Sulfated Fucose-Containing Polysaccharides from Brown Algae

Department of Chemistry, University of Puget Sound, Tacoma, WA 98416. The cell wall of ..... Calculated from gas chromatograms where total area under ...
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15 Sulfated Fucose-Containing Polysaccharides from Brown Algae: Structural Features and Biochemical Implications D A R R E L L G.

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Department of Chemistry, University of Puget Sound, Tacoma, W A 98416

The cell w a l l of p l a n t cells is an intriguing biochemical system t h a t continues to defy definitive c h a r a c t e r i z a t i o n . Peter Albersheim and his co-workers r e c e n t l y have completed the most comprehensive c h a r a c t e r i z a t i o n of the composition of the wall of t i s s u e c u l t u r e d cells ( 1 , 2 , 3 ) . In a d d i t i o n , Albersheim has suggested a model f o r the structural arrangement of these polymers in the w a l l and in some cases, has suggested specific r o l e s f o r particular p o l y s a c c h a r i d e s (4). N e v e r t h e l e s s , the exact r o l e s of the v a r i o u s f r a c t i o n s is not well understood. Moreover, information on the site and pathways of b i o s y n t h e s i s of w a l l polymers is very imcomplete (5,6). The cell w a l l s of algal cells have a unique composition and a l s o p r e s e n t , in certain systems, a unique o p p o r t u n i t y f o r the study of cell w a l l p o l y s a c c h a r i d e b i o s y n t h e s i s and assembly. The fertilized eggs o f the brown a l g a Fucus appear to be ideal f o r the study of cell w a l l composition, b i o s y n t h e s i s , and c o n t r o l of cell w a l l formation ( 7 , 8 , 9 ) . W a l l - l e s s eggs can be fertilized in a c o n t r o l l e d manner and the development of the wall f o l l o w e d in the synchronously developing, p o p u l a t i o n (10). The composition of the c e l l w a l l i n the brown algae (Phaeophyceae) i s made up of two f i b r i l l a r or s t r u c t u r a l polymers, c e l l u l o s e and a l g i n i c a c i d , and m a t r i x polymers. These m a t r i x polymers are a complex array of f u c o s e - c o n t a i n i n g s u l f a t e d p o l y saccharides (11). Mian and P e r c i v a l (12) concluded t h a t these polymers represented a spectrum from h i g h u r o n i c a c i d low s u l f a t e c o n t a i n i n g polymers to a r e l a t i v e l y pure fucan s u l f a t e . The abundance of s u l f a t e d p o l y s a c c h a r i d e s i n a l l three c l a s s e s of marine a l g a e , Chlorophyceae (green), Rhodophyceae ( r e d ) , Phaeophyceae (brown), w h i l e being e s s e n t i a l l y absent i n land p l a n t s , has i n v i t e d s p e c u l a t i o n as to the p h y s i o l o g i c a l f u n c t i o n of these components. The two most commonly suggested r o l e s are (1) involvement w i t h s e l e c t i v e c a t i o n i c t r a n s f e r i n the s a l i n e medium; (2) p r e v e n t i o n of d e s i c c a t i o n when the p l a n t s are exposed to drying c o n d i t i o n s such as low t i d e s (13). A recent r e p o r t of the presence of a s u l f a t e d p o l y s a c c h a r i d e as a major 0-8412-0426-8/78/47-077-225$05.00/0 © 1978 American Chemical Society

Schweiger; Carbohydrate Sulfates ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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component i n the c e l l w a l l of a h a l o p h i l i c b a c t e r i a (14) lends support to these suggestions. However, d e f i n i t i v e data on the s p e c i f i c biochemical r o l e of s u l f a t e i n these polymers has been lacking. This r e p o r t w i l l examine the composition of the s u l f a t e d f u c o s e - c o n t a i n i n g p o l y s a c c h a r i d e s from brown algae i n terms of t h e i r s t r u c t u r a l c h a r a c t e r i s t i c s and p o s s i b l e r o l e s i n c e l l development and c e l l w a l l formation and f u n c t i o n .

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P o l y s a c c h a r i d e Composition and S t r u c t u r e The e a r l y work on the chemical composition of the f u c o s e c o n t a i n i n g polymers i n brown algae centered on the m a t e r i a l c a l l e d " f u c o i d a n " , a hygroscopic s u l f a t e d p o l y s a c c h a r i d e f r a c t i o n f i r s t i s o l a t e d by K y l i n i n 1913. This m a t e r i a l seemed to be u b i q u i t o u s i n brown a l g a e , but the most e x t e n s i v e s t u d i e s were done on m a t e r i a l i s o l a t e d from Fucus v e s i c u l o s u s and AscophyHum nodosum by d i l u t e a c i d or water e x t r a c t i o n . Although small amounts of x y l o s e and g a l a c t o s e were always found a s s o c i a t e d w i t h " f u c o i d a n " , i t was g e n e r a l l y considered to be a unique substance. I t s b a s i c s t r u c t u r e was shown by Conchie and P e r c i v a l (15) to be composed p r i m a r i l y o f L-fucopyranose u n i t s l i n k e d a - ( l 2) w i t h s u l f a t e groups p r i m a r i l y l o c a t e d a t p o s i t i o n 4. Small amounts of s i n g l e u n i t branches at C-3 and C-4 a l s o were detected (15,16,17). Larsen, Haug and P a i n t e r (18) showed t h a t crude a l g i n i c a c i d p r e p a r a t i o n s from Ascophyllum nodosum were contaminated by s m a l l amounts o f a f u c o s e - c o n t a i n i n g m a t e r i a l which was not " f u c o i d a n . " This m a t e r i a l was named a s c o p h y l l a n and was composed of fucose (25%), x y l o s e (26%), g l u c u r o n i c a c i d (27%) and 13% s u l f a t e . Traces of g a l a c t o s e and mannose a l s o were found. Data from p a r t i a l base h y d r o l y s i s of the molecule suggested a u r o n i c a c i d "backbone" w i t h branches c o n t a i n i n g x y l o s e and f u c o s e . L a t e r work (19) l e d t o the i s o l a t i o n of a s i g n i f i c a n t q u a n t i t y of 3-0-3-D-xylopyxanosyl-L-fucose from a m i l d a c i d h y d r o l y z a t e of a s c o p h y l l a n . This l e d to the c o n c l u s i o n t h a t both sugars were p a r t of the same branched c h a i n . P e r c i v a l (20,21) a l s o i s o l a t e d and p a r t i a l l y c h a r a c t e r i z e d a s u l f a t e d polymer f r a c t i o n from A. nodosum which contained fucose (49%), x y l o s e (10%) and g l u c u r o n i c a c i d (11%). These data i n d i cate t h a t t h i s was a d i f f e r e n t polymer f r a c t i o n from a s c o p h y l l a n . I t was shown to be a h i g h l y branched s t r u c t u r e w i t h end-group and (1-+4)-linked x y l o s e , end group and ( l - * 2 ) - l i n k e d fucose (some u n i t s were s i n g u l a r l y branched at p o s i t i o n 3 or 4 and some doubled branches were a l s o i n d i c a t e d ) and ( l - * 4 ) - l i n k e d g l u c u r o n i c a c i d . 3-0-(3-D-Glucopyranosyluronic a c i d ) - L - f u c o s e was a s i g n i f i cant s t r u c t u r a l e n t i t y and evidence f o r (l->3)-linked fucose was a l s o found. S u l f a t e groups were l o c a t e d on p o s i t i o n 4 of fucose residues. Larsen, e t . a l . (22) confirmed t h a t a s c o p h y l l a n was not the only nonfucoidan fucose polymer i n brown a l g a e . They showed !f

fl

Schweiger; Carbohydrate Sulfates ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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Sulfated

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t h a t both F\ v e s i c u l o s u s and A. nodosum contained other p o l y saccharide "complexes" and suggested t h a t perhaps " f u c o i d a n " i t s e l f d i d not e x i s t on these algae i n the n a t i v e s t a t e . More r e c e n t l y , P e r c i v a l s group s t u d i e d the carbohydrates i n s e v e r a l species o f brown algae from d i f f e r e n t f a m i l i e s of p l a n t s (12,23,24). They were unable t o separate the f u c o s e - c o n t a i n i n g f r a c t i o n s i n t o unique e n t i t i e s but d i d accomplish s e p a r a t i o n i n t o a s e r i e s o f f r a c t i o n s ranging from a n e a r l y pure fucan s u l f a t e t o one w i t h high u r o n i c a c i d and low s u l f a t e . These were considered to be a m o r e - o r - l e s s continuous spectrum o f s t r u c t u r e s . A l l species s t u d i e d had e s s e n t i a l l y the same p a t t e r n and the s t r u c t u r a l data suggested the same h i g h l y branched c h a r a c t e r i s t i c s as p r e v i o u s l y r e p o r t e d f o r " f u c o i d a n " from F_. v e s i c u l o s u s (15) and the glucuronoxylofucan from A. nodosum (20,21). One o f the s p e c i e s , Desmarestia a c u l e a t a , was shown t o have a much higher p r o p o r t i o n o f g a l a c t o s e i n i t s " f u c a n s " (24), p a r t i c u l a r l y i n the m a t e r i a l e x t r a c t e d w i t h d i l u t e a l k a l i . This crude e x t r a c t had g a l a c t o s e , fucose, x y l o s e and g l u c u r o n i c a c i d i n the molar p r o p o r t i o n s o f 2:1:0.13:1.7. The g a l a c t o s e was D-galactose and m e t h y l a t i o n s t u d i e s i n d i c a t e d i t was present i n the polymer p r i m a r i l y as end-group and (1-K5)-linked r e s i d u e s . S t i l l other v a r i a t i o n s i n composition have been found i n p a r t i a l l y p u r i f i e d e x t r a c t s from Padina pavonia (25). In l i g h t o f the apparent complexity o f the f u c o s e - c o n t a i n i n g polymers i n brown algae, there i s a need f o r a more d e f i n i t i v e c h a r a c t e r i z a t i o n o f these polymers i n order t o study t h e i r p o s s i b l e r o l e i n c e l l development. Medcalf and Larsen (2(3,27) have r e examined the f u c o s e - c o n t a i n i n g s u l f a t e d p o l y s a c c h a r i d e s from two w e l l - s t u d i e d brown a l g a l s p e c i e s , AscophyHum nodosum and Fucus v e s i c u l o s u s . An attempt was made t o assure minimum degradation during e x t r a c t i o n i n order t o i n c r e a s e the r e l i a b i l i t y of the chemical and b i o l o g i c a l c o r r e l a t i o n s . E x t r a c t i o n o f d r i e d weed w i t h water a t pH 2 o r 0.1M EDTA a t pH 7.5 gave i d e n t i c a l r e s u l t s as shown by both f r e e boundary e l e c t r o p h o r e s i s at pH 2 and pH 7 and c e l l u l o s e a c e t a t e e l e c t r o p h o r e s i s a t pH 7.5. Previous work had shown t h a t the f u c o s e - c o n t a i n i n g polymers were a l l r e l a t i v e l y h i g h l y charged w i t h u r o n i c a c i d and/or s u l f a t e groups. Thus, e l e c t r o p h o r e s i s should be a r e l i a b l e technique t o i n d i c a t e the complexity o f the e x t r a c t s and t o f o l l o w the success o f attempts to separate the mixtures i n t o i n d i v i d u a l components. A f r a c t i o n a t i o n procedure developed by Larsen, et_ al^. (18,22) was adapted f o r use i n t h i s study. I t i n v o l v e d f r a c t i o n a l p r e c i p i t a t i o n w i t h ethanol o f polymer s o l u t i o n s c o n t a i n i n g v a r y i n g conc e n t r a t i o n s o f magnesium o r calcium c h l o r i d e . C e l l u l o s e acetate e l e c t r o p h o r e s i s was used t o f o l l o w s m a l l s c a l e t r i a l f r a c t i o n a t i o n s . P r e p a r a t i v e s c a l e f r a c t i o n a t i o n s were done using optimum c o n d i t i o n s and the f r a c t i o n s c h a r a c t e r i z e d by both f r e e boundary (pH 2) and c e l l u l o s e a c e t a t e (pH 7.5) e l e c t r o p h o r e s i s . Isolated f r a c t i o n s were r e f r a c t i o n a t e d u n t i l f r e e boundary e l e c t r o p h o r e s i s i n d i c a t e d a t l e a s t 95% homogeneity. 1

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General f r a c t i o n a t i o n schemes f o r the crude e x t r a c t s from A. nodosum and F. v e s i c u l o s u s are shown i n Figure 1. Figures 2, 3, 4, and 5 show the r e s u l t s of the p u r i f i c a t i o n e f f o r t s as i n d i c a t e d by e l e c t r o p h o r e s i s . Four e l e c t r o p h o r e t i c a l l y pure and d i s t i n c t f r a c t i o n s were i s o l a t e d from A. nodosum (Frac. 1-4, Table I) and two from F. v e s i c u l o s u s (Frac." 1-2, Table I I ) . In each case an a d d i t i o n a l mixed f r a c t i o n was o b t a i n e d . However, the homogenious f r a c t i o n s accounted f o r almost 90% of the crude e x t r a c t s from each s p e c i e s . Therefore, these f r a c t i o n s represent unique p o l y mer molecules present i n these algae which can be e x t r a c t e d w i t h very d i l u t e a c i d . They are probably present as m a t r i x polymers i n the c e l l w a l l . Quatrano (28) has shown t h a t d i l u t e a c i d e x t r a c t s from the c e l l w a l l s of 24 hour embryos of these species have an e l e c t r o p h o r e t i c p a t t e r n very s i m i l a r to those shown here. Tables I and II give the a n a l y t i c a l data f o r these f r a c t i o n s . F r a c t i o n 1 from Ascophyllum was very s i m i l a r i n composition to ascophyllan and was i d e n t i c a l i n e l e c t r o p h o r e t i c m o b i l i t y to an ascophyllan sample i s o l a t e d p r e v i o u s l y (18). H y d r o l y s i s of t h i s f r a c t i o n w i t h d i l u t e base f o l l o w e d by d i a l y s i s and a n a l y s i s f o r fucose, u r o n i c a c i d and unsaturated u r o n i c a c i d gave the r e s u l t s shown i n Table I I I . A s i g n i f i c a n t p o r t i o n of the remaining u r o n i c a c i d and the unsaturated u r o n i c a c i d produced v i a 3 - e l i m i n a t i o n were i n the d i a l y z a b l e f r a c t i o n w h i l e the fucose remained essent i a l l y i n the n o n - d i a l y z a b l e p o r t i o n . These data suggested t h a t the m a j o r i t y of the fucose molecules were i n r e l a t i v e l y large fragments t h a t d i d not c o n t a i n u r o n i c a c i d . This i s c o n s i s t e n t w i t h a molecular s t r u c t u r e having a m a j o r i t y of the u r o n i c a c i d u n i t s i n a main chain to which are attached f u c o s e - c o n t a i n i n g s i d e c h a i n s . While a complete s t r u c t u r a l a n a l y s i s has not been done, these data p l u s the e a r l i e r data of Larsen (19) suggest t h a t a s i g n i f i c a n t p o r t i o n of the ascophyllan s t r u c t u r e can be represented as shown i n Figure 6. The nature of the linkages i n the main u r o n i c a c i d chain and the l i n k a g e of fucose t o xylose have not been determined f o r a s c o p h y l l a n . The s t r u c t u r e i n Figure 6 uses i n f o r m a t i o n obtained by P e r c i v a l (21) f o r these l i n k a g e s . The a s c o p h y l l a n - l i k e polymer i s o l a t e d by Medcalf and Larsen (27) contained mannuronic and g u l u r o n i c a c i d i n a d d i t i o n to g l u c u r o n i c a c i d i n c o n t r a s t to both the data of P e r c i v a l (20, 21) and e a r l i e r work by Larsen, e t . a l . (18,22) where only g l u c u r o n i c a c i d was d e t e c t e d . T h i s o b s e r v a t i o n was confirmed by Larsen (29) on a new Ascophyllum sample. F u r t h e r work i s needed to a s c e r t a i n whether these u r o n i c a c i d s are p a r t of a s i n g l e molecule, or whether there are s e v e r a l types o f a s c o p h y l l a n - l i k e molecules, having the same b a s i c s t r u c t u r e , but d i f f e r i n g i n the r e l a t i v e amounts of each u r o n i c a c i d . F r a c t i o n 1 from Fucus was s i m i l a r to a s c o p h y l l a n i n e l e c t r o p h o r e t i c m o b i l i t y . I t d i f f e r e d somewhat i n composition, p r i m a r i l y i n an i n c r e a s e d fucose content. However, i t s p r o p e r t i e s suggested a s t r u c t u r e c l o s e l y r e l a t e d to t h a t given f o r a s c o p h y l l a n . F r a c t i o n s 2 and 3 from Ascophyllum and F r a c t i o n 2 from Fucus

Schweiger; Carbohydrate Sulfates ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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were complexes which could be e a s i l y hydrolyzed i n very d i l u t e a c i d at 80° t o two new p o l y s a c c h a r i d e s . These polymers could be d i s t i n g u i s h e d e l e c t r o p h o r e t i c a l l y as shown i n F i g u r e 7 f o r the Ascophyllum f r a c t i o n s . The two new polymers were separated by f r a c t i o n a l p r e c i p i t a t i o n w i t h ethanol from a M g C l s o l u t i o n . Compositional data f o r these f r a c t i o n s are shown i n Table IV. The i n s o l u b l e (slower) f r a c t i o n was very s i m i l a r t o ascophyllan i n both e l e c t r o p h o r e t i c m o b i l i t y and composition. However, o n l y mannuronic a c i d could be detected i n s i g n i f i c a n t amounts (27). Base h y d r o l y s i s o f the complex from Ascophyllum gave a p a t t e r n of d i a l y z a b l e and n o n - d i a l y z a b l e fucose, u r o n i c a c i d and unsat­ urated u r o n i c a c i d which was the same as found f o r a s c o p h y l l a n (Table I I I ) . F r a c t i o n 2 from Fucus gave very s i m i l a r e l e c t r o ­ p h o r e t i c r e s u l t s a f t e r d i l u t e a c i d h y d r o l y s i s . The major d i f f e r ­ ence between the "complexes" from the two species seemed t o be that the Fucus f r a c t i o n , on h y d r o l y s i s , gave more o f the f a s t e r moving fucan polymer and l e s s of the a s c o p h y l l a n - l i k e f r a c t i o n . No f r a c t i o n from e i t h e r species corresponding t o " f u c o i d a n " could be detected i n the o r i g i n a l e x t r a c t . I t was concluded t h a t " f u c o i d a n " i s not a n a t i v e polymer i n these s p e c i e s , but i s a product o f the h y d r o l y s i s o f the fucose r i c h "complexes." The two complex f r a c t i o n s from Ascophyllum were shown by a time-course study o f the a c i d h y d r o l y s i s a t 75 t o g i v e a gradual i n c r e a s e i n the m o b i l i t y o f the o r i g i n a l band and a simultaneous gradual i n c r e a s e i n the c o n c e n t r a t i o n o f the slower component (Figure 8 ) . These data were i n t e r p r e t e d t o suggest t h a t the fucan p o r t i o n formed the backbone o f the molecule, and the a s c o p h y l l a n ­ l i k e components were attached as branches by a c i d - l a b i l e l i n k ­ ages. The v a r i o u s complexes d i f f e r from each other only i n the number o f a s c o p h y l l a n - l i k e molecules attached t o the fucan back­ bone. A proposed s t r u c t u r e i s shown i n Figure 9. The fucan i s shown w i t h α - (1 2 ) - l i n k a g e s based on data from Conchie and P e r c i v a l (15) . The nature o f the l i n k a g e between the two p o l y ­ saccharides i n the complex i s not known. Medcalf and Larsen (27) showed i t d i d not i n v o l v e a p e p t i d e b r i d g e . F r a c t i o n 4 from Ascophyllum had a n e u t r a l sugar composition q u i t e d i f f e r e n t from any f r a c t i o n p r e v i o u s l y r e p o r t e d f o r t h i s s p e c i e s . I t contained about equal amounts o f fucose and g a l a c ­ t o s e , some u r o n i c a c i d , and 14% s u l f a t e . Only two g a l a c t o s e r i c h polymers p r e v i o u s l y have been i d e n t i f i e d i n brown algae (24,30) and o n l y P e r c i v a l and Young (24) have p u b l i s h e d s t r u c ­ t u r a l d a t a . T h e i r f r a c t i o n , i s o l a t e d by a l k a l i n e e x t r a c t i o n , was d e s c r i b e d e a r l i e r . Medcalf, Schneider and Barnett (31) r e i s o l a t e d f r a c t i o n 4 i n h i g h l y p u r i f i e d form by repeated f r a c t i o n ­ a t i o n w i t h ethanol from CaCl~ s o l u t i o n s . I t gave a s i n g l e band on e l e c t r o p h o r e s i s and had trie composition shown i n Table V. The u r o n i c a c i d was shown t o be g l u c u r o n i c and only t r a c e s o f n e u t r a l sugars other than g a l a c t o s e and fucose were found i n the p u r i f i e d m a t e r i a l . The g a l a c t o s e was shown by D-galactose o x i ­ dase t o be p r i m a r i l y L - g a l a c t o s e . Only two major methylated 2

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Schweiger; Carbohydrate Sulfates ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

Schweiger; Carbohydrate Sulfates ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

Figure 1.

insoluble ( F r a c t . 2) insoluble (Fract.3)

Γ insoluble ( F r a c t . 5)

(0.8*

CTAB

soluble

soluble n e u t r a l glucan 0.5 v o l . ethanol

1 v o l . ethanol

1 v o l . ethanol

soluble (Fract.4)

5 vol. ethanol

2

insoluble i n 0.02 M C a C l )

Fractionation scheme used for the isolation of electrophoretically pure fractions from A. nodosum dilute acid extracts

insoluble ( F r a c t . 1)

1 v o l . ethanol

insoluble (0.75% i n 0.03 M M g C l J I — Ζ

2

(0.5% i n 0.05 M MgCl )

CRUDE EXTRACT

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Schweiger; Carbohydrate Sulfates ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

Figure 1.

soluble ( F r a c t . 3)

CTAB

soluble

soluble ( n e u t r a l glucan)

1 v o l , ethanol

insoluble ( F r a c t . 2)

Fractionation scheme used for the isolation of electrophoretically pure fractions from F'. vesiculosus dilute acid extracts

insoluble ( F r a c t . 1)

0.7 v o l . ethanol

2

insoluble (0.5% i n 0.05 M MgCl )

2

(0.5% i n 0.05 M MgCl )

CRUDE EXTRACT

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232

CARBOHYDRATE SULFATES

Acid Extract

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Fract. 1

Fract. 2

Fract. 3

A-

Fract. 4 Figure 2. Ascending pattern at pH = 2 of the original extract and purified fractions from A. nodosum

Fract. 5

Acid Extract J Fract. 1 I Fract. 2 I Fract. 3 Figure 3. Cellulose acetate electrophoresis, toluidine Blue stain, at pH = 7.5 of the original extract and purified fractions from A. nodosum

|Fract.4 j Fract. 5

Schweiger; Carbohydrate Sulfates ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

MEDCALF

15.

Sulfated

Fucose-Containing

Polysaccharides

233

Acid Extract

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Fract. 1

Fract. 2

A y0

Fract. 3

ι



Figure 4. Ascending pattern at pH = 2 of the original extract and puri\mmtk fied fractions from F . vesiculosus

j Acid Extract I Fract. 1

Figure 5. Cellulose acetate electrophoresis, toluidine Blue stain, at pH = 7.5 of the origi­ nal extract and purified frac­ tions from F . vesiculosus

I Fract. 2 I Fract. 3

TABLE I

MAJOR FRACTIONS FROM A. nodosum Protein .(%)

Uronic Acid (%)

S u l - Fuf a t e cose (%) (%)

Neutral Sugar d i s t r i b u t i o n ^ Fuc X y l Mann Gal Glu (%)

Frac­ tion

Yield (%)

1

31

3.6

26.4

12.8

15

37

29

21

3

11

2

29

2.4

15.8

20.9

33

73

11

10

2

5

3

18

1.8

6.4

25.2

40

81

9

4

2

4

4

11

8.5

7.1

14.7

19

34

14

15

27

10

5

11

3.1

7.4

8.1

36

71

7

4

14

4

a

Based on recovered m a t e r i a l o n l y . C a l c u l a t e d from gas chromatograms where t o t a l area under the f i v e peaks equals 100%.

Schweiger; Carbohydrate Sulfates ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

234

CARBOHYDRATE

SULFATES

TABLE I I , MAJOR FRACTIONS FROM F. v e s i c u l o s u s

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Fraction

Yield (%)

Protein (%)

1

21

6.3

2

66

5.4

3

13

3.0

Uronic Acid (%)

21.9 5.6 13.9

S u l - Fuf a t e cose (%) (%)

Neutral ^ Sugar d i s t r i b u t i o n Fuc X y l Mann Gal Glu (%)

18,.1

50

25.4

48,,1

70

12.7

40,.4

70

4.1

17

4

14

7

4

8

11

7

9

5

9

15

Based on recovered m a t e r i a l o n l y . C a l c u l a t e d from gas chromatograms where t o t a l area under the f i v e peaks equals 100%.

TABLE III* COMPOSITION OF FRACTIONS 1 AND 2 AFTER BASE HYDROLYSIS Total Carbohydrate (mg/ml sample)

Fucose (mg/ml sample)

Uronic Acid (mg/ml sample)

Unsaturated Uronic A c i d (mg/ml sample)

Fraction 1 0.12

0.242

0.0

0.13

0.206

0.042

0.62

0.11

0.153

0.028

Original

0.44

0.22

0.128

0.0

Base-hydrolyzed

0.46

0.23

0.098

0.028

Base-hydrolyzeddialyzed

0.38

0.20

0.067

0.017

Original

0.70

Base-hydrolyzed

0.76

Base-hydrolyzeddialyzed

Fraction 2

Schweiger; Carbohydrate Sulfates ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

Schweiger; Carbohydrate Sulfates ACS Symposium Series; American Chemical Society: Washington, DC, 1978. Figure 6.

Structural features of ascophyllan

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236

CARBOHYDRATE SULFATES

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Figure 7. Cellulose acetate electrophoresis, toluidine Blue stain, at pH = 7.5; (A) ascophyllan; (B) Fraction 2; (C) Fraction 2, hydrolyzed; (D) Fraction 3; (E) Fraction 3, hydrolyzed; (F) hydrolyzed Fraction 2, ethanol-insoluble fraction; (G) hydrolyzed Fraction 2, ethanol-soluble fraction

TABLE IV,FRACTIONS FROM MILD ACID HYDROLYSIS OF A. nodosum COMPLEX

Yield " (%) 3

Fraction

Protein (%)

Uronic Acid (%)

Fucose (%)

Neutral ^ sugar d i s t r i b u t i o n Fuc X y l Mann Gal Glu (%)

2(complex 1)

--

2.4

15.8

33

73

11

10

2

5

Insoluble

32

6.8

24.1

11

28

22

28

6

17

Soluble

68

1.6

4.1

42

77

8

8

1

6

Based on recovered m a t e r i a l o n l y . C a l c u l a t e d from gas chromatograms where the t o t a l area under the f i v e peaks equals 100%.

Figure 8. Cellulose acetate electrophoresis, toluidine Blue stain, at pH = 7.5. Mild acid hydrolysis (0.02M HCl, 75°C) of Fraction 2.

Schweiger; Carbohydrate Sulfates ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

MEDCALF

Sulfated

Fucose-Containing

Polysaccharides

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15.

Figure 9.

Structural features of a fucan "complex

Schweiger; Carbohydrate Sulfates ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

237

CARBOHYDRATE

238

SULFATES

sugars were found a f t e r h y d r o l y s i s o f the u r o n i c a c i d reduced and methylated polymer; 2,3,4-tri-0-methylfucose and 3,6,-di-0-methylg a l a c t o s e . Much smaller amounts o f 2,3,4,6-tetra-0-methylglucose and mono-methylfucose were a l s o detected. These data, along with data on p e r i o d a t e o x i d a t i o n from both the o r i g i n a l and d e s u l f a t e d polymers, suggested the p a r t i a l s t r u c t u r e shown i n Figure 10.

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Biochemical Role The e a r l y stages of growth o f f e r t i l i z e d Fucus zygotes are shown i n Figure 11 ( 7 ) . A l o c a l i z e d protuberance or r h i z o i d appears 12-16 hours a f t e r f e r t i l i z a t i o n . T h i s i s subsequently p a r t i t i o n e d from the r e s t o f the c e l l by the f i r s t d i v i s i o n at 20-24 hours. The r h i z o i d c e l l becomes the s i t e o f attachment to the substratum (32) and c o n s i d e r a b l e data has been accumulated to show t h a t t h i s c e l l i s both m o r p h o l o g i c a l l y and chemically d i s t i n c t from the other c e l l o f the t w o - c e l l e d embryo (7,32^33). One o f the d i s t i n g u i s h i n g c h a r a c t e r i s t i c s o f the r h i z o i d c e l l i s i t s accumulation o f a s u l f a t e d f u c o s e - c o n t a i n i n g p o l y s a c c h a r i d e . The p r o p e r t i e s o f t h i s polymer f r a c t i o n and i t s r o l e i n r h i z o i d formation and c e l l d i f f e r e n t i a t i o n have been s t u d i e d e x t e n s i v e l y by Quatrano and co-workers (32,34,35,36) . ^ Both a u d i o r a d i o g r a p h i c techniques with S, and t o l u i d i n e Blue 0 c y t o c h e m i c a l l y , showed that the s u l f a t e d fucan i n i t i a l l y i s observed around the r h i z o i d - h a l f o f the nucleus r a d i a t i n g t o ward the s i t e o f r h i z o i d i n i t i a t i o n , and e v e n t u a l l y becomes l o c a l i z e d i n the r e g i o n o f the c e l l w a l l protuberance. After c e l l d i v i s i o n t h i s m a t e r i a l i s found i n the c e l l w a l l o f the r h i z o i d c e l l (32,37). Quatrano and Crayton (32) showed that s u l f a t i o n o f the preformed polymer began a t about the time o f rhizoid initiation. I t was p o s t u l a t e d that the s y n t h e s i s o f the h i g h l y charged fucan s u l f a t e might be the cause o f p o l a r i t y i n the zygote and lead to r h i z o i d formation. However, some very elegant f u r t h e r work by Crayton, Wilson and Quatrano (34) proved that the establishment o f a p o l a r a x i s and i n i t i a t i o n o f r h i z o i d formation could occur even though s u l f a t i o n o f the fucan polymer was prevented. Very r e c e n t l y , Hogsett and Quatrano (36) i s o l a t e d a p l a n t l e c t i n from R i c i n u s seeds which would b i n d to both s u l f a t e d and d e s u l f a t e d fucans. T h i s l e c t i n was g a l a c t o s e s p e c i f i c , but showed a f f i n i t y f o r the fucans found i n Fucus. These authors a t t r i b u t e t h i s b i n d i n g to g a l a c t o s e r e s i d u e s a s s o c i a t e d with these f r a c t i o n s . By a t t a c h i n g a f l o u r e s c e n t dye to the l e c t i n , they were able to use i t as a cytochemical marker f o r the fucan polymers. The data obtained c l e a r l y showed that the u n s u l f a t e d fucan does not accumulate i n the r h i z o i d r e g i o n . The polymer was a v a i l a b l e but s u l f a t i o n was r e q u i r e d f o r i t s l o c a l i z a t i o n and i n c o r p o r a t i o n i n t o the c e l l w a l l o f the r h i z o i d c e l l . T h i s appears to be the f i r s t c l e a r documentation o f a s p e c i f i c b i o chemical r o l e f o r s u l f a t e groups i n polymers o f brown algae. The a v a i l a b i l i t y o f synchronously developing populations o f

Schweiger; Carbohydrate Sulfates ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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15.

M E D C A L F

Sulfated

Fucose-Containing

239

Polysaccharides

ft

Ο

ο

on

< ρ

"ο ρ. "δ Ο

ο "S «+~. Ο co

H—,

8

I Ο Où

S)

Schweiger; Carbohydrate Sulfates ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

CARBOHYDRATE SULFATES

240

TABLE V*GENERAL CHARACTERIZATION OF THE GALACTOFUCAN POLYMER

Molar r a t i o

fa]π

~

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S u l f a t e (%)

5 4

15

°

Galactose

1.0

Fucose

1.1

Uronic A c i d (%)

8

Xylose

0.1

P r o t e i n (%)

7

Mannose

0.1

Glucose

trace

Experimental Marine Biology Figure 11. Stages in the development of Fucus embryos. (A) 0-12 hr, (B) 12-16 hr, (C) 16-20 hr, (D) 30-36 hr. X600 (7). (Photomicrographs courtesy of G. B. Bouck)

Schweiger; Carbohydrate Sulfates ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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15.

MEDCALF

Sulfated

Fucose-Containing

241

Polysaccharides

Fucus zygotes from eggs which have no d e t e c t a b l e c e l l w a l l p r o v i d e s a unique system f o r the study o f w a l l formation (8,9). The appearance o f v a r i o u s w a l l f r a c t i o n s can be f o l l o w e d and these data may suggest s p e c i f i c r o l e s f o r i n d i v i d u a l w a l l p o l y s a c c h a r i d e s . Quatrano and Stevens (9) used t h i s system t o f o l l o w w a l l development i n Fucus v e s i c u l o s u s zygotes. C e l l w a l l formation could be observed as e a r l y as 10 t o 15 minutes a f t e r f e r t i l i z a t i o n . However, w a l l b i r e f r i n g e n c e was not observed u n t i l 60 minutes a f t e r f e r t i l i z a t i o n . Complete b i r e f r i n g e n c e ( a l l the p o p u l a t i o n ) d i d not occur f o r f o u r hours. The w a l l was composed of a l g i n a t e and c e l l u l o s e i n about equal amounts a t 30 minutes (Figure 12). Fucans were not detected u n t i l f o u r hours a f t e r f e r t i l i z a t i o n when they represented about 25-30% o f w a l l carbohydrate. While the t o t a l carbohydrate i n the w a l l continued t o i n c r e a s e , p a r t i c u l a r l y during r h i z o i d e l o n g a t i o n (16-24 h o u r s ) , the p r o p o r t i o n o f the major p o l y s a c c h a r i d e groups remained r e l a t i v e l y constant. Embryos a t 24 hours c o n s i s t e d o f 60% a l g i n a t e , 20% c e l l u l o s e and 20% f u c o s e - c o n t a i n i n g polymers. The fucans were composed o f two e l e c t r o p h o r e t i c a l l y d i s t i n c t f r a c t i o n s ; F , a slow moving component, which very l i k e l y was the a s c o p h y l l a n - l i k e F r a c t i o n 1 from Fucus d e s c r i b e d by Medcalf and Larsen (26); and V^> a f a s t e r moving f r a c t i o n having a h i g h e r fucose content than F^. This corresponded t o the complex ( F r a c t i o n s 2 and 3) r e p o r t e d by Medcalf and Larsen (26,27). The two fucan polymers d i d not appear i n developing w a l l s a t the same t i m e . Only F. was detected p r i o r t o 12 hours a f t e r f e r t i l i z a t i o n . By 12 nours, the developing w a l l acquired F which corresponded t o the f r a c t i o n which was s u l f a t e d and a s s o c i a t e d w i t h r h i z o i d f o r m a t i o n . The r o l e these polymers p l a y i n the developing w a l l i s only s p e c u l a t i o n a t t h i s time. However, Quatrano and Stevens (9) have suggested t h a t the appearance o f F^ as the e a r l i e s t m a t r i x component i n the developing w a l l , c o i n cident w i t h the a c q u i s i t i o n o f s t r u c t u r a l i n t e g r i t y and b i r e f r i n gence o f the w a l l , argues f o r i t s b a s i c s t r u c t u r a l r o l e i n w a l l assembly and f u n c t i o n . F which appeared a t the time o f r h i z o i d f o r m a t i o n , and which had t o be s u l f a t e d before i t was i n c o r p o r a t e d (37) may be r e s p o n s i b l e f o r c e l l adhesion. Zygotes i n which s u l f a t i o n o f F was i n h i b i t e d , formed r h i z o i d s but d i d not adhere to the substratum (32). The requirements o f s u l f a t e groups f o r adhesion i s c o n s i s t e n t w i t h data suggesting t h a t the presence o f a n i o n i c groups a t the surface p l a y s an important r o l e i n barnacle adhesion (38). The data on the p o l y s a c c h a r i d e s found i n the primary c e l l w a l l of s u s p e n s i o n - c u l t u r e d sycamore c e l l s has been proposed as a model by which t o compare i n f o r m a t i o n on c e l l w a l l s from other systems (_3). While a l g a l systems have unique f e a t u r e s , such as s t r u c t u r a l polymers other than c e l l u l o s e and high degrees o f s u l f a t i o n , there i s a s i m i l a r i t y o f s t r u c t u r e s which can be noted. Table VI i s an attempt t o show p o s s i b l e r e l a t i o n s h i p s between the d e t a i l e d w a l l i n f o r m a t i o n from sycamore c e l l s (4)and the data 2

Schweiger; Carbohydrate Sulfates ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

242

CARBOHYDRATE SULFATES

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70+

TIME A F T E R F E R T I L I Z A T I O N

(HRS.)

Plant Physiology Figure 12. Changes in the major polysaccharide composition of isolated cell walls from F. vesiculosus at different times after fertilization (9)

TABLE V I . COMPARISON OF CELL WALL POLYSACCHARIDES

% o f T o t a l Carbohydrate Sycamore C e l l s

a

Brown Algae

Structural Polymers

C e l l u l o s e , 27%

C e l l u l o s e , 20% A l g i n i c A c i d , 60%

Pectic

Arabinogalactan, 22% Rhamnogalactan, 18%

Glucuronoxylofucan ( a s c o p h y l l a n - l i k e ) 9% Glucuronogalactofucan, 1%

Xyloglucan, 23%

Fucan complexes, 10%

Polymers

Matrix Polymers

Taken from Albersheim (4) Composite o f data from Medcalf and Larsen (26) and Quatrano and Stevens (!9) .

Schweiger; Carbohydrate Sulfates ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

15.

MEDCALF

Sulfated

Fucose-Containing

Polysaccharides

243

obtained by Medcalf and Larsen (26) and Quatrano and Stevens (9) from brown algae. While the proposed r e l a t i o n s h i p s are over­ s i m p l i f i e d , they do suggest the p o s s i b i l i t y that p l a n t c e l l w a l l s , from whatever the source, i n c l u d i n g algae, have more s i m i l a r i t i e s than may p r e v i o u s l y have been recognized. A l g i n i c a c i d may serve both s t r u c t u r a l and p e c t i c r o l e s in the brown algae and thus could be l i s t e d in both f r a c t i o n s in the t a b l e .

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Summary The complicated nature o f the s u l f a t e d f u c o s e - c o n t a i n i n g p o l y s a c c h a r i d e s from brown algae have been reviewed. For Asco­ phyllum nodosum and Fucus v e s i c u l o s i s , a s e r i e s of e l e c t r o p h o r e t i c a l l y homogeneous f r a c t i o n s have been i s o l a t e d and p a r t i a l l y c h a r a c t e r i z e d . These i n c l u d e a xylofucoglucuronan (ascophyllan or a s c o p h y l l a n - l i k e ) , complexes which have a s c o p h y l l a n - l i k e mole­ cules attached t o a fucan backbone, and in Ascophyllum, a novel glucuronogalactofucan. Information on the biochemical r o l e o f major f u c o s e - c o n t a i n i n g polymers in Fucus have been reviewed. Work by Quatrano and co-workers i n d i c a t e d that these polymers have an important r o l e in c e l l wall development. The fucan com­ p l e x e s , when s u l f a t e d , are l o c a l i z e d in the r h i z o i d c e l l and are r e q u i r e d f o r adhesion o f the embryo t o the substratum. Literature Cited (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11)

(12)

Talmadge, K.W., K. Keegstra, W.D. Bauer, and P. Albersheim, Plant P h y s i o l . , (1973), 51, 158. Bauer, W.D., K.W. Talmadge, K. Keegstra, and P. Albersheim, Plant P h y s i o l . , (1973), 51, 174. Keegstra, Κ., K.W. Talmadge, W.D. Bauer, and P. Albersheim, Plant P h y s i o l . , (1973), 51, 188. Albersheim, P., in "Plant Biochemistry, 3rd ed.", J. Bonner and J.E. Varner, eds., P. 225, Academic Press, N.Y., 1976. C h r i s t p e e l s , M.J., Ann. Rev. Plant P h y s i o l . , (1976), 27, 19. Northcote, D.H., in "Plant Carbohydrate Biochemistry", J.B. Pridham, ed., P. 165, Academic Press, N.Y., 1974. Quatrano, R.S., in "Experimental Marine B i o l o g y " , R. M a r i s c a l , ed., P. 303, Academic Press, N.Y., 1974. Novotny, A.M. and M. Forman, P l a n t a , (1975), 122, 67. Quatrano, R.S. and P.T. Stevens, Plant P h y s i o l . , (1976), 58, 224. Ley, A.C. and R.S. Quatrano, B i o l . B u l l . , (1973), 145, 446. Percival, E. and R.M. McDowell, "Chemistry and Enzymology of Marine A l g a l P o l y s a c c h a r i d e s " , p. 176, Academic Press, London, 1967. Mian, A . J . and E. Percival, Carbohydr. Res., (1973), 26, 133.

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244

(13)

(14) (15)

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(16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) (31) (32) (33) (34) (35) (36) (37) (38)

CARBOHYDRATE

SULFATES

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RECEIVED February 6, 1978.

Schweiger; Carbohydrate Sulfates ACS Symposium Series; American Chemical Society: Washington, DC, 1978.