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Screening for plant lectins by latex agglutination tests
Phytochnistry, Vol. 30, No. 12, pp. 4005 4009, 1991 Printed in Great Britain.
SCREENING
0031-9422/91 63.00+0.00 Pcrgamon Press plc
FOR PLANT LECTINS
BY LATEX AGGLUTINATION
TESTS
RAJNI KAUL, JOHN READ* and Bo MAI-TIASSON Department of Biotechnology, Chemical Center, Lund University, Post Box 124, S-221 00 Lund, Sweden; *Department of Biochemistry, University of Zimbabwe, P.O. Box MP 167, Mount Pleasant, Harare, Zimbabwe (Received 9 May 1991)
Key Word Index-Lectin; latex agglutination.
Abstract-The latex agglutination test has been applied as a detection system for lectins, the method being especially useful in locations where the dependence on blood for hemagglutination tests could be minimised. The binding of various glycoproteins and sugars individually to the latex particles facilitated the agglutination with lectins having varying sugar specificities. The glycoproteins used were ovalbumin, horseradish peroxidase, porcine mucin and fetuin, while N-acetylglucosamine, N-acetylgalactosamine comprised the sugars used for binding to latex. The sensitivity of the latex agglutination tests was comparable with that of hemagglutination tests. Sugar binding specificity of the lectins could also be determined by inhibition of the agglutination in the presence of corresponding free sugars. The method proved to be useful in screening crude seed extracts for the presence of lectins.
INTRODUCTION
Lectins are being increasingly used for the investigation of complex carbohydrate structures on cell surfaces and proteins, purification of carbohydrate-containing polymers, blood typing, mitogenic stimulation of lymphocytes etc. Cl]. These carbohydrate-binding proteins are found widely distributed in nature, an extremely good source being the seeds of plants especially legumes [2, 31. The first biological activity to be recognised for lectins was their capacity to agglutinate erythrocytes, some lectins being blood group and/or subgroup specific. Even now, the most popular procedure for the detection of lectins is the hemagglutination test using animal or human erythrocytes, or both, despite the development of more refined procedures like affinity electrophoresis, and those based on the ability of lectins to precipitate glycoconjugates and polysaccharides [4, S]. However, quite often laboratories in some countries lack the facilities for proper handling and testing of human blood and thus depend on animal blood for routine laboratory studies. During our studies on the screening for lectins from Zimbabwean flora, we found it necessary to have a detection system which would be robust and convenient to use as a field test. Substitution of erythrocytes as the agglutinating material with polystyrene latex beads having bound glycoconjugates as demonstrated by Pongor and Riedl [6] could be one of the viable alternatives. Latex agglutination tests are being applied for diagnosis of various infectious states. The principle behind the diagnostic tests is that the antibody coated latex particles agglutinate when in contact with a sample containing the appropriate antigen. As the lectin-saccharide interactions are analogous with those between antibody and antigen, the latex agglutination tests could be modified suitably for the detection of lectins. Pongor and Riedl have reported the agglutination with concanavalin A (Con A), wheatgerm agglutinin (WGA),
and soyabean agglutinin (SBA), of latex particles bound to blood group A+H substance (BGA), which was purified from hog stomach [6]. In our laboratory, we have attempted to study the possibility of using other glycoprotein (sugar)-latex preparations for detection of lectins and the feasibility of applying the latex agglutination tests for screening of plant seed extracts for the presence of lectins.
RESULTSAND DISCUSSION
Based on their carbohydrate binding specificity lectins have been broadly classified into five groups: glucose/mannose group, N-acetyl glucosamine group, N-acetyl galactosamine/galactose group, L-fucose group, and sialic acid group [4]. Even within these groups, lectins differ in their affinity with the monosaccharides and/or oligosaccharides, and also with respect to their anomeric specificity. The binding of lectins to different glycoproteins is, therefore, determined by the glycan structure present in the latter. These glycoproteins when fixed to the latex beads should be able to agglutinate in the presence of the complementary lectin. The binding to a glycoprotein with a defined carbohydrate structure would be valuable in providing information about binding specificity of the lectin. Of course, agglutination with latex particles having just a bound sugar would make the identification of binding specificity of a lectin much more simple and precise. Latex agglutination siudies with standard lectins In the studies presented here, the glycoproteins, ovalbumin, horseradish peroxidase (HRP), mucin, and fetuin were used for coating the latex beads by physical adsorption. Mucin was also covalently coupled to the latex. The first two proteins were chosen because of their low cost,
R. KAUL et al.
4006
especially that of ovalbumin, and their easy availability. The amount of ovalbumin and HRP adsorbed to the latex in terms of the protein was estimated to be 0.25-0.3 mgml-’ of the final latex suspension. The results of the agglutination tests with different standard lectins are presented in Table I. Chicken ovalbumin contains mannose and N-acetylglucosamine residues linked as a single oligosaccharide to the polypeptide chain [7]. The agglutination of latexovalbumin with Con A, WGA, Phaseolus vulgaris agglutinin (PHA), and Pisum lectin agreed with the known specificity of these lectins for the above sugars. HRP has been known to bind Con A and the enzyme-lectin interactions have been employed in several purification strategies [S]. HRP bound to the latex served as a good detection system for glucose/mannose binding lectins. It was observed that the sensitivity of agglutination was improved upon covalently linking mucin to carboxylated latex. Hence, this preparation was used for the present studies. It was seen to agglutinate with Con A, WGA, Tetragonolobus and Dolichos lectins, however, soya and peanut lectins showed a negative response. According to an earlier report, latex-BGA, which is a
f
o-
,
,
0 075 0.15 mg HRP
Fig.
,
,
,
,
0.3
0.6
1.2
2.4
product of mucin, reacted positively with Con A, WGA and SBA [6]. The coupling of N-acetylglucosamine (NAcGlc), Nacetylgalactosamine (NAcGal), and lactosamine to latex proved extremely useful in agglutinating lectins having binding specificity to the corresponding sugars. Hence, Glycine max and Aruchis hypogea lectin which were difficult to detect with the latex-glycoprotein preparations, agglutinated efficiently the NAcGal/lactosamine and lactosamine bound latex respectively (Table 1). The lectin from Limulus polyphemus having sialic acid specificity, was able to agglutinate latex beads with bound fetuin. It was also possible to carry out the inhibition of latex agglutination with sugars specific for the respective lectins in the same manner as is done for hemagglutination. Sensitivity
Various concentrations of the glycoproteins were used for adsorption to latex to find the optimal preparation giving good sensitivity of detection in a limited time period In all the cases, 0.5-0.6 mg glycoprotein added to 50~1 of 10% latex suspension which gave a I ml final preparation (see Experimental), was found to be the best. The minimum detection limit for Con A with different latex-HRP preparations is shown in Fig. 1. The latex suspension with 0.3 and 0.6 mg HRP/ml could be agglutinated with 4 pg Con A ml- * within 2 hr. Though the preliminary tests with adsorption of HRP to bigger sized latex beads (0.8 pm) increased the sensitivity of detection to 2 pg Con A ml- I, smaller latex beads (0.46 pm) were preferred for studying agglutination, because of the tendency of bigger particles to settle down on the plate which made it difficult to read the results. The number of double dilutions of some lectins giving agglutination with different preparations are shown in Table 2. The sensitivity of latex agglutination tests was comparable with that of hemagglutination. It was, however, noticed that when the multidish plates were rocked during the agglutination test, the sensitivity of detection
.
loaded/50 microlit. 10% latex
I. Effect ofdifferent HRP concentrations adsorbed on the sensitivity of defection of Con A.
Table
to latex
1. Latex agglutination
tests for tectin detection Latex preparation
Lectin
for positive
Glucose/mannose group Canavalia ensijormis (ConA)* Pisum satioum* N-Acetylglucosamine group Triticum uubare (WGA) N-Acetylgalactosamine/Galactose Glycine mclx (SBA) Dolichos b~f7orus Arachis hypogea+ Phaseolus uulgaris (PHA) L-Fucose group Tetragonolobus purpureas Sialic acid group Limu/u.s polyphemu.9 *Agglutination
Ovalbumin/HRP/mucint Ovalbumin/HRP Ovalbumin/mucint/NAcGlc group NAcGal/lactosamine mucint/NAcGal lactosamine ovalbumin
Mucint Fetuin
test was done in the presence
tMucin was covalently coupled to latex.
of
Ca2’/MnZf/Mg2+
agglutination
Screening of plant lectins
increased considerably. For example, in case of HRP bound to latex it was possible to obtain agglutination with 11-12 double dilutions of Con A as compared to eight when the plates were kept stationary. This amounts to 7-14 x lo-l4 mol Con A based on its M, of 104000. This level of sensitivity, of course, may not apply to all lectin-glycoprotein combinations. Detection
of lectins in crude extracts
of seeds
The latex preparations were used for the detection of lectins from the crude extracts of seeds. Initially, seeds known to contain lectins like Triticum vulgare, Canaoalia ensiformis, Artocarpus integrifolia and Glycine max were
Table 2. Comparison of number of double dilutions of lectins giving positive agglutination with latex and erythrocytes
Lectin Pure lectin’ Con A Pisum lectin WGA Dolichos lectin SBA Crude extracts T. vulgare C. ensiformis A. inregrifolia
Hemagglutination
Latex agglutination (ligand used)
9 I 9 3 7
10 4 10 8 10
ND ND ND
> 6 (ovalbumin) > 8 (ovalbumin) r 8 (ovalbumin)
*The starting concentration 0.5 mg ml _ ‘. ND: not determined.
(ovalbumin) (HRP) (ovalbumin) (NAcGal) (NAcGal)
of the standard
Table
lectins
3. Agglutination
was
4007
tried. The response of the agglutination was extremely positive, e.g. latex-ovalbumin reacted positively with all the extracts except the soyabean which agglutinated the latex-NAcGal/lactosamine. Moreover, several-fold dilution of the extract could give agglutination with the coated latex. This is important in case of any interference originating from the crude material. Any non-specific agglutination could also be detected by testing the extract with latex-BSA. Later, 16 different seed extracts from Zimbabwean flora were screened for the presence of lectins using this method, and comparisons were also made with hemagglutination tests. The response obtained with the latex agglutination and hemagglutination test with the various extracts, and also the results of the sugar inhibition studies are given in Table 3. Of the plant extracts under investigation, the presence of &tins in Eryrhrina lysistemon and Calpurnia aurea [9, lo] has previously been reported. In the present studies, only five extracts showed positive hemagglutination including the two mentioned above. The extracts exhibited no blood group specificity, however, Calpurnia aurea seemed to prefer the A+ erythrocytes. Moreover, the latter agglutinated the RBCs only in the presence of metal ions, and the agglutination was enhanced by trypsinization of the blood cells. Even Combretum zeyheri was seen to require metal ions to show hemagglutination. Inhibition studies with sugars could confirm the specificity of only Erythrina species and C. aurea for galactose and NAcGal. Latex agglutination tests resulted in positive response with six extracts. Feretia aeruginescens and C. zeyheri extracts which agglutinated the RBCs did not give any noticeable latex agglutination. Because the sugar specificity of these extracts could not be confirmed by hemagglutination test, it is difficult at this stage to evaluate
results with extracts from Zimbabwean Agglutination/sugar
flora inhibition
----.--.
Family
RBC
Anthocleisto grandijiora Calpwnia aurea
Laoganiaceae Papilionoideae
+ /NAcGal,
Combretum paniculatum Combretum zeyheri Crotolaria capensis Domboyea burgessiae Erythrina abyssinica
Combretaceae Combretaceae Papilionoideae Sterculiaceae Papilionoideae
+I’ + /NAcGal,
Gal
Erythrino lysistemon
Papilionoideae
+/NAcGal
Gal
Feretia oeruginescens Friesodielsia obovata Halleria lucida Mimosa pigra Mundulea sericea Pterocorpus angolensis Strelitzia nicolai Toddaliopsis bremekampii
Rubiaceae Annonaceae Scrophulariaceae Mimosoideae Papilionoideae Papilionoideae Strelitziaazae Rutaceae
+I* -
Plant
spp.
Latex (ligand)
Gal
+(ovalbumin, mucin, NAcGal)/NAcGal, NacGlc +(ovalbumin, mucin, NAcGal, lactosamine)/ NAcGal, Gal +(ovalbumin, muck, NAcGal, lactosamine)/ NAcGAI, Gal -
--
*The sugars, glucose. mannose, galactose, fucose, NAcGlc, tNon-specific agglutination observed (details in the text).
+(ovalbumin)/+ + (ovalbumin, mucin, N AcGal, lactosamine)/NAcGal, Gal -
-t +(ovalbumin, and NAcGal
HRP, mucin, NAcGlc)/Glc,
Man, NAcGlc
did not give any visible inhibition of agglutination.
R. KAUL er (I/.
4008
the results. On the other hand, Mimosa pigra extract showed a non-specific agglutination with latex-BSA preparation. An interesting observation was that the Pterocarpus angolensis extract which in a study in Zimbabwe, has been shown to agglutinate erythrocytes from umbilical cord and new born children [l 11, and in our laboratory was unable to agglutinate adult A, B, or 0 erythrocytes, readily agglutinated latex beads coated with ovalbumin, horseradish peroxidase and porcine mucin. Furthermore, inhibition studies carried out using ovalbumin/HRPlatex showed that this lectin was inhibited by glucose, mannose and NAcGlc, as had been reported for cord erythrocytes by Moore [I]. Additionally, DomhnJea hurgessiae extract tested positive to latex agglutination test only in the presence of metal ions, and the agglutination could be inhibited by NAcGlc and NAcGal. Anthocleista grand(flora extract showed a rather weak agglutination with ovalbuminlatex, however, its sugar binding specificity could not be ascertained. From the studies presented above, it is evident that latex agglutination tests could be good substitutes for hemagglutination tests for lectin detection. The potential of the glycoproteins for use in latex agglutination tests is highly dependent on their ability to bind properly to the latex beads, and the accessibility of their sugar moiety for interaction with the lectin. On the other hand, sugars require a functional group for coupling to the latex particles. The latex particles are extremely stable in the freezedried form, and it is convenient to use them for field tests. The tests could be used satisfactorily to investigate new lectins and also avoid or detect the problems associated with haemolysins and non-specific (tanin based) agglutination that can cause investigations to over report the presence of lectins in crude plant extracts. The method was found to be sensitive, useful for sugar specificity studies, and could be used to isolate small amounts of the lectins for gel electrophoresis.
EXPERIMENTAL rVfateria/s. The following materials were obtained from Sigma: latex beads (polystyrene, 0.46 pm diameter, 10% solids content), carboxylate modified latex beads (0.4 pm diameter), ovalbumin (crude), horseradtsh peroxidase (type II, 175 U mg-’ solid), mucin (from porcine stomach), fetuin (from fetal calf serum), NI-ethyl-3-(3acetylglucosamine, N-acetyl galactosamine, dimethyl aminopropyl) carbodiimide, Jack beans (Canacalin ensiformis). concanavalin A, wheat germ agglutinin. lectins from Arachis
hypogeo,
Dolichos
bijorus,
Pisum
satirwn.
Phaseolus
and Terragonolohus purpureas. Artocarpus integrijolia (Jack fruit) seeds were procured from a market in Bangalore, India, whereas other plant seeds were obtained from the Botanical gardens, Harare, Zimbabwe. Soyabean agglutinin and lactosamine were prepd in the laboratory according to standard procedures [l2, 131. All other chemicals were of reagent grade quality. Preparation of seed extracrs. The seeds were crushed in a grinder prior to defatting. The ground material was defatted by mixing with heptane (5 ml g- * seed) for 2 hr at room temp. The heptane layer was filtered off through a sintered glass funnel, and the solid was dried overnight in a fume hood. It was then extracted with 0.9% saline (5 ml) for 1 hr at room temp. The tiulgaris
extract was filtered, and later clarified by centrifugation 15 mm at 10000 rpm.
at 4’ for
Physical udsorprion of glycoprofeins on latex beads. To 50 ~1 of 10% latex beads in a tube was added 400 PI of diluted (I : I) glycine buNer saline (0.17 M glycine. 0.9% NaCl and 0.004% NaN,, pH 7.3). I mg of a glycoprotein (ovalbumin. horseradish peroxldase, porcine mucin. fctuin) in 400 ~1 of 0.9% saline. The nixt. was incubated for 1 hr at 37 with intermIttent shaking. Subsequently, it was centrifuged at 10000 rpm for 7 mm. washed once with glycine buffer sahne (GBS) and once with GBS containing 0.1% bovine serum albumin (GAS-BSA). Latex particles coated only with BSA served as a control. Covalent
coupling
I$ glpcoprorems
and wnmo
sugars to car-
Porcine mucin and some derivatizcd sugars like N-acetylglucosaminc (NAcGlc). N-acetylgalactosamine (NAcGal) and lactosamme were coupled to the carboxylated latex by water-sol. carbodiimide. The coupling was carried out either by preactlvating the latex with I-ethyl-3-(3dimethylaminopropyl) carbodiimlde (EDC) [l4], or by incubating the glycoprotcin,‘sugar with the latex directly in the presence of the carbodiimide [IS]. According lo the first method, 50~1 of the latex beads were washed with I ml of borate buffer saline (BBS, 0.02 M borate-NaOH buffer, pH 8 m 0.17 M NaCI). After centrifugation the pellet was resuspended in IO0 ~1 BBS, lo which was then added 100~1 BBS containing 5 mg EDC. The suspension was vortexed at room temp. for I hr, centrifuged, and the pellet resuspended in 50 /iI BBS. To this was then added S mg glycoprotein or sugar in 0.5 ml BBS. After overmght shaking a! 4’. the latex was washed once with 2.5 ml GBS-0.1% Tween 20, and then twice with CBS-BSA. The particles were finally suspended in I ml GBS-BSA. Alternatively, 50 ~1 of latex suspension was washed with 0.5 M NaCI, and then suspended in 50 ~1 H,O at pH 4.5. To this was then added lOO/~l sugar:glycoproteln (5 mg) and 50~1 EDC (1 mg). both in H,O. pH 4.5. The mixt. was incubated overnight at room temp., centrifuged, washed twice with H,O and once with CBS-BSA. and finally suspended in I ml GBS-BSA. Latex agglutination fesfs. In a well of a multidish (Nunc, Denmark) (plate equipped with 12;24 wells, used for cell culture), I5 /II of the latex-glycoprotcin (sugar) suspension was mixed with an equal volume of saline containing lectin. The plate was covered and observed for agglutination up lo I -2 hr. The sensitivity of the level of detection of lectins was studied by double dilution of the lectin (I mgml ‘) with saline several times, and noting the least diluuon giving visible agglutination. The agglutination test was also carried out in the presence of Ca*‘, Mn*+, and Mg’+ rons at a concn of 2 mM. except for the Pisum lectin where the ion concn was maintained at 50 mM. A blank in which the coated latex was incubated with saline instead of the lectin was always included during the test. The possibility of non-specific agglutination was seen by incubating the known lectin or the plant extract with the BSA coated latex suspension. Sugar inhibition oj /arex agglufinaflon. Latex agglutmation with lectins and extracts was also performed in the presence of sugars--glucose. mannosc. galactosc, NAcGal. NAcGlc. or fucase. During such tests IO 111of the coated latex suspension was added to a mixt. of 10 ~1 lectin and IO ~1 sugar soln. The maximal sugar concn obtamed in the mixt. was I67 mM. The inhibition of the agglutination reaction by a sugar was taken as an evidence for the sugar specificity of the lectm. Hemagglucination tesrs. Hemagglutinating activity of the lectins and the plant extracts was also measured by the double dilution method, in which 50 /II of 2% RBC suspension (isolated from human blood group A,‘B,!O + , and washed with saline) was added to 50 ~1 of successively dilute lectin soln in a microtiter boxy/are
modified
lorex
huuds.
Screening for plant lectins plate. Agglutination was noted within 1 hr. Sometimes, trypsinized erythrocytes were required for the agglutination test. In such cases, the erythrocytes were treated with trypsin (0.1%) prior to the test, by a standard procedure [16]. Hemagglutination test was also carried out in the presence of metal ions in the same way as for the latex agglutination test. Sugar inhibition tests were also performed in case of seed extracts. Protein assay. Protein concentration was estimated by BioRad method using BSA as the standard. AcknowIedgemenrs-The financial support provided by Swedish Agency for Cooperation with Developing Countries (SAREC) is gratefully acknowledged. The authors are also thankful to Elisabeth Bjurulf and Roswita Schmitz for their excellent technical assistance, and to Eva Linne for providing purified soyabean agglutinin and lactosamine.
REFERENCES Lis, H. and Sharon, N. (1986) in The Lectfnr (Linier, I. E., Sharon, N. and Goldstein, 1. J., eds), p. 293. Academic Press, Florida. Kocourek, J. (1986) in The Pectins (Linier, 1. E., Sharon, N. and Goldstein, I. J., eds), p. 1. Academic Press, Florida. Strosberg, A. D., Buffard, D.. Lauwercys, M. and Foriers, A.
4009
(1986) in The Pectins (Linier, I. E., Sharon, N. and Goldstein, I. J., eds), p. 249. Academic Press, Florida. 4. Goldstein, 1. J. and Poretz, R. D. (1986) in The Pectins (Linier, 1. E., Sharon, N. and Goldstein, I. J.. eds), p. 33. Academic Press, Florida. Horejsi, V. and Kocourek, J. (1974) Biochim. Biophys. Acta 336,338. Pongor, S. and Reidl, Z. (1983) AM/. Biochem.129, 51. Huang, C.-C., Mayer, Jr., H. E. and Montgomery, R. (1970) Carbohydrate Res. 13. 127. Lanner, M., Bergquist, R., Car&m, J. and Huldt, G. (1978) in Afinity Chromatography (Hoffman-Ostenhof, O., ed.), p. 237. Pergamon Press, Oxford. 9. Lis, H., Joubert, F. J. and Sharon, N. (1985) Phytochemistry 24, 2803.
Bird, G. W. G. (1957) Nature 180, 657. Moore, H. H. (1977) M.Sc. Thesis. University of Rhodesia. Vretblad, P. (1976) Biochim. Biophys. Acta 434, 169. Honda, S., Suzuki, S. and Kakehi, K. (1987) J. Ckromut. 396, 93. 14. Masson, P. L., Cambiaso, C. L., Collet-Cassart, D., Magnusson, C. G. M., Richards, C. B. and Sindic, C. J. M. (1981) Methods Enrymol. 74, 106. Handbook, Pharmacia Fine 15. Affinity Chromatography Chemicals, Sweden. 16. Lis, H. and Sharon, N. (1972) Methods Enzymol. 28, 360. 10. 11. 12. 13.
SCREENING
0031-9422/91 63.00+0.00 Pcrgamon Press plc
FOR PLANT LECTINS
BY LATEX AGGLUTINATION
TESTS
RAJNI KAUL, JOHN READ* and Bo MAI-TIASSON Department of Biotechnology, Chemical Center, Lund University, Post Box 124, S-221 00 Lund, Sweden; *Department of Biochemistry, University of Zimbabwe, P.O. Box MP 167, Mount Pleasant, Harare, Zimbabwe (Received 9 May 1991)
Key Word Index-Lectin; latex agglutination.
Abstract-The latex agglutination test has been applied as a detection system for lectins, the method being especially useful in locations where the dependence on blood for hemagglutination tests could be minimised. The binding of various glycoproteins and sugars individually to the latex particles facilitated the agglutination with lectins having varying sugar specificities. The glycoproteins used were ovalbumin, horseradish peroxidase, porcine mucin and fetuin, while N-acetylglucosamine, N-acetylgalactosamine comprised the sugars used for binding to latex. The sensitivity of the latex agglutination tests was comparable with that of hemagglutination tests. Sugar binding specificity of the lectins could also be determined by inhibition of the agglutination in the presence of corresponding free sugars. The method proved to be useful in screening crude seed extracts for the presence of lectins.
INTRODUCTION
Lectins are being increasingly used for the investigation of complex carbohydrate structures on cell surfaces and proteins, purification of carbohydrate-containing polymers, blood typing, mitogenic stimulation of lymphocytes etc. Cl]. These carbohydrate-binding proteins are found widely distributed in nature, an extremely good source being the seeds of plants especially legumes [2, 31. The first biological activity to be recognised for lectins was their capacity to agglutinate erythrocytes, some lectins being blood group and/or subgroup specific. Even now, the most popular procedure for the detection of lectins is the hemagglutination test using animal or human erythrocytes, or both, despite the development of more refined procedures like affinity electrophoresis, and those based on the ability of lectins to precipitate glycoconjugates and polysaccharides [4, S]. However, quite often laboratories in some countries lack the facilities for proper handling and testing of human blood and thus depend on animal blood for routine laboratory studies. During our studies on the screening for lectins from Zimbabwean flora, we found it necessary to have a detection system which would be robust and convenient to use as a field test. Substitution of erythrocytes as the agglutinating material with polystyrene latex beads having bound glycoconjugates as demonstrated by Pongor and Riedl [6] could be one of the viable alternatives. Latex agglutination tests are being applied for diagnosis of various infectious states. The principle behind the diagnostic tests is that the antibody coated latex particles agglutinate when in contact with a sample containing the appropriate antigen. As the lectin-saccharide interactions are analogous with those between antibody and antigen, the latex agglutination tests could be modified suitably for the detection of lectins. Pongor and Riedl have reported the agglutination with concanavalin A (Con A), wheatgerm agglutinin (WGA),
and soyabean agglutinin (SBA), of latex particles bound to blood group A+H substance (BGA), which was purified from hog stomach [6]. In our laboratory, we have attempted to study the possibility of using other glycoprotein (sugar)-latex preparations for detection of lectins and the feasibility of applying the latex agglutination tests for screening of plant seed extracts for the presence of lectins.
RESULTSAND DISCUSSION
Based on their carbohydrate binding specificity lectins have been broadly classified into five groups: glucose/mannose group, N-acetyl glucosamine group, N-acetyl galactosamine/galactose group, L-fucose group, and sialic acid group [4]. Even within these groups, lectins differ in their affinity with the monosaccharides and/or oligosaccharides, and also with respect to their anomeric specificity. The binding of lectins to different glycoproteins is, therefore, determined by the glycan structure present in the latter. These glycoproteins when fixed to the latex beads should be able to agglutinate in the presence of the complementary lectin. The binding to a glycoprotein with a defined carbohydrate structure would be valuable in providing information about binding specificity of the lectin. Of course, agglutination with latex particles having just a bound sugar would make the identification of binding specificity of a lectin much more simple and precise. Latex agglutination siudies with standard lectins In the studies presented here, the glycoproteins, ovalbumin, horseradish peroxidase (HRP), mucin, and fetuin were used for coating the latex beads by physical adsorption. Mucin was also covalently coupled to the latex. The first two proteins were chosen because of their low cost,
R. KAUL et al.
4006
especially that of ovalbumin, and their easy availability. The amount of ovalbumin and HRP adsorbed to the latex in terms of the protein was estimated to be 0.25-0.3 mgml-’ of the final latex suspension. The results of the agglutination tests with different standard lectins are presented in Table I. Chicken ovalbumin contains mannose and N-acetylglucosamine residues linked as a single oligosaccharide to the polypeptide chain [7]. The agglutination of latexovalbumin with Con A, WGA, Phaseolus vulgaris agglutinin (PHA), and Pisum lectin agreed with the known specificity of these lectins for the above sugars. HRP has been known to bind Con A and the enzyme-lectin interactions have been employed in several purification strategies [S]. HRP bound to the latex served as a good detection system for glucose/mannose binding lectins. It was observed that the sensitivity of agglutination was improved upon covalently linking mucin to carboxylated latex. Hence, this preparation was used for the present studies. It was seen to agglutinate with Con A, WGA, Tetragonolobus and Dolichos lectins, however, soya and peanut lectins showed a negative response. According to an earlier report, latex-BGA, which is a
f
o-
,
,
0 075 0.15 mg HRP
Fig.
,
,
,
,
0.3
0.6
1.2
2.4
product of mucin, reacted positively with Con A, WGA and SBA [6]. The coupling of N-acetylglucosamine (NAcGlc), Nacetylgalactosamine (NAcGal), and lactosamine to latex proved extremely useful in agglutinating lectins having binding specificity to the corresponding sugars. Hence, Glycine max and Aruchis hypogea lectin which were difficult to detect with the latex-glycoprotein preparations, agglutinated efficiently the NAcGal/lactosamine and lactosamine bound latex respectively (Table 1). The lectin from Limulus polyphemus having sialic acid specificity, was able to agglutinate latex beads with bound fetuin. It was also possible to carry out the inhibition of latex agglutination with sugars specific for the respective lectins in the same manner as is done for hemagglutination. Sensitivity
Various concentrations of the glycoproteins were used for adsorption to latex to find the optimal preparation giving good sensitivity of detection in a limited time period In all the cases, 0.5-0.6 mg glycoprotein added to 50~1 of 10% latex suspension which gave a I ml final preparation (see Experimental), was found to be the best. The minimum detection limit for Con A with different latex-HRP preparations is shown in Fig. 1. The latex suspension with 0.3 and 0.6 mg HRP/ml could be agglutinated with 4 pg Con A ml- * within 2 hr. Though the preliminary tests with adsorption of HRP to bigger sized latex beads (0.8 pm) increased the sensitivity of detection to 2 pg Con A ml- I, smaller latex beads (0.46 pm) were preferred for studying agglutination, because of the tendency of bigger particles to settle down on the plate which made it difficult to read the results. The number of double dilutions of some lectins giving agglutination with different preparations are shown in Table 2. The sensitivity of latex agglutination tests was comparable with that of hemagglutination. It was, however, noticed that when the multidish plates were rocked during the agglutination test, the sensitivity of detection
.
loaded/50 microlit. 10% latex
I. Effect ofdifferent HRP concentrations adsorbed on the sensitivity of defection of Con A.
Table
to latex
1. Latex agglutination
tests for tectin detection Latex preparation
Lectin
for positive
Glucose/mannose group Canavalia ensijormis (ConA)* Pisum satioum* N-Acetylglucosamine group Triticum uubare (WGA) N-Acetylgalactosamine/Galactose Glycine mclx (SBA) Dolichos b~f7orus Arachis hypogea+ Phaseolus uulgaris (PHA) L-Fucose group Tetragonolobus purpureas Sialic acid group Limu/u.s polyphemu.9 *Agglutination
Ovalbumin/HRP/mucint Ovalbumin/HRP Ovalbumin/mucint/NAcGlc group NAcGal/lactosamine mucint/NAcGal lactosamine ovalbumin
Mucint Fetuin
test was done in the presence
tMucin was covalently coupled to latex.
of
Ca2’/MnZf/Mg2+
agglutination
Screening of plant lectins
increased considerably. For example, in case of HRP bound to latex it was possible to obtain agglutination with 11-12 double dilutions of Con A as compared to eight when the plates were kept stationary. This amounts to 7-14 x lo-l4 mol Con A based on its M, of 104000. This level of sensitivity, of course, may not apply to all lectin-glycoprotein combinations. Detection
of lectins in crude extracts
of seeds
The latex preparations were used for the detection of lectins from the crude extracts of seeds. Initially, seeds known to contain lectins like Triticum vulgare, Canaoalia ensiformis, Artocarpus integrifolia and Glycine max were
Table 2. Comparison of number of double dilutions of lectins giving positive agglutination with latex and erythrocytes
Lectin Pure lectin’ Con A Pisum lectin WGA Dolichos lectin SBA Crude extracts T. vulgare C. ensiformis A. inregrifolia
Hemagglutination
Latex agglutination (ligand used)
9 I 9 3 7
10 4 10 8 10
ND ND ND
> 6 (ovalbumin) > 8 (ovalbumin) r 8 (ovalbumin)
*The starting concentration 0.5 mg ml _ ‘. ND: not determined.
(ovalbumin) (HRP) (ovalbumin) (NAcGal) (NAcGal)
of the standard
Table
lectins
3. Agglutination
was
4007
tried. The response of the agglutination was extremely positive, e.g. latex-ovalbumin reacted positively with all the extracts except the soyabean which agglutinated the latex-NAcGal/lactosamine. Moreover, several-fold dilution of the extract could give agglutination with the coated latex. This is important in case of any interference originating from the crude material. Any non-specific agglutination could also be detected by testing the extract with latex-BSA. Later, 16 different seed extracts from Zimbabwean flora were screened for the presence of lectins using this method, and comparisons were also made with hemagglutination tests. The response obtained with the latex agglutination and hemagglutination test with the various extracts, and also the results of the sugar inhibition studies are given in Table 3. Of the plant extracts under investigation, the presence of &tins in Eryrhrina lysistemon and Calpurnia aurea [9, lo] has previously been reported. In the present studies, only five extracts showed positive hemagglutination including the two mentioned above. The extracts exhibited no blood group specificity, however, Calpurnia aurea seemed to prefer the A+ erythrocytes. Moreover, the latter agglutinated the RBCs only in the presence of metal ions, and the agglutination was enhanced by trypsinization of the blood cells. Even Combretum zeyheri was seen to require metal ions to show hemagglutination. Inhibition studies with sugars could confirm the specificity of only Erythrina species and C. aurea for galactose and NAcGal. Latex agglutination tests resulted in positive response with six extracts. Feretia aeruginescens and C. zeyheri extracts which agglutinated the RBCs did not give any noticeable latex agglutination. Because the sugar specificity of these extracts could not be confirmed by hemagglutination test, it is difficult at this stage to evaluate
results with extracts from Zimbabwean Agglutination/sugar
flora inhibition
----.--.
Family
RBC
Anthocleisto grandijiora Calpwnia aurea
Laoganiaceae Papilionoideae
+ /NAcGal,
Combretum paniculatum Combretum zeyheri Crotolaria capensis Domboyea burgessiae Erythrina abyssinica
Combretaceae Combretaceae Papilionoideae Sterculiaceae Papilionoideae
+I’ + /NAcGal,
Gal
Erythrino lysistemon
Papilionoideae
+/NAcGal
Gal
Feretia oeruginescens Friesodielsia obovata Halleria lucida Mimosa pigra Mundulea sericea Pterocorpus angolensis Strelitzia nicolai Toddaliopsis bremekampii
Rubiaceae Annonaceae Scrophulariaceae Mimosoideae Papilionoideae Papilionoideae Strelitziaazae Rutaceae
+I* -
Plant
spp.
Latex (ligand)
Gal
+(ovalbumin, mucin, NAcGal)/NAcGal, NacGlc +(ovalbumin, mucin, NAcGal, lactosamine)/ NAcGal, Gal +(ovalbumin, muck, NAcGal, lactosamine)/ NAcGAI, Gal -
--
*The sugars, glucose. mannose, galactose, fucose, NAcGlc, tNon-specific agglutination observed (details in the text).
+(ovalbumin)/+ + (ovalbumin, mucin, N AcGal, lactosamine)/NAcGal, Gal -
-t +(ovalbumin, and NAcGal
HRP, mucin, NAcGlc)/Glc,
Man, NAcGlc
did not give any visible inhibition of agglutination.
R. KAUL er (I/.
4008
the results. On the other hand, Mimosa pigra extract showed a non-specific agglutination with latex-BSA preparation. An interesting observation was that the Pterocarpus angolensis extract which in a study in Zimbabwe, has been shown to agglutinate erythrocytes from umbilical cord and new born children [l 11, and in our laboratory was unable to agglutinate adult A, B, or 0 erythrocytes, readily agglutinated latex beads coated with ovalbumin, horseradish peroxidase and porcine mucin. Furthermore, inhibition studies carried out using ovalbumin/HRPlatex showed that this lectin was inhibited by glucose, mannose and NAcGlc, as had been reported for cord erythrocytes by Moore [I]. Additionally, DomhnJea hurgessiae extract tested positive to latex agglutination test only in the presence of metal ions, and the agglutination could be inhibited by NAcGlc and NAcGal. Anthocleista grand(flora extract showed a rather weak agglutination with ovalbuminlatex, however, its sugar binding specificity could not be ascertained. From the studies presented above, it is evident that latex agglutination tests could be good substitutes for hemagglutination tests for lectin detection. The potential of the glycoproteins for use in latex agglutination tests is highly dependent on their ability to bind properly to the latex beads, and the accessibility of their sugar moiety for interaction with the lectin. On the other hand, sugars require a functional group for coupling to the latex particles. The latex particles are extremely stable in the freezedried form, and it is convenient to use them for field tests. The tests could be used satisfactorily to investigate new lectins and also avoid or detect the problems associated with haemolysins and non-specific (tanin based) agglutination that can cause investigations to over report the presence of lectins in crude plant extracts. The method was found to be sensitive, useful for sugar specificity studies, and could be used to isolate small amounts of the lectins for gel electrophoresis.
EXPERIMENTAL rVfateria/s. The following materials were obtained from Sigma: latex beads (polystyrene, 0.46 pm diameter, 10% solids content), carboxylate modified latex beads (0.4 pm diameter), ovalbumin (crude), horseradtsh peroxidase (type II, 175 U mg-’ solid), mucin (from porcine stomach), fetuin (from fetal calf serum), NI-ethyl-3-(3acetylglucosamine, N-acetyl galactosamine, dimethyl aminopropyl) carbodiimide, Jack beans (Canacalin ensiformis). concanavalin A, wheat germ agglutinin. lectins from Arachis
hypogeo,
Dolichos
bijorus,
Pisum
satirwn.
Phaseolus
and Terragonolohus purpureas. Artocarpus integrijolia (Jack fruit) seeds were procured from a market in Bangalore, India, whereas other plant seeds were obtained from the Botanical gardens, Harare, Zimbabwe. Soyabean agglutinin and lactosamine were prepd in the laboratory according to standard procedures [l2, 131. All other chemicals were of reagent grade quality. Preparation of seed extracrs. The seeds were crushed in a grinder prior to defatting. The ground material was defatted by mixing with heptane (5 ml g- * seed) for 2 hr at room temp. The heptane layer was filtered off through a sintered glass funnel, and the solid was dried overnight in a fume hood. It was then extracted with 0.9% saline (5 ml) for 1 hr at room temp. The tiulgaris
extract was filtered, and later clarified by centrifugation 15 mm at 10000 rpm.
at 4’ for
Physical udsorprion of glycoprofeins on latex beads. To 50 ~1 of 10% latex beads in a tube was added 400 PI of diluted (I : I) glycine buNer saline (0.17 M glycine. 0.9% NaCl and 0.004% NaN,, pH 7.3). I mg of a glycoprotein (ovalbumin. horseradish peroxldase, porcine mucin. fctuin) in 400 ~1 of 0.9% saline. The nixt. was incubated for 1 hr at 37 with intermIttent shaking. Subsequently, it was centrifuged at 10000 rpm for 7 mm. washed once with glycine buffer sahne (GBS) and once with GBS containing 0.1% bovine serum albumin (GAS-BSA). Latex particles coated only with BSA served as a control. Covalent
coupling
I$ glpcoprorems
and wnmo
sugars to car-
Porcine mucin and some derivatizcd sugars like N-acetylglucosaminc (NAcGlc). N-acetylgalactosamine (NAcGal) and lactosamme were coupled to the carboxylated latex by water-sol. carbodiimide. The coupling was carried out either by preactlvating the latex with I-ethyl-3-(3dimethylaminopropyl) carbodiimlde (EDC) [l4], or by incubating the glycoprotcin,‘sugar with the latex directly in the presence of the carbodiimide [IS]. According lo the first method, 50~1 of the latex beads were washed with I ml of borate buffer saline (BBS, 0.02 M borate-NaOH buffer, pH 8 m 0.17 M NaCI). After centrifugation the pellet was resuspended in IO0 ~1 BBS, lo which was then added 100~1 BBS containing 5 mg EDC. The suspension was vortexed at room temp. for I hr, centrifuged, and the pellet resuspended in 50 /iI BBS. To this was then added S mg glycoprotein or sugar in 0.5 ml BBS. After overmght shaking a! 4’. the latex was washed once with 2.5 ml GBS-0.1% Tween 20, and then twice with CBS-BSA. The particles were finally suspended in I ml GBS-BSA. Alternatively, 50 ~1 of latex suspension was washed with 0.5 M NaCI, and then suspended in 50 ~1 H,O at pH 4.5. To this was then added lOO/~l sugar:glycoproteln (5 mg) and 50~1 EDC (1 mg). both in H,O. pH 4.5. The mixt. was incubated overnight at room temp., centrifuged, washed twice with H,O and once with CBS-BSA. and finally suspended in I ml GBS-BSA. Latex agglutination fesfs. In a well of a multidish (Nunc, Denmark) (plate equipped with 12;24 wells, used for cell culture), I5 /II of the latex-glycoprotcin (sugar) suspension was mixed with an equal volume of saline containing lectin. The plate was covered and observed for agglutination up lo I -2 hr. The sensitivity of the level of detection of lectins was studied by double dilution of the lectin (I mgml ‘) with saline several times, and noting the least diluuon giving visible agglutination. The agglutination test was also carried out in the presence of Ca*‘, Mn*+, and Mg’+ rons at a concn of 2 mM. except for the Pisum lectin where the ion concn was maintained at 50 mM. A blank in which the coated latex was incubated with saline instead of the lectin was always included during the test. The possibility of non-specific agglutination was seen by incubating the known lectin or the plant extract with the BSA coated latex suspension. Sugar inhibition oj /arex agglufinaflon. Latex agglutmation with lectins and extracts was also performed in the presence of sugars--glucose. mannosc. galactosc, NAcGal. NAcGlc. or fucase. During such tests IO 111of the coated latex suspension was added to a mixt. of 10 ~1 lectin and IO ~1 sugar soln. The maximal sugar concn obtamed in the mixt. was I67 mM. The inhibition of the agglutination reaction by a sugar was taken as an evidence for the sugar specificity of the lectm. Hemagglucination tesrs. Hemagglutinating activity of the lectins and the plant extracts was also measured by the double dilution method, in which 50 /II of 2% RBC suspension (isolated from human blood group A,‘B,!O + , and washed with saline) was added to 50 ~1 of successively dilute lectin soln in a microtiter boxy/are
modified
lorex
huuds.
Screening for plant lectins plate. Agglutination was noted within 1 hr. Sometimes, trypsinized erythrocytes were required for the agglutination test. In such cases, the erythrocytes were treated with trypsin (0.1%) prior to the test, by a standard procedure [16]. Hemagglutination test was also carried out in the presence of metal ions in the same way as for the latex agglutination test. Sugar inhibition tests were also performed in case of seed extracts. Protein assay. Protein concentration was estimated by BioRad method using BSA as the standard. AcknowIedgemenrs-The financial support provided by Swedish Agency for Cooperation with Developing Countries (SAREC) is gratefully acknowledged. The authors are also thankful to Elisabeth Bjurulf and Roswita Schmitz for their excellent technical assistance, and to Eva Linne for providing purified soyabean agglutinin and lactosamine.
REFERENCES Lis, H. and Sharon, N. (1986) in The Lectfnr (Linier, I. E., Sharon, N. and Goldstein, 1. J., eds), p. 293. Academic Press, Florida. Kocourek, J. (1986) in The Pectins (Linier, 1. E., Sharon, N. and Goldstein, I. J., eds), p. 1. Academic Press, Florida. Strosberg, A. D., Buffard, D.. Lauwercys, M. and Foriers, A.
4009
(1986) in The Pectins (Linier, I. E., Sharon, N. and Goldstein, I. J., eds), p. 249. Academic Press, Florida. 4. Goldstein, 1. J. and Poretz, R. D. (1986) in The Pectins (Linier, 1. E., Sharon, N. and Goldstein, I. J.. eds), p. 33. Academic Press, Florida. Horejsi, V. and Kocourek, J. (1974) Biochim. Biophys. Acta 336,338. Pongor, S. and Reidl, Z. (1983) AM/. Biochem.129, 51. Huang, C.-C., Mayer, Jr., H. E. and Montgomery, R. (1970) Carbohydrate Res. 13. 127. Lanner, M., Bergquist, R., Car&m, J. and Huldt, G. (1978) in Afinity Chromatography (Hoffman-Ostenhof, O., ed.), p. 237. Pergamon Press, Oxford. 9. Lis, H., Joubert, F. J. and Sharon, N. (1985) Phytochemistry 24, 2803.
Bird, G. W. G. (1957) Nature 180, 657. Moore, H. H. (1977) M.Sc. Thesis. University of Rhodesia. Vretblad, P. (1976) Biochim. Biophys. Acta 434, 169. Honda, S., Suzuki, S. and Kakehi, K. (1987) J. Ckromut. 396, 93. 14. Masson, P. L., Cambiaso, C. L., Collet-Cassart, D., Magnusson, C. G. M., Richards, C. B. and Sindic, C. J. M. (1981) Methods Enrymol. 74, 106. Handbook, Pharmacia Fine 15. Affinity Chromatography Chemicals, Sweden. 16. Lis, H. and Sharon, N. (1972) Methods Enzymol. 28, 360. 10. 11. 12. 13.