Phytochemrstry, Vol 29, No 6, pp 1745-1749,1990 Pnnted III Great Brltam

ISOLATION

0

003l-9422/90$300+ 0 00 1990Pergamon Press plc

AND CHARACTERIZATION OF A LECTIN FROM THE SEEDS OF DIOCLEALEHMANNI GERARDO

Biochemistry

Laboratory,

PEREZ,*

MARCELA

Department

HERNANDEZ

of Cheinistry

and

Umversldad

ELSA MORA

Naclonal,

Bogota,

Colombia

(Recewed in revzsedform 26 October 1989) Key

Word Index-Dloclea

lehmannt; Legummosae;

seeds; isolation;

characterization;

lectm.

Abstract-Affinity chromatography of the globulin fraction from the seeds of Dioclea lehnzanni on Sephacryl S-200 yielded two lectins, one slightly retarded and another strongly bound. The latter, which was a glucose/mannose specific lectin, was purified and the following properties were determmed: pl, M, of subunits, carbohydrate content, A, ammoacid composition, hemagglutination and inhibition patterns, N-terminal sequence and mitogenic activity. These properties of the lectm were very simdar to those of the Con A and Dioclen grandijZora lectins.

INTRODUCTION

Studies on lectins from the tribe Dlocleae have been focused on the Canavalia genus and Con A (the lectin from Canaval~ enszfirmis) is perhaps the best characterized lectin [l-3]. The Dioclea genus with 30-50 species [4] is widespread in tropical zones and most of the species are found in Central and South America, especially in the Amazonian basin; however comparatively few studies have been carried out on their seeds. In some species physostygmine, a parasympathomimetic alkaloid, has been found [4] and lectins have been detected [S, 63 but, with one exception, these have not been further studied. Recently trypsin inhibitors from seeds of Dioclea grandiflora have been isolated [7]. This species also has a lectin which has been characterized [8] and for which the primary structures of the a, fl and y subunits have been established [9, lo]. The protein is a mannose/glucosebinding lectm with a structure and properties very similar to those of Con A In a preliminary study [l l] a lectin was detected in Dzoclea Iehmanni (DLL), which 1s very abundant in Colombia and of interest as a food source. We describe here the lsolatlon and properties of a lectin from the seeds of Dioclea lehmannz as well as evidence for the presence of another lectin in its seeds. RESULTS AND DISCUSSION

Isolation and purification of the lectin

Preliminary analysis of the seeds revealed a fat content (1.7%) lower than in Canavalia ensijormis (3 6%) [12] which meant that it was not necessary to defat the flour before extraction. The high content of total N (4.83%) suggested a high level of protein; however when the nonprotein nitrogen was determined (37% of total N), the protein content was reduced to 19.1%, being thus very similar to the real protein (19.4%) obtained for C. enszjbrmis (Perez et al., unpublished results). Small scale *Author to whom correspondence should be addressed.

assays showed that the highest yield of albumin plus globulin proteins (71%) was obtained after four extractions with 1% sodium chloride (see Experimental). In all cases the last extract had a higher lectin specific titre than that found in the third extract; however considering the small amount of protein present m the fourth extract, the flour was extracted only four times The lectin was subsequently purified as shown in Table 1. Fractional precipitation with ammonium sulphate [&SO%, 50-70% saturation(s)], showed that at 70%~ the lectm was completely precipitated, as no hemagglutinating activity was observed in the 70%~ supernatant. Affinity chromatography on a Sephacryl S-200 column gave an elution profile with three main peaks and a few minor peaks (Fig. 1).The last major peak (P4) being obtained after addition of 0.1 M glucose. Peaks P2, P3 and P4 exhibited hemagglutinating activity (very famt for P3) against human erythrocytes; control experiments applying one-third and five times the amount of globulins, showed that this result was not due to an overloadmg of the column, but to the existence in Dioclea lehmanni of at least one lectin which was retarded but not firmly bound to the column and which does not require glucose to be eluted. The P4 lectin, on the other hand, behaved like DGL (the lectin from Dioclea grandijlora) [8]. Both the elution profile and the position of the lectin activity remained unchanged when the extraction was carried out in the presence of protease inhibitors (see Experimental), suggesting that it was not likely that the lectin in P2 resulted from proteolytic cleavages during the extraction. Moreira et al. [S] did not observe an additional peak with lectm activity m the nonretained fraction. This was probably due to the different chromatographic conditions used and to the fact that rabbit red blood cells (RBCs), which were used by these authors, are not agglutinated by P2. In dry seeds the amount of P2 was 107 mg per 100 g flour and P4 was 131 mg per 100 g flour. This latter figure agrees well with the reported value (121 mg per 100 g flour) for the equivalent lectm in seeds of Dioclea grandzjlora [8]. As Judged from the specific titre values the extent of purification was 7.8-fold for P2 and 6.2-fold for P4, being similar

1745

G PEREZet al

1746 Table

1 Purrfication

of the lectms from D lehmanni

Volume

Protem

Total protem

Stage

(ml)

(mg ml-‘)

(mg)

1 2 3 4

282 360 370 368

21 7 521 123 0 59 443 9 36

6130 1880 455 217

Fust sahne extract Second sahne extract Third sahne extract Fourth saline extract Pool sahne extracts 5 50-70% (NHJ2 SO, fraction 6 Affimty chromatography Sephacryl S-200 P2 lectm 1 P4 lectm 4

239

450 49 8

0 283 036

2230t

127 179

Specific We* 23 6 3 10 6 50 1360 145 274

113 88 9

* I’he speelk tltre 1s dkfined-as the hemaggltitmatlon tltre divldizd-by the protein concentration solution tThis amount of protem corresponds to the preclpltation of 1320 ml of pooled extracts $77 3 mg of globuhn fraction apphed to the column $81 9 mg of globuhn fraction apphed to the column

_.-0.0-U 0

400

200 ml

Fig 1 Affimty chromatography of the D lehmanm lectm Sephacryl S-200 was used as the affimty medium Elutlon procedure as described m the Experimental

to that recalculated (9.6-fold) from the data of Moreira et al. [S], taking the saline extract as a basis It should be noted however, that in our saline extracts we were measuring the actlvlty of a lectin mixture If we assume that the two lectins present were extracted to the same degree, then the level of purification for each lectm can be estimated as 12-l S-fold Molecular properties of the lectin P4 Alkaline PAGE of P4 on gels of different crosslmkages always gave a poorly defined main band with low mobility and a minor sharp band that migrated with the dye marker Acldlc PAGE of P4 showed two sharp, wellseparated bands with medium mobility that possibly correspond to the a and p/ly chains [8]. These results suggested that P4 IS a basic protein, and this was confirmed by the results of isoelectric focusmg which showed four bands with pls of 8 00, 8 13, 8.30 and 8.42 This

Purlficatron (fold)

Extracted protem (mgg-’ flour) 153 469 114 54

10 19

78 6.2

(mgml-- 'j ot-tiie assayed-

multlbanded pattern IS similar to that previously observed for DGL wlthm a range 8.6-9.0 [S]. A variation m pI values (0.14 2 pH units) between different samples of DLL was noted, similar to that reported m DGL by Ainouz et al. [lo]. Both of the bands on acidic PAGE became stained after treatment with PAS-Schiff reagent mdlcatmg that they were glycoprotems After extensive dialysis the nentral sugar content was found to be 1.7-1.9%. Interestingly no carbohydrate was detected m DGL [8]. SDS-PAGE of P4 gave two bands with M, 25 300f400 and 14 000 + 200, these figures agree closely with those obtained by Morelra et al. [S] for the a and /? subunits of DGL. In our experimental conditions, we were not able to observe a separate band for the y subunit as reported by Amouz et al. [lo]. It is likely that the b and y subunits ran as a single band m our medium-crosslmked gels N-Terminal analysis of P4 revealed two sequences, one of them was Ala-Asp-Thr-Ile-Val-Ala-Val which was identical to the N-terminal regions of Con A [ 131 and the a and b chains of DGL [9, lo] and the other was Ser-Ile-Ala-Asp which was identical with the N-terminal region of the y cham of DGL [lo]. Further sequence studies of DLL have provided evidence for the presence of a y chain (Perez and Richardson, unpublished results) and therefore the P4 lectm 1s composed of a, fl and y chains m an analogous manner to that displayed by DGL. The attempts to determme the M, of the native protein by gel filtration using BloGel P-100, Ultrogel AcA 54 or Sephadex G-200 (m the presence of 0 1 M glucose), were unsuccessful. In all cases the protein eluted at an abnormally high volume (calculated M, < SOOO),suggesting that hydrophobic mteractlons had occurred between the protein and the supports; occaslonally a minor peak was observed at a posItion corresponding to an M, 55 000-60000. Human RBCs were agglutmated unspecifically by the P4 lectm and by the P2 lectm, the latter showmg a higher specific titre (Table 1); 5 mM CaL+ and 5 mM Mn* + only doubled the tltre of each lectin, which IS within the experlmental error of the method. A similar result was obtained with P4 and rabbit erythrocytes Prehmmary metal analysis of P4 Indicated that significant amounts of

Lectin from Dioclea lehmannt

1747

Table 2. Agglutination of human and animal erythrocytes by Dioclea lectins D. grandijlora

D. glycrnordes

D malacocarpa

D megacarpa

RBCs

P2

D lehmanm

P4

PI

CSI

c51

El

Human Rabbit Chlcken cow Rat Monkey Mice Dog Horse Sheep

113.1*

n.d. 768$

nd.

88 9* +4 nd

n.d

+1 +1 +1 +l +1 n.d.

n.d. n.d. n.d. n.d. n.d. n.d. n.d n d.

8t

183

1024t l6t

75.03 nd nd n.d n.d. n.d. n.d.

n.d. n.d. n.d. n.d. n.d 2048t

n.d. n d. n.d. n d. n.d.

nd

n.d.

*Specific tltre (see Table 1). tTitre of sahne extract $Hemagglutmatmg umts/mg (as defined m refs [6, 81). t4 Strong agglutmation. tl Weak agglutmatlon

Table 3 Carbohydrate

mlubitlon of agglutination lectm (P4)

by the

D. lehmanm

Carbohydrate D-Glucose

o-Mannose D-Fructose L-Sorbose 2-Deoxyglucose N-Acetylglucosamme Methyl-a-D-mannoside Methyl-a-D-glucoslde Methyl-P-D-glucoslde p-Nltrophenyl-a-D-glucoslde D-!hCrOSe

D-Palatmose D-Trehalose D-Melezltose

Mmimal mlnbltory concentration (mM)

Relative inhibitory actlvlty

50 50 50 25-50 100 100-150 625 12.5 150 0.013 100 25 125 25

2-1 0.5 0.54 3 8 4 0.3 3846 0.5 2 4 2

The relative mhlbitory activity 1s calculated as the ratlo MIC of glucose/MIC of tested sugar The followmg sugars were not mhlbltory at concentrations (mM) gven in parentheses: D-arabmose (150), D-fucose (150), D-galactose (150), D-XylOSC (150), D-&ICOSaIIIinC (150), Dgalactosamme (lOO), N-acetylgalactosamine (lOO), Nacetylneurammlc acid (150), D-galacturomc acid (150), mannitol (150), methyl-p-D-galactoslde (l50), methyl-a-D-galactoslde (150), p-mtrophenyl-a-D-galactoslde (25), D-cellobiose (lOO), Dgentiobiose (lOO), D-hCtOSC (150), D-meliblose (150), D-raffinose (100).

CaZ+ and Mn2+ were bound to the protein, and extensive dialysis against EDTA and 0.1 M acetic acid did not abolish Its hemagglutinating activity as a specific titre of 9.5 was observed. Lectm P2 did not agglutinate rabbit RBCs in the presence or absence ofCa2+ and Mn2+. Cow RBCs were not agglutinated by the P4 lectin which reacted with RBCs from monkey, mice, dog and horse.

The few data available (Table 2) shows that the Dzoclea lectins differ in their ability to agglutinate animal RBCs, suggesting that minor variations in the carbohydrate binding site should exist between them. The P4 lectin was shown to belong to the glucose/mannose group as the agglutination of 0+ RBCs was inhibited to a similar extent by these sugars (Table 3). An interesting furanose binding was also demonstrated, as fructose and sorbose were both inhibitors. Substitution at C-2 lowers the RIA as shown by the values obtained for 2deoxyglucose and N-acetylglucosamine. The importance of free 4-OH and 6-OH groups was apparent from the data obtained with the oligosaccharides and a marked preference for the a-anomeric form was also clear. The elevated inhibitory activity of p-nitrophenyl EDglucoside (RIA = 3846) suggested its binding to the site adjacent to the carbohydrate specific site, as in Con A [ 11, and also demonstrates the specificity for the sugar moiety, as the galactoside analogue was not inhibitory. Overall there was a close similanty between the carbohydrate inhibition patterns of DLL and Con A, the only lectin from the tribe Diocleae for which systematic data is available. Therefore it is likely that the sugar binding site of P4 is very similar to that of Con A. The ammo acid analysis of DLL (Table 4) showed a similar proportion of charged and polar residues (49%) compared to the hydrophobic ones (5 1%). In agreement with its lower PI, the lysine and histidine content appeared to be lower than that found for the tl subunit of DGL [lo] or for Con A [13] With the exception of l/2 cys the values for the rest of the aminoacids were very close to those reported for DGL and Con A. The presence of two l/2 cys is unique to DLL among the lectins from the tribe Diocleae and may be important in maintaining the structure of the protein. The value of M,, on the basis of aminoacid composition, (23 980) agreed well with that calculated from SDS-PAGE (24 845) taking into account the carbohydrate content. The P4 lectin had an absorption value of Ah; cm 14.85 at 280 nm which was slightly higher than those found for Con A (13.7 ref. [ 141) or DGL (12.0 ref. [8]) in spite of their similar content of aromatic amino acids. When assayed for mitogenic activity DLL was able to

G. PEREZ et al

1748 Table

LYS HIS Arg Asp Thr Ser Glu Pro GlY Ala Cyst. Val Met Be Leu Tyr Phe Trp§

4

Ammo

acrd composttton

of the D lehmannt lectm (P4)

mol AA 100 000 g protein

Calculated

restdues mol - 1 Integer

Resrdues mol- ’ Con A* DGLt

20 50 1200 28 10 131 80 87 90 11250 44.96 35 80 64 40 62.55 848 58 50 4.86 5604 7415 26 73 4000

5.09 298 6.98 32.74 2183 27 96 1117 891 1600 15.54 2.10 14.53 121 1392 1842 663 993 5 26

12 6 6 32 19 31 12 II 16 19 0 16 2 15 18 7 11 4

Nearest

5 3 7 33 22 28 11 9 16 16 2 15 1 14 18 7 10 5

10 5 5 65 325 19 33 11 11 17 165 0 17 1 15 5 18 7 12 45

*Data taken from ref [13] tData taken from ref [9] j.Determmed as CySO, §Determmed colortmetncally. Calculattons are based on a M, 25 300 wtth 1 8% carbohydrate.

Table 5 Mttogemc

actrvity

of D. lehmannr lectm

Protem

D lehmannr lectin

PHA P vulgarzs

(pg ml-‘)

% transformed cells

7.5 15.7 23.5 314 174

305 3 50 5 70 3.90 1020

stimulate blast transformatton in lymphocyte cultures from monkey (Aotus Iemurinus griceimembra), rodent (Proechzmrs sp.) and man. The results obtained with human lymphocytes wrth DLL and the phytohemaglutinm (PHA) from Phaseolus vulgaris are shown in Table 5. A maximum of 5.7% transformed cells was obtained at 23.5 pg P4 ml- ‘, that IS 57% of the maxtmal activtty of PHA, indtcatmg that DLL was a potent mttogen. It is noteworthy that for Con A the dose-response curve reaches a plateau [15] while for DLL it passed through a maximum, suggesting that the lectin was cytotoxtc at high concentratrons. The compartson of the structural and functional properties of the P4 lectin with the other characterized lectins from the tribe Diocleae, points to a closer stmtlarity wtth Con A due perhaps to the fact that more data is available for this lectm Although there are minor differences (1.e. presence of carbohydrate and Cys m DLL), the srmrlar properties of the lectms of the tribe Diocleae reinforces the proposal [9] that they may be useful to delimit this tribe. In this regard it will be of interest to determme the primary structure of the P4 lectm

EXPERIMENTAL

Matermls. The seeds of D lehmannz (voucher COL 185000) were collected at Guateque (km 27), Cundmamarca. Phosphorylase b, BSA, ovalbumm, soybean trypsm mhlbttor and a-lactalbumm (all from Pharmacra) were used as standards m SDS-PAGE. The reagents for sequence determmatton were all sequencer grade The rest of the materrals were as m ref. [16] Prelrmmary analyses were done by the methods crted m ref. Cl71 Non-protern nrtrogen was determined by extracting 1 5 g of finely ground seeds wtth 10% CCI,CO,H acid (10 ml) at room temp wtth sturmg for 2 hr After centrrfugmg (10000 g, 20 mm), the pellet was re-extracted ( x 3) the supernatants were pooled and the N content determmed as m ref [16] Isolatton and pur$catron To determine the extraction condtttons, small scale assays (1 5 g undefatted flour) were done by extracting x 4 at 2, 4 and 8 hr (see ref [ 181) wrth yields of 48.3, 58 8 and 71% of total protem respectively and with hemagglutmatmg tltres (1st extract) of 64,128 and 256 Accordmgly 40 g of flour were extracted for 8 hr with 1% NaCl(1 10, w/v) or with a buffer contammg protease mhtbttors (ref [18]), the rest of the extraction procedure was as m ref [18] Fractronal pptn with O-50% and 50-70% satd (NH&SO, was done as m ref [16] The dtalysed 70% satd fraction was freeze-dried and CA 500 mg were dissolved m 1% NaCl and clarrfied by centrtfugatlon The soln was apphed to a column (2 x 73 cm) packed with Sephacryl S-200 equrhbrated and eluted (10 ml hr _ ‘) wrth 1% NaCl After elutron of the non-retamed material (peaks Pl, P2 and P3), 0 1 M Glc was added The peak (P4) eluted with this soln, was exhaustrvely dtalysed against 1% NaCl or 50 mM NH,OAc and freeze-dried Agglutmatlon and mhrbttlon assays and protem, electrophoresu, carbohydrate and ammo acid determrnutlons were performed by the methods cited m ref [16]

Lectin from Dioclea lehmann~ M, ojsubunrts were determined by PAGE-SDS employmg the Tris-HCI buffers and the gels described m ref. [19], using a T&z stacking gel and a T,,Cz 6 separating gel After runnmg 90 mm at 200 V the gels were stamed and destained accordmg to ref Cl91. pl. A 3.5-10 pH gradient was used with the method cited m ref. Cl61. Ammo acid sequences of the N-terminal region were determmed as in ref. [ZO] Mnogentc actrulty was assayed accordmg to ref. [21] Acknowledgements-We wish to thank Dr R. Jaramilllo (ICN, Umversidad National) for his assistance with the botanical classificatton, Dr J. Santome (Umverstdad Buenos Aires) for the ammo acid analysis, Dr M. Bueno (INAS, Bogota) for her help with the assays ofmitogenesrs and Dr M. Richardson (University of Durham) for the trammg m the sequencmg techniques and his kmd revtsron of the manuscript This work was supported by the Orgamzacton de Estados Amencanos and the Departamento de Qmmica (Umversidad National). REFERENCES

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1749

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