Plant Physiol.

(1987) 85, 566-569

0032-0889/87/85/0566/04/$01.00/0

Leaves of the Orchid Twayblade (Listera ovata) Contain a Mannose-Specific Lectin'

Received for publication May 28, 1987 and in revised form July 14, 1987

ELs J. M. VAN DAMME2, ANTHONY K. ALLEN, AND WILLY J. PEUMANS*

Laboratorium voor Plantenteelt, Katholieke Universiteit Leuven, Kardinaal Mercierlaan 92, B-3030 Leuven, Belgium. (E.J.M.VD., W.J.P.); and Department of Biochemistry, Charing Cross and Westminster Medical School, Hammersmith, London W6 8RF, United Kingdom (A.K.A.)
filtrate was used as a source of LOA3 for affinity chromatography. Affinity Chromatography on Immobilized Mannose. The crude A new lectin was isolated from leaves of the twayblade (Listera ovata). filtrate of twayblade leaves was applied to a column of immobiIt is a diuneric protein built up of two subunits of M, 12,500. This lectifn, lized mannose (Selectin 10 from Pierce Chemical Co., Rockford, which is the first to be isolated from a species of the family Orchidaceae, IL), equilibrated with 1 M (NH4)2SO4. Unbound protein was exhibits exclusive specificity towards nmannose. washed off with 1 M (NH4)2SO4 until the A280 of the effluent fell below 0.01. Then the lectin was desorbed with 20 mM (unbuffered) DAP. Ion Exchange Chromatogaphy on Mono-Q. After the affinitychromatography step the lectin was further purified by anion exchange chromatography using a Pharmacia FPLC system (Pharmacia, Uppsala, Sweden). The lectin fractions collected At present over 100 plant lectins have been isolated and after affinity chromatography were dialyzed against 20 mm DAP characterized with respect to their biochemical, physicochemical, (pH 9.0) and applied to a column of Mono-Q type HR 5/5 and carbohydrate binding properties (7, 8). Although most of (Pharmacia), equilibrated with the same buffer. After the column these proteins have been isolated from dry seeds it is now well had been washed with 4 ml of 20 mM DAP (pH 9.0) elution was established that other plant parts also contain lectins as they have performed using a linear salt gradient (20 ml, 0-0.4 M NaCl) in been found in vegetative tissues such as leaves, stems, roots, bark, DAP. Peak fractions were collected and used for further experitubers, and rhizomes (2, 4, 6, 12, 15). Detailed studies of a ments. Assays and Analysis Methods. Hemagglutination assays were number of lectins from different vegetative tissues have shown that some of them are very similar to the seed lectins present in carried out in small glass tubes containing, in 0.1 ml final volume, the same species (e.g. leaf lectins of Griffonia simplicifolia, Dol- 80 gl of a 1 % suspension of rabbit trypsin-treated erythrocytes ichos biflorus) (6, 1 1); others, however, are completely different and 20 ,ul of crude extract or lectin solution. Agglutination was from all known seed lectins and occur apparently exclusively in determined visually (with the unaided eye) after 1 h at room vegetative tissue (e.g. lectins from rhizomes of Bryonia dioica, temperature. The carbohydrate specificity of the lectin was determined with Urtica dioica) (12, 13). In the present paper we describe the isolation and partial characterization of a mannose-specific lectin a series of simple sugars (glucose, galactose, glucosamine, galacfrom leaves of twayblade (Listera ovata), a representative of the tosamine, N-acetylglucosamine, N-acetylgalactosamine, mannose, lactose, melibiose, fucose, arabinose, ribose, fructose, treplant family Orchidaceae. halose, sorbose, xylose, sucrose, maltose, and sorbitol) and glycoproteins (thyroglobulin, ovomucoid, fetuin, and asialofetuin). MATERIALS AND METHODS Lectin preparations were analyzed by SDS-PAGE using a disconMaterial. Leaves of Listera ovata (twayblade) were collected tinuous system (10) on 12.5 to 25% (w/v) acrylamide gradient from flowering plants around the first half of May and used gels. To enhance the resolution of the polypeptide bands, the lectin was reduced and alkylated. Purified lectin (0.5 mg/ml in immediately or stored at -80C. Extraction and Purification of the L ovata Agglutinin. Leaves water) was made 0.05% (v/v) with respect to ,3-mercaptoethanol of L. ovata were cut into small pieces and homogenized with a and heated in a boiling water bath for 1 min. After cooling, the blender in 5 volumes (v/w) of a solution of 1 M (NH4)2SO4 solution was made 0.1 M in iodoacetamide (with a 1 M solution) containing 10 mm thiourea. The homogenate was squeezed and incubated for 30 min at 37C. The reaction was stopped through a double layer of cheesecloth and centrifuged (15 min, with mercaptoethanol (final concentration, 1 M). The amino acid composition of the proteins was determined 20,000g). The resulting supernatant was frozen at -20C, then thawed. The precipitate which formed as a result of the freezing after hydrolysis under N2 in 3 M toluene-p-sulfonic acid at 10C and thawing was removed by centrifugation as before. Finally, for 24 and 72 h and appropriate correction factors derived for the cleared supernatant was filtered through filter paper (What- destruction or slow release of amino acids. Glucosamine was man 3 MM) to remove any particulate material. This crude determined on the analyses after hydrolysis under N2 in 3 M toluene-p-sulfonic acid at 100C (1). Cystine was determined as ' Supported by grants from the National Fund for Scienfific Research cysteic acid after performic-acid oxidation of the protein and
ABSTRACr

(Belgium), of which W. P. is a Senior Research Associate. 2 Supported by a Fellowship of the Belgian Instituut tot Aanmoediging van het Wetenschappelijk Onderzoek in Nijverheid en Landbouw.

3 Abbrevations: LOA, Listera ovata agglutinin; DAP, 1,3 diaminopropane; FPLC, fast protein liquid chromatography. 566

TWAYBLADE (LISTERA OVA TA) AGGLUTININ

Table I. Carbohydrate Specificity of Crude Extracts and Purified LOA Isolectins Minimal Concentration of Sugar or Glycoproteinb Sugar'/ Purified lectind Crude Glycoprotein extractc LOA II LOA I 6 mM 6 mM 6 mM Mannose 500 gg/ml 500 Ag/ml 500 ,g/ml Fetuin 100 .g/ml 100 yg/ml Asialofetuin 100 4g/ml 500 Mg/ml 500 ,sg/ml 500 ,g/ml Ovomucoid 5 lAg/ml 5 Ag/ml 5 ,g/ml Thyroglobulin aAll other sugars tested (cf. "Materials and Methods") were not b Minimal concentration inhibitory at concentrations below 100 mM. required for 50% inhibition of the agglutination activity in assays with c The titer of the crude extract trypsin-treated rabbit erythrocytes. d The final concentrations of LOA I and LOA II were 10 was o00.

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FIG. 2. Ion exchange chromatography of LOA on a Mono-Q column. Chromatogram of total affinity-purified LOA; b, chromatogram of LOA I purified by repeated chromatography on a Mono-Q column; c, chromatogram of LOA II purified by repeated chromatography on a Mono-Q column. Experimental details are described in the "Materials and Methods" section.
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FIG. 1. Affinity chromatography of LOA on immobilized mannose. A partially purified extract of twayblade leaves (250 g fresh weight) was applied to a column of immobilized mannose (10 ml bed volume). Unbound protein was eluted with 1 M (NH4)2SO4 until the A280 fell below 0.01 and the lectin was desorbed with 20 mm DAP. Fractions of 5 ml were collected and the A2M and agglutination titer determined (with trypsin-treated rabbit erythrocytes). The overall yield was 105 mg of LOA.

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subsequent hydrolysis at 1 lOC in constant-boiling HCI (9). Amino acids were measured on a Locarte Mini amino acid analyzer by reaction with ninhydrin; details of the elution programs which were used have been given elsewhere (2, 3). Neutral sugars were determined after methanolysis and trimethylsilylation by GLC (5). The instrument which was used was a Varian 3300 fitted with a capillary column (WCOT fused silica 25 m x 0.32 mm i.d. column with a coating of CP-SIL8 CB from Chrompack, Middelburg, The Netherlands).
RESULTS Purification of the L. ovata Agglutinin. Preliminary experiments with crude extracts of twayblade leaves indicated that they contain readily detectable amounts of an agglutinating factor. Indeed, when tested with trypsin-treated rabbit erythrocytes, titers of extracts prepared by homogenizing leaves in 10 volumes of PBS, went up to 100. To work out a purification procedure the carbohydrate specificity of the agglutinin was determined using a series of simple sugars. Since these experiments indicated that mannose was the

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FIG. 3. SDS-PAGE of LOA isolectins. Lanes a and b, LOA I and LOA II, respectively. Lane RI, Mr reference proteins (lysozyme [Mr 14,300], soya-bean trypsin inhibitor [Mr 21,000], carbonic anhydrase [M, 30,000], ovalbumin [Mr 45,000], BSA [Mr 67,000], and phosporylase [Mr 93,000]). Lane R2, Mr reference proteins (myoglobin [intact, Mr 17,201], myoglobin I + II [Mr 14,632], myoglobin I [Mr 8,235], myoglobin II [Mr 6,383]).

only inhibiting saccharide (Table I) mannose coupled to agarose affinity matrix for the purification of the LOA. Following the purification scheme described in the "Materials and Methods" section, LOA could be purified in a simple step with almost complete recovery. Virtually all the initial agglutination activity was recovered in the fraction desorbed with DAP (Fig. 1). It should be mentioned here that LOA can be desorbed equally well with mannose. However, since high concentrations (0.5 M) are required, and in addition the lectin elutes in a large
was chosen as an

568

VAN DAMME ET AL.

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Plant Physiol. Vol. 85, 1987

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FIG. 4. Gel filtration of LOA I and LOA II on Sephadex G-100. Purified LOA isolectins were chromatographed on a Sephadex G-100 column (28 x 1.6 cm) in PBS (a) or PBS containing 0.1 M mannose (b). M, reference proteins were hemoglobin (Mr 65,000), Cyt c (Mr 12,500), and vitamin B12 (Mr 1,400). Their elution position was determined by measuring the A420. The elution position of LOA was traced by determining the agglutination titer of the fractions. LOA I and LOA II (chromatographed in separate runs) eluted at exactly the same position.

FIG. 5. Gel filtration of LOA I and LOA II on a Superose 12 column. Purified LOA isolectins were chromatographed on a Pharmacia Superose 12 (type HR 10/30) column in PBS (a) or in PBS containing 0.1 M mannose (b). The elution position of the Mr reference proteins (aldolase [Mr 160,000], BSA [(Mr 67,000], ovalbumin (Mr 45,000], chymotrypsin [Mr 25,000], and Cyt c [Mr 12,500]) was determined in a parallel run (as well in PBS as in PBS with 0.1 M mannose) to avoid interactions between the lectins and the reference glycoproteins. LOA I and LOA II (chromatographed in separate runs) eluted at exactly the same position.

volume, nonspecific desorption with DAP is therefore more practical. Moreover, LOA preparations desorbed with either mannose or DAP are equally pure as far as can be judged by SDS-PAGE (results not shown). Besides immobilized mannose, fetuin-agarose also can be used as an affinity matrix for the purification of LOA although the capacity of the latter is considerably lower. A final remark concerns the use of 1 M (NH4)2SO4 as a medium for homogenization and affinity chromatography. This medium offers a double advantage: first, the polyphenol oxidase activity (which is abundant in twayblade leaves) is drastically reduced; and second, LOA binds much stronger to the affinity column than in a commonly used buffer such as PBS. LOA is a mixture of two isolectins. Ion exchange chromatography of the affinity-purified LOA on a Mono-Q anion exchanger column yielded two sharp peaks designated as LOA I and LOA II in accordance with their elution position (Fig. 2a). Each isolectin was recycled on the Mono-Q column till it yielded a single symmetrical peak (Fig. 2, b and c). LOA I and LOA II represent 33 and 67% of the total lectin content, respectively. Molecular Structure of LOA I and LOA II. The molecular structure of both LOA isolectins was analyzed by SDS-PAGE, gel filtration, and sucrose-gradient centrifugation. As shown in figure 3, both isolectins yielded a polypeptide band of Mr 12,500 upon SDS-PAGE. The M, of native LOA was determined by gel filtration of LOA

I and LOA II on Sephadex G- 100 and Superose 12. Both isolectins eluted with an apparent Mr of 3,400 on Sephadex G-100 and 5,600 on Superose 12 (Figs. 4a and 5a). Since these M, values are lower than the apparent Mr of the lectin polypeptides determined by SDS-PAGE, interactions between LOA and both types of matrices became obvious and gel filtration experiments were repeated in the presence of 0.1 M mannose. Under these conditions LOA isolectins eluted with an apparent M, of 12,000 and 14,000 from a Sephadex G-100 and Superose 12 column, respectively, which indicated that in the presence of this specific sugar the interactions between lectin and the gel matrices were reduced (Figs. 4b and Sb). Because of the uncertainty inherent to Mr estimations based on gel filtration LOA was also centrifuged in a sucrose gradient. From their sedimentation position (relative to that of M, marker proteins) a Mr between 20,000 and 25,000 was calculated for both native LOA isolectins (Fig. 6). These results, taken together with the SDS-PAGE pattern, indicate that both LOA I and LOA II are dimers composed of two identical subunits of M, 12,500. Amino acid analyses revealed that LOA I and LOA II have a similar amino acid composition typified by a high content of asparagine/aspartic acid, glutamine/glutamic acid, glycine and serine. LOA lacks methionine and histidine (Table II). Sugar determination of purified LOA indicated no covalently bound carbohydrate, suggesting that the lectin is not a glycoprotein.

TWAYBLADE (LISTERA OVATA) AGGLUTININ

569

0.4

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thyroglobulin; asialofetuin inhibited the agglutination at a 5-fold lower concentration than native fetuin. As far as can be concluded from hapten inhibition assays both LOA isolectins behave identically with respect to their carbohydrate-binding specificity.
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DISCUSSION

Leaves of twayblade (Listera ovata) contain a lectin which can

easily be purified by affinity chromatography on immobilized mannose. This lectin is a mixture of two different molecular forms which can be separated by ion exchange chromatography on a Mono-Q column. Both isolectins are dimers of two identical sized subunits of Mr 12,500 and each isolectin exhibits the same agglutination properties and carbohydrate-binding specificity. The sugar-binding specificity of LOA is unique in that it binds exclusively mannose. This is in contrast to other mannosespecific lectins from dicots such as concanavalin A, pea, and lentil lectin, which bind glucose almost equally well as mannose. Furthermore, the greater inhibitory power of thyroglobulin over the other glycoproteins indicates that the twayblade lectin preferentially binds to a high-mannose structure. Recently another monocot lectin with exclusive specificity towards mannose has been isolated from snowdrop (Galanthus nivalis) bulbs (14). However, unlike LOA, the latter lectin has no higher affinity for mannose oligosaccharides than for the monomer of mannose. Another difference from these mannose-specific monocot lectins is that the snowdrop lectin does not bind to fetuin-agarose whereas LOA can readily be isolated on this affinity matrix. LOA is the first lectin to be isolated from a species belonging to the family Orchidaceae and it differs in specificity and structure from all other lectins so far described. The absolute specificity towards mannose and especially towards mannose oligosaccharides is unique and might be quite useful in glycoconjugate research.
LITERATURE CITED
1. ALLEN AK, A NEUBERGER 1975 The quantitation of glucosamine and galactosamine in glycoproteins after hydrolysis in p-toluene-sulphonic acid. FEBS Lett 60: 76-80 2. ALLEN AK, NN DESAI, A NEUBERGER, JM CREETH 1978 Properties of potato lectin and the nature of its glycoprotein linkages. Biochem J 171: 665-674 3. ASHFORD D, NN DESAI, AK ALLEN, A NEUBERGER, MA O'NEILL, RR SELVENDRAN 1982 Structural studies of the carbohydrate moieties of lectins from potato (Solanum tuberosum) tubers and thorn apple (Datura stramon-

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08

0.4

0.2

40

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44

12

16

Fraction number

FIG. 6. Sucrose-density gradient centrifugation of LOA isolectins. Purified LOA isolectins were centrifuged in a linear 12 to 38% (w/v) sucrose gradient for 30 h at 45,000 rpm in a Beckman SW 50.1 rotor. Gradients were fractionated into 0.2 ml portions. The position of the Mr markers (hemoglobin [Hb, Mr 65,000] and Cyt c [Cyt c, Mr 12,500]) was determined by measuring the A420 of the fractions. LOA isolectins were traced by determining the agglutination activity of the fractions.

Table II. Amino Acid Composition of LOA I and LOA II Amino Acid LOA I LOA II

mol %
Asx Thr Ser Glx Pro

Gly
Ala

Cys
Val Met Ile Leu Tyr Phe His Lys Arg

15.8 7.4 10.4 9.6 1.9 12.5 6.0 2.0 6.4 0.0 4.6 10.5 4.0 1.0 0.0 1.3 6.7

16.6 7.7 9.4 9.4 5.0 11.7 6.1 2.0 5.3 0.0 4.1 11.2 3.3 0.8 0.0 0.6 6.9

ium) seeds. Biochem J 201: 199-208 4. BROEKAERT WF, M NsiMBA-LUBAKI, B PEETERS, WJ PEUMANS 1984 A lectin
from elder (Sambucus nigra L.) bark. Biochem J 221: 163-169 5. CHAMBERS RE, JR CLAMP 1971 An assessment of methanolysis and other factors used in the analysis of carbohydrate containing molecules. Biochem J 125: 1009-1018 6. ETZLER ME, C BORREBAECK 1980 Carbohydrate-binding activity of a lectinlike glycoprotein from stems and leaves of Dolichos biflorus. Biochem Biophys Res Commun 96: 92-97 7. ETZLER ME 1985 Plant lectins. Molecular and biological aspects. Annu Rev Plant Physiol 36: 209-234 8. GOLDSTEIN U, CE HAYES 1978 The lectins: carbohydrate-binding proteins of plants and animals. Adv Carbohydr Chem Biochem 35: 127-340 9. HIRs CHW 1967 The oxidation of ribonuclease with performic acid. J Biol
Chem 219: 611-621 10. LAEMMLI UK 1970 Cleavage of the structural proteins during the assembly of the head of bacteriophage T4. Nature 277: 680685 11. LAMB JE, S SHIBATA, IJ GOLDSTEIN 1983 Purification and characterization of

Carbohydrate-binding Specificity and Agglutination Properties of LOA I and LOA II. Purified LOA isolectins readily agglutinate rabbit erythrocytes, the minimal concentration required for agglutination of trypsin-treated and untreated cells being 1.6 and 12.5 gg/ml respectively, for both isolectins. Human red blood cells, irrespective of their blood group, were not agglutinated even at lectin concentrations as high as 2 mg/ml. Hapten inhibition assays carried out with both LOA isolectins confirmed that from all sugars which were tested only mannose inhibits the agglutination activity, the concentration required for 50% inhibition being 6 mM (Table I). From the glycoproteins we tested, thyroglobulin proved to be the most potent inhibitor, being effective at a concentration as low as 5 ,g/ml (Table I). Ovomucoid and fetuin were 100 times less potent inhibitors than

Griffonia simplicifolia leaf lectins. Plant Physiol 71: 879-887

12. PEUMANS WJ, M DE LEY, WF BROEKAERT 1984 An unusual lectin from

stinging nettle (Urtica dioica) rhizomes. FEBS Lett 177: 99-103

13. PEUMANS WJ, M NsiMBA-LUBAKI, AR CARLIER, E VAN DRIESSCHE 1984 A lectin from Bryonia dioica root stocks. Planta 160: 222-228 14. VAN DAMME EJM, AK ALLEN, WJ PEUMANS 1987 Isolation and characterization of a lectin with exclusive specificity towards mannose from snowdrop

(Galanthus nivalis) bulbs. FEBS Lett 215: 140-144

15. WAXDAL MJ 1974 Isolation, characterization, and biological activities of five

mitogens from pokeweed. Biochemistry 13: 3671-3676