Circular Dichroism and Saccharide-Induced Conformational

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Circular Dichroism and Saccharide-Induced Conformational Transitions of Soybean Agglutinin MICHAEL W. THOMAS, JEANNE E. RUDZKI, EARL F. WALBORG, JR., and BRUNO JIRGENSONS The University of Texas Cancer Research Center, Department of Biochemistry, M. D. Anderson Hospital and Tumor Institute, Houston, TX 77030

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Lectins, sugar-binding proteins, have become powerful molecular probes to investigate the structure, topography and dynamics of cell-surface saccharide determinants (1). The utility of these proteins in the study of the surface properties of a variety of cell types has stimulated renewed interest in the determination of the molecular basis of their saccharide specificity. Furthermore lectins provide relatively simple models for the investigation of noncovalent interactions between saccharides and proteins. Since the discovery of soybean agglutinin (SBA) by Liener in soybean (Glycine max) extracts (2), a number of papers (3-5) have been published on its isolation, characterization, and sugar-binding specificity. SBA i s a glycoprotein comprised of four peptide subunits of approximately 30,000 daltons each (4). Two different types of subunits have been demonstrated by electrophoresis in the presence of sodium dodecyl sulfate (6). This lectin interacts specifically with 2-acetamido-2-deoxy-D-galactose (GalNAc) and galactose (Gal) (4) and possesses two saccharide binding sites per 120,000 daltons (5). Circular dichroism (CD) has been utilized to investigate the effect of saccharides on the conformation of lectins in solution. CD has demonstrated saccharide-induced conformational changes in the lectins from Canavalia ensiformis (7), Dolichos biflorus (8), Ricinus communis (9), and Triticum vulgaris (10). The present study uses circular dichroism to assess the secondary structure of SBA and to measure conformational transitions induced by the saccharides which bind to this lectin. These and previous studies w i l l contribute to a clearer understanding of the unique properties of these sugar-binding proteins. Materials and Methods Partially purified soybean hemagglutinin was prepared from untoasted soybean flour (Soyafluff 200W, Central Soya, Chicago, IL) according to the method of Liener (11). Final purification was achieved by chromatography on hydroxylapatite, prepared Current address: Research Division, Science Park, Smithville, TX 78957. 0-8412-0466-7/79/47-088-067$05.00/0 © 1979 American Chemical Society 1

Goldstein; Carbohydrate-Protein Interaction ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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according t o T i s e l i u s e t a l . (12). The f r e e z e - d r i e d p r o t e i n was d i s s o l v e d i n Ca 2- and M g ^ - f r e e phosphate b u f f e r e d s a l i n e (CMFPBS), pH 7.5, and i n s o l u b l e residue removed by c e n t r i f u g a t i o n . P r o t e i n concentrations were determined using an e x t i n c t i o n coef f i c i e n t of 1.28 a t 280 nm f o r a 0.1% s o l u t i o n of SBA i n a 1.0 cm cuvette (5). Polyacrylamide s l a b g e l e l e c t r o p h o r e s i s of SBA was performed i n 10% acrylamide g e l s i n the presence o f 0.1% sodium dodecyl s u l f a t e (SDS). Samples i n 2% SDS and 5% 3-mercaptoethanol were heated a t 100° C f o r 2 min. p r i o r t o a p p l i c a t i o n t o the g e l . S t a i n i n g was c a r r i e d out w i t h 0.05% Coomassie B r i l l i a n t Blue R-250 i n 7% a c e t i c a c i d . The s p e c i f i c hemagglutination a c t i v i t y of SBA was determined w i t h r a b b i t e r y t h r o c y t e s by the method o f Smith et_ a l . (13). One hemagglutination u n i t (HAU) i s defined as the minimum amount of l e c t i n necessary t o a g g l u t i n a t e e r y t h r o c y t e s . The a b i l i t y of saccharides t o i n t e r a c t w i t h the l e c t i n was d e t e r mined by t h e i r i n h i b i t i o n of l e c t i n - i n d u c e d a g g l u t i n a t i o n of rab b i t e r y t h r o c y t e s , according t o the method o f Smith e t a l . (13). One hemagglutination i n h i b i t i o n u n i t (HAIU) i s d e f i n e d as the min imum amount of saccharide necessary to i n h i b i t completely three hemagglutination u n i t s of l e c t i n . The purest a v a i l a b l e sugars were obtained from v a r i o u s sources : 2-acetamido-2-deoxy-D-glucose (GlcNAc), GalNAc, and 2-acetamido-2-deoxy-D-mannose (ManNAc) from Sigma Chemical Co., St. L o u i s , M i s s o u r i , and D-Gal and l a c t o s e from F i s h e r S c i e n t i f i c Co., F a i r Lawn, New J e r s e y . CD recordings were made on a Durrum-Jasco Model CD-SP Dichrograph, improved by D. P. Sproul of Sproul S c i e n t i f i c Instruments, Tucson, A r i z o n a . The s e n s i t i v i t y s c a l e s e t t i n g was 2 χ 10"^ d i c h r o i c absorbance per 1 cm on the recorder c h a r t . Spectra were measured a t p r o t e i n concentrations o f 0.48 mg/ml (1.0-cm c e l l ) i n the r e g i o n above 250 nm and 0.046 mg/ml (0.1-cm c e l l ) below 250 nm. A mean r e s i d u e weight of 109 was c a l c u l a t e d from the amino a c i d a n a l y s i s of SBA ( 5 ) . These data are expressed i n terms of mean r e s i d u e e l l i p t i c i t i e s [θ], i n degrees •cm* dmol""-^. A l l recordings were performed i n CMF-PBS, pH 7.5, a t 25 - 2° C and were repeated two o r three times. CMF-PBS was prepared according to Cronin e t a l . (14). A DuPont Model 310 curve r e s o l v e r was used to r e s o l v e CD curves i n t o gaussian bands. +

R e s u l t s and D i s c u s s i o n P r e p a r a t i o n and C h a r a c t e r i z a t i o n of SBA. P a r t i a l l y p u r i f i e d SBA was i s o l a t e d from 700 g of untoasted soybean f l o u r e s s e n t i a l l y as described by L i e n e r (11) f o l l o w i n g e x t r a c t i o n o f l i p i d s w i t h petroleum e t h e r . The procedure was c a r r i e d out through the step i n v o l v i n g d i a l y s i s a g a i n s t 60% ethanol a t -15° whereupon a p r e c i p i t a t e formed. This p r e c i p i t a t e d crude hemagglutinin (2 grams) was submitted t o chromatography on h y d r o x y l a p a t i t e as described by L i s et a l . (3). A summary of the p u r i f i c a t i o n of SBA i s presented i n Table I . L i s et^ ad. (15) reported the p o s s i b l e presence o f i s o l e c t i n s of SBA based on i t s r e s o l u t i o n i n t o m u l t i p l e components by


6.

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69

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chromatography on DEAE-cellulose. Although i t was p o s s i b l e t o o b t a i n chromatographic patterns s i m i l a r t o those reported by L i s and co-workers, homogeneity of these i s o l e c t i n s could not be demonstrated by rechromatography under the same c o n d i t i o n s . TABLE I PURIFICATION OF SBA

Purification Step H 0 Extract 2

40-70% (NH ) S0 4

2

4

ppt.

60% ethanol d i a l y s i s ppt. Hydroxylapatite chromatography a c t i v e peak

Weight Recovery mg 170,000

Specific Activity HAU/mg 600

Total Activity HAU χ 1 0 " 100

5,700

6,500

37

2,000

12,000

25

80,000

23

290

6

SBA, prepared as described h e r e i n , possessed an u l t r a v i o l e t (UV) a b s o r p t i o n spectrum and an e x t i n c t i o n c o e f f i c i e n t comparable to those p r e v i o u s l y reported ( 5 ) . Polyacrylamide g e l e l e c t r o p h o r e s i s i n the presence of SDS r e s o l v e d SBA i n t o two c l o s e l y spaced peptide bands comparable t o those reported by Lotan et_ a l . ( 6 ) . SBA e x h i b i t e d a s p e c i f i c a c t i v i t y of 80,000 HAU/mg. Secondary S t r u c t u r e of SBA. SBA showed a negative CD band centered a t 225 nm and a p o s i t i v e band a t 197 nm ( F i g . l a ) . Reso l u t i o n of t h i s curve i n t o gaussian bands y i e l d e d maxima a t 197, 217, 226, and 233 nm w i t h [θ] values o f 8900, -2400, -2900, and -900, r e s p e c t i v e l y . The amount of 3 s t r u c t u r e (26%) was estimated u s i n g the band a t 217 nm, according t o Chen e t a l . (16), t a k i n g the value of -9200 f o r the 3 standard. No second Cotton e f f e c t or trough could be discerned i n the r e g i o n of 208 nm i n d i c a t i n g the absence of any a p p r e c i a b l e α - h e l i c a l conformation. However, the p o s i t i v e band a t 196-198 nm i s c h a r a c t e r i s t i c of a high content of the p l e a t e d sheet conformation (17-19). Assessment of the 3 - s t r u c t u r a l content of t h i s p r o t e i n must be taken w i t h some r e s e r v a t i o n due to the general u n c e r t a i n t y i n q u a n t i t a t i o n o f t h i s conformation from CD s p e c t r a . I t i s known t h a t the o p t i c a l a c t i v i t y o f the 3 p l e a t e d sheet depends on the length and width o f the sheet (18) as w e l l as s o l v e n t e f f e c t s . Moreover, the CD e f f e c t s of peptide groups t h a t are n e i t h e r i n h e l i c a l or 3 pleated sheet regions are l i t t l e i n v e s t i g a t e d . According t o CD data the l e c t i n s


70


INTERACTION

i s o l a t e d from Canavalia e n s i f o r m i s ( 7 ) , Dolichos b i f l o r u s ( 8 ) , Pisum sativum (20), Robinia pseudoacacia ( 8 ) , R i c i n u s communis ( 9 ) , and Bandeiraea s i m p l i c i f o l i a (21) have a high content of the β conformation. The l e c t i n from T r i t i c u m v u l g a r i s appears t o be an exception having only 12% 3 s t r u c t u r e (10). CD Band Fine S t r u c t u r e of SBA i n the Near UV. Figure l b shows the CD spectrum of SBA i n the 250-320 nm s p e c t r a l zone. This r e g i o n i s c h a r a c t e r i z e d by a s m a l l negative band a t 300-310 nm, p o s i t i v e peaks a t 294 and 288 nm, and a broad p o s i t i v e r e g i o n a t 265-285 nm. There are crossover p o i n t s a t 300 and 259 nm. The bands a t 300-310 and 294 nm a r e probably due t o tryptophan w h i l e the band a t 288 nm i s due t o the t y r o s i n e chromophore ( 2 2 ) . The broad r e g i o n (265-285 nm) i s c h a r a c t e r i s t i c of the overlapping v i b r o n i c v i c i n a l i n t e r a c t i o n s of the aromatic chromophores ( 2 2 ) . This near UV spectrum i s very s i m i l a r t o that of the a-g-galactop y r a n o s y l - b i n d i n g l e c t i n i s o l a t e d from Bandeiraea s i m p l i c i f o l i a seeds (21). Since the soybean l e c t i n i s devoid of c y s t i n e (3,5, 15) a l l o f the CD bands i n the near UV r e g i o n a r i s e from v i c i n a l e f f e c t s of the aromatic chromophores. Saccharide S p e c i f i c i t y of SBA. The saccharide s p e c i f i c i t y of SBA was determined by hemagglutination i n h i b i t i o n assay (13). Of the saccharides t e s t e d , GlcNAc and ManNAc e x h i b i t e d l e s s than 2 HAIU/ymol. However other saccharides t e s t e d possessed the f o l l o w i n g hemagglutination i n h i b i t o r y a c t i v i t i e s : l a c t o s e , 20; D-Gal, 30; and GalNAc, 1000 HAIU/pmol. The r e l a t i v e a c t i v i t i e s are s i m i l a r t o those reported by L i s et_ a_l. (4) and suggest that an equa t o r i a l 2-acetamtdo group and an a x i a l 4-0H group of the g a l a c t o pyranosyl r i n g a r e important i n the b i n d i n g of saccharides t o the l e c t i n . Furthermore, the f a c t that d i s a c c h a r i d e s of GalNAc and D-Gal were not s i g n i f i c a n t l y b e t t e r i n h i b i t o r s than the c o r r e sponding monosaccharides (4) suggests that the saccharide b i n d i n g r e g i o n o f SBA may be no l a r g e r than the s i z e o f a monosaccharide. E f f e c t s of Saccharides on Conformation of SBA. None of the saccharides i n v e s t i g a t e d a f f e c t e d the CD spectrum o f SBA i n the f a r UV (190-250 nm) s p e c t r a l zone. However s i g n i f i c a n t sacchar i d e - i n d u c e d e f f e c t s were observed a t 265-290 nm, the r e g i o n i n which the aromatic chromophores d i s p l a y t h e i r Cotton e f f e c t s . These observations are i n accord w i t h the e s t a b l i s h e d i d e a that weak aromatic t r a n s i t i o n s can gain o r l o s e i n t e n s i t y through v i b r o n i c v i c i n a l i n t e r a c t i o n s which p r a c t i c a l l y do not a f f e c t the backbone peptide chromophores. The r e s u l t s are compiled i n Table II. Conformational t r a n s i t i o n s were induced by GalNAc and to a much l e s s e r extent by l a c t o s e and D-Gal. Several concentrations of GalNAc were u t i l i z e d t o e s t a b l i s h the minimal saccharide con c e n t r a t i o n r e q u i r e d t o produce the maximal conformational t r a n s i t i o n . GalNAc a t a c o n c e n t r a t i o n 1 mM induced maximal t r a n s i t i o n s


6.

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Figure 1. CD spectrum of SBA in the (a) far-UV and (b) near-UV regions. The lectin concentration was 0.046 mg/ml in the far-UV region and 0.48 mg/ml in the near-UV region. The light path length was 1 mm below 250 nm and 1 cm above 250 nm. The solid curves were constructed from four recordings each. The far-UV region was resolved into gaussian bands using the curve resolver. Solvent, CMF-PBS, pH 7.5. Bars indicate maximum deviation from mean. Comparable CD patterns were obtained using SBA, prepared by the method of Lotan et al. (6), obtained commercially from Miles Laboratories, Elkhart, Indiana.


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i n SBA as evidenced by a l t e r a t i o n s i n the CD spectrum a t 260-290 nm ( F i g . 2 ) . The saccharide e f f e c t was most pronounced a t 282 nm where SBA showed a mean r e s i d u e e l l i p t i c i t y [θ] i n degrees Όη^· dmo^." o f 56 ΐ 2 i n the absence o f s a c c h a r i d e . Α [θ] v a l u e of 4 4 - 2 was obtained i n the presence of 1 mM GalNAc. These e x p e r i ments were performed under c o n d i t i o n s i n which there were a v a i l able 125 molecules of saccharide per saccharide b i n d i n g s i t e . A d d i t i o n of 0.1 mM GalNAc, 25 mM l a c t o s e , o r 25 mM D-Gal t o SBA y i e l d e d [θ] values o f 50, 49, and 50, r e s p e c t i v e l y . See Table I I . The a d d i t i o n of 25 mM GlcNAc o r ManNAc, which do not bind t o SBA, d i d not i n f l u e n c e l e c t i n conformation (Table I I ) . By assuming that the l e c t i n b i n d i n g s i t e s are s a t u r a t e d a t those saccharide c o n c e n t r a t i o n s which y i e l d the maximal e f f e c t on the CD spectrum, i t was p o s s i b l e t o u t i l i z e the CD data t o c a l c u l a t e an a s s o c i a t i o n constant f o r the GalNAc/SBA i n t e r a c t i o n (7 χ 1 0 l i t e r . m o l e " ) . This value compares reasonably w e l l t o that reported by Lotan et a l . (5) u s i n g e q u i l i b r i u m d i a l y s i s and g e l f i l t r a t i o n (3 χ 10^ liter«mo l e " ) . 3

1

1

TABLE I I SACCHARIDE-INDUCED ALTERATIONS IN CD OF SBA

Saccharide

Conc. (mM)

b

294 nm

[û^sugar 288 nm

/ [θ] 282 nm

η ο

a

sugar 252 nm

GlcNAc

25

1.00

1.02

1.00

0.97

ManNAc

25

1.00

1.06

1.04

1.00

Lactose

25

0.94

0.91

0.88

0.97

D-Gal

25

0.94

0.88

0.89

0.96

GalNAc

0.01 0.1 1 25

0.94 0.96 0.94 0.92

0.95 0.92 0.85 0.86

0.98 0.89 0.73 0.76

0.97 0.97 1.00 1.00

[θ] The e f f e c t s a r e expressed as r a t i o s of ^ s u g a r / n o sugar at 294, 288, 282, and 252 nm. Maximum d e v i a t i o n from mean 4%. In a l l recordings the b a s e l i n e s were run w i t h s o l u t i o n s o f the saccharides i n CMF-PBS, pH 7.5. The saccharides exhib i t e d no CD bands i n the near UV. b) Concentration of the saccharide i n the l e c t i n - s a c c h a r i d e s o l u t i o n . In a l l cases, the c o n c e n t r a t i o n of the l e c t i n was 0.48 mg/ml (4yM). S o l v e n t , CMF-PBS, pH 7.5. u

J


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Circular Dichroism

73

We conclude that the interaction of the lectin with GalNAc, D-Gal, or lactose resulted in an alteration of the asymmetric environment of aromatic side-chain chromophores present at the surface of the lectin molecule. According to the CD data (Table II and Fig. 2), the most likely chromophores which are involved in the SBA-saccharide interactions are tyrosine and tryptophan side chains. The CD changes at 282 and 288 nm probably involve tyrosine and those at 294 nm, tryptophan. Similar effects on aromatic side chain chromophores have been seen during saccharide binding to the lectins from Canavalia ensiformis (7) and Triticum vulgaris (10). Since there are overlaps of the various chromophore effects over the whole near-UV zone, a more definitive assignment of the CD bands requires additional investigation. Acknowledgements This work was supported by grants from The Robert A. Welch Foundation (G-051), The George and Mary Josephine Hamman Founda tion and The Paul and Mary Haas Foundation. Abstract Conformational studies on soybean agglutinin (SBA) were performed using circular dichroism (CD). SBA exhibited a CD spectrum characterized by a small negative band at 300-310 nm, positive peaks at 294 and 288 nm, a broad positive region at 265-285 nm, a negative band centered at 224 nm and a positive peak at 197 nm. Analysis of the far ultraviolet CD bands indi cated approximately 26% pleated sheet (β) and no evidence of α-helix. 2-Acetamido-2-deoxy-D-galactose (GalNAc) at a concen tration 1 mM induced maximal conformational transitions in SBA (4μΜ) as evidenced by alterations in the CD spectrum at 260-290 nm. The saccharide effect was most pronounced at 282 nm where SBA showed a mean residue e l l i p t i c i t y [θ] in degrees•cm •dmol of 56 ±2 in the absence of saccharide. Α [θ] value of 44 ±2 was obtained in the presence of 1 mM GalNAc. Addition of 0.1 mM GalNAc, 25 mM lactose, or 25 mM D-galactose to SBA (4μM) yielded [θ] values of 50, 49, and 50, respectively. 2-Acetamido-2-deoxyD-glucose and 2-acetamido-2-deoxy-D-mannose, which do not bind to SBA, did not influence lectin conformation. According to the CD spectra, the polypeptide chain backbone of the lectin was not affected by interaction with the saccharides. 2

Literature Cited 1. 2. 3.

Sharon, N. and L i s , H. Science (1972) 177, 949-959. Liener, I. E. and Pollansch, M. J. J . Biol. Chem. (1952) 197, 29-36. L i s , H . , Sharon, Ν., and Katchalski, E. J . Biol. Chem. (1965) 241, 684-689.


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260

280

INTERACTION

300

X,nm

Figure 2. Effect of GalNAc on the conformation of SBA. The optical path length was 1 cm, the concentration of the lectin was 0.48 mg/ml, and the concentration of the saccharide was 1 mM. ( ) lectin without sugar; ( ) lectin with sugar. Solvent, CMF-PBS, pH 7.5. Bars indicate maximum deviation from mean.


6. THOMAS ET AL. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

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L i s , H., Sela, B-A., Sachs, L . , and Sharon, N. Biochim. Biophys. Acta (1970) 211, 582-585. Lotan, R., Siegelman, H. W., L i s , H . , and Sharon, N. J. Biol. Chem. (1974) 249, 1219-1224. Lotan, R., Cacan, R., Cacan, M., Debray, H . , Carter, W. G., and Sharon, N. FEBS Letters (1975) 57, 100-103. Pflumm, M. Ν., Wang, J. L . , and Edelman, G. M. J. Biol. Chem. (1971) 241, 4269-4370. Pere, M., Bourrillon, R., and Jirgensons, B. Biochem. Biophys. Acta (1975) 393, 31-36. Shimazaki, K . , Walborg, E. F., J r . , and Jirgensons, B. Arch. Biochem. Biophys. (1975) 169, 731-736. Thomas, M. W., Walborg, E. F . , J r . , and Jirgensons, B. Arch. Biochem. Biophys. (1977) 178, 625-630. Liener, J. E. J. Nutr. (1953) 49, 527-539. Tiselius, Α., Hjerten, S., and Levin, O. Arch. Biochem. Biophys. (1956) 65, 132-155. Smith, D. F . , Neri, G., and Walborg, E. F . , Jr. Biochemistry (1973) 12, 2111-2118. Cronin, A. P., Biddle, F . , and Saunders, F. K. Cytobios (1970) 2, 225-231. L i s , H., Fridman, C., Sharon, N . , and Katchalski, E. Arch. Biochem. Biophys. (1966) 117, 301-309. Chen, Y-H., Yang, J. T., and Chau, K. H. Biochemistry (1974) 13, 3350-3359. Jirgensons, B. "Optical Activity of Proteins and Other Macromolecules", (1973) 2nd ed., pp. 77-122, Springer-Verlag, Berlin. Woody, R. W. Biopolymers (1966) 8, 669-683. Balcerski, J. S., Pysh, E. S., Banora, Η. Μ., and Toniolo, C. J. Am. Chem. Soc. (1976) 98, 3470-3473. Bures, L . , Entlicher, G., and Kocourek, J. Biochim. Biophys. Acta (1972) 285, 235-242. Lönngren, J., Goldstein, I. J., and Zand, R. Biochemistry (1976) 15, 436-440. Strickland, E. H. CRC Crit. Rev. Biochem. (1974) 2, 113-174.

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September 8, 1978.


Circular Dichroism and Saccharide-Induced Conformational

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