) was grown on Harrold’s M40 agar slants con-taining (in g/L): malt extract 20.0, yeast extract 5.0, sucrose400.0 and agar 30.0. Aspergillus sclerotiorum (MTCC 1008)

was grown on malt extract agar slants containing (in g/L):malt extract 10.0, peptone 3.0 and agar 30.0. Aspergillusmanginii (MTCC 1283) was grown on potato dextrose agarslants containing (in g/L): potato extract (200 g scrubbed,diced, boiled to prepare extract and filtered through dou-ble layered muslin cloth), dextrose 20.0, agar 30.0 with pHadjusted to 5.6. Aspergillus gorakhpurensis (MTCC 547)was grown on Czapek malt agar slants containing (in g/L):sodium nitrate 3.0, potassium chloride 0.5, magnesium sul-phate 0.5, ferrous sulphate 0.01, dipotassium hydrogen or-thophosphate 20.0, sucrose 30, malt extract 40 and agar30. All the strains were cultivated by inoculating agar discs(5 mm diameter) covered with mycelia into their respectiveliquid medium (100 mL) in Erlenmyer flasks (250 mL), fol-lowed by incubation under stationary conditions at 30◦C for5, 7 and 9 days. The cultures were also grown on solidifiedagar plates containing 3% agar and incubated at 30◦C for 7days to investigate their lectin activity.

Preparation of lectin extractsFungal mycelia from submerged cultures were harvestedfrom broth by filtration. Mycelial mat from agar plateswas obtained by scrapping it from the surface of solidi-fied agar. Mycelium from both cultures were washed thor-oughly with distilled water and phosphate buffered saline(PBS, 0.1 M, pH 7.2), pressed dry and used as the sourceof lectin. Fungal extracts were prepared for determinationof intracellular lectin activity as described earlier (Singhet al. 2008). Mycelium free culture broth was centrifuged(3,000×g, 10 min, 4◦C) and also assayed for extracellularlectin activity.

Conidial lectin activityConidia were obtained by scrapping the mycelia of 7 dayold cultures grown on agar plates. The mycelia was sus-pended in PBS (0.1 M, pH 7.2) in test tube containing glassbeads and vortexed at room temperature for 10 min. Thesuspension containing conidia was decanted and centrifuged(1,500×g) for 5 min. The pellet was re-suspended in PBSand absorbance was adjusted to 0.6 (app. 108 conidia/mL)at 620 nm using spectrophotometer (Shimadzu UV-1700,Japan). Conidial suspensions in aliquots of 2 mL were sub-jected to sonication at an acoustic power of 200 W for 5 minwith 30 s pulse on and off each using an ultrasonicator (VCX130, Sonics & Materials Inc., USA) in an ice bath. Soni-cated conidial suspension was centrifuged (8,000×g) at 4◦Cfor 10 min and supernatant obtained was analysed for lectinactivity.

Fungal growth vs. lectin activityLectin positive cultures were incubated over 5–12 days andlectin activity was tested at 24 h intervals. Erlenmyer flasks(250 mL) containing 100 mL medium were inoculated with5 mm agar discs covered with mycelia to ensure uniformityof the inoculum and incubated at 30◦C under stationarycondition in a BOD incubator. The same amount of biomasswas taken for each of the cultures over the days to determinethe lectin activity.

Erythrocyte preparationHuman and animal blood was collected in Alsever’s solu-tion containing (in g/L): sodium chloride 4.2, glucose 20.5,sodium citrate 8.0 and pH 6.1 in the ratio 1:2. Erythrocytesuspension (2%, v/v) was prepared in PBS as described ear-lier (Singh et al. 2008). The prepared erythrocyte suspensionwas used to detect lectin activity.

Lectin activity from aspergilli 17

Enzymatic modification of erythrocytesA 10% (v/v) suspension of blood type O erythrocytes inPBS was treated with neuraminidase (0.2 IU/mL; Sigma,USA), while 10% (v/v) suspensions of blood type O andpig erythrocytes were treated with protease (2 mg/mL; ICN,USA) as described previously (Singh et al. 2009a). Lectinactivity was detected using enzymatically modified erythro-cytes.

Haemagglutination assayLectin activity was determined using human and animalerythrocytes as described previously (Singh et al. 2008).Briefly, crude lectin sample (20 µL) was serially dilutedwith PBS and then 2% (v/v) suspension of enzyme treatedand untreated erythrocytes was added in 96-well U-bottommicrotitre plates (Tarsons Products Pvt. Ltd., India). Theplates were incubated at room temperature for 30 min andstabilized at 4◦C for 1 h. Agglutination was monitored vi-sually. The activity was expressed as a titre, which was areciprocal of the highest two-fold dilution exhibiting positiveagglutination.

Haemagglutination inhibition assayTo investigate the inhibition of lectin-induced agglutination,haemagglutination-inhibition tests were performed. Appro-priately diluted lectin preparation was incubated with sugarstock solution in equal ratio at room temperature for 1 h.Double amount of 2% (v/v) erythrocyte suspension wasadded and incubated at room temperature for 30 min andthen stabilized at 4◦C for 2–3 h. Button formation in thepresence of carbohydrate indicated specific interaction be-tween the two, while mat formation indicated non-specificcarbohydrate. Minimum inhibitory concentration (MIC) ofeach of the specific sugars was determined by two-fold se-rial dilution of the sugar stock solution prepared in PBS(0.1 M, pH 7.2). MIC was defined as the minimum inhibitoryconcentration required for inhibition of lectin-mediatedhaemagglutination. Carbohydrates tested include D-ribose,L-rhamnose, D-raffinose, D-xylose, L-fucose, D-fructose, D-mannitol, D-arabinose, L-arabinose, D-galactose, D-glucose,D-mannose, D-sucrose, D-maltose, D-lactose, chondroitin-6-sulphate, inositol, meso-inositol, D-trehalose dihydrate,D-glucosamine hydrochloride, D-galactosamine hydrochlo-ride, D-glucuronic acid, D-galacturonic acid, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, N-acetylneurami-nic acid, N-glycolylneuraminic acid, 2-deoxy-D-glucose, 2-deoxy-D-ribose, fetuin, thiodigalactoside, inulin, bovinesubmaxillary mucin, porcine stomach mucin, asialofetuin,pullulan, melibiose, starch, dextran and γ-globulin. Simplesugars were tested at a final concentration of 100 mM, whilecomplex sugars and glycoproteins were tested at 1 mg/mLconcentration.

Partial purification of A. carbonarius lectinLectin was partially purified by ammonium sulphate pre-cipitation, dialysis and ultrafiltration. To the crude mycelialextract (10 mL) ammonium sulphate (10-100%) was addedin small fractions with continuous stirring on an ice bath.It was kept overnight at 4◦C. Precipitates were centrifuged(3,000×g, 10 min, 4◦C) and the pellet was dissolved in PBS(0.1 M, pH 7.2). The dissolved precipitates were dialysedextensively against PBS for 24 h using Snake Skin Dialy-sis tubing (10 kDa, Pierce Biotech, Rockford, USA). Thedialysate was further concentrated by ultracentrifugationusing an Amicon ultracentrifugal device (cutoff 10 kDa, Mil-lipore USA). Lectin activity was determined at each stepand also protein content was analysed by the method of

Lowry et al. (1951). Purification factor (purification fold)is defined as specific activity of lectin after a purificationstep/specific activity of lectin before that step. Recoveryyield (%) is defined as the total amount of lectin after apurification step/total amount of lectin before that step.Specific activity (titre/mg) is the lectin activity of the sam-ple/protein content of the sample (mg/mL).

Characterization of partially purified lectinfrom A. carbonariusInfluence of pH and temperature on haemagglutination oflectinOptimum pH for lectin activity was determined by carry-ing out the haemagglutination assay at pH 4.5–9.0 usingdifferent buffers. pH stability of the lectin was estimatedby incubating partially purified lectin (50 µL) with buffers(450 µL) at pH 1.5-12.5 in different aliquots at 4◦C. Lectinactivity was assayed at 0 h, 2 h, 4 h and 24 h of incuba-tion. Treated samples were neutralized prior to estimationof lectin activity. The buffers used were 0.1 M glycine-HClbuffer (pH 1.5–3.5), 0.1 M sodium acetate-acetic acid buffer(pH 4.0–5.0), 0.1 M phosphate buffer (pH 5.5–6.0), 0.1 MTris-HCl buffer (pH 6.5–8.0) and 0.1 M glycine-NaOH buffer(pH 9.0–12.5). Each sample was compared to control (sam-ple incubated in PBS) and expressed in terms of percentagerelative activity in comparison to control.

Optimum temperature for lectin activity was deter-mined by carrying out agglutination assays at different tem-peratures (4, 20, 25, 30, 35 and 40◦C). Thermal stability oflectin was assessed by incubating different aliquotes over atemperature range of 25–100◦C with increments of 5◦C ina water bath for 10 min. Treated samples were chilled inice bath and assayed for lectin activity. Lectin activity wasexpressed in terms of percentage relative activity in relationto control (sample incubated at 4 ◦C).

Effect of denaturantsPartially purified lectin was incubated with an equal volumeof urea (1–4 M), thiourea (1–4 M), and guanidine-HCl (1–4 M) in PBS at 4◦C for 24 h. Control samples were incubatedwith an equal volume of PBS at the same temperature. Ag-glutination assay was carried out at 0, 2, 4, and 24 h usinghuman type O erythrocyte suspension (2%, v/v). Lectin ac-tivity at any concentration at a given time was expressed aspercentage relative activity compared to control.

Results and discussion

Lectin activity in Aspergillus speciesSixteen species of Aspergillus were screened for lectinactivity and only five species were found lectin posi-tive (Table 1). Amongst them Aspergillus fischeri ex-hibited lectin activity only in the undiluted extractof 7 day old mycelia from submerged culture. Hu-man erythrocytes of blood type B, AB, O and rab-bit erythrocytes were found susceptible to this lectin.None of the species showed extracellular lectin activ-ity in the mycelium free broth. The cultures whichdid not exhibit lectin activity with native erythrocyteswere also screened using enzymatically modified bloodtype O erythrocytes. Enzymatically modified erythro-cytes were also not found susceptible to any of the ex-tracts from these species. Intracellular fungal lectins


Table 1. Lectin activity of Aspergillus species.a

5 days growth (titre) 7 days growth (titre) 9 days growth (titre)Species

A B AB O Rb Go Sh Pg A B AB O Rb Go Sh Pg A B AB O Rb Go Sh Pg

A. acristatus 16 16 16 16 16 2 2 8 32 32 32 32 32 8 4 8 8 8 8 8 8 2 2 4A. carbonarius 32 8 8 32 32 -Ve 8 8 32 8 8 32 32 -Ve 8 8 16 4 4 16 16 -Ve 4 4A. panamensis 4 32 16 16 32 -Ve 2 2 4 32 16 16 32 -Ve 2 2 -Ve -Ve -Ve -Ve -Ve -Ve -Ve -VeA. fischeri -Ve -Ve -Ve -Ve -Ve -Ve -Ve -Ve -Ve LA LA LA LA -Ve -Ve -Ve -Ve -Ve -Ve -Ve -Ve -Ve -Ve -VeA. gorakhpurensis 64 32 32 32 32 4 16 4 64 32 32 32 32 4 16 4 64 32 32 32 32 4 16 4

a A, B, AB, O: human blood type erythrocytes; Rb: rabbit erythrocytes; Go: goat erythrocytes; Sh: sheep erythrocytes; Pg: pigerythrocytes. LA: lectin activity only in undiluted extract. -Ve: no hemagglutination.

have been previously reported from Penicillium griseo-fulvum, Penicillium thomii (Singh et al. 2009a), Peni-cillium chrysogenum (Francis et al. 2011), and Fusar-ium solani (Khan et al. 2007). In the previous studyon screening of aspergilli for lectin activity, A. niger,A. versicolor, A. nidulans and A. ruglosus have shownthe presence of intracellular mycelial lectin activities,while A. flavus, A. clavatus, A. aureus, A. awamori,A. foetidus and A. spinulosus did not exhibit any lectinactivity (Singh et al. 2008). Lectin activity has alsobeen reported from the culture filtrate of Macrophom-ina phaseolina (Bhowal et al. 2005). Lectins from Scle-rotium rolfsii (Swamy et al. 1999) and Fusarium solani(Khan et al. 2007) have been reported to agglutinateonly enzyme-treated erythrocytes.Aspergillus lectins displayed a broad biological ac-

tion spectrum. Agglutination of all the human bloodtypes and also of rabbit, pig, sheep and goat erythro-cytes was observed. Human erythrocytes were agglu-tinated relatively equally by the four lectins suggest-ing that they are non-specific in nature and fall in thecategory of panagglutinins. They might interact withsaccharide units other than blood group determinantspresent on the surface of erythrocytes. Non-specificlectins have also been reported from Macrophominaphaseolina (Bhowal et al. 2005) and Anixiopsis sterco-raria (Chabasse & Robert 1986). Lectins from A. nidu-lans and A. niger have been reported to agglutinate allhuman, pig, rat and mice erythrocytes, while no agglu-tination was exhibited with sheep and goat red bloodcells (Singh et al. 2008). Lectin activity from A. fumiga-tus has been determined by using only rabbit red bloodcells (Troncin et al. 2002). Lectins from P. griseofulvumand P. thomii were reported to be blood group specific(Singh et al. 2009a). A. acristatus lectin equally agglu-tinated all four human blood types, but showed lowertitres with animal erythrocytes. Lectin from A. gorakh-purensis exhibited slight preference to blood group Aerythrocytes over type B and O. Blood group A ery-throcytes have N-acetyl-D-galactosamine (GalNAc) asthe terminal saccharide unit. Inhibition of this lectinby N-acetyl-D-galactosamine further confirms this pref-erential interaction. Lectins with similar blood groupspecificity have also been reported from Rhizoctonia sp.(Mwafaida et al. 2004) and Phaeolapiota aurea (Kawag-ishi et al. 1996). A. panamensis lectin had higher affin-ity for human blood type B and rabbit erythrocytes.

Lectin from A. carbonarius was inhibited by deoxysugar L-fucose and was also found to show higher titrewith blood type O as compared to blood type B ery-throcytes.Due to differential growth pattern of moulds on so-

lidified and liquid media, mycelia from agar plates andflask cultures were harvested after 7 days of incubation.They were investigated for the occurrence of lectin ac-tivity using human blood type O erythrocytes. Mycelialextracts of A. acristatus, A. carbonarius and A. pana-mensis from solidified medium showed four times lowerlectin activity (titre 8, 8, 16, respectively) than corre-sponding broth cultures (titre 32, 32, 64, respectively).Agar plate culture of A. panamensis exhibited onlymarginal lectin activity (titre 2) as compared to cul-ture grown in liquid medium (titre 16). A. fischeri ex-hibited no lectin activity on agar plate culture. Lowerlectin titres from agar plate cultures corroborate ourearlier findings on lectins from Aspergillus sp. (Singhet al. 2008) and Penicillium sp. (Singh et al. 2009a).Pleurotus cornucopiae has been reported to synthesizea mycelial lectin in a developmental stage specific man-ner. This lectin was found localized on the surface of thegrowing mycelia on solidified medium, whereas myceliafrom the broth cultures did not show any lectin activ-ity (Oguri et al. 1996). Fungal cultures in broth did notproduce as many spores as the mycelia grown on so-lidified medium, indicating that the lectin activity wasnot spore specific. This observation was further con-firmed by analysing lectin activity from conidial ex-tracts. None of the conidial extracts from lectin pos-itive cultures shown lectin activity. This suggests thatthe lectin is localized only in the mycelia. Comparablelectin activity has been reported in the resting coni-dia and mycelia of A. fumigatus (Tronchin et al. 2002).Lectin activity has also been reported from the conidiaor ascospores of Anixiopsis stercoraria and Chrysospo-rium keratinophilum (Chabasse & Robert 1986).

Influence of enzymatic treatment of erythrocytes onlectin activityProtease treatment of human blood type O erythro-cytes enhanced the agglutination titre of A. acristatuslectin. This indicates that there is a cryptantigen whichis de novo exposed by removing the protein (glyco) coatfrom the erythrocyte surface. An increase in lectin ac-tivity of A. panamensis (titre 8) and A. gorakhpuren-


Fig. 1. Effect of enzymatic treatment of blood group O erythrocytes on lectin activity from Aspergillus species.

sis (titre 16) was observed when protease-treated pigerythrocytes were used, while the lectin activity ofA. acristatus and A. carbonarius remained unchanged.Porcine erythrocytes contain significant amounts of N-glycoylneuraminic acid and do not contain detectableamounts of O-acetylated sialyl residues. Protease treat-ment of pig erythrocytes might have reduced the sterichindrance on the blood cells and as a result the N-glycoylneuraminic containing sugar chains on the cellscould be recognized by the lectin (Kobayashi et al.2004). Neuraminidase is a glycoside hydrolase enzymethat cleaves the glycosidic linkages of sialic acids. Re-moval of neuraminic acids exposes the penultimategalactosyl residues on the surface of RBCs. Lectins fromA. gorakhpurensis, A. panamensis and A. carbonariusshowed more than two-fold increase in haemaggluti-nation titre when neuraminidase-treated blood groupO erythrocytes were used for haemagglutination assay(Fig. 1). However, the lectin activity of A. acristatus re-mained unchanged. Sugar inhibition profile of this lectinshows that sialylated glycoproteins (porcine stomachmucin and bovine submaxillary mucin) are strong in-hibitors of this lectin. Human erythrocytes besides hav-ing Neu5Ac also contain di-, tri- and tetra-O-acetylatedNeu5Ac as well as N-glycoylneuraminic acid (Neu5Gc)and may exhibit the same titre as of untreated erythro-cytes (Bhowal et al. 2005).

Carbohydrate specificity of Aspergillus lectinsThe minimum inhibitory concentrations of carbohy-drate specificity for Aspergillus lectins are summarizedin Table 2. Lectin from A. panamensis was strongly in-hibited by L-fucose (0.0487 mM), while higher concen-tration of this deoxy sugar was inhibitory to A. acrista-tus (3.125 mM) and A. carbonarius (25 mM) lectins.Fucose specific lectins have been reported from otheraspergilli (Singh et al. 2008, 2010b; Matsumara etal. 2007), which suggests a high instance of fucose

specificity amongst Aspergillus sp. lectins. Mannosespecificity was exhibited by A. panamensis lectin. Amannose-specific lectin has been reported from P.chrysogenum. Mannose binding lectins are consideredto be biologically important because mannose is dis-tributed in microorganisms and animals including in-sects (Francis et al. 2011). A. carbonarius and A. pana-mensis lectins showed affinity for D-arabinose, whereaslectin from A. acristatus was able to distinguish D-and L-isomers of arabinose. Moreover, D-arabinose wasfound a more potent inhibitor. Such lectins may beof value for investigating lectin-carbohydrate interac-tions and factors determining sugar specificity (Wang& Ng 2005). Porcine stomach mucin, containing O-linked glycans, was found to inhibit all the four As-pergillus lectins. A. acristatus lectin was also specificfor both mucins tested, i.e. bovine submaxillary mucinand porcine stomach mucin. The minimum concentra-tion required for complete inhibition was 0.0039 mg/mLfor bovine submaxillary mucin and 0.0078 mg/mL forporcine stomach mucin. Mucin specific lectins havebeen reported from Arthrobotrys oligospora (Rosen etal. 1992), Aspergillus sp. (Singh et al. 2010b) andPenicillium sp. (Singh et al. 2009a). Fetuin containingboth O-glycosidically and N-glycosidically linked sugarchains, was found to be a strong inhibitor of the lectinfrom A. acristatus. Since asialofetuin was found to benon-inhibitory, N-acetyl neuraminic acid may play animportant role in the interaction of this lectin with fe-tuin. Of the two free sialic acids tested, N-acetyl neu-raminic acid showed stronger inhibition as compared toN-glycolylneuraminic acid. Sialoglycoproteins showedgreater affinity for this lectin as compared to free sialicacid, suggesting its higher affinity for linked sialic acids.N-glycolylneuraminic was more specific for A. gorakh-purensis and A. panamensis lectins. This observationcan be supported by the findings that lectin activ-ity from these lectins is inhibited by porcine stomach


Table 2. Carbohydrate inhibition profile of Aspergillus lectins.a

Minimum inhibitory concentration (MIC)Sugar

A. acristatus A. gorakhpurensis A. panamensis A. carbonarius

D-ribose NI 100 mM 3.125 mM 12.5 mML-rhamnose NI 100 mM 25 mM 50 mMD-raffinose NI NI 25 mM NID-xylose NI NI NI NIL-fucose 3.125 mM NI 0.0487 mM 25 mMD-arabinose NI NI 0.39 mM 25 mMD-galactose 25 mM NI NI NID-glucose NI 25 mM NI NID-fructose NI 25 mM 6.25 mM NID-mannitol NI 12.5 mM NI NIL-arabinose 100 mM 25 mM NI NID-maltose NI NI NI NID-lactose NI NI NI NID-mannose NI NI 25 mM NID-sucrose NI 100 mM 50 mM 12.5 mMChondroitin-6-sulphate NI 0.0625 mg/mL NI 0.12 mg/mLInositol 1.0 mg/mL NI 0.0625 mg/mL NIMeso-inositol NI NI 6.25 mM NID-trehalose dihydrate NI 6.25 mM NI NID-glucosamine HCl 100 mM 50 mM NI NID-galactosamine HCl 100 mM NI NI 0.625 mMD-glucuronic acid 100 mM NI NI NID-galacturonic acid NI NI 100 mM NIN-acetylneuraminic acid 6.25 mM 100 mM 50 mM 25 mMN-glycolylneuraminic acid 25 mM 1.56 mM 6.25 mM 50 mMN-acetyl-D-glucosoamine NI NI NI NIN-acetyl-D-galactosamine NI 0.0975 mM NI 6.25 mM2-deoxy-D-glucose NI NI NI NI2-deoxy-D-ribose NI 25 mM 0.39 mM NIFetuin 0.0312 mg/mL 0.25 mg/mL 0.5 mg/mL 0.0625 mg/mLAsialofetuin NI NI NI NIBovine submaxillary mucin 0.0039 mg/mL NI NI 6.25 mg/mLPorcine stomach mucin 0.0078 mg/mL 0.0002 mg/mL 0.00195 mg/mL 125 mg/mLPullulan NI 0.25 mg/mL NI NIThiodigalactoside NI NI NI NIMelibiose NI NI NI 50 mMStarch NI 0.5 mg/mL NI NIGammaglobulin NI NI 0.5 mg/mL NIDextran NI 1.0 mg/mL NI NI

a NI: non inhibitory.

mucin in which more than 90% sialic acids are NeuGc(Kobayashi et al. 2004). A. carbonarius lectin showedhigh specificity for N-acetylneuraminic acid and sialo-glycoproteins fetuin and bovine submaxillary mucin. N-acetyl neuraminic acid and sialic acid rich glycoproteinslike bovine submaxillary mucin, thyroglobulin, fibrino-gen and fetuin have been reported to inhibit the A. fu-migatus lectin (Tronchin et al. 2002). Both the mucinsand asialofetuin have been reported to be strong in-hibitors of A. niger and A. nidulans lectin (Singh et al.2009b,2010c). N-acetyl-D-galactosamine specificity wasexhibited by lectins from A. gorakhpurensis and A. car-bonarius. GalNAc specific lectins have been reportedfrom Ciborinia camelliae (Otta et al. 2002) and Grifolafrondosa fruiting bodies (Kawagishi et al. 1990). Thissupports the increase in lectin titre upon neuraminidasetreatment. Galactose containing residues might serveas receptors for this lectin. Out of the various polysac-charides tested, pullulan, dextran and starch inhibitedthe activity of A. gorakhpurensis lectin. Chondroitin-6-sulphate composed of GlcA(β1→3) GalNAc6SO−

3 link-

ages showed strong affinity for A. gorakhpurensis lectin.Only a few fungal lectins including Xylaria hypoxylon(Liu et al. 2006) and Fusarium solani (Khan et al. 2007)have been reported to be inhibited by biopolymers.

Growth vs. lectin activityAll the Aspergillus sp. expressed lectin activity after the5th day of cultivation. A. gorankhpurensis exhibited aconstant lectin titre during 5-8 days of cultivation, af-ter which it declined sharply. Lectins from A. acrista-tus, A. carbonarius and A. panamensis showed maxi-mum activity between 5–7 days of cultivation (Fig. 2).Even though biomass increases with culture age, cor-responding increase in lectin activity was not observedbeyond a particular level, suggesting that lectin activ-ity is not a function of growth rate alone. The resultsare in agreement with earlier reports on Penicilliumlectins (Singh et al. 2009a). Lectin activity has been re-ported in six-day old cultures of A. niger and reachingits maximum on the 9th day of cultivation (Singh etal. 2009b). A. nidulans has also been reported to ex-


Fig. 2. Lectin activity as a function of growth of A. acristatus (a), A. carbonarius (b), A. gorakhpurensis (c) and A. panamensis (d).

Table 3. Summary of partial purification of A. carbonarius lectin.

Sample Total titre Total protein (mg) Specific activity (Titre/mg) Purification fold Recovery yield (%)

Crude 1600 173.16 9.24 1 100Precipitate 1280 82.0 15.60 1.68 80Dialysate 832 80.6 10.32 1.20 52Ultrafiltrate 640 22.5 28.4 2.75 40

press lectin activity after 6 days of growth (Singh etal. 2010c). Developmental regulation of lectin activityhas also been reported in Sclerotium rolfsii (Swamy etal. 2004) and Rhizoctonia solani (Kellens & Peumans1991). The agglutination activity from Sclerotium rolf-sii has been found dependent on the age of the cultureand optimal activity has been reported after 5 days ofcultivation and was lost after 7 days of growth (Baraket al. 1985).

Partial purification of A. carbonarius lectinAmongst the five species of aspergilli found lectin posi-tive, A. carbonarius is a primary producer of ochratoxinand possesses teratogenic, immunosuppressive and car-cinogenic properties. Ochratoxin is a mycotoxin whichaffects crops, such as tree nuts, coffee and wine grapes,and also causes the human disease Balkan nephropathy(Cabanesa et al. 2002). Keeping this in view, lectin fromA. carbonarius was selected for partial purification andcharacterization.A. carbonarious lectin was precipitated at 40% sat-

uration of ammonium sulphate, with a recovery yield of80% exhibiting 1.68-fold purification. The correspond-

ing specific activity was 15.60 titre/mg. Dialysed sam-ples yielded 52% activity. The dialysate was furtherconcentrated by ultrafiltration and 2.75-fold purifica-tion was achieved. Partial purification of A. carbonariuslectin is summarized in Table 3. Lectin from A. oryzaehas been reported to be precipitated at 30–75% satu-ration of ammonium sulphate (Matsumara et al. 2007).Lectins from A. niger, A. rugulosus, A. nidulans andA. versicolor were found to be completely precipitatedat 40–50 % saturation (Singh et al. 2008).

Characterization of partially purified lectin from A. car-bonariusInfluence of pH and temperature on lectin activityLectin activity was found to be low at acidic pH, whilemaximum titre was obtained with PBS adjusted topH 7.5–8.0, thereafter showing a decline in lectin ac-tivity. Lectin was found to be 100% stable within apH range of 7.0–8.0 even after 24 h, while at pH 5about 95% of the activity was lost within 2 h of in-cubation and no activity was observed after 4 h. AtpH 8.5, 75% activity was lost after 2 h of incuba-tion (Fig. 3). This shows that the lectin was more un-


Fig. 3. Influence of pH on lectin activity of A. carbonarius. CurveA – optimal pH; curve B – pH stability profile after 24 h incuba-tion.

Fig. 4. Influence of temperature on lectin activity of A. carbona-rius. Curve A – temperature optima; curve B – thermal stabilityprofile.

stable at acidic pH as compared to the alkaline pHrange.Optimum temperature for lectin activity was ob-

served to be 35–40◦C. Higher temperatures were moredetrimental to lectin activity with no activity beyond50◦C. Lectin was found to be 100% stable up to 40 ◦C,while at temperature above 50◦C, there was a 100%loss in lectin activity (Fig. 4). A. nidulans lectin hasbeen reported to be completely stable within pH 5.0–8.0and temperature at or below 40◦C (Singh et al. 2011c),whereas lectin from A. terricola was stable within a pHrange of 7.0–10.5 and remained unaffected upon incu-bation at 70◦C for 2.5 h.

Effect of denaturantsLectin activity was not affected in the presence of ureaup to 4 h. However, a prolonged incubation with ureawas accompanied by a loss of 50% activity at all the con-centrations of denaturant, as compared to control incu-bated with PBS (0.1 M, pH 7.2). Lectin resisted 24 hincubation with 1 M thiourea, while higher concentra-tions lowered the activity by 50% after 24 h. Guanidine-HCl had no effect on activity of A. carbonarius lectinand 100% activity was recovered after prolonged incu-

bation with different concentrations of guanidine-HCl.

ConclusionsIt is evident from the present study that genus As-pergillus represents a rich source of mycelial lectins.High incidence of mucin specific lectins from Aspergillussp. suggests that porcine stomach mucin can be ex-ploited for single-step affinity purification of theselectins. A. panamensis and A. acristatus exhibited in-frequent specificities to mannose and arabinose, respec-tively. Identification and characterization of glycocon-jugates from various sources can be based on the carbo-hydrate specificity studies of these lectins. Lectin fromA. carbonarius was 2.75-fold purified and it was foundto be stable within a pH range of 7.0–8.0 and at tem-perature below 40◦C. New lectins from Aspergillus sp.would be added in the database of fungal lectins andcan also be exploited for mitogenic potential and theirapplications in biomedical research.

Acknowledgements

Authors are thankful to Head, Department of Biotechnol-ogy for providing necessary laboratory facilities to executethis work. The use of infrastructure generated under FISTProgramme, Department of Science and Technology, Gov-ernment of India, New Delhi is also duly acknowledged.

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Received June 1, 2013Accepted October 11, 2013

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New lectins from aspergilli and their carbohydrate specificity