Gangliosides as Receptors for Cholera Toxin, Tetanus Toxin, and

21 Gangliosides as Receptors for Cholera Toxin, Tetanus Toxin, and Sendai Virus LARS SVENNERHOLM and P. FREDMAN Department of Neurochemistry, St. Jörgen Hospital, University of Göteborg, S-422 03 Hisings, Backa, Sweden

Downloaded by TUFTS UNIV on June 3, 2018 | https://pubs.acs.org Publication Date: July 31, 1980 | doi: 10.1021/bk-1980-0128.ch021

H. ELWING, J. HOLMGREN, and Ö. STRANNEGÅRD Institute of Medical Microbiology, University of Göteborg, S-413 46 Göteborg, Sweden The carbohydrate residues of cell surfaces have been implicated in many cell functions such as cell-cell interactions, immunological specificity, and receptors for bacterial toxins, viruses and hormones. Cell surface carbohydrates reside in both glycoproteins and glycolipids, and it is conceivable that some cell surface properties are determined by carbohydrate groups whether these groups are components of glycolipids and glycoproteins or not. The ABO-blood group substances are an example of glycoconjugates which are both glycolipids and glycoproteins. The gangliosides have recently attracted considerable interest as receptors, not only for bacterial toxins, but also for Sendai virus, interferon and some glycoprotein hormones (1). The major basic carbohydrate structure of gangliosides, the gangliotetraose moiety, has been considered unique for this group of lipids, but future work might show that the same structure might reside in glycoproteins, and then explain findings which are at present difficult to understand. The possibility of gangliosides serving as receptors for toxins was speculated upon as far back as the 1950s, but was only recently confirmed by work on cholera toxin. This toxin is readily isolated, its structure is fairly well known, and it is relatively stable during storage or when radioactively labelled. The receptor ganglioside, GM1, is the parent ganglioside of the major brain gangliosides. Except for ganglioside GM2 it is the most stable of all known gangliosides, and it occurs abundantly in almost a l l mammalian plasma membranes. The studies of the interaction between ganglioside GM1 and cholera toxin might therefore be a prototype for ganglioside studies. In our own studies the use of highly purified gangliosides and newly developed quantitative binding assays produced results which allowed detailed prediction of the recognition-specific structures of gangliosideGM1for cholera toxin.

0-8412-0556-6/80/ 47-128-373$5.00/ 0 © 1980 American Chemical Society Sweeley; Cell Surface Glycolipids ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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W i t h t h i s s y s t e m a s a p o i n t o f d e p a r t u r e we a d o p t e d t h e s a m e procedures f o r our s t u d i e s of the i n t e r a c t i o n s between gangliosides and t e t a n u s t o x i n and S e n d a i v i r u s . Isolation

of

gangliosides

H o m o g e n i z e d human b r a i n t i s s u e was e x t r a c t e d t w i c e w i t h t w e n t y v o l u m e s o f c h l o r ο f o r m - m e t h a n o l - w a t e r ^:8:3 ( f i n a l solvent r a t i o ) . The g a n g l i o s i d e s were s e p a r a t e d f r o m o t h e r l i p i d s b y phase p a r t i t i o n - w a t e r was a d d e d t o t h e t o t a l l i p i d e x t r a c t t o g i v e a f i n a l chloroform-methanol-water volume r a t i o of The upper p h a s e was e v a p o r a t e d t o d r y n e s s , d i s s o l v e d i n w a t e r , d i a l y s e d a g a i n s t s e v e r a l changes of water f o r t h r e e d a y s , and evaporated to d r y n e s s i n a r o t a t i n g e v a p o r a t o r . The g a n g l i o s i d e s d i s s o l v e d i n chloroform-methanol-water were s e p a r a t e d a c c o r d i n g t o t h e i r n u m b e r o f s i a l i c a c i d s o n a new a n i o n e x c h a n g e r e s i n , S p h e r o s i l - D E A E - D e x t r a n , c o n s i s t i n g of porous g l a s s beads covered with c r o s s - l i n k e d DEAE-Dextran, with a discontinuous gradient of potassium acetate i n methanol. From the f i v e major g a n g l i o s i d e f r a c t i o n s o b t a i n e d - w i t h one t o f i v e s i a l i c a c i d s - i n d i v i d u a l g a n g l i o s i d e s were i s o l a t e d by s i l i c a g e l chromatography on columns o r t h i n - l a y e r p l a t e s . T h e i n d i v i d u a l g a n g l i o s i d e s wei'3 p u r i f i e d t o h a v e a c a r b o h y d r a t e c o m p o s i t i o n , w h i c h w a s a t l e a s t 99% pure. G a n g l i o s i d e G T l a a n d G Q l b w e r e a t l e a s t 95% p u r e , a n d G P l c c o n t a m i n a t e d w i t h 10% G P l b . T h e m e t h o d s u s e d f o r t h e d e t e r m i n a t i o n o f t h e p u r i t y of i n d i v i d u a l g a n g l i o s i d e s have r e c e n t l y been d e s c r i b e d


U:8:5.6.

60:30:U.5

(2). Ligand

methods

Ganglioside-ELISA method. T h e g l y c o l i p i d (0.1 o r 0.2 ml) i s attached to the polystyrene t e s t tubes (or to the w e l l s of m i c r o t i t e r t r a y s ) by i n c u b a t i o n a t room t e m p e r a t u r e o v e r n i g h t . U n o c c u p i e d b i n d i n g s i t e s on t h e p l a s t i c s u r f a c e a r e b l o c k e d by i n c u b a t i o n w i t h 1% s e r u m a l b u m i n . T h e t u b e s a r e t h e n i n c u b a t e d f o r 1-5 h a t room t e m p e r a t u r e w i t h d i l u t i o n s o f t h e t e s t samples ( u s u a l l y 2 h f o r n o n - a n t i b o d y l i g a n d s and 5 h f o r a n t i b o d i e s ) in b u f f e r pH s u p p l e m e n t e d w i t h 1% s e r u m a l b u m i n o r i n t h e c a s e o f a n t i b o d i e s w i t h 0.05% T w e e n 20. Unbound m a t e r i a l i s r e m o v e d b y r i n s i n g t h e t u b e s w i t h b u f f e r c o n t a i n i n g 0.05% Tween 20. Bound n o n - a n t i b o d y l i g a n d s a r e t h e r e a f t e r demonstrated by s e q u e n t i a l i n c u b a t i o n s o f t h e t u b e s w i t h : ( l ) s p e c i f i c a n t i b o d y , (2) anti i m m u n o g l o b u l i n c o u p l e d t o a l k a l i n e p h o s p h a t a s e , a n d (3) nitrop h e n y l - p h o s p h a t e s u b s t r a t e (3_). The t e c h n i q u e i s i l l u s t r a t e d i n F i g u r e 1.

7.0-7-2

The w a t e r c o n d e n s â t i o n - o n - s u r f a c e (VCS) m e t h o d . In this method, o r i g i n a l l y elaborated for studying antigen-antibody r e a c t i o n s (k) t h e g a n g l i o s i d e i s a t t a c h e d t o t h e i n n e r b o t t o m

Sweeley; Cell Surface Glycolipids ACS Symposium Series; American Chemical Society: Washington, DC, 1980.


21.

SVENNERHOLM ET A L .

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s u r f a c e o f p o l y s t y r e n e P e t r i d i s h e s ( d i a m e t e r 3.5 cm; Heger P l a s t i c s A B ) , a n d u n o c c u p i e d b i n d i n g s i t e s b l o c k e d w i t h serum a l b u m i n i n t h e same m a n n e r a s f o r t h e g a n g l i o s i d e - E L I S A t e s t . A f t e r r i n s i n g w i t h 0.15 M NaCl, melted agar a t a concentration of 1% c o n t a i n i n g 0.01% serum a l b u m i n i s poured i n t o t h e d i s h e s f o r m i n g , a f t e r c o n g e l a t i o n , a 2.5 mm t h i c k a g a r l a y e r . W e l l s o f 5 mm d i a m e t e r a r e t h e n p u n c h e d i n t h e g e l a n d f i l l e d (50 y l ) w i t h different concentrations of the test ligand (e.g. cholera toxin). A f t e r d i f f u s i o n f o r 20 h a t r o o m t e m p e r a t u r e t h e a g a r g e l i s r i n s e d o f f and t h e p l a s t i c s u r f a c e washed w i t h d i s t i l l e d water and d r i e d . T h e d i s h i s t h e n p l a c e d u p s i d e down o v e r a c o n t a i n e r filled w i t h w a t e r a t 60°C. A f t e r e x p o s u r e t o w a t e r v a p o u r f o r o n e m i n u t e t h e d i s h i s removed and c o v e r e d . A c o n d e n s a t i o n p a t t e r n c o n s i s t i n g o f l a r g e c o n f l u e n t water drops i s t h e n formed i n a c i r c u l a r zone where a s p e c i f i c g a n g l i o s i d e - l i g a n d r e a c t i o n has t a k e n p l a c e . T h i s hydrophilic pattern contrasts to that f o r the remainder of the s u r f a c e where o n l y s m a l l c o n d e n s a t i o n drops a r e formed i n d i c a t i n g a d i s t i n c t l y l e s s h y d r o p h i l i c s u r f a c e ( F i g u r e 2). Hemadsorption binding assay. Identification of gangliosidebound v i r u s b y hemadsorption i s performed i n P e t r i d i s h e s i n c u b a t ed w i t h g a n g l i o s i d e s , egg a l b u m i n a n d v i r u s as f o r t h e w a t e r c o n d e n s a t i o n m e t h o d . A f t e r r e m o v i n g u n b o u n d v i r u s , to the d i s h i s added

5 ml of a 1ί suspension of guinea p i g e r y t h r o c y t e s i n 0.15 M N a C l . A f t e r 15 m i n u t e s o f s e d i m e n t a t i o n t h e u n a d s o r b e d e r y t h r o c y t e s a r e washed o f f and t h e hemadsorption p a t t e r n i n s p e c t e d . Permanent r e c o r d i n g o f t h e hemadsorption i s o b t a i n e d by contact copying o f the surface on photographic paper w i t h t h e use of m o n o c h r o m a t i c l i g h t a t U05 n m . I n t e r a c t i o n between

cholera

t o x i n a n d g a n g l i o s i d e GMI

I n 1971 v a n H e y n i n g e n a n d c o - w o r k e r s (5.) d e s c r i b e d t h a t a c r u d e g a n g l i o s i d e m i x t u r e i n a c t i v a t e d c h o l e r a t o x i n . We d e m o n s t r a t ed t h a t t h e i n a c t i v a t i o n was c a u s e d b y a s p e c i f i c r e a c t i o n between t h e t o x i n a n d a s i n g l e g a n g l i o s i d e G M I (6_). G M I i n h i b i t e d t h e b i o l o g i c a l e f f e c t s o f c h o l e r a t o x i n down t o e q u i m o l a r w i t h t o x i n , a n d i n c o n t r a s t t o a l l o t h e r s u b s t a n c e s t e s t e d , GMI g a n g l i o s i d e g a v e a s p e c i f i c p r e c i p i t a t i o n band w i t h c h o l e r a t o x i n i n Ouchterlony d o u b l e d i f f u s i o n - i n - g e l t e s t s . I n d e p e n d e n t l y , C u a t r e c a s a s (7.) a n d K i n g a n d v a n H e y n i n g e n {Q) d e s c r i b e d G M I g a n g l i o s i d e a s t h e s u b stance r e a c t i n g strongest with cholera t o x i n but w i t h less s p e c i f i c i t y than i n our study, probably because t h e ganglioside p r e p a r a t i o n s t h e y worked w i t h were n o t p u r e . Subsequent s t u d i e s i n s e v e r a l l a b o r a t o r i e s have p r o v i d e d f u r t h e r e v i d e n c e t h a t g a n g l i o s i d e GMI i s t h e n a t u r a l b i o l o g i c a l receptor for cholera toxin: l ) Studies of various c e l l types, including small intestinal mucosal c e l l s of d i f f e r e n t species, demonstrated a d i r e c t r e l a t i o n s h i p b e t w e e n t h e c e l l c o n t e n t o f GMI a n d t h e number o f t o x i n


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GLYCOLIPIDS

Enzymesubstrate Anti-rabbit — lgG«enzyme

Figure 1.

Schematic of the gangliosideELISA method

Cholera toxin(CT)

Water

Figure 2. Schematic of the vapor condensation-on-surface method


21.

SVENNERHOLM ET AL.

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as

Receptors

377


m o l e c u l e s t h a t t h e c e l l s c a n b i n d (9., 10). 2) E x o g e n o u s G M I g a n g l i o s i d e c a n b e i n c o r p o r a t e d i n t o t h e c e l l membrane a n d t h e n a c t a s a f u n c t i o n a l r e c e p t o r . T h i s was f i r s t s h o w n b y C u a t r e c a s a s ( l l ) who o b s e r v e d a n i n c r e a s e d b i n d i n g c a p a c i t y and l i p o l y t i c responsiveness of f a t c e l l s which had been soaked i n GMI. U s i n g t r i t i u m - l a b e l l e d G M I - g a n g l i o s i d e , Holmgren e t a l . (9_) d e m o n s t r a t e d i n c o r p o r a t i o n o f G M I i n t o s m a l l i n t e s t i n a l e p i t h e l i a l membrane a n d showed t h a t t h e i n c r e a s e i n GMI was associated with a corresponding increase i n the capacity of the i n t e s t i n e t o b i n d c h o l e r a t o x i n as w e l l as i n an i n c r e a s e d s u s c e p t i b i l i t y of the gut t o the d i a r r h e o g e n i c a c t i o n of the t o x i n (9.). I n c o r p o r a t i o n o f g a n g l i o s i d e GMI i n t o t r a n s f o r m e d c e l l s d e f i c i e n t on t h i s g a n g l i o s i d e r e s t o r e d c e l l responsiveness to cholera toxin (12). 3) P r e t r e a t m e n t o f c e l l m e m b r a n e s w i t h c h o l e r a t o x i n s p e c i f i c a l l y b l o c k e d t h e membrane GM1-ganglioside from r e a c t i n g w i t h galactose oxidase (13). h) I n c u b a t i o n o f e n z y m e - s u s c e p t i b l e t i s s u e s w i t h Vibrio cholerae s i a l i d a s e i n c r e a s e d t h e number o f b i n d i n g s i t e s p r o p o r t i o n a l t o t h e i n c r e a s e i n GMI g a n g l i o s i d e , a s w e l l a s i n c r e a s e d c e l l u l a r s e n s i t i v i t y to the toxin (9.). 5) C h e m i c a l m o d i f i c a t i o n s o f c h o l e r a t o x i n s b y m e a n s o f v a r i o u s reagents were c o n s i s t e n t l y found t o a f f e c t b i n d i n g t o c e l l s a n d t o GMI g a n g l i o s i d e t o t h e same e x t e n t (lU). A c e t y l s p h i n g o s i n e - G M 1 , i n w h i c h t h e f a t t y a c i d o f GMI was r e p l a c e d by an a c e t y l group, had r o u g h l y t h e same a b i l i t y a s i n t a c t GMI t o r e a c t w i t h c h o l e r a t o x i n i n c l u d i n g f o r m a t i o n o f a p r e c i p i t a t i o n l i n e i n agar g e l . Oligosaccharide-GM1 ( g a n g l i o t e t r a ose) d e v o i d o f b o t h the f a t t y a c i d and t h e s p h i n g o s i n e and t h e r e fore unable to form micelles could not p r e c i p i t a t e c h o l e r a t o x i n but e f f e c t i v e l y i n h i b i t e d the p r e c i p i t a t i o n r e a c t i o n s between t o x i n a n d G M I , p r o v i d e d t h a t i t was i n a 5-fold m o l a r e x c e s s t o the t o x i n E s s e n t i a l l y s i m i l a r r e s u l t s were o b t a i n e d by S t a e r k e t a l . ( ΐ β ) . The c h o l e r a t o x i n m o l e c u l e i s composed o f p r o b a b l y 5 l i g h t r e c e p t o r - b i n d i n g s u b u n i t s (Β) and a s t r u c t u r a l l y u n r e l a t e d " h e a v y " e f f e c t o r s u b u n i t ( A ) (17, 18). Since the f i v e l i g h t B - s u b u n i t s a r e i d e n t i c a l each t o x i n m o l e c u l e c a n be e x p e c t e d t o b i n d up t o 5 m o l e c u l e s o f GMI, an a s s u m p t i o n s u p p o r t e d by S a t t l e r e t a l . (19) a n d F i s h m a n e t a l . (20) who f o u n d t h a t a c h o l e r a t o x i n m o l e c u l e b i n d s a t m o s t U-6 gangliotetraose molecules.

(]_5).

The f i r s t s u g g e s t i o n t h a t g a n g l i o s i d e s may p l a y a r o l e a s r e c e p t o r f o r t e t a n u s t o x i n was made b e f o r e t h e b i o c h e m i c a l s t r u c t u r e o f t h e g a n g l i o s i d e s w e r e f u l l y k n o w n (21, 22). To d e m o n s t r a t e f i x a t i o n of t e t a n u s t o x i n by g a n g l i o s i d e s van Heyningen and M i l l e r (22) used boundary e l e c t r o p h o r e s i s and u l t r a c e n t r i f u g a t i o n . They found t h a t t h e g a n g l i o s i d e s GDlb and GTIb had t h e g r e a t e s t t o x i n b i n d i n g c a p a c i t y . T h e same s p e c i f i c i t y o f g a n g l i o s i d e s t r u c t u r e was a l s o f o u n d f o r b i n d i n g a t l o w t o x i n a n d g a n g l i o s i d e c o n c e n t r a t i o n s . I n t h i s c a s e f i x a t i o n was s t u d i e d b y f o l l o w i n g t h e b i n d i n g o f t o x i n t o g a n g l i o s i d e made i n s o l u b l e b y c o m p l e x i n g i t w i t h


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c e r e b r o s i d e . The i n s o l u b l e receptor and the t o x i n were c e n t r i f u g e d out. Later work using t h i s technique and described i n Ledeen and Mellanby (23.) showed that the t o x i n - b i n d i n g c a p a c i t y of g a n g l i o s i d e GDlb was about 8 times that of g a n g l i o s i d e GTIb and 20-80 times b e t t e r than GMI and GDla. Table I . A comparison of the a b i l i t y of d i f f e r e n t gangliosides i n cerebroside-ganglioside complexes t o b i n d tetanus t o x i n ( 23 )


Ganglioside

Smallest amount o f g a n g l i o s i d e necessary to bind 10 LD^ Q

ng GMI GDla GDlb GTIb

20 5.5 0.25 2.0

Ledley et a l . (2k), who measured the c a p a c i t y o f various g a n g l i o s i d e s to i n h i b i t tetanus t o x i n b i n d i n g to t h y r o i d membranes, found the order o f reactivity of the various t e s t e d g a n g l i o s i d e s to be GTl-GDlb>GM1-GDla>GM2. In c o n t r a s t , H e l t i n g et a l . (25) reported GMI to be e q u a l l y e f f e c t i v e as GDlb i n b i n d i n g tetanus t o x i n . The aim of our s t u d i e s was to d e f i n e the degree of s t r u c t u r e s p e c i f i c i t y i n the b i n d i n g o f tetanus t o x i n t o gang l i o s i d e s and thus i d e n t i f y the presumed o l i g o s a c c h a r i d e r e c o g n i t i o n s t r u c t u r e o f the r e c e p t o r . The b i n d i n g of tetanus t o x i n t o p l a s t i c - a t t a c h e d g a n g l i o s i d e s was determined u s i n g e i t h e r gangl i o s i d e - E L I S A or VCS techniques f o r q u a n t i t a t i o n of the t o x i n bound. F i g u r e 3 shows the r e s u l t s obtained with the g a n g l i o s i d e ELISA method. In c o n t r a s t to c h o l e r a t o x i n , which i n the same system was bound only to GMI, tetanus t o x i n bound s i g n i f i c a n t l y to s e v e r a l g a n g l i o s i d e s . The strongest r e a c t i o n s were obtained with GTIb, GQlb and GDlb c l o s e l y followed by GTla. D i s t i n c t l y lower but s t i l l strong a c t i v i t y was seen with GMI ( d i f f e r e n c e to e.g. GDlb: ρ 5

>25

>25

GMI

>5 >5 >5

>25 >25 >25

>25 25

5 >5 >5

>25 >25

GM2 GM3 GDla GDlb

GD3 GTla GTIb

2

2

n.t.

0.1

GQlb GPlc

2.8

2

n.t.

1

0.9

25 25 n.t.

5

2.8

2.8

0.1

0.05

0.01

0.1

0.05

0.02

n.t.

= not t e s t e d from dogfish. Approx.

10%

of

NeuAc was a l s o

0-acetylated


the i n -


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Figure 7. Specific binding of Sendai virus to GQlb ganglioside and its inhibition by antiserum as demonstrated with (a) the VCS method and (b) hemadsorption (see text). A: virus in fourfold serial dilutions in buffer; B: in lower-titer antiserum, 1%; C: in the same serum, 10%; D: in higher titer antiserum, 1%; this serum, 10%.

GTla

^—O-^—e-Ceramide Τ

GQlb

- BB- -CC e r a m i d e E[ ^ rr--CCHH^^h— Τ

GP1c

Τ

DK>-r}--B-Ceramide

f

Ψ Ύ

Figure 8. Ganglioside receptors for Sendai virus. |, Glucose; •, galactose; O, galactosamine; Y, NeuAc.


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v e s t i g a t e d whether the spot-forming m a t e r i a l would bind e r y t h r o cytes s e c o n d a r i l y (hemadsorption). This was shown to be the case. In each instance i n which the water condensation method gave positive results a parallel t i t r a t e d plate displayed specific hemadsorption f o r the same p o s i t i o n s (Figure Τ b ) . The hemadsorp t i o n method was s l i g h t l y more s e n s i t i v e than the water condensa t i o n technique a l l o w i n g d e t e c t i o n o f a k- to l 6 - f o l d higher v i r u s d i l u t i o n . Immune serum was shown t o s p e c i f i c a l l y i n h i b i t the bind i n g o f Sendai v i r u s to e.g. GQlb as examined by e i t h e r o f the two methods . A q u a n t i t a t i v e l y more p r e c i s e method to compare the b i n d i n g a f f i n i t y of Sendai v i r u s f o r the v a r i o u s g a n g l i o s i d e s was to a t t a c h the g a n g l i o s i d e s spotwise i n s e r i a l d i l u t i o n s to the p l a s t i c and then incubate the whole p l a t e with v i r u s ( g a n g l i o s i d e spot a s s a y ) . The minimal e f f e c t i v e c o a t i n g c o n c e n t r a t i o n of each g a n g l i o s i d e f o r b i n d i n g Sendai v i r u s could then be determined with e i t h e r the water condensation or the hemadsorption methods (Table II). The r e s u l t s confirmed those obtained with the v i r u s spot assay showing t h a t the v i r u s a f f i n i t y f o r GQlb and GPlc was about 50- t o 100-fold higher than f o r GDla and GTIb and >500-fold higher than f o r any of the other t e s t e d substances (Table I I ) . Thus i t i s c l e a r that Sendai v i r u s has a very strong b i n d i n g tendency to GQlb which seems to exceed that o f tetanus t o x i n to i t s "receptor" g a n g l i o s i d e s and a c t u a l l y approach the b i n d i n g strength of c h o l e r a t o x i n to GMI. Conversely to the s i t u a t i o n with tetanus t o x i n the s i a l i c a c i d r e s i d u e s extending from the t e r m i n a l galactose are the c r i t i c a l ones f o r b i n d i n g (Figure 8). One such residue i s an absolute requirement (compare GTIb and GDla with GDlb) and a d i s i a l o s y l group i n t h i s p o s i t i o n apparent l y confers maximal b i n d i n g c a p a c i t y (GQlb^GTla-GPlc). However, a l s o the Ν-acetylgalactosamine r e s i d u e (or the chain l e n g t h as such) i n the backbone seems to c o n t r i b u t e markedly to the "receptor" s t r u c t u r e , as i n d i c a t e d by the f a c t t h a t GD3 had only minimal b i n d i n g c a p a c i t y i n s p i t e o f possessing a d i s i a l o s y l group l i n k e d to a t e r m i n a l g a l a c t o s e . Sendai v i r u s has been shown to have the strongest a f f i n i t y f o r g a n g l i o s i d e s with the common t e r m i n a l end sequence: NeuAca2->8NeuAca2->3Gaiei^3GalNAc^ Some a f f i n i t y was a l s o shown by g a n g l i o s i d e s GDla and GTIb, with the same carbohydrate sequence but l a c k i n g the t e r m i n a l NeuAc. The sequence NeuAca2->3Gal3l->3GalNAc a l s o e x i s t s i n some g l y c o p r o t e i n s , i . e . g l y c o p h o r i n , the predominant g l y c o p r o t e i n of human erythrocytes. In a recent paper (31) i t was demonstrated t h a t the removal of s i a l i c a c i d from human erythrocytes with V i b r i o cholerae s i a l i d a s e a b o l i s h e d hemagglutination by Sendai v i r u s . Hemagglutin a t i o n t i t e r s were r e s t o r e d s e l e c t i v e l y by the i n c o r p o r a t i o n of NeuAc with β-galactoside a2->3sialyltransferase which has a s t r i c t



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substrate specificity for the Gal31-*3GalNAc sequence. It is conceivable that the natural receptor binding structure for Sendai virus has only one terminal NeuAc residue, but it is also possible that some of the oligosaccharide moieties received a disialosyl linkage during the incubation -with the specific sialyltransferase. Glycophorin is generally assumed to be the erythrocyte receptor of myxoviruses, primarily because purified glycophorin effectively inhibits agglutination of erythrocytes by most viruses (32), and it is the major glycoprotein of red cell membranes. This membrane may contain also minute amounts of ganglioside GQlb or one of the other two high affinity gangliosides for Sendai virus. In this case they may serve as the Sendai virus receptor. The study by Paulson et al. (31) has not ruled out this possibility, since their analytical procedure for products from reaction with specific sialyltransferases does not exclude the existence of gangliosides. A clear understanding of the interaction between a virus and the cell surface receptors will require an exact knowledge about the oligosaccharide structure. A glycoprotein has in general a relatively large number of oligosaccharide moitiés, which might differ considerably in composition. This disadvantage does not exist for glycolipids. By the development of the new methods for the separation of gangliosides with homogenous carbohydrate moieties and sensitive ligand methods, a sensitive tool has been created for the elucidation of the receptor structure, irrespective of whether the receptor is a glycoprotein or a glycolipid. Acknowledgements. The costs of the studies were defrayed by grants from the Swedish Medical Research Council (3X-62T and 16X-3382).

Literature Cited 1. Fishman, P.H.; Brady, R.O.: Biosynthesis and function of gangliosides. Science, 1976, 194, 906-9105. 2. Svennerholm, L. Structure and biology of cell membrane gangliosides, in 43rd Novel Symposium, Ouchterlony, Ö. and Holmgren, J., Eds. "Cholera and Related Diarrheas. Molecular Aspects on a Global Health Problem"; Karger: Basel, 1979, in press. 3. Holmgren, J.; Svennerholm, A.-M.: Enzyme-linked immunosorbent assays for cholera serology. Infect. Immun., 1973, 5, 662-667. 4. Elwing, H.; Nilsson, L.-Å.; Ouchterlony, Ö.: Visualization principles in thin-layer immunoassays (TIA) on plastic surfaces. Int. Archs. Allergy Appl. Immun., 1976, 51, 757-762.


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5. Van Heyningen, W.E.; Carpenter, C.C.J.; Pierce, N.F.; Greenough, W.B.: Deactivation of cholera toxin by ganglioside. J. Infect. Dis., 1971, 124, 415-418. 6. Holmgren, J.; Lönnroth, I.; Svennerholm, L . : Tissue receptor for cholera exotoxin: Postulated structure from studies with GM1-ganglioside and related glycolipids. Infect. Immun., 1973, 8, 208-214.


7. Cuatrecasas, P.: Interaction of Vibrio cholerae enterotoxin with cell membranes. Biochemistry, 1973, 12, 3457-3558. 8. King, C.A.; van Heyningen, W.E.: Deactivation of cholera toxin by a sialidase-resistant monosialosyl-ganglioside. J. Infect. Dis., 1973, 127, 639-647. 9. Holmgren, J.; Lönnroth, I.; Månsson, J.-E.; Svennerholm, L.: Interaction of cholera toxin and membraneGM1ganglioside of small intestine. Proc. Natl. Acad. Sci., (U.S.A.), 1975, 72, 2520-2524. 10. Hansson, H.-A.; Holmgren, J.; Svennerholm, L . : Ultrastructural localization of cell membraneGM1ganglioside by cholera toxin. Proc. Natl. Acad. Sci., (U.S.A.), 1977, 74, 3782-3786. 11. Cuatrecasas, P.: Gangliosides and membrane receptors for cholera toxin. Biochemistry, 1973, 12, 3558-3566. 12. Moss, J.; Fishman, P.H.; Manganiello, V.C.; Vaughan, M.; Brady, R.O.: Functional incorporation of ganglioside into intact cells: Induction of choleragen responsiveness. Proc. Natl. Acad. S c i . , (U.S.A.), 1976, 73, 1034-1037. 13. Mullin, B.R.; Aloj, S.M.; Fishman, P.H.; Lee, G.; Kohn, L.D.; Brady, R.O.: Cholera toxin interactions with thyrotropin receptors on thyroid plasma membranes. Proc. Natl. Acad. Sci., (U.S.A.), 1976, 73, 1679-1683. 14. Holmgren, J.; Lönnroth, I.: Cholera toxin and the adenylate cyclase-activating signal. J. Infect. D i s . , 1976, 133, 64-74. 15. Holmgren, J.; Månsson, J.-E.; Svennerholm, L . : Tissue receptor for cholera enterotoxin: Structural requirements ofGM1ganglioside in toxin binding and inactivation. Medical Biology, 1974, 52, 229-233. 16. Staerk, J.; Ronneberger, H.J.; Wiegandt, H.; Ziegler, W.: Interaction of ganglioside G and its derivatives with choleragen. Eur. J. Biochem., 1974, 48, 103-110. Gtetl


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17. Lönnroth, I.; Holmgren, J.: Subunit structure of cholera toxin. J. Gen. Microbiol., 1973, 76, 417-427. 18. Holmgren, J.; Lönnroth, I.: Structure and function of enterotoxins and their receptors, in 43rd Nobel Symposium, Ouchterlony, Ö. and Holmgren, J., Eds. "Cholera and Related Diarrheas. Molecular Aspects on a Global Health Problem"; Karger: Basel, 1979, in press.


19. Sattler, J.; Schwarzmann, G.; Staerk, J.; Ziegler, W.; Wiegandt, H.: Studies of the ligand binding to cholera toxin. Hoppe-Seyler's Z. physiol. Chem., 1977, 385, 159-163. 20. Fishman, P.H.; Moss, J.; Osborne, J.C.: Interaction of choleragen with the oligosaccharide of gangliosideGM1:Evidence for multiple oligosaccharide binding sites. Biochemistry, 1978, 17, 711-716. 21. Van Heyningen, W.E.: Tentative identification of the tetanus toxin receptor in nervous tissue. J. Gen Microbiol., 1959, 20, 310-320. 22. Van Heyningen, W.E.; Miller, P.A.: The fixation of tetanus toxin by ganglioside. J. Gen. Microbiol., 196l, 24, 107-119. 23. Ledeen, R.W.; Mellanby, J.: Gangliosides as receptors for bacterial toxins, in "Perspectives in Toxicology", Bernheimer, Α., Ed.; John Wiley and Sons (New York), 1977, pp. 15-42. 24. Ledley, F.D.; Lee, G.; Kuhn, L.D.; Habig, W.H.; Hardegree, M.C. Tetanus toxin interactions with thyroid plasma membranes. Im plications for structure and function of tetanus toxin re ceptors and potential pathophysiological significance. J. Biol. Chem., 1977, 252, 4049-4055. 25. Helting, T.B.; Zwisler, O.; Wiegandt, H.: Structure of tetanus toxin. II. Toxin binding to ganglioside. J. Biol. Chem., 1977, 194-198. 26. Van Heyningen, W.E.; Mellanby, J.: The effect of cerebrosides and other lipids on the fixation of tetanus toxin by gang lioside. J. Gen. Microbiol., 1968, 52, 447-454. 27. Mellanby, J.; Whittaker, V.P.: The fixation of tetanus toxin by synaptic membranes. J. Neurochem., 1968, 15, 205-208. 28. Mellanby, J.; Morgan, I . G . , cited by Ledeen R.W. and Mellanby, J.: Gangliosides as receptors for bacterial toxins, in "Per spectives in Toxicology", Bernheimer, Α., Ed.; John Wiley and Sons (New York), 1977, pp. 15-42.



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29. Helenius, A.; Morein, B.; Fries, E.; Simons, K.; Robinson, P.; Shirrmacher, V.; Terhorst, C.; Strominger, J.L.: Human (HLA-A and HLA-B) and murine (H-2K and H2-D) histocompatibility antigens are cell surface receptors for Semliki Forest virus. Proc. Natl. Acad. S c i . , (U.S.A.), 1978, 75, 3846-3850. 30. Haywood, A.M.: Characteristics of Sendai virus receptors in a model membrane. J. Mol. Biol., 1974, 83, 427-436.


31. Paulson, J . C . ; Sadler, J . E . ; H i l l , R.L.: Restoration of specific myxovirus receptors to asialoerythrocytes by i n corporation of sialic acid with pure sialyltransferases. J . Biol. Chem., 1979, 254, 2120-2124. 32. Bächi, T . ; Deas, J.E.; Howe, C.: Virus-erythrocyte membrane interactions, in "Cell Surface Reviews, Virus Infection and the Cell Surface", Poste, G., and Nicholson, G.L., Eds.; North-Holland (Amsterdam), 1977, Vol. 2, pp. 83-127. RECEIVED

December 10, 1979.


Gangliosides as Receptors for Cholera Toxin, Tetanus Toxin, and

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