Tomato lectin is located predominantly in the locular fluid of ripe tomatoes

Plan t Science, 48 ( 1987 ) 71--78

71

Elsevier Scientific Publishers Ireland Ltd.

TOMATO LECTIN IS LOCATED PREDOMINANTLY IN THE LOCULAR FLUID OF RIPE TOMATOES

ROBERTA K. MERKLE and RICHARD D. CUMMINGS*

Department of Biochemistry, University of Georgia, Athens, GA 30602 (U.S.A.) (Received August 20th, 1986) (Revision received October 15th, 1986) (Accepted October 20th, 1986) We have investigated the localization of the tomato lectin within Mill. All of the hemagglutinating activity was found in the fluid and ation of the locular material produced a clear, gel-free extract from to apparent homogeneity in one step by affinity chromatography on a

the ripe fruit of Lycopersicon esculentum placental tissues of the locules. Centrifugwhich the t o m a t o lectin could be purified column of ovomucoid-Sepharose 4B.

Key words: Lycopersicon esculentum; tomato lectin; hemagglutinin; affinity chromatography

Introduction The lectin from the c o m m o n tomato Lycopersicon esculentum Mill. is a non-specific hemagglutinin with regard to blood type [1]. Kilpatrick [1,2] reported that hemagglutinating activity in the tomato is contained largely in the fruit, particularly the juice. Therefore, procedures for purifying tomato lectin employ juice as starting material. However, it has been noted by several investigators that the purification of tomato lectin from tomato juice or extracts is complicated by the presence of components in the extracts which form gels and greatly reduce the flow rates of affinity columns [3,4]. We also encountered these problems in attempts to purify the lectin. These observations prompted us to reexamine the location of the lectin within various tissues and corn-

*To whom all correspondence should be addressed at: Department of Biochemistry, Boyd Graduate Studies Research Center, University of Georgia, Athens, GA 30602, U.S.A. Abbreviations: PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis

partments of the tomato fruit. We have found that greater than 80% of the tomato lectin is contained in the locular fluid and gel of the fruit with the remaining activity in the placental tissue. The lectin is n o t present in significant amounts in the pulp or pericarp. These findings have allowed us to modify a simple and rapid method for isolating tomato lectin [5] without the interference of non-lectin and gel-forming components in the starting extracts. Materials and methods Materials Ripe tomatoes of different varieties were purchased from local groceries and fruit stands. The Rutgers variety of tomatoes were kindly supplied by Mr. A. Falkenhein of Falkenhein Farms in Marissa, Illinois. Ovomucoid (Trypsin inhibitor, Type III-O), tomato lectin, chitin, gel filtration molecular weight markers, Sephacryl S-200 and CNBractivated Sepharose 4B were obtained from Sigma Chemical Co. Protein standards (high molecular weight) for gel electrophoresis were obtained from Bio-Rad. Datura stramonium agglutinin was purified by the

0618-9452/87/$03.50 © Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

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method of Kilpatrick and Yeoman [6] with the modification described by Cummings and Kornfeld [7]. N,N',N"-triacetylchitotriose and N,N',N",N'"-tetraacetylchitotetraose was prepared from chitin by the procedure of Rupley [8].

Preparation of Ovomucoid-Sepharose 4B Affinity Column The ovomucoid affinity resin was prepared from ovomucoid (trypsin inhibitor, Sigma, type III-O) and CNBr-activated Sepharose 4B (Sigma) as described by Nachbar and Oppenheim [5]. The ligand was coupled at an efficiency of 86% such that the final coupling density of the affinity resin was 26 mg of ovomucoid per ml of gel. The column was equilibrated in PBS containing 6.7 mM KH2PO4 and 150 mM NaC1 at pH 7.4.

Hemagglu tination assay The presence o f lectin in the extracts was detected by a modification of the m e t h o d of Kornfeld et al. [9] using a 10% (v/v) suspension of human blood type A erythrocytes. One hemagglutinating unit was defined as that amount causing visible agglutination in 3 rain. Specific activity was expressed as units per milligram of protein as measured by the method of Lowry et al. [10].

Preparation of extracts from the components of whole tomatoes A whole tomato was rinsed in deionized water and divided into its separate components by first removing and reserving the skin, and then dissecting the remaining parts by hand as described below. After removal of the skin the t o m a t o was cut in half transversely (Fig. 1) [11]. The fluid from all the locules was removed gently with a spatula to minimize scraping of the tomato pulp. This fluid was combined and is referred to as 'locular fluid'. The placental material was dissected o u t carefully to avoid inclusion of radial wall tissue. The inner and outer wall pericarp tissues were combined separately from the radial wall. The

tissues, including the reserved skin were minced finely, and the respective volumes measured. The locular fluid was centrifuged at 30 000 X g for 15 min to effect separation into 3 layers: a clear yellow supernatant fraction ('locular supernatant'), a gel-tissue layer ('locular gel'), and a seed layer. The skin, locular gel, locular seeds and the pericarp tissues were homogenized separately using a Polytron. A small amount of PBS was added to the skin and to the seeds to allow homogenization.

Preparation of extracts of the tomato lectin

for purification

Two to five kilograms of store-bought or garden-grown tomatoes were rinsed then halved transversely. The locular material was removed gently by hand and kept on ice. The total volume was measured, and the hemagglutination activity and titer and protein content were determined. The remaining locular fluid (including seeds) was centrifuged at 30 000 X g (16 000 rev./min in a Beckman JA 21 rotor) for 15 min. The clear yellow supernatant fractions were pooled and the volume measured. This liquid was then neutralized by adding 10 N NaOH dropwise while stirring. At this stage, the locular supernatant could be stored frozen for at least 2 months w i t h o u t detectable loss of hemagglutinating activity. The supernatant was applied to an ovomucoid-Sepharose 4B affinity column (25 ml of ovomucoidSepharose 4B in a 60-ml disposable syringe) as described by Nachbar and Oppenheim [5] (see below). Twelve-milliliter fractions were collected at a flow rate of 7.5 ml/min and the absorbance at 280 nm and the hemagglutinating activity were monitored as the effluent was collected to avoid overloading the column. The column was then washed with PBS at the same flow rate as before and the effluent was monitored until the absorbance at 280 nm was below 0.03. The column was then washed with at least two column volumes of 6.7 mM KH2PO4 (pH 7.4) contain-

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Fig. 1. Transverse section of mature t o m a t o fruit (based on Ref. 11). The parts of the fruit body are indicated by the arrows: 1, epidermis; 2, inner wall of the pericarp; 3, outer wall of the pericarp; 4, placental tissue; 5, radial wall of pericarp; 6, locular cavity with jelly-like parenchyma around the seeds.

ing 1 M NaC1, then with at least two column volumes of PBS before beginning the elution. Elution of t o m a t o lectin was effected by the passage of PBS containing 10 mg/ml of an approximately equal mixture of N,N',N". triacetylchitotriose and N,N',N",N'"-tetraacetylchitotraose. Five-milliliter fractions were collected during the elution at a flow rate of 5.5 ml/min. The absorbance of the eluant at 280 nm was monitored during the elution, but hemagglutinating activity could not be assayed due to the presence of the hapten sugar. The fractions that exhibited an absorbance at 280 nm greater thal~ 0.04 were pooled and concentrated on an Amicon YM10 membrane to 10--20 ml. The Amicon filtrate containing the N,N',N"-triacetylchitotriose

and N,N',N",N%tetraacetylchitotetraose was recovered and was reused for other column elutions. The concentrated preparation of lectin was dialyzed overnight against 4 1 of PBS, pH 7.0 with two changes of buffer. The column chromatography as well as the concentration and dialysis steps were carried o u t at 4°C within a 24-h period.

Polyacrylamide Gel Electrophoresis SDS-polyacrylamide gel electrophoresis was performed according to the m e t h o d of Laemmli [12] using 5, 7, 7.5, 10 or 12% gels. The samples were subjected to electrophoresis in a Bio-Rad Protean Slab Gel Electrophoresis Cell cooled b y circulating tap water. The protein bands were stained

74

with Coomassie brilliant blue, and the apparent Mr values were estimated by using a mixture of protein standards containing myosin (200 000), ~-galactosidase (116 250), phosphorylase B (92 500), bovine serum albumin (66 200) and ovalbumin (45 000).

Gel Filtration Chromatography Five milligrams of tomato lectin in 3.2 ml of PBS, pH 7.0 was applied to a Sephacryl S-200 column (2.5 × 78 cm) that had been previously equilibrated in the same buffer. The column was eluted with the same buffer at a flow rate of 20 ml/h. Fractions (3 ml) were monitored for protein by absorbance at 280 nm and for hemagglutinating activity by the assay described above. The column was calibrated with the molecular weight markers, ~-amylase (200 000), alcohol dehydrogenase (150 000), bovine serum albumin (66 000), carbonic anhydrase (29 000) and cytochrome c (12 400). Results and discussion

In order to determine the location of the tomato lectin within the fruit of the tomato, a whole tomato was separated into its various components (Fig. 1), and extracts were prepared from each of these

Table I.

components as described in Materials and Methods. The greatest amount of hemagglutinating activity was found in the locular supernatant, with some activity associated with the locular gel, and a smaller amount associated with placental extract (Table I). N o activity could be detected in extracts of the pericarp tissue, skin and seeds. The locular supematant was then used as the starting material for the purification of the tomato lectin. This fraction from 4 kg of tomatoes was applied to an ovomucoidSepharose column (2.7 × 44 cm), and the column was washed and eluted as shown in Fig. 2. Chromatography was not complicated by the formation of gels in the applied extract, and the elution rate did not decrease. The hapten-eluted samples that exhibited an absorbance at 280 nm greater than 0.04 were pooled (fractions 79--87), concentrated by ultrafiltration and then dialyzed. After dialysis the hemagglutinating titer was found to be 1:256 with a final yield of 11.4 mg of lectin protein. The final yield of activity in relation to the activity in the locular extract was 70% with a 168-fold purification (Table II). This entire procedure was repeated with 4.5 kg of another batch of tomatoes with similar results (data not shown). To investigate the effect of the variety of tomato on

L o c a l i z a t i o n o f h e m a g g l u t i n a t i n g activity in t o m a t o tissue e x t r a c t s .

Tissue

Wt. (g)

Extracted vol. (ml)

Hemagglutination titer

Total units in e x t r a c t

Whole t o m a t o Locular supernatant L o c u l a r gel L o c u t a r seeds Placenta O u t e r wall p e r i c a r p I n n e r wall p e r i c a r p Radial wall Skin

258.0

--

-1/8 1/16 0 1/4 0c

-17 6 0 0 (64) b 5 6 0 0 (20) 0 4 4 0 0 (16) 0c

39.3 a 16.2 128.2 24.6 42.6 ND

22.0 3.5 ND 11.0 120.0 c 25 5

0 0

0 0

aThe components of the locular fluid (supernatant, gel and seeds) were weighed together. bNumbers in parentheses represent the percentage of the total hemagglutinating activity for each component. CThe outer and inner wall pericarp extracts were combined. N D, Not determined.

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Fig. 2. Affinity chromatography of tomato hemagglutinating activity on an ovomucoid-Sepharose-4B column. A locular extract (478 ml) of tomatoes was applied to the column, and the column was then washed and eluted as described in Materials and methods. The arrows indicate the beginning of elution with: 1, PBS; 2, 6.7 mM KH=PO4 (pH 7.4) containing 1M NaCI; 3, PBS; 4, an equal mixture of N,N',N".triacetylehitotriose and N,N',N",N'"-tetraacetylchitotetraose (10 mg/ml). A2a0 represents the absorbance at 280 nm.

t h e yield, a similar e x t r a c t was p r e p a r e d f r o m t h e R u t g e r s v a r i e t y o f t o m a t o . With a s t a r t i n g locular s u p e m a t a n t o f 5 2 0 m l f r o m 4 kg o f t o m a t o e s w e w e r e able t o achieve a 2 3 0 - f o l d p u r i f i c a t i o n w i t h a 78% yield resulting in 11.6 m g o f lectin. Purif i c a t i o n o f t h e lectin f r o m several d i f f e r e n t b a t c h e s o f t o m a t o e s (4 k g / b a t c h ) r e s u l t e d

in yields o f 1 0 - - 3 2 m g w i t h 5 5 - - 2 3 0 - f o l d purification. T h e a f f i n i t y - p u r i f i e d t o m a t o lectin was subjected to electrophoresis under reducing c o n d i t i o n s on S D S - p o l y a c r y l a m i d e gels (Fig. 3). E v e n w h e n t h e gel lanes w e r e o v e r l o a d e d (30 #g o f p r o t e i n ) a single p r o t e i n b a n d was visible b y C o o m a s s i e staining. T h e p o s i t i o n

Table II. Summary of purification of hemagglutinating activity in locular extracts of tomato by affinity chromatography on ovomucoid-Sepharose 4B. Purification step

Vol, (ml)

Hemagglutination (units/ml)

From 4.0 kg tomatoes: I. Locular extract (without seeds)

II. Locular supernatant III. Affinity purified on ovomucoid-Sepharose, Concentrated by ultrafiltration

Total units

Total protein (rag)

Hemagglutination yield

Purification (fold)

(units/rag protein )

(%)

550

800

440 000

2750

160

478

800

382 400

1052

364

100 87

12

25 600

307 200

11.4

26,947

70

1 2.3

168

76

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2

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5

6

200-

116 93-

45-. Fig. 3. SDS-polyacrylamide gel electrophoresis of purified tomato lectin. Thirty #g samples of protein in buffer containing 0.0625 M Tris--HC1 (pH 6.8), 10% (v/v) glycerol, 2% (w/v) SDS, 0.00125% (w/v) bromophenol blue, and 5% (v/v) 2-mercaptoethanol were applied to an SDS-polyacrylamide gel (7%) and subjected to electrophoresis for 5 h at 30 mA. Lane 1, molecular weight standards (kDa), Lane 2, D. stramonium agglutinin, Lanes 3--5, t o m a t o lectin from three separate purifications. Lane 6, commercial t o m a t o lectin. Molecular weights were estimated by comparison wth the protein standards shown. Gels were stained with Coomassie Brilliant Blue.

of the t o m a t o lectin band corresponded to an apparent Mr of 138 000 on a 7% polyacrylamide gel. We have noted a slight variation in apparent

M r among

various

electrophoretic

separations (using different concentrations of polyacrylamide ranging from 5 to 12%). These M r values ranged from 115 000 to 150 000 with an average Mr of 130 000. Under non-reducing conditions, the t o m a t o lectin appeared as an ill-defined band near the top of the gel (data n o t shown). Kilpatrick et al. [3] reported an M r of 68 000 for the t o m a t o lectin subjected to electrophoresis on a gradient gel for 16 h. However, they also demonstrated that the apparent molecular weight of the lectin was much higher than this when the electro-

phoresis time was only 3 h. As shown in Fig. 3 and as observed in gels of other percentages of acrylamide (data n o t shown), the electrophoretic mobility o f t o m a t o lectin prepared in our laboratory was identical to that of commercially available t o m a t o lectin. When the hemagglutinin from D. stramonium was subjected to electrophoresis concurrently with t o m a t o lectin, the apparent subunit Mr's w e r e 37 000 and 50 000 which is in agreement with the values in the literature of 40 000 and 45 000 [6] (Fig. 3). The molecular weight of the native t o m a t o lectin was estimated by gel filtration using a column of Sephacryl S-200 (Fig. 4). The lectin activity eluted in a single symmetrical peak which corresponded to the peak of protein determined by absorbance at 280 nm. The elution volume of the lectin was identical to that of the calibration standard ~-amylase (200 000), which is in exact agreement with the results of Kilpatrick [1]. Portions of each fraction in this peak were subjected to electrophoresis on an SDSpolyacrylamide gel (Fig. 4, inset) and the Mr of each fraction was found to be 138 000, identical to the purified lectin and the same as our previous results. These results suggest that the t o m a t o lectin exists as a dimer in solution, b u t it is possible that the apparent molecular weight of the lectin as determined by gel filtration is also anomalously affected by the large a m o u n t of carbohydrate since the lectin contains 50% carbohydrate b y weight [4,5]. It should be noted, however, that Nachbar et al. [4] determined that tomato lectin has a molecular weight of 71 000 by sedimentation equilibrium centrifugation even in the presence of the hapten N,N'N"-triacetylchitotriose and therefore concluded that the lectin exists as a monomer. Our results demonstrate that the greatest amount of hemagglutinating activity is present in the locular fluid and gel of the tomato. It is interesting to note that the tomato lectin shares many properties in c o m m o n with the lectin from D. stramonium,

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Fig. 4. Size exclusion chromatography of tomato lectin on Sephacryl S-200 followed by SDS-polyacrylamide gel electrophoresls of fractions exhibiting hemagglutinating activity. A 5-mg sample of purified tomato lectin was applied, and the column was eluted as described in Materials and Methods. Fractions were assayed for absorbance at 280 n m (o o) and the fractions yielding positive hemagglutinating activity were then titered (c o). The elution volumes of standards are indicated by the arrows: 1, blue dextran; 2, ~-amylase (200 000); 3, alcohol dehydrogenase (150 000); 4, bovine serum albumin (66 000); 5, carbonic anhydrase (29 000); and 6, cytochrome c (12 400). The inset shows an SDS-polyacrylamide gel of the Sephacryl S-200 fractions that exhibited hemagglutinating activity. Samples (11 #g protein) of each fraction (fractions 56--64, lanes 2--10) and of the starting affinity purified tomato lectin (lane 11) were subjected to electrophoresis under reducing conditions in a 7.5% polyacrylamide gel. The molecular weights were estimated by comparison with the protein standards shown (kDa). Gels were stained with Coomassie BrilliantBlue.

including the inhibition of hemagglutination by chitin-derived oligomers and immunological cross-reactivity with an antibody raised against the tomato lectin [2]. However, the lectin from D. stramonium is present in the seeds of the fruits [6], whereas the tomato lectin is contained in the placental tissue and locular fluid surrounding the seeds. The biological function and significance of the tomato lectin is not understood. In our own and other studies [1,2,4,5], it has been shown that tomato lectin has a high affinity for chitin and oligomers of N-acetyl D-glucoseamine. These polymers exist in insects and microorganisms that m a y attack

the seeds and other parts of the tomato fruit. Although purely speculative, it may be that tomato lectin confers resistance on the fruit to such pathogens. The supematant fraction obtained after centrifugation on the locular fluid and gel from ripe tomatoes can be directly applied to the affinity adsorbent. By several criteria the lectin isolated by this procedure appears homogeneous. The ease of preparation allows the rapid purification of large amounts of lectin, since the affinity adsorbent is easily regenerated and the haptenic sugars required for elution may be recovered and reused repeatedly.

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The lectin thus prepared can be coupled to cyanogen bromide-activated Sepharose 4B immediately after concentrating the affinity purified material; the presence of haptenic sugars is not a complication, since these are normally included in the coupling to Sepharose to prevent coupling of the lectin through its binding site. The haptenic sugars may be easily removed by a simple filtration step. The immobilized lectin retains its hemagglutinating activity and affinity for animal cell glycoconjugates (Merkle and Cummings, manuscript in preparation). Kilpatrick et al. have used immobilized tomato lectin to isolate lymphocyte surface glycoproteins [13]. The ready availability of large amounts of tomato lectin should promote further investigations of the oligosaccharide-binding specificity of the lectin and allow inclusion of immobilized tomato lectin in the general technique of serial lectin affinity chromatography for the separation and analysis of animal cell glycoconjugates. Acknowledgments This investigation was supported by PHS Grant Number R01CA37626, awarded by the National Cancer Institute. R.K.M. was supported by the Public Health Service, National Researgh Service Award Number 1F32CA07795-01 from the National Cancer Institute, DHHS. We thank Ms. Leila Oertel for photo-

graphs of the gels, and Ms. Yvonne Bowers and Ms. Lisa Bright for their assistance in the initial attempts to purify the tomato lectin. We thank Ms. Jacque Marlowe and Ms. Annette Burrell for preparation of the manuscript. References 1 D.C. Kilpatrick, Biochem. J., 185 (1980) 269. 2 D.C. Kilpatrick, Tomato (Lycopersicon esculentum) lectin and serologically related molecules, in: I.J. Goldstein and M.E. Etzler (Eds.}, Chemical Taxonomy, Molecular Biology, and Function of Plant Lectins, Alan R. Liss, Inc., N.Y., 1983, p. 63. 3 D.C. Kilpatrick, J. Weston and S.J. Urbaniak, Anal. Biochem., 134 (1983} 205. 4 M.S. Nachbar, J.D. Oppenheim and J.O. Thomas, J. Biol. Chem., 255 (1980) 2056. 5 M.S. Nachbar and J.D. Oppenheim, Methods Enzymol., 83, Part D (1982) 363. 6 D.C. Kilpatrick and M.M. Yeoman, Biochem. J., 175 (1978) 1151. 7 R.D. Cummings and S. Kornfeld, J. Biol. Chem., 259 (1984) 6253. 8 J.A. Rupley, Biochim. Biophys. Acta, 83 (1964) 245. 9 R. Kornfeld, W.T. Gregory and S. Kornfeld, Methods Enzymol., 28, Part B (1972) 344. 10 O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, J. Biol. Chem., 193 (1951) 265. 11 G.E. Hobson and J.N. Davies, The tomato, in: A.C. Hulme (Ed.), The Biochemistry of Fruits and Their Products, Vol. 2, Academic Press, N.Y., 1971, p. 437. 12 U.K. Laemmli, Nature (London}, 271 (1970) 680. 13 D.C. Kilpatrick, C. Graham and S.J. Urbaniak, Scand. J. Immunol., 24 (1986) 11.