BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS

A lectin with antifungal and mitogenic activitiesfrom red cluster pepper (Capsicum frutescens) seeds

Patrick H. K. Ngai & T. B. Ng

Received: 21 June 2006 /Revised: 15 September 2006 /Accepted: 18 September 2006 / Published online: 3 November 2006# Springer-Verlag 2006

Abstract A monomeric mannose/glucose-binding lectin,with a molecular mass of 29.5 kDa and an N-terminalsequence GQRELKL showing resemblance to that of thelectin-like oxidized low-density lipoprotein receptor fromthe rabbit, has been isolated from the seeds of red clusterpepper Capsium frutescens L. var. fasciculatum. Theprotocol involved anion exchange chromatography ondiethylamino ethanol-cellulose and Q-Sepharose and fastprotein liquid chromatography on Mono Q. Its hemaggluti-nating activity toward rabbit erythrocytes was inhibited byD-mannose and glucose, specifically. The activity wasstable from 0 to 40°C, reached a maximum at pH 7 and8, and was potentiated by Ca2+ and Mn2+ ions. The lectinshowed strong mitogenic activity toward spleen cellsisolated from BALB/c mice. The mitogenic activity, whichreached a peak at a lectin concentration of 0.27 μM, wasinhibited specifically by D(+)-mannose. The lectin wascapable of inhibiting the germination of Aspergillus flavusand Fusarium moniliforme spores and hyphal growth in thetwo fungi.

Keywords Lectin . Antifungal . Red pepper . Seeds

Introduction

Stillmark (1888) first noted that castor bean extract wascapable of agglutinating erythrocytes. Elfstrand (1898)introduced the term “hemagglutinin” to designate proteins

with hemagglutinating activity. The term “lectin” was usedby Renkonnen (1948) to denote proteins that selectivelyagglutinate blood cells of a particular blood group in theABO blood group system. Lectins have now been isolatedfrom a diverse number of organisms encompassing fungi,bacteria, plants, invertebrates, and vertebrates (Knibbs et al.1991; Pusztai 1991; Tateno et al. 1998; Wang et al. 1996;Inamori and Saito 1999; Leteux and Chai 2000). Lectinshave attracted the attention of numerous researchers byvirtue of the potentially exploitable activities that theymanifest including antiproliferative, antitumor, immuno-modulatory (Wang et al. 1996, 2000, Yu et al. 2001; SinghBains et al. 2005), antifungal (Ye et al. 2001), antiviral (Ooiet al. 2000), and anti-insect (Machuka and Oladapo 2000)activities.

Lectins can be divided into different groups in accor-dance with their carbohydrate-binding specificities. Thegroups include mannose-binding, mannose- and glucose-binding, galactose-binding, N-acetylgalactosamine-binding,N-acetylglucosamine-binding, and sialic acid-binding lec-tins. The mannose- and glucose-binding group is repre-sented by concanavalin A (Agrawal and Goldstein 1967)and chestnut lectin (Nomura et al. 1998). The mannose-binding group comprises lectins from the monocot familiesincluding Amaryllidaceae (Ooi et al. 2000), Alliaceae (Lamand Ng 2001), Orchidaceae (Van Damme et al. 1994), andLiliaceae (Singh Bains et al. 2005). In spite of the fact thatpepper is an ingredient of many dishes, no lectin haspreviously been isolated from pepper. The intent of thepresent study was to isolate a lectin from seeds of the redcluster pepper. This work reports the isolation of apreferentially mannose-binding lectin from the seeds ofthe pepper that belongs to the family. The isolation ofpepper lectin adds to the list of compounds produced by thepepper, which comprises capsaicin, renowned for its

Appl Microbiol Biotechnol (2007) 74:366–371DOI 10.1007/s00253-006-0685-y

P. H. K. Ngai : T. B. Ng (*)Department of Biochemistry, Faculty of Medicine,The Chinese University of Hong Kong,Shatin, New Territories,Hong Kong, Chinae-mail: b021770@mailserv.cuhk.edu.hk

extensive application in neuroscience research (Szolcsanyiand Bartho 2001); capsanthin; and solanine. It is perhapsnoteworthy that in traditional Chinese medicine pepper isused for the treatment of rheumatism, loin pain, frost bite,furunculosis, anorexia, indigestion, and gastrointestinaldistension.

Materials and methods

Purification of lectin

Dried red cluster pepper Capsicum frutescens L var.fasciculatum (459 kg) from Thailand, about half the sizeof the little finger, was purchased from a local supermarket.It was ground into a pulp in a Waring blender. The seedswere sieved out, weighed, and then soaked in distilledwater for 2 days before homogenization in distilled water,which had been adjusted to pH 9. The homogenate wasfiltered through cheesecloth and allowed to stand at 4°Covernight. The suspension was centrifuged at 10,000×g for30 min and the resulting supernatant was lyophilized. Thelyophilized powder was dissolved in 50 mM NH4HCO3

(pH 9.2) and applied to a column of diethylamino ethanol(DEAE)-cellulose (Sigma) (2×2.5 cm). After elution ofunadsorbed proteins, the column was eluted sequentiallywith 200 mM, 300 mM, 500 mM, and 1 M NH4HCO3 atpH 9.2. All chromatographic fractions were tested forhemagglutinating activity as described below. The activefraction was then applied to a Q-Sepharose (AmershamBiosciences) column (8×25 cm), which had been pre-equilibrated with 10 mM Tris–HCl buffer (pH 7.2). Afterremoval of unadsorbed proteins, the column was elutedwith a linear concentration (0–0.1 M) gradient of NaCl inthe Tris–HCl buffer. The fraction with hemagglutinatingactivity was dialyzed and lyophilized before being separat-ed by fast-protein liquid chromatography (FPLC) on aMono Q (Amersham Biosciences) column, which had beenpre-equilibrated, and was then eluted with 50 mMNH4HCO3 (pH 9.2). After elution of the unadsorbedproteins, adsorbed proteins were eluted with a lineargradient of 0–0.1 M NaCl in the NH4HCO3 buffer. Thepeak containing hemagglutinating activity represented thepurified pepper lectin.

Estimation of molecular mass and determinationof N-terminal sequence

The purified lectin was subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE)for molecular mass determination in accordance with theprocedure of Laemmli and Favre (1993). Gel filtration onan FPLC-Superdex 75 column, which had been calibrated

with molecular mass markers (Amersham Biosciences),was also conducted to determine the molecular mass of thelectin. The N-terminal sequence of the lectin was deter-mined by using a Hewlett-Packard HP G1000A Edmandegradation unit and an HP 1000 high-performance liquidchromatography system.

Assay of hemagglutinating activity

In the assay for hemagglutinating activity, a serial twofolddilution of the lectin solution in microtiter U-plates (50 μl)was mixed with 50 μl of a 2% suspension of rabbit redblood cells in phosphate-buffered saline (pH 7.2) at 20°C.The results were read after about 1 h when the blank hadfully sedimented to form a dot at the bottom of the well andthe lectin-treated red blood cells had clumped together. Thehemagglutination titer, defined as the reciprocal of thehighest dilution exhibiting hemagglutination, was definedas one hemagglutination unit. Specific activity is thenumber of hemagglutination units per milligram of protein(Wang et al. 2000).

The tests to investigate the inhibition of lectin-inducedhemagglutination by various carbohydrates were performedin a manner analogous to the hemagglutination test. Serialtwofold dilutions of D-(+)-mannose, L-(−)-mannose,D-(+)-glucose, D-(+)-glucosamine, D-(+)-mannosamine, D-(+)-galactosamine, D-(+)-raffinose, α-D(+)-melibiose,α-(+)-lactose, and L-rhamnose were prepared in phos-phate-buffered buffer. All of the dilutions were mixed withan equal volume (25 μl) of the lectin (three hemagglutina-tion units). The mixture was allowed to stand for 30 min atroom temperature and then was mixed with 50 μl of a 2%rabbit erythrocyte suspension. The minimum concentrationof the sugar in the final reaction mixture which completelyinhibited three hemagglutination units of the lectin prepa-ration was calculated (Wang et al. 2000).

For thermal stability studies, the purified lectin wasincubated in a water bath for 30 min at various temper-atures: 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100°C, andthen cooled to 20°C. Hemagglutinating assay was carriedout as described above.

For pH stability studies, the purified lectin was notdemetallized by treatment with EDTA incubation in a waterbath at 20°C for 30 min at various pH values from 1 to 14by using HCl and NaOH solutions. The pH was thenadjusted to 7.0. Assay of hemagglutinating activity of lectinwas carried out as described above.

For metal binding site analysis, the purified lectin wasdemetallized by treatment with EDTA before incubation ina water bath at 20°C for 30 min in the presence of chloridesof various metals: NaCl, CaCl2, KCl, MgCl2, MnCl2,FeCl2, FeCl3, and NH4Cl, all at 25 mM. Hemagglutinatingassay then followed.

Appl Microbiol Biotechnol (2007) 74:366–371 367

Assay of inhibitory activity on hyphal growth

Antifungal activity was determined by using a hyphalgrowth assay using Aspergillus flavus and Fusariummoniliforme. Hyphal culture liquid (100 μl) was inoculatedinto a culture tube containing 900 μl of potato dextrosebroth (Sigma). Then, 50 μl of lectin was added to the liquid

culture tube and the mixture was incubated at 25°C for24 h. Buffer was used as control. The turbidity reflectinghyphal growth was measured at 560 nm using a Milton RoySpectronic 3000 spectrophotometer. The percent inhibitionof hyphal growth was calculated using the followingformula:

Inhibition %ð Þ of hyphal growth ¼ Absorbance of control� Absorbance of lectin�treated sample

Absorbance of control� 100%

Assay of inhibitory activity on spore germination

The assay of spore germination inhibition was carried outas follows: Fungi were grown on potato dextrose agar(PDA) (Sigma) slants for 7 days. Spores were thenharvested from the slants and suspended in sterile 10 mMTris–HCl buffer (pH 7.2) containing 1% (v/v) Tween 20.The concentration of spores in the suspension was adjustedto 1.0×105 spores/ml. An aliquot of freshly prepared sporesuspension (100 μl) was placed in a Petri dish containingsolid PDA. The lectin sample (100 μg/ml) was applied tosterile filter disks placed equidistantly in the Petri dish. ThePetri dish was incubated at 25–28°C for 48 h. The zonesizes around each disk were measured.

Assay of mitogenic activity toward splenocytes

The assay of mitogenic activity was performed as describedby Wang et al. (1996).

Results

Five absorbance peaks resulted when the pepper seedextract was fractionated on DEAE-cellulose (Fig. 1). OnlyD2, the peak eluted with 200 mM NH4HCO3 (pH 9.2),contained hemagglutinating activity. When D2 was subse-quently chromatographed on Q-Sepharose, it was fraction-

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Fig. 2 Ion exchange chromatography of fraction D2 on a Q-Sepharose column

368 Appl Microbiol Biotechnol (2007) 74:366–371

ated into three adsorbed peaks: Q1, Q2, and Q3 (Fig. 2).Hemagglutinating activity resided only in Q2. Q2 wasresolved into two adsorbed peaks, a large peak, M1, and asmall peak, M2, upon FPLC on MonoS (Fig. 3). M2 wasdevoid of hemagglutinating activity and M1 constitutedpurified pepper seed lectin. About 10-fold purification oflectin activity was achieved using this procedure (Table 1).The lectin was obtained with a yield of 8 mg from 459 gseeds. The lectin exhibited a single band with a molecularmass of 29.5 kDa in SDS-PAGE (Fig. 4). Its N-terminalsequence was GQRELKL. It demonstrated similarity to C-terminal fragments of viral RNA polymerases and the N-terminal sequence of rabbit lectin-like oxidized low-densitylipoprotein receptor. The motif is also found in many otherproteins (Table 2).

A small decrease in the hemagglutinating activity of thelectin was observed when the incubation temperature wasincreased to 30°C (10% reduction) and then to 40°C (15%reduction). At 50°C, 60% of the activity remained, whereasat 60°C, only about 10% hemagglutinating activity wasdetectable. Beyond 60°C, the activity was completelyabolished (data not shown). Maximal hemagglutinatingactivity was observed at pH 7 and 8. Between pH 8 and 9,

full or almost full activity was observed. At pH 6 and 10,about 82 and 62% of the activity remained, respectively. AtpH 5 and 11, only about 30% of the activity was retained.Further decrease or increase in pH to pH 4 and 12 resultedin indiscernible activity (data not shown). Of the variouschlorides tested, including NH4Cl, NaCl, KCl, MgCl2,CaCl2, MnCl2, and FeCl3, only those of calcium andmanganese enhanced the activity of the lectin by twofold(data not shown). The lectin exhibited a strong inhibitoryeffect on growth and spore germination in the fungi F.moniliforme and A. flavus. However, its effect on otherfungal species including Fusarium graminearum, Fusariumsolani, Physalospora piricola, and Botrytis cinerea wasnegligible. At a dose of 1 mg lectin/ml, (65±5)% and (80±5)% inhibition of hyphal growth for F. moniliforme and A.flavus, respectively, was observed. At 100 μg lectin/ml, thewidths of inhibition zone in the spore germination test inthe two fungi were 4±0.4 and 5±0.4 mm (mean±SD, n=3),respectively.

The mitogenic activity of the lectin toward mousesplenocytes is shown in comparison with Con A in Fig. 5.It can be seen that Con A was more potent. Among thesugars tested, D-(+)-mannose was the most potent in

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Fig. 3 Fast protein liquid chromatography of fraction Q2 on a MonoQ column. Flow rate: 2 ml/min. Slanting lines across chromatogramrepresent two linear concentration gradients of NaCl (0–0.3 and 0–1 M, respectively) used to elute the column

Table 1 Lectin (hemaggluti-nating) activities and yields atvarious stages of purification

Chromatographicfraction

Specificactivity(titre/mg)

Purificationfold

Total protein(mg)

Total activity(titre/mg ×103)

Recovery(%)

Crude extract(from 459 g seeds)

315 1 210 66 100

D2 (after DEAE) 590 1.9 88 52 79Q2 (after Q-Sepharose) 1,785 5.7 15 27 41M1 (After FPLC) 2,990 9.5 8 24 36

Fig. 4 SDS-PAGE of purifiedCapsicum frutescens lectin.Lane S: C. frutescens lectin.Lane M: Amersham Biosciencesmolecular mass markers

Appl Microbiol Biotechnol (2007) 74:366–371 369

inhibiting hemagglutination induced by the lectin. D-(+)-glucose was the second most potent. The minimum sugarconcentrations required to inhibit three hemagglutinatingunits of the lectin were 31 mM for D-(+)-mannose; 125 mMfor D-(+)-glucose; 250 mM for L-(−)-mannose, D-(+)-galactose, and α-L(−)-fucose; and >1,000 mM for D(+)-glucosamine, D(+)-galactosamine, D-(+)-mannosamine,D-(+)-raffinose, α-D(+)-melibiose, α-(+)-lactose, and L-rhamnose.

Discussion

The red pepper is a favorite vegetable both in the West andin the Orient. Several compounds have been isolated fromthe red pepper, including the well-known capsaicin withanalgesic activity, the pigment capsanthin, and the bitterpoisonous alkaloid solanine with narcotic and insecticidalproperties. However, to date, very few proteinaceousconstituents have been reported. In order to acertainwhether red cluster pepper lectin is endowed with someexploitable or novel characteristics, an attempt was made topurify the lectin.

The lectin from red cluster pepper seeds possesses an N-terminal sequence unlike those of previously reported

lectins. Its sugar binding specificity is similar to concanav-alin A in that they are both glucose- and mannose-binding.However, pepper seed lectin also binds galactose andfucose, albeit less avidly. Pepper seed lectin differs fromlectins of the Amaryllidaceae, Alliaceae, Orchidaceae, andLiliaceae, which are exclusively mannose-binding. Themolecular mass of pepper seed lectin families is similar tothat of a Con A subunit. The pepper seed lectin displays aslightly weaker mitogenic activity than Con A on mousesplenocytes. Pepper seed lectin resembles lectins from redkidney bean, potato tuber, and Urtica diocia (Gozia et al.1993; Broekaert et al. 1998; Ye et al. 2001) in possessingantifungal activity. Its antifungal activity is species-specifictoward F. moniliforme and A. flavus. Activity toward F.solani, F. graminearum, and Physalospora pricola ismeager.

The thermostability and pH stability characteristics oflectins are known to differ from lectin to lectin (Wang et al.1996). However, themostability and pH stability studieshave not been conducted on all aforementioned mannose-binding lectins. The hemagglutinating activity of pepperseed lectin is thermolabile and pH-sensitive. This investi-gation disclosed that red pepper lectin was stable at pH 8and up to 30°C, beyond which a loss of lectin activity wasobserved. The ability of Ca2+ and Mn2+ ions to potentiatethe hemagglutinating activity of pepper seed lectin isconsistent with a similar observation on Con A and thecommon practice of including the two aforementionedcations in buffer used to keep Con A Sepharose or inbuffer for affinity chromatography on Con A Sepharose.

Pepper seed lectin possesses an N-terminal amino acidsequence that is not found in other lectins. It isinteresting to note that it resembles the N-terminalsequence of rabbit lectin-like oxidized low-density lipo-protein receptor-1 and the C-terminal sequences of someviral polymerases. Nevertheless, it resembles Con A tosome extent in its carbohydrate-binding specificity,activation by Ca2+ and Mn2+ ions, and mitogenic activitytoward murine splenocytes.

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Fig. 5 [Methyl-3H] thymidine incorporation (cpm) by mouse spleno-cytes (index of mitogenic activity) in response to pepper seed lectinand Con A. Data represent mean of triplicates. SD was <5% for alldata points

Table 2 Comparison of N-ter-minal sequence of pepper seedlectin with other proteins(results of BLAST search)

Identical amino acids areunderlined

Total no. of aminoacids

Sequence

Pepper seed lectin 1 GQRELKL 7Lectin-like oxidized LDL receptor (Oryctoaguscuniculus)

278 2 QRELK 6

Transcription factor (Oryza sativa) 443 15 GERELKL 21RNA polymerase (Nipah virus) 2,244 1,875 GQRELKL 1,881Polymerase (Hendra virus) 2,244 1,875 GQRELRL 1,881RNA polymerase beta subunit (humanparainfluenza virus 3)

2,233 1,840 GQRELK 1,845

Large polymerase subunit L 1845 (bovineparainfluenza virus 3)

2,233 1,840 GQRELK 1845

370 Appl Microbiol Biotechnol (2007) 74:366–371

There are only a few lectins known to possess antifungalactivity (Gozia et al. 1993; Broekaert et al. 1998; Ye et al.2001). Pepper seed lectin is one of them. Its antifungal andmitogenic activities are potentially exploitable. Its sugar-binding characteristics are distinctive: a variety of mono-saccharides including galactose and fucose in addition tomannose are able to inhibit, albeit less potently thanmannose, its hemagglutinating activity. Pepper seed lectinis, thus, an interesting lectin in some aspects. It isinteresting to note that lectin activity is not found in theseeds of bell pepper and long pepper, which are varietiesclosely related to the red cluster pepper.

Acknowledgments The award of a direct grant by the MedicinePanel, Chinese University of Hong Kong Research Committee and theskilled secretarial assistance of Ms. Fion Yung, Ms. Grace Chan, andMs. Christine Chung are gratefully acknowledged.

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