Affinity sorbent for galactose specific lectins consisting of deproteinized plant cell wall ghosts

Plant Science, 82 (1992) 29-35 Elsevier Scientific Publishers Ireland Ltd.

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Affinity sorbent for galactose specific lectins consisting of deproteinized plant cell wall ghosts K e r s t i n L u c k a, R . E h w a l d ", P. Z i s k a b a n d H . K o p p i t z c aHumboldt-Universitiit zu Berlin, Fachbereieh Biologie, lnstitut fiir Pl~mzenphysiologie und Zellbiologie, 0-1040 Berlin. lnvalidenstr. 42. l~lnstitut fiir lmmunpr@arate und Niihrmedien, 0-1120 Berlin. Klement-Gottwald-Allee 317. "Perm.~elect Gmh ll. 0-1017 Berlin. Air Stralau 52-54 (German)').

(Received June 3rd, 1991: revision received November 19th, 1991: accepted November 19th, 1991~

Purified plant cell wall ghosts with high pectin content were investigated for affinity sorption of the galactosc specific plant Icctins from Rieinus eommunis L., Glycine max (L.) Merr. and Viseum album L. The experiments were carried out with the previously described material Permselect prepared from suspension cultured ceils of Chenopodium album L. which has been nriginally developed for permeation chromatography, and a similar material obtained from parenchyma of cabbagehead IBrassica oh,racea L.). After a pretreatment with 2% Na2CO 3 at room temperature, both cell wall materials showed strongly increased capacity for affinity binding of galactose specific lectins from the crude extracts. Ricin (RCA21 is the Rieinus lectin preferently bound to pretreated Permselect. Results are discussed with respect to cell wall composition and porosity as well as practical application of Pcrmselcct for purification of galactose specific lectins. Key words: affinity chromatography: affinity sorbent; lectins: galactose: cell wall composition: cell wall ghost: cell wall porosity: vesicular packing material: Permselect

Introduction

Material and Methods

Lectins are generally isolated from crude extracts or biological fluids by affinity chromatography using immobilized glycoligands. The choice of the affinity sorbent is critical for the efficiency and economy of the purification process. A stationary phase consisting of deproteinized plant cells (i.e. cell wall microcapsules or plant cell wall ghosts) obtained from a higher plant suspension culture has been applied as vesicular packing material (VP) for permeation chromatography [1,2]. We tested this material and a comparable preparation from parenchyma of cabbagehead as affinity sorbents for galactose specific lectins because most terminal nonreducing sugar units in the matrix of dicot primary cell walls are Dgalactose and L-arabinose [3,4].

Affinity sorbents The previously described VP Permselect [1] prepared from cell clusters of a suspension culture of Chenopodium album L. was used. A similar VP was prepared from tissue slices of white cabbagehead (Brassica oleracea L. var. capitata) by the earlier described method using a porcine pancreas multienzyme preparation for decomposition of the included unpermeable or insoluble cell contents [5]. Briefly, for this purpose lipids and pigments were extracted from cabbagehead tissue particles (edge length about 0.5 mm) with 96'7, acidified ethanol (containing phosphoric acid, 0.1% v/v). The material was then incubated in a 0.2% buffered solution (pH 6.5) of 'Trypsin zur Zellzucht' (Biochemie Bernd Belger, Kleinmachnow, Germany) for 2 days, washed extensively with 0.5% NaCI solution, water and ethanol and stored either in ethanol or dried under vacuum from ethanol. VPs from both species were used as

Correspondence to," R. Ehwa[d, Humboldt-Universitfit zu Berlin, Fachbereich Biologie, Institut f/Jr Plfanzenphysiologie und Zellbiologie, lnvalidenstr. 42, 0-1040 Berlin, Germany.

0168-9452/92/$05.00 ~5 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

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affinity sorbent in an untreated and modified variant. The modification was carried out by mixing one volume of 4°/° sodium carbonate solution with one volume of a suspension of the untreated material in distilled water (10 g dry material/I), incubating the mixture for 2 h, neutralization with acetic acid and washing the particles with 1% NaC1 solution (all steps at room temperature, 23°C). Lectin containing crude extracts Defatted Castor bean (Ricinus communis L.) flour and powder from dry mistletoe branches ( Viscum album L.) were extracted by suspension of 3 g dry materials with I 0 ml 0.1 M sodium chloride or 50 mM Tris-HC1 (pH 8) and incubating the suspension for 8 h at 4°C. The yield of extracted lectins was increased by grinding the paste in a mortar. In case of mistletoe, in most experiments thiourea was added to a concentration of 0.01 M. Seeds of soybean (Glycine max L. Merr.) were soaked in water overnight and then homogenized. The homogenate was dispersed in 0.1 M NaCI (30 g/100 ml) and incubated at 4°C for 5 h. Extracts were obtained by either filtration and washing the material with 0.1 M NaCI solution on cellulose acetate filter Synpor 3 (Chemapol Praha) or two centrifugation steps (each 30 min at 10 000 x g). Affinity chromatography Short beds prepared from a slurry of the VP in aqueous buffer (1-5 cm bed length) covered with filter paper were perfused with the crude protein preparation, the washing buffer and lactose solution used for elution. Flow was driven by gravity with a height difference of 30 cm. Sugar solutions and buffers contained NaN 3 (0.05%). Eluate fractions were analyzed for protein content by turbidity measurement after precipitation with 7.5% trichloroacetic acid. Electrophoresis SDS-PAGE was performed by the PhastSystem (Pharmacia) in PhastGel gradient 8-25.

Results

Preliminary experiments with commercial lectin preparations (not shown) proved that significant amounts of wheat germ agglutinin could not be specifically eluted by 0.1 M N-acetylglucosamin from Permselect and that the capacity for binding of Concanavalin A (mannose and glucose specific) to this sorbent was small (7 mg/g dry material) in comparison to that of (galactose specific) Ricinus agglutinin. The erythrocyte agglutinating activity was almost completely (>90%) removed from diluted crude protein extracts of Ricinus communis, Viscrnm album and Glycine max by their filtration through packages of the VPs until beginning saturation of the capacity. The capacity of the

Table I, Lectins eluted with lactose from vesicular cell wall preparations that had been perfused with saturating a m o u n t s of crude extract and washed. Mean values in bold type. Package volume: 1 ml; package dry weight: 8-15 mg; loading: 10 ml of crude extract containing about 500 mg protein of Ricinus communis or 23 ml containing about 100 mg protein of Viscum album, respectively, washing with buffer and elution with lactose solution as described in legend of Fig. I. Affinity sorbent

Lectins eluted with lactose mg/g dry material

Viscum Chenopodium album cell wall particles Untreated 9.5

Treated with Na2CO 3

300 314

Brassica oleracea cell wall particles Untreated

Ricinus

40 30 27 32 390 410 430 466 424

80 80 80

Treated with Na2CO 3

Gel permeation chromatography Self packed columns of Sephadex G 150 were used in connection with a refractometric index detector R I D K (Czechoslovakia) and a peristaltic pump.

280 285 282 Sepharose 4 B

31,8

31

VP for affinity sorption of galactose specific lectins was strongly increased by a treatment of the cell wall particles with 2% sodium carbonate at room temperature. The effect of this treatment for the binding of Ricinus and Viscum lectins is shown in Table I. The pretreated material was tested for its suitability as affinity sorbent for purification of galactose specific lectins. Beds of the VP which had been perfused with crude extracts and washed thereafter with an excess of buffer, released no detectable amounts of protein. The bound lectins (from seeds of Ricinus communis and Glycine max or branches of Viscum album) were eluted by 0.1 M lactose with peak maximum near to the breakthrough of the lactose front (Fig. 1). The binding capacity of pretreated Permselect for the two Ricinus lectins reaches values of nearly the half of the particle dry weight. In spite of the low concentration of the solid phase in the VPs, the lectin binding capacity per volume of pretreated materials is high in comparison to that of Sepharose 4B (Table I). The proteins specifically eluted by 0.1 M lactose from a Permselect bed loaded with mistletoe extract showed a single peak in gel permeation chromatography with Sephadex G 150 superfine at a Kay-value of 0.21, fitting to the molar mass of Eluted lectin (mg)

8r,

VAA1, the main Viscum album agglutinin (115-120 kDa). SDS-PAGE showed the 4 bands described in Refs. 6 and 7: the AB complex at 65 kDa, Al, A2 at approx. 30 kDa, and B chain 34 kDa (Fig. 2a). The hemagglutinating activities of the purified lectins were in all cases increased in comparison to the crude protein solution used for loading the Permselect column (castor bean agglutinin about 12 times, mistletoe and soybean lectins more than 300 times). Electrophoretograms of Ricinus lectin containing fractions eluted from Permselect by lactose showed the bands of Ricinus agglutinin at 120 kDa and Ricinus toxin at 60 kDa (the latter being the major band). Two peak fractions according to the above mentioned molecular masses are also observed in gel permeation chromatography (GPC) of the lactose eluate with Sephadex G 150 (Fig. 2b). The specific hemagglutinating activity of the purified RCAt was about 40-times higher than the specific activity of the crude extract. The ratio between RCAl and RCA 2 in the lactose eluate of a non-saturated Permselect column - - assumed to represent the ratio in the crude extract - - was determined by gel permeation chromatography as 0.43. RCAI/RCA2 ratios obtained from the lactose eluate of a column perfused with an excess of crude extract (about 5-times the volume which is Eluted lectin (rag)

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Fig. 1. Elution of galactose specific lectins from the pretreated vesicular packing material Permselect. Small columns were perfused with saturating amounts of crude extract and washed with buffer. At zero elution with 0.1 M lactose (in buffer) and collection of eluate fractions was started. Columns: 5 ml, 16 mm i.d. × 25 mm in case of Ricinus communis, 1 ml, 9 mm i.d. × 15 mm in the other cases. Washing: 90 ml of 20 mM sodium phosphate, 100 mM NaCI, 0.05% NaN 3 (pH 7.8) in case of Ricinus, 50 ml 0.05 M Tris buffer of pH 7.5 in other cases. Arrows: Breakthrough of lactose (elution volume equals one bed volume).

32

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6 Elution volume (ml)

RCA1

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Elution volume (ml) Fig. 2. Fractionation on Sephadex G 150 (superfine) and SDS-PAGE of lectins eluted from Permselect by lactose. (a) Viseum album lectins, Column: II mm i.d. × 132 ram. Sample: 0.2 ml of lectin peak fraction eluted from Permselect by lactose as shown in Fig. I. Eluent: 20 mM NaC1, 10 mM Tris-HCl (pH 7.3), 0.01% NaN 3, 5 mM lactose, Vo, exlusion volume. Designation of electrophoretic bands as described in Refs. 6 and 7. (b) Ricinus communis lectins. Column: 11 mm i.d. × 118 mm. Sample: 0.4 ml of pooled lectin fractions eluted from Permselect as shown in Fig. I. Eluent: phosphate buffer (pH 7.8) (20 mM sodium phosphate, 100 mM NaCI, 0.05% NAN3). Vo, exclusion volume. Electrophoresis: on the left side lactose eluate from Permselect, on the right side crude extract of Rieinus communis in 50 mM Tris-HC1 (pH 8).

33

necessary to saturate the package) were below 0.12. If RCA 2 is preferentially bound to a Permselect package, RCA] should be enriched in the filtrate relative to RCA2 until the composition of bound lectins is in equilibrium with that of the extract. For checking this point, l-ml fractions of a crude Ricinus extract (5.3 mg protein/mi) were filtered through a column of pretreated Permselect (package volume 1 ml, height 2 cm). While the first filtrate fractions showed no activity, in the fifth fraction hemagglutinating activity reached half of the titer of the crude preparation. The lectins occurring in the fourth and fifth fractions were bound to a second column (1 ml package volume) and, after the necessary washing procedure, desorbed with lactose. They consisted mainly of RCA I (61%). The lactose eluate of the first column contained mainly RCA 2 (93%). A lactose concentration of 0.02 mM was insufficient to dissociate significant amounts of Ricinus lectins from a saturated bed. At sugar concentrations below 0.1 M, the volume necessary for complete elution was dependent on the sugar

1

Eluted leetin (rng)

concentration, at a sugar concentration below 0.01 M elution was incomplete even in 10 packages volumes (Fig. 3).

Discussion Lectin binding capacity and cell wall structure Certainly both the terminal galactose and arabinose in the cell wall matrix are important for lectin binding. Branched oligosaccharide chains consisting of these sugars are main components of cell wall glycoproteins (extensin, arabinogalactan proteins) and heteropolysaccharides, e.g. rhamnogalacturonan I and II, xyloglucans, arabinoxylans and glucuronoarabinoxylans [3]. The investigated lectins belong to a group of plant lectins with an affinity to non-reducing ~-galactose residues which is higher than that to free galactose [8]. It is known, that the affinity of the Ricinus lectins for L-arabinose is little lower than that for D-galactose [9]. Cross-linked arabinogalactan has earlier been used for lectin purification [10]. As shown in this paper, arabinose and galactose at

Eluted lectin (mg) 2

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Fig. 3. Elution of Ricinus lectins from a Permselect package in dependence of lactose concentration. (a) Elution profiles at different concentrations of lactose. Lectins were eluted with lactose containing buffer (4 mM sodium phosphate, 20 mM NaCI, 0.05% NaN 3) from 1 ml beds of the pretreated packing material which had been loaded with crude extract (50 mg protein) and washed with 40 ml buffer (20 mM sodium phosphate, 100 mM NaCI, 0.05% NaN 3, pH 7.8). Eluate fractions (1 ml) were collected for protein determination. Lactose concentration: O, 0.5 mM: +, 10 mM. (b) Total lectin amount eluted with 10 packages volumes of lactose solution at different concentrations of lactose.

34 Table II. Comparison of the specific activities a of the lectin crude extracts and the lectin fractions purified by Permselect. Packages of Permselect pretreated mith sodium carbonate in the case of mistletoe (5 ml, 20 x 16 mm; loaded with 225 mg extract protein), and in the cases af soybean and castor bean ( 1 ml, I0 x 13; loaded with 600 mg and 300 mg extract protein) were washed with phosphate- or tris-buffer (pH 8) (20 m M Na2HPO 4, 0.1 M NaC1, 0.05% N a N 3 or 0.05 M Tris, 0.1 M NaC1, 0.05% NAN3) and than filtrated with 0.1 M lactosebuffer. The protein peak fractions (130-150% of package volume) were analyzed for the specific activity after removal of lactose by gel filtration with Sephadex G 25. Tested solution, protein conc.

Mistletoe (Viscum album) crude extract, 0.51 mg/ml Solution of purified mistletoe lectin, 0.026 mg/ml Soybean (Glycine max) crude extract, 30 mg/ml Solution of purified soybean lectin, 0.13 mg/ml Castor bean (Ricinus communis) crude extract, 3.38 mg/ml Solution of purified agglutinin (RCAI), 0.3 mg/ml

Titer

Spec. act. (ml/mg)

1

2

20

770

280

9

400

3100

1800

530

2000

6700

aTiter of hemagglutination of sialidase-treated h u m a n erythrocytes (blood group O) referred to the protein concentration of the tested solution.

recommended for purification of galactose specific lectins. It shows selective, reversible and reproducible affinity binding of galactose specific lectins from crude preparations and is stable in the pH range 3-10. When lactose is used for lectin elution, its concentration should be 0.1 M or higher in order to get concentrated lectin eluates. At this concentration, most of the bound lectin may be obtained in about one bed volume. Referred to dry weight of the affinity sorbent, the observed lectin binding capacity is very high (0.2 g/g in case of mistletoe lectins and 0.4 g/g in case of Ricinus lectins). The low concentration of the solid phase in chromatographic beds of Permselect (20-25 g dry wt./1), so important for use of the purified cell wall system in permeation chromatography, is not necessary for its application as affinity sorbent. Further improvement in concentration of eluted lectins would be reached, if it were possible to reduce the high liquid absorption capacity of the cell wall particles.

Acknowledgement This project was supported by the Biotechnology program of the Bundesministerium ffir Forschung und Technologie. Authors thank Prof. Dr. med. habil. Dr. rer. nat. H. Franz, Institut ffir Immunprfiparate und N~ihrmedien, Berlin, for helpful discussions.

References their native site in the cell wall may be likewise applied, especially if the wall is treated in such way that the penetration of protein into the polysaccharide matrix is facilitated. The exclusion limit of the cell wall matrix is increased by treatment with sodium carbonate from 6.5 nm to more than 10 nm [2]. This means, that the investigated lectins are excluded from the native cell wall but can permeate in the pretreated material. The change in porosity explains why the alkaline pretreatment has increased the lectin binding capacity.

Practical application The chromatographic packing material Permselect prepared from suspension cultured cell clusters and treated with sodium carbonate may be

1

2

3

4

5 6

R. Ehwald, G. Fuhr, M. Olbrich, H. G6ring, R. Kn6sche and R. Kleine, Chromatography based on membrane separation with vesicular packing material. Chromatographia, 28 (1989) 561-564. R. Ehwald, P. Heese and U. Klein, Determination of size limits of membrane separation in vesicle chromatography by fractionation of polydisperse dextran. J. Chromatogr., 542 (1991) 239-245. M. McNeil, A.G. Darvill, S.C. Fry and P. Albersheim, Structure and function of the primary cell walls of plants. Annu. Rev. Biochem., 53 (1984) 625-663. S.C. Fry, Cross linking of matrix polymers in the growing cells of Angiosperms. Annu. Rev. Plant Physiolol., 37 (1986) 167-186. R. Ehwald and G. Fuhr, Vesikul~ires Trenn-, Frill- und Tr~igermaterial, Patent D D 247 570 (1987). R. Samtleben, M. Kiefer and P. Luther, Characterization of the different lectins from Viscum album (mistletoe) and

35 their strucutral relationships with the agglutinins from Abrus precatorius and Ricinus communis, in: T.C. BogHansen and J. Breborowicz (Eds.), Lectins: Biology, Biochemistry, Clinical Biochemistry, Vol. 4, Proc. 6th. Lectin Meeting, Poznan 1984, De Gruyter, Berlin, 1985, pp. 617-626. H. Franz, Viscaceae lectins~ in: H. Franz (Ed.), Advances in Lectin Research, Vol. 2, VEB Verlag Volk und Gesundheit, Berlin, 1989, pp. 28-59. I.J. Goldstein and R.D. Poretz, Isolation, physicochemical characterization and carbohydrate-binding specificity

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oflectins, in: I.E. Liener, N. Sharon, l.J. Goldstein (Eds.), The Lectins - - Properties, Functions, and Applications in Biology and Medicine, Academic Press, Orlando, 1982. pp. 119-196. G.L. Nicolson, J. Blaustein and M.E. Etzler, Characterization of two plant lectins from Ricinus communis and their quantitative interaction with murin lymphoma. Biochemistry, 13 (19741 196-198. T. Majumdar and A. Surolia, Cross-linked arabinogalactan: A new affinity matrix for the purification of Ricinus comrnunis lectins. Experientia, 34 (1978) 979-980.