Purification and partial characterization of a lectin from Datura innoxia seeds

Phytochemistry,Vol. 34, No. 2, pp. 343 348, 1993 printed in Great Britain.

0031-9422/93 $6.00+0.00 © 1993PergamonPress Ltd

PURIFICATION AND PARTIAL CHARACTERIZATION OF A LECTIN FROM DATURA INNOXIA SEEDS STEFANA-MARIAPETRESCU, ANDREI-JOSEPETRESCUand HAROLD E. F. RUDIGER* Institute of Biochemistry, Splaiul Independentei 296, 79651 Bucharest, Romania; *Institut fiir Pharmazie und Lebensmittelchemie, Am Hubland, D-8700 Wiirzburg, Germany (Receioed in revisedforra 16 February 1993)

Key Word Index--Datura innoxia; Solanaceae; purification; lectins; isolectins.

A b s t r a c t - - A haemagglutinin has been purified from the seeds of Datura innoxia. The purification procedure included

affinity chromatography on chitin followed by ion-exchange chromatography on DEAE-cellulose, and allowed the separation of three isoforms of the lectin. The isoforms differ in specific agglutinating activities and in subunit patterns, consisting of different combinations of two major polypeptides with apparent M,s of 49 and 60-67 × 103. By immunoprecipitation it was shown that the main lectin partially resembles other Solanaceae lectins from D. stramonium seeds and potato tubers, but that they are not completely identical. The D. innoxia lectin has a high affinity for N-acetylglucosamine oligomers.

INTRODUCTION Extracts from many plants possess haemagglutinating activity due to the presence of lectins, i.e. proteins that specifically recognize and bind sugar residues Ell. Lectins especially from the Leguminosae, Gramineae and Solanaceae have been characterized most thoroughly. Solanaceae lectins have been isolated from the seeds of Jimson weed [2], from potato tubers [3] and from fruits of tomato [4], potato E5] and tamarillo E6]. In contrast to lectins from Leguminosae which are rather similar with respect to their molecular structure but very heterogeneous in sugar specificities, Solanaceae lectins are similar both in structure and in sugar specificities. Firstly, they have unusual amino acid compositions with high contents of hydroxyproline, serine, glycine and cysteine Ell, and are highly glycosylated, with up to 50% carbohydrate in the potato lectin E3] and 40% in the Jimson weed lectin C7]. Secondly, they all bind specifically to oligomers of N-acetylglucosamine (GIcNAc). Some differences have been reported in their subunit structures [8]. Datura innoxia L. is a further member of the thorn apple family. Probably, as with D. stramonium, its original habitat was Central America where it was used as a medicinal plant, now it has spread over many countries. In the Old World it was first introduced as an ornamental plant, but is now cultivated in southeastern Europe for the production of scopolamine and other alkaloids [9]. Extracts from its seeds have been shown to agglutinate human blood cells El0, 1 ll. In this report, We describe the isolation of lectins from the seeds of D. innoxia. They agglutinate animal and human blood cells irrespective of the ABH (ABO) system. The main lectin interacts with GlcNAc oligomers and

specifically binds to chitin and fetuin. The thermal stability is similar to that reported for the other Solanaceae lectins. RESULTS AND DISCUSSION

Purification of Datura innoxia seed aoglutinin (DIA)

Crude seed extract was subjected to affinity chromatography on chitin (Fig. la). By elution with 0.1M HOAc, a main fraction and two smaller ones were obtained. A fourth minor fraction which was eluted by 0.5 M HOAc was not studied further. The fractions eluted by 0.1 M HOAc displayed uniform, patterns consisting of four bands at 82, 54, 49 and 41 x 10aM, (Fig. lb). They therefore most probably arise due to inhomogeneities in column packing rather than to the presence of isolectins. Only two of these bands (at 49 and 41 x 10a M, are shared by a chitin purified Datura stramonium seed agglutinin (DSA) preparation (Fig. 2b, lane 4). Additionally, DSA contains two further bands at 32 and 26 x l0 a M,. Broekaert et al. E8] found that in DSA, bands at 45 and 40 x 10aM, belong to the lectin, whereas the other two bands are carbohydrate binding, but non-agglutinating proteins which copurify with the lectin. Apparently such proteins are absent from D. innoxia seeds. The main fraction (Fig. la) was precipitated with ammonium sulphate between 30 and 60 % saturation, and further purified by ion exchange chromatography on DEAE-cellulose. The elution pattern, together with the SDS-PAGE analysis is shown in Fig. 2. Within the gradient, two agglutinating fractions were obtained. The first one (A) which was eluted at 0.2 M NaC1 displayed 343

344

S.-M. PETRESCUet al.

A28C 1.0-

A280 0.1-

A

a

_ /M NoCl .......~i o.s

B

I

0.5

25

Froction no.

0,05-

0.25

t'

5o

4'0

20

60 Froction no.

66 45

66, /.,5 ,

24 14.3

24 14.3

1 1

2

3 /4

Fig. 1. (a) Affinity chromatography of D. innoxia extract. Extract (62 ml) containing 353 mg protein was applied to a column (3.1 x 23 cm) of chitin. The column was washed with water, 1 M NaCI and again with water until the A2ao was at ca 0.03 (not shown). Elution was started with 0.1 M and continued with 0.5 M HOAc as indicated by arrows A and B, respectively. Fractions of 10 ml 5 min- 1 were collected. (b) SDS--PAGE of fractions of Fig. la. Lane 1" fractions 7-13; 2: fractions 21-26; 3: fractions 30-38; 4: partially purified DSA.

mainly one band at 49 x 10a M, in SDS-PAGE and an additional minor one at 82 x 10s M, (Fig. 2b, lane 1). The second fraction (B) appeared at about 0.45 M NaCl and consisted ofessentiaUy the same bands (Fig. 2b, lane 2). This suggests that the isolectins A and B are homodimeric in nature and that they are formed by two monomers of identical or very similar M,. The faint 82 x 103 M, band probably represents residual non-dissociated dimer. The subunits are not linked by disulphide bridges as they are in DSA, since SDS-PAGE either in the presence or absence of 2-mercaptoethanol gave the

2

3

Fig. 2. (a) Ion-exchange chromatography of D. innoxia lectin after affinity chromatography. The main fraction of Fig. la (8.5 ml) containing 15.8 mg protein was applied to a column (2.5 x 20 cm) of DEAE-cellulose. The column was washed with 500 ml of 0.1 M Tris-HCl buffer pH 9.4, then a linear gradient formed from 250 ml of Tris buffer and 250 mi 1.0 M NaCI in Tris buffer was applied (interrupted line). Fractions of 5 ml 5 minwere collected. (b) SDS-PAGE of fractions of Fig. 2a. Lane 1: fraction A; 2: fraction B; 3: fraction C. same patterns (Fig. 3). In fraction B (Fig. 2b, lane 2) further bands in the 60-67 x 103 M, region are seen which are absent from fraction A. After elution with 0.5 M NaCI, a third peak (C) appears which exclusively consists of these multiple 60-67 x 10a M, bands (Fig. 2b, lane 3). All peaks from ion-exchange chromatography behave as lectins. From peak A to C, the specific haemagglutinating activities decrease (titre/A2so = 1070, 360 and 100 for A, B and C, respectively). Interestingly enough, all three isolectins are secreted into the imbibition medium from intact developing seeds. Isolectin C is found in the medium from the first until the fifth day of incubation, whereas the other isolectins are secreted only during the first 24 hr (results not shown). The physiological signifi-

Datura innoxia leetin

cance of this release and the different kinetics are still obscure. Table 1 shows the purification protocol. The procedure resulted in a 12-13-fold overall purification for fraction A. In electrophoresis under non-denaturing conditions at p H 8.3, all fractions from DEAE-cellulose migrate as single bands (not shown). By hydrophobic chromatography, Broekaert et al. resolved the D. stramonium lectin into three isolectins. They demonstrated that the isolectins consist both of hetero or homo combinations of two monomers of 40 and 45 [8], or 28 and 32 x 10s M, [12].

i

345

Gel filtration

By gel filtration on Sephadex G-200, fraction A was resolved into two peaks corresponding to Mrs of 88 and 35x 103 M, (not shown). Both were identical in S D S - P A G E consisting of only one band at 49 x 103 Mr (Fig. 4). The 35 x 103 M r value in gel filtration apparently results from a slight retardation on the column, since M, values lower than 49 x 103M, were not observed in SDS-PAGE. This therefore suggests the existence of a monomeric and dimeric form of the lectin, the monomer weakly interacting with the Sephadex matrix. A similar structure was reported for the D. stramonium lectin [12]. Both forms of DIA separated by gel filtration exhibit haemagglutinating activity with specific activities (titre/A2so) of 1070 and 540 for the 88 and 35 x 10a M, proteins, respectively. In contrast to DIA, DSA gave only one peak at ca 63 x 10aM, on the same gel filtration

66-45-2/-,-.3,

1 2 Fig. 3. Electrophoreses of DSA and DIA. SDS-PAGE according to Laemmli [221, but under non-reducing conditions. Lane 1: DIA (fraction A from Fig. 2a); 2: partially purified DSA.

1

Fig. 4. SDS-PAGE of peaks obtained by gel fltration. Lane 1: first peak at 88 x 103M,; 2: second peak at 35 x 103M,.

Table 1. Purification of D. innoxia lectin from 10 g of seed powder Purification step Crude extract Afl~n. chrom. frs 7-13 frs 21-26 frs 30-38 frs 42-49 Ion exchange chromatogr. (fr. A)

Protein (mg) 353 15.84 8.25 1.1 7.83 5.3

2

Agglutinin amount (titre × ml)

Yield (%)

Specific activity

29760

100

84

8160") 6400 t 1280 6400

75

515 776 1160 817

6.1 9.2 13.8 9.7

5682

52

1072

12.8

Purific. factor 1

346

S.-M. PETRESCUet al. Table 2. Chemical composition of the D. innoxia lectin

Hypro Asx Thr Ser Glx Pro Gly Ala Cys Val Met Ile

DIA (this work)

DSA (ref. [12])

9.3 7.2 5.3 10.8 9.0 7.3 13.7 4.7 13.1 2.2 0.9 2.2

11.3 6.8 6.6 13.2 9.5 7.4 13.2 4.5 15.0 1.3 0.0 0.3

DIA (this work) Leu Tyr Phe His Lys Trp Arg Ara Gal

DSA (ref. [12])

3.1 0.9 1.9 0.7 3.0 1.4 3.3 106 16

1.3 2.1 0.5 0.0 1.1 2.4 3.7 144 11

Amino acid contents are given in mol %, carbohydrates as residues per one lectin molecule (88 x 103 M,).

column, similar to the value of 67 x 103 M, found by Desai et aL [13] after gel filtration on Sepharose 4B.

Table 3. Inhibition of haemagglutinating activity by carbohydrates and giycoproteins

Agglutination

Carbohydrate/ glycoprotein

Concentration required for 50 % inhibition

(GIcNAc)2 (GIcNAc)3 (GIcNAc)4 Fetuin Ovomucoid Mucin Asialomucin

1.25 mM 0.6 mM 0.6 mM 0.03 mg ml- 1 0.015 nag ml- 1 0.03 mg ml- 1 0.015 mg ml-

Datura innoxia seed extracts agglutinate rabbit as well as human erythrocytes. This activity did not depend upon the presence of bivalent metal ions such as Ca 2 + and Mg 2+ (results not shown). Chemical composition In Table 2, the amino acid and carbohydrate compositions of DIA are given and compared with those of DSA. On the whole, the compositions are similar. Both lcctins contain considerable amounts of hydroxyproline and are rich in serine, glutamic acid/glutamine, glycine and cysteine. In detail, however, some differences occur. In particular, DIA contains methionine and histidine which are lacking in DSA. Striking differences are also found among the minor amino acids valine, isoleucine, leucine, tyrosine, phenylalanine and lysine. DIA is a glycoprotein containing as much as 25% of neutral carbohydrate. Arabinose is the predominant sugar, followed by galactose (Table 2). The values found lie in between those reported by others for DSA [12, 13]. Since DIA tolerates heating for 10 rain at 80 ° without a measurable loss in activity, the glycosylation is probably responsible for the thermal stability. High thermal stability has also been observed for other Solanaceae lectins

Unless stated otherwise, carbohydrates are of D-configuration. GIcNAc, giucosamine, N-acetylgalactosamine, a-methylglucopyranose, L-fucose, L-xylose, N-acetylneuraminic acid and melibiose were not inhibitory up to 200 raM.

[14].

ive than N,N'-diacetylchitobiose. The monomer, GIcNAc, does not inhibit even at high concentrations. Thus, the carbohydrate binding site appears to be best adapted to a chain of at least three #(1-~4)-linked N-acetylglucosamine units. Similar sugar specificities were reported for DSA [12], potato [15], tomato [4] and tamarillo [6] lectins. The glycoproteins tested, fetuin, ovomucoid and hog gastric mucin, were also inhibitors for DIA at low concentrations. Desiaiylation of mucin improved its inhibitory potency by a factor of two.

Carbohydrate binding specificity

lmmunochemical experiments

The sugar specificity of DIA was tested by haemagglutination inhibition of the affinity purified preparation (Fig. la) by carbohydrates and glycoproteins (Table 3). Of the carbohydrates tested, only GIcNAc oligomers were inhibitory, N,N',N"-triacetylchitotriose and N,N',N",N'"-tetraacetylchitotetraose being more effect-

As shown by double immunodiffusion (Fig. 5a), antiSolanum tuberosum tuber agglutinin (STA) antibody not only reacted with STA but also with DIA. Between antiSTA and STA there could be observed two lines of precipitation representing two populations of antibodies, probably one directed against the glycosylated portion

Datura innoxia lectin

Fig. 5. Double immunodiffusion of purified DIA, DSA and STA against anti-STA (a) and anti-DIA (b). Wells 1: DIA; 2: STA; 3: DSA.

and the other one against the non-glycosylated portion of the lectin as reported by Allen [15]. DIA presents confluent lines only with one of these populations of antibodies suggesting total identity with only one of the STA moieties. Both Datura lectins react with the anti-DIA antibody (Fig. 5b). The spur emerging from the well with DSA towards the DIA well indicates that only partial identity exists between the two Datura lectins. Conclusion Datura stramonium and D. innoxia, though belonging to the same genus and being very similar in alkaloid content, contain lectins that differ significantly. In view of the fact that the biological role of plant lectins is still a matter of discussion, it will be necessary to further characterize DIA isoforms with respect to chemical struc! ture, immunological properties, intracellular location and to potential natural targets.

EXPERIMENTAL

Materials. Mono- and disaccharides, raffinose, fetuin, asialofetuin, ovalbumin, hog gastric mucin, chitin and reference proteins for gel filtration and electrophoresis were obtained from Sigma. DEAE-cellulose was purchased from Whatman and Sephadex G-200 from Pharmacia. Chitin was purified by washing with HCI and N a O H as described by van Driessche et al. [16]. Seeds of D. innoxia were obtained from the Institute of Plant Protection at F u n d u l e a (Romania). A partially affinity purified preparation of D. sframonium aggiutinin (DSA) was kindly donated by Dr W. J. Peumans (Leuven). All other reagents were of the highest purity available.

347

Hog gastric mucin was partially desialylated by treatment with 0.075 M H2SO 4 for 90 min at 80 ° similar to the method of Svennerholm [17]. Methods. Agglutination assays were performed with human blood group A erythrocytes as previously described [18]. Protein was determined by the method of Lowry etal. [19] using bovine serum albumin as a standard. The carbohydrate content of DIA was determined by the method of Dubois et al. [20]. Oligomers of GlcNAc were prepared by controlled acid hydrolysis of chitin according to Rupley [21]. Purification of DIA. Seeds of D. innoxia were ground and defatted x 3 by 1 hr treatment with CH2CI 2. The dry powder was extracted for 1 hr at room temp. with 10 vol. of 0.5 M HCI containing 0.01 M EDTA and 5 mM thiourea. After centrifugation (176000, 30min), the supernatant was brought to p H 5.0 with 1 M N a O H and the soln recentrifuged. The clear supernatant was subjected to affinity chromatography on chitin (Fig. la). The lectin-containing frs were combined and further subjected to (NH4)2SO 4 fractionation at 30, 60 and 90% satn. After each fractionation step, the suspension was stirred for 1 hr and the precipitate collected by centrifugation. The pellet of the precipitation at 30-60% satn which contained the lectin was resuspended in a minimal vol. of 25 mM Tris-HC1, pH 9.4, dialysed exhaustively against the same buffer for 48 hr and centrifuged at 17600 0 for 30min. The supernatant was subjected to ion-exchange chromatography on DEAE-cellulose (Fig. 2a). The lectin activity occurred in 3 frs (tubes 29-36, 47-52 and 55-59). They were combined separately, dialysed and coned by ultrafiltration. Gel electrophoresis. S D S - P A G E was performed in polyacrylamide gels (12.5% T, 2.6% C) under reducing and non-reducing conditions as described by Laemmli [22]. Bovine serum albumin (66 x 10 a M,), hen ovalbumin (45 x 103 M,), bovine trypsinogen (24 x 103 M,) and chicken egg white lysozyme (14.3 x 10a M,) were used as references. For electrophoreses under non-denaturing conditions the protocol of Davis [23] was followed. Gel filtration. Separation of oligomeric lectin aggregates was achieved by gel filtration on a Sephadex G-200 column (0.80 x 90 cm) in 0.1 M NaOAc buffer, pH 5.0. Frs of I ml were collected and analysed for A2s 0 and haemagglutinating activity. The column was calibrated with rabbit muscle aldolase (158 x 103 M,), bovine serum albumin (66 x 103 M,), hen ovalbumin (45 x 10a M,), bovine chymotrypsinogen (25 x 10a M,) and horse heart cytochrome c (12.5 x 10 a M,). Chemical composition. Samples were subjected to hydrolysis in 6 M HC1 or 4 M methane sulphonic acid and the amino acids determined after ion exchange chromatography using a Spectra Physics 8100 analyser as described in ref. [24]. Monosaccharides were determined by GC, as described previously [25]. Immunological methods. Antibodies against DIA and STA were raised in rabbits by initial immunization with 0.5 mg of purified DIA or STA in 0.5 ml of phosphate buffer saline (PBS) containing complete Freunds adjuv-

348

S.-M. PETItESCtJet al.

ant (Difco). A booster injection of 0.5 mg of DIA was given after 2 weeks using incomplete Freunds adjuvant (Difco). Serum was collected 3 weeks after the initial injection. Radial double immunodiffusion was performed by the Ouchterlony method [26] in 1% agarose gels prepared in PBS pH 7.4 containing 1% (GlcNAc)n. Acknowledgements--We gratefully acknowledge the support from the Deutscher Akademischer Austauschdienst, the Federation of the European Biochemical Societies, the Deutsche Forschungsgemeinschaft and the Fends der Chemischen Industrie. Thanks are also due to Prof. J. Montreuil, Prof. Bouguelet, C. Brassard and C. AIonso for performing amino acid and carbohydrate analyses, to Dr W. Peumans for giving us a sample of DSA, and to Dr L. Buzila for help in raising antisera.

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9. Cucu, V. and Paun, E. (1968) Planta Med. 16, 338. 10. Boyd, W. C. and Reguera, R. M. (1949) J. 1mmunol. 62, 333. 11. Mogos, S. M., Stefan, E., Hulea, S. A., Andras, M., Trif, M., Pop, A., Buzila, L., Dihoru, G., Roman, N. and Motas, C. (1989) Rev. Roum. Biochim. 26, 139. 12. Broekaert, W. F., Allen, A. K. and Peumans, W. J. (1987) Fedn Eur. Biochem, Socs Letters 220, 116. 13. Desai, N. N., Allen, A. K. and Neuberger, A. (1981) Biochem. J. 197, 345. 14. Kilpatrick, D. C. (1983) in Chemical Taxonomy, Molecular Biology and Function of Plant Lectins (Goldstein, I. J. and Etzler, M. E., eds), p. 63. Alan R. Liss, New York. 15. Allen, A. K. (1983) in Chemical Taxonomy, Molecular Biology and Function of Plant Lectins (Goldstein, I. J. and Etzler, M. E.,eds), p. 71. Alan R. Liss, New York. 16. van Driessche, E., Beeckmans, S., Dejaegere, R. and Kanarek, L. (1983) Lectins, Biology, Biochemistry, Clinical Biochemistry 3, 629. 17. Svennerholm, L. (1958) Acta Chem. Scand. 12, 547. 18. Gebauer, G., Schiltz, E., Schimpl, A. and Riidiger, H. (1979) Hoppe-Seyler's Z. Physiol. Chem. 360, 1727. 19. Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193, 265. 20. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A. and Smith, F. (1956) Analyt. Chem. 28, 350. 21. Rupley, J. A. (1964) Biochim. Biophys. Acta 83, 245. 22. Laemmli, U. K. (1971) Nature 227, 680. 23. Davis, B. J. (1964) Ann. N. Y. Acad. Sci. 121, 404. 24. Roughly, P. J. and White, R. J. (1980) J. Biol. Chem. 255, 217. 25. Chambers, R. E. and Clamp, J. R. (1971) Biochem. J. 125, 1009. 26. Ouchterlony, O. (1949) Acta Pathol. Microbiol. Scand. 26, 507.