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On the interaction of Ricinus communis lectin with plant amyloids
148 Biochimica et Biophysica Acta, 354 (1974) 148--151
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands BBA 27433 ON THE INTERACTION OF R I C I N U S C O M M U N I S LECTIN WITH PLANT AMYLOIDS
J.P. VAN WAUWE, F.G. LOONTIENS and C.K. DE BRUYNE Laboratorium voor Algemene en Biologische Scheikunde, Rijksuniversiteit van Gent, K.L. Ledeganckstraat 35, B-9000 Gent (Belgium)
(Received January 29th, 1974)
Summary The interaction of R i c i n u s c o m m u n i s lectin with amyloids from tamarind, balsamine and capucine was demonstrated using the quantitative precipitin m e t h o d and immunodiffusion technique. Hapten inhibition indicate that amyloids are very reactive in coprecipitating with the lectin.
Introduction Recently we have reported the capacity of R i c i n u s c o m m u n i s lectin to coprecipititate with galactomannans [ 1 ], most probably by binding the terminal non-reducing a-D-galactopyranosyl units of these polysaccharides. Only a weak interaction between the lectin and the larch arabinogalactan was demonstrated, whereas charged and linear galactose-containing polymers, such as carregeenans, did n o t form a precipitate with the lectin. It was suggested that the precipitation reaction between the divalent [1,2] R. c o m m u n i s lectin and branched polysaccharides, fulfilling specific stereochemical requirements is analogous to an antibody--antigen system. This paper deals with a study of the interaction of the R. c o m m u n i s lectin with amyloids, water-soluble polysaccharides, giving a blue-staining reaction with iodine [3]. The techniques of coprecipitation, hapten inhibition and immunodiffusion are used. Materials and Methods The R. c o m m u n i s lectin was purified as described [4]. Quantitative precipitin studies and hapten inhibition experiments were performed as previously reported [ 1 ]. Immunodiffusion on cellulose acetate membranes was carried out as described [5]. The amyloids from tamarind ( T a m a r i n d u s indica), balsamine ( I m p a t i e n s
149
balsamina), and capucine (Tropaeolum majus) were gifts of Dr P. Le Dizet. Alfalfa galactomannan (Medicago sativa) was isolated as described [6]. D-Lactose was a product of UCB, Belgium, and methyl ~-D-galactopyranoside was prepared as published [7]. Escherichia coli ~-galactosidase was purchased from Boehringer. Results and Discussion The precipitation curves of the amyloids with the lectin are presented in Fig. 1. The structure of amyloids consists of a linear chain of ~-l,4-1inked D-glucopyranose units which carries 1,6-1inked single a-D-xylopyranose residues and 2-O-~-D-galactopyranosyl-~-D-xylopyranose units [3,8]. For the amyloids from tamarind and capucine, molar ratios of D-galactose: D-glucose: D-xylose were shown to be 1:3:2. However, for the balsamine amyloid, the molar ratios were 1:4:2 (Le Dizet, P., personal communication). Typical precipitin-like curves are obtained when increasing amounts of amyloid are incubated with a constant quantity of the lectin. The region of maximal turbidity was reached by adding 150--200 ~g of tamarind and capucine amyloids, whereas about 450 pg of balsamine amyloid was needed to obtain the same effect. This relatively weak interaction of balsamine amyloid with the lectin cannot only be explained by its lower galactose content, but probably also by its slightly different branching degree (Le Dizet, P., personal communication) or molecular weight [9] as compared with the other amyloids. In order to demonstrate the binding strength between the combining sites of the R. communis lectin and the amyloids, the concentrations of methyl-fl-Dgalactopyranoside and D-lactose required to inhibit the formation of protein-polysaccharide aggregates were examined. Table I shows that for the R. communis lectin--amyloid systems, much higher concentrations of low molecular weight carbohydrates are necessary for 50% inhibition than in the case of the lectin--alfalfa galactomannan system.
0.81
I
:
~
i
0.6
!|
°.'
0.2 i
i
,I
I
i
p| ~ l y l ~ r ~ l h N t
Fig. 1. T u r b i d i m e t r i c a l r e a c t i o n o f R. c o m m u n i s l e c t i n as a f u n c t i o n o f p o l y s a c c h a r i d e c o n c e n t r a t i o n . R e a c t i o n m i x t u r e ( p H 7 . 0 ) c o n t a i n e d 0 . 0 1 6 M s o d i u m p h o s p h a t e b u f f e r - - 0 . 1 M NaC1, p r o t e i n ( 0 . 7 5 r a g ) in a t o t a l v o l u m e o f 3 m l . e , t a m a r i n d a m y l o i d ; o, b a l s a m i n e a m y l o i d ; 4 c a p u c i n e a m y l o i d .
150 TABLE I I N H I B I T I O N OF T H E R. COMMUNIS
L E C T I N - - P O L Y S A C C H A R I D E P R E C I P I T A T I O N BY 50%
A t y p i c a l i n c u b a t i o n m i x t u r e c o n t a i n e d l e c t i n ( 7 5 0 ttg), a m y l o i d (at a c o n c e n t r a t i o n giving m a x i m a l t u r b i d i t y ) a n d i n h i b i t o r in a t o t a l v o l u m e o f 3.0 ml. T h e final m i x t u r e ( p H 7.0) was 0.1 M in NaC1 a n d 0.016 M with respect to sodium phosphate buffer. Compound
m M r e q u i r e d f o r 50% i n h i b i t i o n
D-lactose Methyl-fl-D-galactoside Methyl-fl-D-galact oside/D-lac tose
Tamarind amyloid
Capacine amyloid
Balsamine amyloid
Alfalfa galactomannan
0.32 0.85 2.6
0.44 1.05 2.4
0.29 0.80 2.7
0.050 0.16 3.2
The binding moiety of the amyloids (2-O-fl-D-galactosyl-D-xylopyranosyl unit) appears to represent more favourable configurational features necessary for interaction with the R. c o m m u n i s lectin than the a-D-galactosyl single-side chains of the alfalfa galactomannan. Furthermore, after incubation of capucine amyloid (10 mg in 5 ml 0.1 M sodium phosphate buffer containing lmM MgC12 ) with fl-galactosidase (0.15 units) [10,11] at 25°C for 24 h, the polysaccharide failed to coprecipitate with the lectin. This finding is an additional proof for the correctness of the structure of capucine amyloid as proposed by Le Dizet [8], as methyl-D-xylopyranosides are known to be non-inhibitors of polysaccharides--R, c o m m u n i s lectin systems. (Van Wauwe, J.P., unpublished). The ratio of the molar quantities of D-lactose and methyl-fl-D-galactoside required to produce 50% inhibition are quite similar in the amyloid and alfalfa galactomannan systems. This may be an indication that the fl-D-galactopyranosyl unit of amyloids and the a-D-galactopyranosyl unit of alfalfa galactomannan combine at the same binding site of the R. c o m m u n i s protein. The immunodiffusion patterns on cellulose acetate membranes of the R. c o m m u n i s lectin and the amyloids are shown in Fig. 2. All polysaccharides are found to be homogenous forming a single band after diffusion. We recommend this method for gaining information on the homogeneity of polysaccharide preparations, containing amyloids or galactomannans.
I
5,
~ .2
x/ •3
Fig. 2. I m m u n o d i f f u s i o n p a t t e r n s o n c e l l u l o s e a c e t a t e m e m b r a n e s . C e n t r a l well: R . c o m m u n i s l e c t i n (5 m g / m l ) Well 1, t a m a r i n d a m y l o i d (1.5 m g / m l ) ; Well 2, b a l s a m i n e a m y l o i d (1 m g / m l ) ; Well 3, e a p u c i n e a m y l o i d (1.5 rag/m1); Well 4, alfalfa g a l a c t o m a n n a n (1 m g / m l ) ; Well 5, saline c o n t r o l . 2o~zl s a m p l e s w e r e applied on the membranes. After diffusion (20 h at room temperature), precipitates were stained with 0 , 0 0 2 % n i g r o s i n in 2% a c e t i c a c i d .
151
'Acknowledgement We thank Dr P. Le Dizet for generous samples of amlyloids. References 1 Van Wauwe, J.P., Loontiens, F.G. and De Bruyne, C.K. (1973) Biochim. Biophys. Acta 313, 99--105 2 0 l s n e s , S., Saltvedt, E. and Pihl, A. (1974) J. Biol. Chem. 249, 803--810 3 AspinaU, G.O. (1970) Polysaecharides, Pergamon Press Ltd, New Y ork 4 Tomita, W., Kuxokawa, T., Onozaki, K., Ichiki, N., Osawa, T. and Ukita, T. (1972) Experientia 28, 84---85 5 Wi|liams, C.A. and Chase, M.W. (1971) Methods in I m m u n o l o g y and I m m u n o c h e m i s t r y , Academic Press, New Y o r k and L o n d o n 6 Andrews, P., Hough, L. and Jones, J.K. (1952) J. Am. Chem. Soc. 74, 4029--4034 7 De Bruyne, C.K. and Van der Groen, G. (1972) Carbohydr. Res. 25, 59---65 8 Le Dizet, P. (1972) Carbohydx. Res. 24, 505--509 9 So, L.L. and Goldstein, I.J. (1969) J. Immunol. 102, 53--57 10 Loontiens, G.G., Wallenfels, K. and Weft, R. (1970) Eur. J. Biochem. 14, 138--149 11 Van der Groen, G., Wouters-Leysen, J., Yde, M. and De Bruyne, C.K. (1973) Eur. J. Biochem. 38, 122--129
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands BBA 27433 ON THE INTERACTION OF R I C I N U S C O M M U N I S LECTIN WITH PLANT AMYLOIDS
J.P. VAN WAUWE, F.G. LOONTIENS and C.K. DE BRUYNE Laboratorium voor Algemene en Biologische Scheikunde, Rijksuniversiteit van Gent, K.L. Ledeganckstraat 35, B-9000 Gent (Belgium)
(Received January 29th, 1974)
Summary The interaction of R i c i n u s c o m m u n i s lectin with amyloids from tamarind, balsamine and capucine was demonstrated using the quantitative precipitin m e t h o d and immunodiffusion technique. Hapten inhibition indicate that amyloids are very reactive in coprecipitating with the lectin.
Introduction Recently we have reported the capacity of R i c i n u s c o m m u n i s lectin to coprecipititate with galactomannans [ 1 ], most probably by binding the terminal non-reducing a-D-galactopyranosyl units of these polysaccharides. Only a weak interaction between the lectin and the larch arabinogalactan was demonstrated, whereas charged and linear galactose-containing polymers, such as carregeenans, did n o t form a precipitate with the lectin. It was suggested that the precipitation reaction between the divalent [1,2] R. c o m m u n i s lectin and branched polysaccharides, fulfilling specific stereochemical requirements is analogous to an antibody--antigen system. This paper deals with a study of the interaction of the R. c o m m u n i s lectin with amyloids, water-soluble polysaccharides, giving a blue-staining reaction with iodine [3]. The techniques of coprecipitation, hapten inhibition and immunodiffusion are used. Materials and Methods The R. c o m m u n i s lectin was purified as described [4]. Quantitative precipitin studies and hapten inhibition experiments were performed as previously reported [ 1 ]. Immunodiffusion on cellulose acetate membranes was carried out as described [5]. The amyloids from tamarind ( T a m a r i n d u s indica), balsamine ( I m p a t i e n s
149
balsamina), and capucine (Tropaeolum majus) were gifts of Dr P. Le Dizet. Alfalfa galactomannan (Medicago sativa) was isolated as described [6]. D-Lactose was a product of UCB, Belgium, and methyl ~-D-galactopyranoside was prepared as published [7]. Escherichia coli ~-galactosidase was purchased from Boehringer. Results and Discussion The precipitation curves of the amyloids with the lectin are presented in Fig. 1. The structure of amyloids consists of a linear chain of ~-l,4-1inked D-glucopyranose units which carries 1,6-1inked single a-D-xylopyranose residues and 2-O-~-D-galactopyranosyl-~-D-xylopyranose units [3,8]. For the amyloids from tamarind and capucine, molar ratios of D-galactose: D-glucose: D-xylose were shown to be 1:3:2. However, for the balsamine amyloid, the molar ratios were 1:4:2 (Le Dizet, P., personal communication). Typical precipitin-like curves are obtained when increasing amounts of amyloid are incubated with a constant quantity of the lectin. The region of maximal turbidity was reached by adding 150--200 ~g of tamarind and capucine amyloids, whereas about 450 pg of balsamine amyloid was needed to obtain the same effect. This relatively weak interaction of balsamine amyloid with the lectin cannot only be explained by its lower galactose content, but probably also by its slightly different branching degree (Le Dizet, P., personal communication) or molecular weight [9] as compared with the other amyloids. In order to demonstrate the binding strength between the combining sites of the R. communis lectin and the amyloids, the concentrations of methyl-fl-Dgalactopyranoside and D-lactose required to inhibit the formation of protein-polysaccharide aggregates were examined. Table I shows that for the R. communis lectin--amyloid systems, much higher concentrations of low molecular weight carbohydrates are necessary for 50% inhibition than in the case of the lectin--alfalfa galactomannan system.
0.81
I
:
~
i
0.6
!|
°.'
0.2 i
i
,I
I
i
p| ~ l y l ~ r ~ l h N t
Fig. 1. T u r b i d i m e t r i c a l r e a c t i o n o f R. c o m m u n i s l e c t i n as a f u n c t i o n o f p o l y s a c c h a r i d e c o n c e n t r a t i o n . R e a c t i o n m i x t u r e ( p H 7 . 0 ) c o n t a i n e d 0 . 0 1 6 M s o d i u m p h o s p h a t e b u f f e r - - 0 . 1 M NaC1, p r o t e i n ( 0 . 7 5 r a g ) in a t o t a l v o l u m e o f 3 m l . e , t a m a r i n d a m y l o i d ; o, b a l s a m i n e a m y l o i d ; 4 c a p u c i n e a m y l o i d .
150 TABLE I I N H I B I T I O N OF T H E R. COMMUNIS
L E C T I N - - P O L Y S A C C H A R I D E P R E C I P I T A T I O N BY 50%
A t y p i c a l i n c u b a t i o n m i x t u r e c o n t a i n e d l e c t i n ( 7 5 0 ttg), a m y l o i d (at a c o n c e n t r a t i o n giving m a x i m a l t u r b i d i t y ) a n d i n h i b i t o r in a t o t a l v o l u m e o f 3.0 ml. T h e final m i x t u r e ( p H 7.0) was 0.1 M in NaC1 a n d 0.016 M with respect to sodium phosphate buffer. Compound
m M r e q u i r e d f o r 50% i n h i b i t i o n
D-lactose Methyl-fl-D-galactoside Methyl-fl-D-galact oside/D-lac tose
Tamarind amyloid
Capacine amyloid
Balsamine amyloid
Alfalfa galactomannan
0.32 0.85 2.6
0.44 1.05 2.4
0.29 0.80 2.7
0.050 0.16 3.2
The binding moiety of the amyloids (2-O-fl-D-galactosyl-D-xylopyranosyl unit) appears to represent more favourable configurational features necessary for interaction with the R. c o m m u n i s lectin than the a-D-galactosyl single-side chains of the alfalfa galactomannan. Furthermore, after incubation of capucine amyloid (10 mg in 5 ml 0.1 M sodium phosphate buffer containing lmM MgC12 ) with fl-galactosidase (0.15 units) [10,11] at 25°C for 24 h, the polysaccharide failed to coprecipitate with the lectin. This finding is an additional proof for the correctness of the structure of capucine amyloid as proposed by Le Dizet [8], as methyl-D-xylopyranosides are known to be non-inhibitors of polysaccharides--R, c o m m u n i s lectin systems. (Van Wauwe, J.P., unpublished). The ratio of the molar quantities of D-lactose and methyl-fl-D-galactoside required to produce 50% inhibition are quite similar in the amyloid and alfalfa galactomannan systems. This may be an indication that the fl-D-galactopyranosyl unit of amyloids and the a-D-galactopyranosyl unit of alfalfa galactomannan combine at the same binding site of the R. c o m m u n i s protein. The immunodiffusion patterns on cellulose acetate membranes of the R. c o m m u n i s lectin and the amyloids are shown in Fig. 2. All polysaccharides are found to be homogenous forming a single band after diffusion. We recommend this method for gaining information on the homogeneity of polysaccharide preparations, containing amyloids or galactomannans.
I
5,
~ .2
x/ •3
Fig. 2. I m m u n o d i f f u s i o n p a t t e r n s o n c e l l u l o s e a c e t a t e m e m b r a n e s . C e n t r a l well: R . c o m m u n i s l e c t i n (5 m g / m l ) Well 1, t a m a r i n d a m y l o i d (1.5 m g / m l ) ; Well 2, b a l s a m i n e a m y l o i d (1 m g / m l ) ; Well 3, e a p u c i n e a m y l o i d (1.5 rag/m1); Well 4, alfalfa g a l a c t o m a n n a n (1 m g / m l ) ; Well 5, saline c o n t r o l . 2o~zl s a m p l e s w e r e applied on the membranes. After diffusion (20 h at room temperature), precipitates were stained with 0 , 0 0 2 % n i g r o s i n in 2% a c e t i c a c i d .
151
'Acknowledgement We thank Dr P. Le Dizet for generous samples of amlyloids. References 1 Van Wauwe, J.P., Loontiens, F.G. and De Bruyne, C.K. (1973) Biochim. Biophys. Acta 313, 99--105 2 0 l s n e s , S., Saltvedt, E. and Pihl, A. (1974) J. Biol. Chem. 249, 803--810 3 AspinaU, G.O. (1970) Polysaecharides, Pergamon Press Ltd, New Y ork 4 Tomita, W., Kuxokawa, T., Onozaki, K., Ichiki, N., Osawa, T. and Ukita, T. (1972) Experientia 28, 84---85 5 Wi|liams, C.A. and Chase, M.W. (1971) Methods in I m m u n o l o g y and I m m u n o c h e m i s t r y , Academic Press, New Y o r k and L o n d o n 6 Andrews, P., Hough, L. and Jones, J.K. (1952) J. Am. Chem. Soc. 74, 4029--4034 7 De Bruyne, C.K. and Van der Groen, G. (1972) Carbohydr. Res. 25, 59---65 8 Le Dizet, P. (1972) Carbohydx. Res. 24, 505--509 9 So, L.L. and Goldstein, I.J. (1969) J. Immunol. 102, 53--57 10 Loontiens, G.G., Wallenfels, K. and Weft, R. (1970) Eur. J. Biochem. 14, 138--149 11 Van der Groen, G., Wouters-Leysen, J., Yde, M. and De Bruyne, C.K. (1973) Eur. J. Biochem. 38, 122--129