Planta
Planta I42, 97 101 (1978)
9 by Springer-Verlag 1978
The Isolation and some Properties of a Leetin (Haemagglutinin) from Cucurbita Phloem Exudate D.D. Sabnis and J.W. H a r t Department of Botany, University of Aberdeen, Aberdeen AB9 2UD, U.K.
Abstract. The occurrence of high haemagglutinating (lectin) activity in p h l o e m exudate f r o m three cucurbit species is reported. The protein responsible for this lectin activity in Cucurbita m a x i m a Duch. has been isolated by cation exchange c h r o m a t o g r a p h y on Sepharose and identified by gel electrophoresis. The lectin showed agglutinating activity at concentrations as low as 0.1 lag/ml. N o sugar, including those transported in the phloem o f these species, interacted with agglutination. The lectin could not be extracted f r o m cucurbit seed, but a p p e a r e d in 5-day old seedlings. The possible role o f a lectin in the sieve element is discussed. Key words: Cucumis - Cucurbita - Haemagglutinin - Lectin - Phloem exudate proteins - Phytohaemagglutinin.
Introduction Lectins (phytohaemagglutinins) are proteins or glycoproteins that bind to c a r b o h y d r a t e groups in a highly selective and specific m a n n e r (Sharon and Lis, 1972; Lis and Sharon, 1973; Liener, 1976). Such lectins have been f o u n d p r e d o m i n a n t l y in the seeds of plants, in particular those o f t h e legumes, but are also f o u n d in other parts such as the roots, leaves and bark (Sharon and Lis, 1972; Callow, 1976). While m u c h w o r k has been published on the effects of lectins on m a m m a l i a n cells, little is k n o w n of their origins or roles in the plants f r o m which they are derived. There is some evidence that lectins m a y function in cell recognition mechanisms that involve the cell wall or glycocalyx, including incompatibility systems (Matsson et al., 1974; Weise and H a y w a r d , 1972), m o r p h o genesis (Rosen et al., 1973) and host plant-microorganism interactions (Albersheim and Anderson, 1971 ; H a m b l i n and Kent, 1973). Recently, however, Kauss and Ziegler (1974) reported lectin activity in sieve tube sap f r o m Robinia
pseudoacacia. The function o f a lectin in a sugar-
conducting channel is of obvious interest. We n o w report on the occurrence of high haemagglutinating activity in p h l o e m exudate f r o m three cucurbit species, Cucurbita maxima, Cucumis sativus and Cucumis melo. The protein c o m p l e m e n t of p h l o e m sap f r o m Cucurbita m a x i m a has been well studied (Beyenbach et al., 1974; Eschrich et al., 1971; Sabnis and Hart, 1976; Sloan et al., 1976; Weber et al., 1974) and we have been able to identify, isolate and study some properties o f the protein responsible for lectin activity in this species.
Materials and Methods Phloem exudate from cut stems of Cucurbita maxima Duch. 'Golden Delicious' (pumpkin), Cucumis sativus L. 'Telegraph' (cucumber) and Cucumis melo L. 'Blenheim Orange' (melon) was collected into 0.9% saline +0.1 M dithiothreitol or 0.14 M 2-mercaptoethanol, to a final concentration of approximately 1.0 mg/ml (Sabnis and Hart, 1976; Sloan et al., 1976). Protein was assayed by the method of Lowry et al. (1951) using bovine serum albumin as the standard. Interference in the protein assays due to the presence of thiol reagents was overcome using the method of Ross and Schatz (1973). Haemagglutination Assay
Serial two-fold dilutions of exudate were tested for lectin activity using a standard haemagglutination assay (Burger, 1974). Typed human erythrocytes were washed several times in saline and used for agglutination tests as 2-5% suspensions. Agglutination was scored by assessing the percentage of single erythrocytes at x 400 magnification. The end-point titre was determined for each assay as the greatest dilution at which agglutination was just visible. Sodium Dodecyt Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
Gel electrophoresis of exudate proteins was carried out in 7.5% polyacrylamide gels containing 0.1% SDS (Weber and Osborn, 1969) and 4M urea. Gels were cast to a length of 68 mm in glass tubes 75 x 6 mm i.d. Electrophoresis was performed at a constant
0032-0935/78/0142/0097/$01.00
98 current of 6.4 mA/gel. After electrophoresis, the gels were fixed in 10% trichloroacetic acid for i h, stained with Coomassie Blue R25(; (Weber and Osborn, 1969) for 1 h, and destained according to Vesterberg (1972), all at 50~ Protein samples were prepared for electrophoresis by adding urea to provide a final concentration of 8 M: the samples were then mixed with equal volumes of 1% SDS in 0.01 M phosphate buffer, pH 7,1, containing 1% 2-mercaptoethanol, and heated to 100~ for 1.5 rain.
D.D. Sabnis and J.W. Hart: Lectin from Cucurbita Phloem Exudate
a
b
c
d
e
Column Chromatography Gel filtration was carried out on Sephadex G-100 in 0.9% NaCI+ 1% 2-mercaptoethanol calibrated with standard proteins (column- 17 x 1.6 cm; void volume -9,0 ml). A 0.5 ml sample of exndate was washed through the column with saline-mercaptoethanol at a flow rate of 40 ml/h; 1.0 ml fractions were collected. To determine the peak of lectin activity, individual fractions were tested for haemagglutinating activity using the dilution assay.
Ion Exchange Chromatography The lectin did not bind to DEAE resin at pH 7.0. Preliminary experiments showed that at this pH the activity was adsorbed to Sephadex CM-50, though not to Sephadex CM-25. The lectin was purified by cation exchange chromatography on a 5.0 ml column of CM Sepharose CL 6B. The column was activated with 50 ml of 2 M NaCI and washed with 50 ml of 0.025 M Tris-HC1 buffer, pH 7.2. A 3.0 ml sample of exudate in 0.025 M Tris buffer + 1% 2-mercaptoethanoi was applied and the c~Ium~ washed with 20 ml of buffer alone to remove acidic proteins and the mercaptoethanol. Elution was carried out in a linear salt gradient of 50 ml buffer +50 ml 0.4 M NaCI. The optical density at 280 nm was monitored and 1.0 ml fractions collected,
Results Samples of exudate f r o m all three p l a n t species were highly active, d e m o n s t r a t i n g h a e m a g g l u t i n a t i n g activity at titres d o w n to 1/2048 after 1 h. A t these endp o i n t dilutions, total p r o t e i n c o n c e n t r a t i o n in the inc u b a t e d drop was a p p r o x i m a t e l y 0.5 gg/ml. N o n e of the exudate samples showed a n y specificity t o w a r d s the m a j o r h u m a n b l o o d groups, A1, A2, B, 0-rhesus negative a n d 0-rhesus positive. The presence of the thiol reagents, dithiothreitol or 2 - m e r c a p t o e t h a n o l , was necessary in order to retard the characteristic gelling of c u c u r b i t exudate (Walker, 1972). T o see if these reagents h a d a n y effects o n exudateLinduced erythrocyte a g g l u t i n a t i o n , exudate from C. m a x i m a was collected into thoroughly degassed saline, in which the proteins are soIuble to a c o n c e n t r a t i o n of a p p r o x i m a t e l y 250 ~g/ m l : such samples were then tested for a g g l u t i n a t i n g activity in the presence a n d absence of thiol reagents. The results indicated that the e n d - p o i n t titre was lowered by half in the presence of the thiol reagents, b u t there was no other recognizable effect o n the a g g l u t i n a t i o n assay.
-H
Fig~. I a-e. SDS-PAGE of proteins in exudate from Cucurbita mccxima. a and d Complete complement of exudate proteins from C. maxima, h Exudate sample after treatment with blood cells to remove lectin (fraction 2). The extra band (t4) is haemoglobin released from the erythrocytes during the centrifugation procedures. e Control, lacking exudate, in which blood cells were subjected to similar washing and centrifugation treatments, e Lectin (fraction 2 protein from C. maxima) purified by CM Sepharose ion exchange chromatography to electrophoretic homogeneity
If the exudate proteins f r o m C. m a x i m a were allowed to gel or precipitate out of solution (by lowering the c o n c e n t r a t i o n of thiol reagent) a n d were r e m o v e d by c e n t r i f u g a t i o n at 3,000 g, lectin activity was lost f r o m the s u p e r n a t a n t fraction. Electrophoresis of the gelled p r o t e i n after several washes in saline which lacked thiol reagents d e m o n s t r a t e d the presence in the gel fraction of all the m a j o r exudate proteins.
Distribution o f Lectin Activity in Cucurbit Seedlings Lectin activity could n o t be detected in extracts of dry seeds of the three c u c u r b i t species. Extracts or diffusates from the cotyledons of g e r m i n a t i n g seeds r e m a i n e d free of lectin activity u n t i l the seedlings were at least five days old. After this period, exudate from the cut h y p o c o t y l or radicle displayed definite haema g g l u t i n a t i n g activity. Electrophoresis of samples
D.D. Sabnis and J.W. Hart: Lectin from Cucurbita Phloem Exudate of exudate from 5-day old seedlings presented protein patterns identical to those obtained from the phloem exudate of mature plants with respect to types of protein subunits as well as their relative concentrations.
Tests for "Hapten' Inhibition
Inhibition by sugars of complex formation between lectins and surface carbohydrate groups of the erythrocytes can give an indication of group specificity. Thus, several sugars were examined for their ability to inhibit agglutination (Lis and Sharon, 1973 ; Kauss and Ziegler, 1974; Burger, 1974) using 0-negative human erythrocytes. The sugars (10-100 mM) were tested against lectin titres which caused approximately half-maximal agglutination or were added to serial dilutions of the lectin. None of the following sugars had any marked inhibitory effect on the rate of agglutination or the end-point titre of cucurbit phloem lectin:-sucrose, N-acetyl-D-galactosamine, N-acetyl-D,glucosamine, D-galactose, D-glucose, Lfucose, D-mannose, D-xylose, D-raffinose, D-stachyose, L-arabinose, D-trehalose, D-melibiose, 2-deoxy-D-glucose, 3-O-methylglucose, p-nitrophenyl-~D-glucoside, p-nitrophenyl-~-D-galactoside. Mixtures of galactose and mannose, galactose and fucose, and mannose and fucose were also ineffective. Control experiments with castor bean lectin and D-galactose provided the expected levels of inhibition, showing that the lack of sugar inhibition with cucurbit lectin was not due to procedures employed.
Isolation of Phloem Lectin from C. maxima
In order for agglutination to occur, the lectin must first bind to saccharide groups on the erythrocyte membrane. Since phloem exudate from C. maxima contains relatively few major proteins (Fig. l a), it was possible to distinguish the protein removed from solution by t h e erythrocytes. One of the major proteins (fraction 1) comprises 60-80% of total exudate protein in this cultivar of C. maxima, while a second (fraction 2) represents another 15-25% (Fig. 1 a). Aliquots of a 50% suspension of thoroughly washed erythrocytes were added to a sample of exudate, and the agglutinated blood was removed by gentle centrifugation. The procedure was repeated until no further agglutination occurred. A final centrifugation removed all suspended blood cells. Control and treated samples were made 8 M with urea and prepared for SDS-PAGE.
99 A comparison of the protein patterns after electrophoresis indicated that while fraction 1 was present in comparable amounts in control and treated samples, fraction 2 was absent in treated preparations of exudate (Fig. i b). The procedure resulted in some release of haemoglobin from the erythrocytes (Fig. 1c). From the concentration of fraction 2 relative to total protein, it can be calculated that the lectin displayed detectable agglutinin activity at a concentration of approximately 0.1 gg/ml. In previous work (Sabnis and Hart, 1976) we reported that the molecular weight (MW) of fraction 2 protein was 22,000 as determined by SDSPAGE. Gel filtration of exudate proteins from C. maxima through Sephadex G-100 showed that the lectin activity was eluted from the column at a position corresponding to a partition coefficient, Kav= 0.58, which approximates to a MW of 20,000. Earlier workers (Beyenbach et al., 1974; Weber et al., 1974) reported that fraction 2 protein possesses a relatively high content of basic amino acids, especially lysine. In the present work, ion exchange chromatography of exudate proteins on a column of CM Sepharose CL 6B yielded a fraction which was eluted at 0.1 M NaC1 and which displayed high optical density at 280 nm, a positive response in the Lowry assay for proteins, and high lectin activity. SDS-PAGE of this fraction revealed a single protein band corresponding to fraction 2 protein (Fig. 1 e).
Discussion
The function of a lectin in the sieve tube is unclear, particularly since the sugar specificity of the lectin has not yet been characterized. Hapten inhibition of haemagglutination is not always possible (Sharon and Lis, 1972). It is unlikely that we have not tested the appropriate sugars, since this would imply that the erythrocyte cell surface possesses very unusual sugars. It has become obvious that saccharide binding sites on lectins are much more complex than may appear from hapten inhibition studies with simple sugars (Lis and Sharon, 1973). Nevertheless, it is of interest that raffinose and stachyose, the sugars that are transported in cucurbit phloem (Webb and Gorham, 1964), do not inhibit haemagglutination. Indeed one could argue that if the lectin was not directly involved in sugar translocation, its presence in the sieve tube might demand particularly high saccharide binding specificity. The evidence clearly demonstrates that fraction 2 protein is a lectin. Thus, we have isolated to electrophoretic purity, a protein from phloem exudate of C. maxima with haemagglutinating activity at a
100 concentration as low as 0.1 ~tg/ml. During agglutination the lectin binds to the erythrocyte surface and is removed from solution. Since dialysed or purified fractions of the lectin remain highly active, cofactors are not involved in the binding reaction. Sephadex gel filtration studies indicate that the Iectin is active as a haemagglutinin in the monomeric form. In its relatively low molecular weight (22,000) and high content of half-cystine residues, the cucurbit lectin is unusual although not unique. Pokeweed mitogen (Reisfeld et al., 1967) and wheat germ agglutinin (Allen et al., 1973) both contain large amounts of half-cystine. Furthermore, wheat germ agglutinin has a molecular weight of 17,000 (Nagata and Burger, 1974), while pokeweed haemagglutinins have molecular weights ranging from 25-31,000 (Waxdal, 1974). Most other purified lectins are k~own to be active as agglutinins in the form of dimers or tetramers, but are comprised of protomeric polypeptide chains ranging from MW 20-30,000. However, the cucurbit lectin does differ from seed lectins so far analysed in its high proportion of basic amino acids and consequently high isoelectric point (Weber et al., 1974; Beyenbach et al., 1974). Despite the immense research effort expended on lectins, the functions of these proteins and glycoproteins in plants remain unknown. For the first time a lectin has now been localized within a single plantcell type modified for a specific function. Furthermore, the relative concentration of lectin in the Cucurbita sieve tube (15-25% of total protein) is also higher than the levels reported for most leguminous seeds which have so far been the principal source of phytohaemagglutinins. It is possible that the lectins play a role in protecting the sugar-rich phloem from bacterial or fungal invasions (suggested to us by Prof. H. Ziegler). Inhibition of fungal growth by wheat germ agglutinin has been reported (Mirelman et al., 1975). Albersheim and co-workers (Albersheim and Anderson, 1971; Albersheim et al., 1969; Anderson and Albersheim, 1972) have proposed an alternative role for lectins in host resistance to pathogenic fungi. They suggest a direct role for lectins in inhibiting fungal cell-wall degrading hydrolases, particularly polygalacturonase, through their ability to bind to carbohydrate moieties. However, with regard to the sieve tube, we consider that a defense mechanism would be more likely to be located outside rather than within the sieve element. An alternative possibility is suggested by the following observations. The characteristic pattern of protein subunits obtained after SDS-PAGE of exudate from C. m a x i m a 'Golden Delicious' remains constant irrespective of the organ (hypocotyl, stem, petiole or fruit) or the age of the tissue from which the exudate is obtained
D.D. Sabnis and J.W~Hart: Lectin from Cucurbita Phloem Exudate (Sabnis and Hart, manuscript in preparation). This is true even for exudate obtained from the hypocotyls of seedlings only 5 or 6 days old. In all these cases, the relative concentration (w/v) of fraction 2 protein remains approximately 25% of that of fraction 1 protein. Since the molecular weights of the two proteins differ by a similar ratio (86,000:22,000; Sabnis and Hart, 1976) it is possible that the two proteins are associated in vivo in a stoichiometric 1 : 1 relationship. If exudate from C. m a x i m a is collected directly onto formvar-coated copper grids, negatively stained and examined under the electron microscope, bundles of P-protein filaments are visible (Kollmann et al., 1970). On exposure to air or buffers lacking thiol reagents, the filaments aggregate into a gel, In the presence of thiol reagents the filaments depolymerise into soluble subunits. Both fraction 1 and franction 2 protein are rich in half-cystine residues (Beyenbach et al., 1974; Weber et al., 1974). Furthermore, Kleinig et al. (1975) have demonstrated the reversible formation of filaments from fraction 1 protein under oxidising conditions. Under such conditions, fraction 2 protein forms amorphous aggregates. In view of these observations, we favour the possibility that, in vivo, fraction 1 protein (in filamentous form) and fraction 2 protein (the lectin) are linked together by disulphide bridges. We suggest that the lectin could serve to anchor the P-protein filaments to glycoprotein or glycolipid components of the sieve element plasma membrane. This would also ensure a parietal distribution of the filaments, facilitating flow of solution through the sieve tube (Evert et al., 1973). The necessity for the P-protein filaments to be anchored during normal sugar translocation in the sieve tube is generally accepted (Cronshaw, 1975). Electron microscopy has not been able to provide evidence for or against a chemical association between filaments and membranes in the sieve element. A lectin bridge might serve this function very well. Sudden release of turgot pressure during damage or excision could possibly break such a weak bond, permitting the plugging of sieve pores by filamentous aggregates. Whether lectins play multifarious roles in the plant, with a unique function in the sieve element, or whether their presence in the phloem is in keeping with some general function of these proteins in cellcell recognition processes, is a problem demanding greater consideration. We gratefullyacknowledgethe adviceand assistance of Dr. Brodie H. Lewis and Mr. R. Main of the Regional Blood Transfusion Service, Aberdeen. We also thank Messrs I. Skene, E. Middleton and J. McRobb for technical assistance.
D.D. Sabnis and J.W. Hart: Lectin from Cucurbita Phloem Exudate
References Albersheim, P., Anderson, A.J.: Proteins from plant cell walls inhibit polygalacturonases secreted by plant pathogens. Proc. Natl. Acad. Sci., USA 68, 1815-1819 (1971) Albersheim, P., Jones, T.M., English, P.D.: Biochemistry of the cell wall in relation to infective processes. Ann. Rev. Phytopathol. 7, 171 i94 (1969) Allen, A.K., Neuberger, A., Sharon, N. : The purification, composition and specificity of wheat germ agglutinin. Biochem. J. 131, 155 162 (1973) Anderson, A.J., Albersheim, P.: Host pathogen interactions V. Comparison of the abilities of proteins isolated from 3 varieties of Phaseolus vulgaris to inhibit the polygalacturonases secreted by three races of Collectotrichum lindemuthianum. Physiol. Plant Pathol. 2, 339 346 (1972) Beyenbach, J., Weber, C., Kleinig, H.: Sieve tube proteins from Cucurbita maxima. Planta 119, 113-124 (1974) Burger, M.M. : Assays for agglutination with lectins. In: Methods in enzymology, pp. 615 621. Vol. 32, Part B. Fleischer, S., Packer, L., eds. New York: Academic Press 1974 Callow, J.A.: Plant lectins. In: Commentaries in plant science, pp. 221 233. Smith, H., ed. Oxford: Pergamon Press 1976 Cronshaw, J. : P-proteins. In: Phloem Transport, pp. 79-115. Aronoff, S., Dainty, J., Gorham, P.R., Srivastava, L.M., Swanson, C.A., eds. New York-London: Plenum Press 1975 Eschrich, W., Evert, R.F., Heyser, W.: Proteins of the sieve tube exudate of Cucurbita maxima. Planta 100, 208 221 (1971) Evert, R.F., Eschrich, W., Eichorn, S.E.: P-protein distribution in mature sieve elements of Cucurbita maxima. Planta 109, 193 210 (1973) Hamblin, J., Kent, S.P.: Possible role of phytohaemagglutinin in Phaseolus vulgaris L. Nature-New Biol. 245, 28-30 (1973) Kauss, H., Ziegler, H.: Carbohydrate-binding proteins from the sieve-tube sap of Robinia pseudoacacia L. Planta 121, 197 200 (1974) Kleinig, H., Thones, J., Dorr, I., Kollmann, R. : Filament formation in vitro of a sieve tube protein from Cucurbita maxima and Cucurbita pepo. Planta 127, 163-170 (1975) Kollmann, R., Dorr, I., Kleinig, H. : Protein filaments-structural components of the phloem exudate. I. Observations with Cucurbita and Nicotiana. Planta 95, 86-94 (1970) Liener, I.E. : Phytohemagglutinins (Phytolectins). Ann. Rev. Plant Physiol. 27, 291-319 (1976) Lis, H., Sharon, N. : The biochemistry of plant lectins (phytohemagglutinins). Ann. Rev. Biochem. 42, 541 574 (1973) Lowry, D.H., Rosebrough, N.J., Farr, N.L., Randall, R.L.: Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275 (1951) Matsson, O., Knox, R.B., Heslop-Harrison, J., Heslop-Harrison, Y. : Protein pellicle of stigmatic papillae as probable recognition site in incompatibility reactions. Nature 247, 298-300 (1974)
101 Mirelman, D., Galun, E., Sharon, N., Lotan, R.: Inhibition of fungal growth by wheat germ agglutinin. Nature 256, 414-416 (1975) Nagata, Y., Burger, M.M.: Wheat germ agglutinin. Molecular characteristics and specificity for sugar binding. J. biol. Chem. 249, 3116 3122 (1974) Reisfeld, R.A., Borjeson, J., Chessin, L.N., Small, P.A. : Isolation and characterisation of a mitogen from pokeweed (Phytolacca americana). Proc. Natl. Acad. Sci. USA 58, 2020 2027 (1967) Rosen, S.D., Kafka, J.A., Simpson, D.L., Barondes, S.H.: Developmentally regulated carbohydrate-binding protein in Dictyostelium discoideum. Proc. Natl. Acad. Sci. USA 70, 2554~557 (1973) Ross, E., Schatz, G.: Assay of protein in the presence of high concentrations of sulfhydryl compounds. Anal. Biochem. 54, 304-306 (1973) Sabnis, D.D., Hart, J.W. : A comparative analysis of phloem exudate proteins from Cucumis melo, Cucumis sativus and Cucurbita maxima by polyacrylamide gel electrophoresis and isoelectric focusing. Planta 130, 211 218 (1976) Sharon, N., Lis, H.: Lectins: cell-agglutinating and sugarspecific proteins. Science 177, 949-959 (1972) Sloan, R.T., Sabnis, D.D., Hart, J.W.: The heterogeneity of phloem exudate proteins from different plants: a comparative survey of ten plants using polyacrylamide gel electrophoresis. Planta 132, 97-102 (i976) Vesterberg, O.: Isoelectric focusing of proteins in polyacrylamide gels. Biochim. Biophys. Acta 257, 11-19 (1972) Walker, T.S.: The purification and some properties of a protein causing gelling in phloem sieve tube exudate from Cucurbita pepo. Biochim. Biophys. Acta 257, 433 444 (1972) Waxdal, M.J. : Isolation, characterization and biological activities of five mitogens from pokeweed. Biochemistry 13, 3671 3677 (1974) Webb, J.A., Gorham, P.R.: Translocation of photosynthetically assimilated C 14 in straight-necked squash. Plant Physiol. 39, 663 672 (1964) Weber, C., Franke, W.W., Kartenbeck, J. : Structure and biochemistry of phloem-proteins isolated from Cucurbita maxima. Exp. Cell Res. 87, 79-106 (1974) Weber, K., Osborn, M.: The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. biol. Chem. 244, 4406-4412 (1969) Weise, L., Hayward, P.C.: On sexual agglutination and matingtype substances in isogamous dieocious Chlamydomonas. III. The sensitivity of sex cell contact to various enzymes. Am. J. Bot, 59, 530 536 (1972)
Received 3 April; accepted 9 May 1978
Planta I42, 97 101 (1978)
9 by Springer-Verlag 1978
The Isolation and some Properties of a Leetin (Haemagglutinin) from Cucurbita Phloem Exudate D.D. Sabnis and J.W. H a r t Department of Botany, University of Aberdeen, Aberdeen AB9 2UD, U.K.
Abstract. The occurrence of high haemagglutinating (lectin) activity in p h l o e m exudate f r o m three cucurbit species is reported. The protein responsible for this lectin activity in Cucurbita m a x i m a Duch. has been isolated by cation exchange c h r o m a t o g r a p h y on Sepharose and identified by gel electrophoresis. The lectin showed agglutinating activity at concentrations as low as 0.1 lag/ml. N o sugar, including those transported in the phloem o f these species, interacted with agglutination. The lectin could not be extracted f r o m cucurbit seed, but a p p e a r e d in 5-day old seedlings. The possible role o f a lectin in the sieve element is discussed. Key words: Cucumis - Cucurbita - Haemagglutinin - Lectin - Phloem exudate proteins - Phytohaemagglutinin.
Introduction Lectins (phytohaemagglutinins) are proteins or glycoproteins that bind to c a r b o h y d r a t e groups in a highly selective and specific m a n n e r (Sharon and Lis, 1972; Lis and Sharon, 1973; Liener, 1976). Such lectins have been f o u n d p r e d o m i n a n t l y in the seeds of plants, in particular those o f t h e legumes, but are also f o u n d in other parts such as the roots, leaves and bark (Sharon and Lis, 1972; Callow, 1976). While m u c h w o r k has been published on the effects of lectins on m a m m a l i a n cells, little is k n o w n of their origins or roles in the plants f r o m which they are derived. There is some evidence that lectins m a y function in cell recognition mechanisms that involve the cell wall or glycocalyx, including incompatibility systems (Matsson et al., 1974; Weise and H a y w a r d , 1972), m o r p h o genesis (Rosen et al., 1973) and host plant-microorganism interactions (Albersheim and Anderson, 1971 ; H a m b l i n and Kent, 1973). Recently, however, Kauss and Ziegler (1974) reported lectin activity in sieve tube sap f r o m Robinia
pseudoacacia. The function o f a lectin in a sugar-
conducting channel is of obvious interest. We n o w report on the occurrence of high haemagglutinating activity in p h l o e m exudate f r o m three cucurbit species, Cucurbita maxima, Cucumis sativus and Cucumis melo. The protein c o m p l e m e n t of p h l o e m sap f r o m Cucurbita m a x i m a has been well studied (Beyenbach et al., 1974; Eschrich et al., 1971; Sabnis and Hart, 1976; Sloan et al., 1976; Weber et al., 1974) and we have been able to identify, isolate and study some properties o f the protein responsible for lectin activity in this species.
Materials and Methods Phloem exudate from cut stems of Cucurbita maxima Duch. 'Golden Delicious' (pumpkin), Cucumis sativus L. 'Telegraph' (cucumber) and Cucumis melo L. 'Blenheim Orange' (melon) was collected into 0.9% saline +0.1 M dithiothreitol or 0.14 M 2-mercaptoethanol, to a final concentration of approximately 1.0 mg/ml (Sabnis and Hart, 1976; Sloan et al., 1976). Protein was assayed by the method of Lowry et al. (1951) using bovine serum albumin as the standard. Interference in the protein assays due to the presence of thiol reagents was overcome using the method of Ross and Schatz (1973). Haemagglutination Assay
Serial two-fold dilutions of exudate were tested for lectin activity using a standard haemagglutination assay (Burger, 1974). Typed human erythrocytes were washed several times in saline and used for agglutination tests as 2-5% suspensions. Agglutination was scored by assessing the percentage of single erythrocytes at x 400 magnification. The end-point titre was determined for each assay as the greatest dilution at which agglutination was just visible. Sodium Dodecyt Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
Gel electrophoresis of exudate proteins was carried out in 7.5% polyacrylamide gels containing 0.1% SDS (Weber and Osborn, 1969) and 4M urea. Gels were cast to a length of 68 mm in glass tubes 75 x 6 mm i.d. Electrophoresis was performed at a constant
0032-0935/78/0142/0097/$01.00
98 current of 6.4 mA/gel. After electrophoresis, the gels were fixed in 10% trichloroacetic acid for i h, stained with Coomassie Blue R25(; (Weber and Osborn, 1969) for 1 h, and destained according to Vesterberg (1972), all at 50~ Protein samples were prepared for electrophoresis by adding urea to provide a final concentration of 8 M: the samples were then mixed with equal volumes of 1% SDS in 0.01 M phosphate buffer, pH 7,1, containing 1% 2-mercaptoethanol, and heated to 100~ for 1.5 rain.
D.D. Sabnis and J.W. Hart: Lectin from Cucurbita Phloem Exudate
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b
c
d
e
Column Chromatography Gel filtration was carried out on Sephadex G-100 in 0.9% NaCI+ 1% 2-mercaptoethanol calibrated with standard proteins (column- 17 x 1.6 cm; void volume -9,0 ml). A 0.5 ml sample of exndate was washed through the column with saline-mercaptoethanol at a flow rate of 40 ml/h; 1.0 ml fractions were collected. To determine the peak of lectin activity, individual fractions were tested for haemagglutinating activity using the dilution assay.
Ion Exchange Chromatography The lectin did not bind to DEAE resin at pH 7.0. Preliminary experiments showed that at this pH the activity was adsorbed to Sephadex CM-50, though not to Sephadex CM-25. The lectin was purified by cation exchange chromatography on a 5.0 ml column of CM Sepharose CL 6B. The column was activated with 50 ml of 2 M NaCI and washed with 50 ml of 0.025 M Tris-HC1 buffer, pH 7.2. A 3.0 ml sample of exudate in 0.025 M Tris buffer + 1% 2-mercaptoethanoi was applied and the c~Ium~ washed with 20 ml of buffer alone to remove acidic proteins and the mercaptoethanol. Elution was carried out in a linear salt gradient of 50 ml buffer +50 ml 0.4 M NaCI. The optical density at 280 nm was monitored and 1.0 ml fractions collected,
Results Samples of exudate f r o m all three p l a n t species were highly active, d e m o n s t r a t i n g h a e m a g g l u t i n a t i n g activity at titres d o w n to 1/2048 after 1 h. A t these endp o i n t dilutions, total p r o t e i n c o n c e n t r a t i o n in the inc u b a t e d drop was a p p r o x i m a t e l y 0.5 gg/ml. N o n e of the exudate samples showed a n y specificity t o w a r d s the m a j o r h u m a n b l o o d groups, A1, A2, B, 0-rhesus negative a n d 0-rhesus positive. The presence of the thiol reagents, dithiothreitol or 2 - m e r c a p t o e t h a n o l , was necessary in order to retard the characteristic gelling of c u c u r b i t exudate (Walker, 1972). T o see if these reagents h a d a n y effects o n exudateLinduced erythrocyte a g g l u t i n a t i o n , exudate from C. m a x i m a was collected into thoroughly degassed saline, in which the proteins are soIuble to a c o n c e n t r a t i o n of a p p r o x i m a t e l y 250 ~g/ m l : such samples were then tested for a g g l u t i n a t i n g activity in the presence a n d absence of thiol reagents. The results indicated that the e n d - p o i n t titre was lowered by half in the presence of the thiol reagents, b u t there was no other recognizable effect o n the a g g l u t i n a t i o n assay.
-H
Fig~. I a-e. SDS-PAGE of proteins in exudate from Cucurbita mccxima. a and d Complete complement of exudate proteins from C. maxima, h Exudate sample after treatment with blood cells to remove lectin (fraction 2). The extra band (t4) is haemoglobin released from the erythrocytes during the centrifugation procedures. e Control, lacking exudate, in which blood cells were subjected to similar washing and centrifugation treatments, e Lectin (fraction 2 protein from C. maxima) purified by CM Sepharose ion exchange chromatography to electrophoretic homogeneity
If the exudate proteins f r o m C. m a x i m a were allowed to gel or precipitate out of solution (by lowering the c o n c e n t r a t i o n of thiol reagent) a n d were r e m o v e d by c e n t r i f u g a t i o n at 3,000 g, lectin activity was lost f r o m the s u p e r n a t a n t fraction. Electrophoresis of the gelled p r o t e i n after several washes in saline which lacked thiol reagents d e m o n s t r a t e d the presence in the gel fraction of all the m a j o r exudate proteins.
Distribution o f Lectin Activity in Cucurbit Seedlings Lectin activity could n o t be detected in extracts of dry seeds of the three c u c u r b i t species. Extracts or diffusates from the cotyledons of g e r m i n a t i n g seeds r e m a i n e d free of lectin activity u n t i l the seedlings were at least five days old. After this period, exudate from the cut h y p o c o t y l or radicle displayed definite haema g g l u t i n a t i n g activity. Electrophoresis of samples
D.D. Sabnis and J.W. Hart: Lectin from Cucurbita Phloem Exudate of exudate from 5-day old seedlings presented protein patterns identical to those obtained from the phloem exudate of mature plants with respect to types of protein subunits as well as their relative concentrations.
Tests for "Hapten' Inhibition
Inhibition by sugars of complex formation between lectins and surface carbohydrate groups of the erythrocytes can give an indication of group specificity. Thus, several sugars were examined for their ability to inhibit agglutination (Lis and Sharon, 1973 ; Kauss and Ziegler, 1974; Burger, 1974) using 0-negative human erythrocytes. The sugars (10-100 mM) were tested against lectin titres which caused approximately half-maximal agglutination or were added to serial dilutions of the lectin. None of the following sugars had any marked inhibitory effect on the rate of agglutination or the end-point titre of cucurbit phloem lectin:-sucrose, N-acetyl-D-galactosamine, N-acetyl-D,glucosamine, D-galactose, D-glucose, Lfucose, D-mannose, D-xylose, D-raffinose, D-stachyose, L-arabinose, D-trehalose, D-melibiose, 2-deoxy-D-glucose, 3-O-methylglucose, p-nitrophenyl-~D-glucoside, p-nitrophenyl-~-D-galactoside. Mixtures of galactose and mannose, galactose and fucose, and mannose and fucose were also ineffective. Control experiments with castor bean lectin and D-galactose provided the expected levels of inhibition, showing that the lack of sugar inhibition with cucurbit lectin was not due to procedures employed.
Isolation of Phloem Lectin from C. maxima
In order for agglutination to occur, the lectin must first bind to saccharide groups on the erythrocyte membrane. Since phloem exudate from C. maxima contains relatively few major proteins (Fig. l a), it was possible to distinguish the protein removed from solution by t h e erythrocytes. One of the major proteins (fraction 1) comprises 60-80% of total exudate protein in this cultivar of C. maxima, while a second (fraction 2) represents another 15-25% (Fig. 1 a). Aliquots of a 50% suspension of thoroughly washed erythrocytes were added to a sample of exudate, and the agglutinated blood was removed by gentle centrifugation. The procedure was repeated until no further agglutination occurred. A final centrifugation removed all suspended blood cells. Control and treated samples were made 8 M with urea and prepared for SDS-PAGE.
99 A comparison of the protein patterns after electrophoresis indicated that while fraction 1 was present in comparable amounts in control and treated samples, fraction 2 was absent in treated preparations of exudate (Fig. i b). The procedure resulted in some release of haemoglobin from the erythrocytes (Fig. 1c). From the concentration of fraction 2 relative to total protein, it can be calculated that the lectin displayed detectable agglutinin activity at a concentration of approximately 0.1 gg/ml. In previous work (Sabnis and Hart, 1976) we reported that the molecular weight (MW) of fraction 2 protein was 22,000 as determined by SDSPAGE. Gel filtration of exudate proteins from C. maxima through Sephadex G-100 showed that the lectin activity was eluted from the column at a position corresponding to a partition coefficient, Kav= 0.58, which approximates to a MW of 20,000. Earlier workers (Beyenbach et al., 1974; Weber et al., 1974) reported that fraction 2 protein possesses a relatively high content of basic amino acids, especially lysine. In the present work, ion exchange chromatography of exudate proteins on a column of CM Sepharose CL 6B yielded a fraction which was eluted at 0.1 M NaC1 and which displayed high optical density at 280 nm, a positive response in the Lowry assay for proteins, and high lectin activity. SDS-PAGE of this fraction revealed a single protein band corresponding to fraction 2 protein (Fig. 1 e).
Discussion
The function of a lectin in the sieve tube is unclear, particularly since the sugar specificity of the lectin has not yet been characterized. Hapten inhibition of haemagglutination is not always possible (Sharon and Lis, 1972). It is unlikely that we have not tested the appropriate sugars, since this would imply that the erythrocyte cell surface possesses very unusual sugars. It has become obvious that saccharide binding sites on lectins are much more complex than may appear from hapten inhibition studies with simple sugars (Lis and Sharon, 1973). Nevertheless, it is of interest that raffinose and stachyose, the sugars that are transported in cucurbit phloem (Webb and Gorham, 1964), do not inhibit haemagglutination. Indeed one could argue that if the lectin was not directly involved in sugar translocation, its presence in the sieve tube might demand particularly high saccharide binding specificity. The evidence clearly demonstrates that fraction 2 protein is a lectin. Thus, we have isolated to electrophoretic purity, a protein from phloem exudate of C. maxima with haemagglutinating activity at a
100 concentration as low as 0.1 ~tg/ml. During agglutination the lectin binds to the erythrocyte surface and is removed from solution. Since dialysed or purified fractions of the lectin remain highly active, cofactors are not involved in the binding reaction. Sephadex gel filtration studies indicate that the Iectin is active as a haemagglutinin in the monomeric form. In its relatively low molecular weight (22,000) and high content of half-cystine residues, the cucurbit lectin is unusual although not unique. Pokeweed mitogen (Reisfeld et al., 1967) and wheat germ agglutinin (Allen et al., 1973) both contain large amounts of half-cystine. Furthermore, wheat germ agglutinin has a molecular weight of 17,000 (Nagata and Burger, 1974), while pokeweed haemagglutinins have molecular weights ranging from 25-31,000 (Waxdal, 1974). Most other purified lectins are k~own to be active as agglutinins in the form of dimers or tetramers, but are comprised of protomeric polypeptide chains ranging from MW 20-30,000. However, the cucurbit lectin does differ from seed lectins so far analysed in its high proportion of basic amino acids and consequently high isoelectric point (Weber et al., 1974; Beyenbach et al., 1974). Despite the immense research effort expended on lectins, the functions of these proteins and glycoproteins in plants remain unknown. For the first time a lectin has now been localized within a single plantcell type modified for a specific function. Furthermore, the relative concentration of lectin in the Cucurbita sieve tube (15-25% of total protein) is also higher than the levels reported for most leguminous seeds which have so far been the principal source of phytohaemagglutinins. It is possible that the lectins play a role in protecting the sugar-rich phloem from bacterial or fungal invasions (suggested to us by Prof. H. Ziegler). Inhibition of fungal growth by wheat germ agglutinin has been reported (Mirelman et al., 1975). Albersheim and co-workers (Albersheim and Anderson, 1971; Albersheim et al., 1969; Anderson and Albersheim, 1972) have proposed an alternative role for lectins in host resistance to pathogenic fungi. They suggest a direct role for lectins in inhibiting fungal cell-wall degrading hydrolases, particularly polygalacturonase, through their ability to bind to carbohydrate moieties. However, with regard to the sieve tube, we consider that a defense mechanism would be more likely to be located outside rather than within the sieve element. An alternative possibility is suggested by the following observations. The characteristic pattern of protein subunits obtained after SDS-PAGE of exudate from C. m a x i m a 'Golden Delicious' remains constant irrespective of the organ (hypocotyl, stem, petiole or fruit) or the age of the tissue from which the exudate is obtained
D.D. Sabnis and J.W~Hart: Lectin from Cucurbita Phloem Exudate (Sabnis and Hart, manuscript in preparation). This is true even for exudate obtained from the hypocotyls of seedlings only 5 or 6 days old. In all these cases, the relative concentration (w/v) of fraction 2 protein remains approximately 25% of that of fraction 1 protein. Since the molecular weights of the two proteins differ by a similar ratio (86,000:22,000; Sabnis and Hart, 1976) it is possible that the two proteins are associated in vivo in a stoichiometric 1 : 1 relationship. If exudate from C. m a x i m a is collected directly onto formvar-coated copper grids, negatively stained and examined under the electron microscope, bundles of P-protein filaments are visible (Kollmann et al., 1970). On exposure to air or buffers lacking thiol reagents, the filaments aggregate into a gel, In the presence of thiol reagents the filaments depolymerise into soluble subunits. Both fraction 1 and franction 2 protein are rich in half-cystine residues (Beyenbach et al., 1974; Weber et al., 1974). Furthermore, Kleinig et al. (1975) have demonstrated the reversible formation of filaments from fraction 1 protein under oxidising conditions. Under such conditions, fraction 2 protein forms amorphous aggregates. In view of these observations, we favour the possibility that, in vivo, fraction 1 protein (in filamentous form) and fraction 2 protein (the lectin) are linked together by disulphide bridges. We suggest that the lectin could serve to anchor the P-protein filaments to glycoprotein or glycolipid components of the sieve element plasma membrane. This would also ensure a parietal distribution of the filaments, facilitating flow of solution through the sieve tube (Evert et al., 1973). The necessity for the P-protein filaments to be anchored during normal sugar translocation in the sieve tube is generally accepted (Cronshaw, 1975). Electron microscopy has not been able to provide evidence for or against a chemical association between filaments and membranes in the sieve element. A lectin bridge might serve this function very well. Sudden release of turgot pressure during damage or excision could possibly break such a weak bond, permitting the plugging of sieve pores by filamentous aggregates. Whether lectins play multifarious roles in the plant, with a unique function in the sieve element, or whether their presence in the phloem is in keeping with some general function of these proteins in cellcell recognition processes, is a problem demanding greater consideration. We gratefullyacknowledgethe adviceand assistance of Dr. Brodie H. Lewis and Mr. R. Main of the Regional Blood Transfusion Service, Aberdeen. We also thank Messrs I. Skene, E. Middleton and J. McRobb for technical assistance.
D.D. Sabnis and J.W. Hart: Lectin from Cucurbita Phloem Exudate
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Received 3 April; accepted 9 May 1978
