Sulfated Fucose-Containing Polysaccharides from Brown Algae

15 Sulfated Fucose-Containing Polysaccharides from Brown Algae: Structural Features and Biochemical Implications D A R R E L L G.

MEDCALF

Downloaded by FUDAN UNIV on January 30, 2017 | http://pubs.acs.org Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch015

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 .


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


15.

MEDCALF

Sulfated

Fucose-Containing

Polysaccharides

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


227



228


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


15.

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Sulfated

Fucose-Containing

Polysaccharides

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


229



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



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


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


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


232


Acid Extract


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


MEDCALF

15.

Sulfated

Fucose-Containing

Polysaccharides

233

Acid Extract


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%.


234

CARBOHYDRATE

SULFATES

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


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. Figure 6.

Structural features of ascophyllan


236



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.


MEDCALF

Sulfated

Fucose-Containing

Polysaccharides


15.

Figure 9.

Structural features of a fucan "complex


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.


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



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)



240

TABLE V*GENERAL CHARACTERIZATION OF THE GALACTOFUCAN POLYMER

Molar r a t i o

fa]π

~


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)



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


242



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) .


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 .


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.


244

(13)

(14) (15)


(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

Percival, E. and R.S. 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. 23, Academic Press, London, 1967. Steber, J. and K.H. S c h l e i f e r , Arch. M i c r o b i o l . , (1975), 105, 173. Conchie, J. and E.G.V. Percival, J. Chem. soc., (1950), 827. Côté, R.H., J. Chem. Soc., (1959), 2248. O ' N e i l l , A.N., J. Am. Chem. soc., (1954), 76, 5074. Larsen, B., A. Haug, T.H. P a i n t e r , Acta Chem. Scand., (1966), 20, 219. Larsen, B. Acta Chem. Scand., (1967), 21, 1395. Percival, E., Carbohydr. Res., (1968), 7, 272. Percival, E., Carbohydr. Res., (1971), 17, 121. Larsen, B., A. Haug, and T.J. P a i n t e r , A c t a . Chem. Scand., (1970), 24, 3339. Mian, A.J. and E. Percival, Carbohydr. Res., (1973), 26, 147. Percival, E. and M. Young, Carbohydr. Res., (1974), 32, 195. Abdel-Fattah, A.F. and M. Endrees, Phytochem., (1977), 16, 939. Medcalf, D.G. and B. Larsen, Carbohydr. Res., in p r e s s . Medcalf, D.G. and B. Larsen, Carbohydr. Res., in p r e s s . Quatrano, R.S., unpublished r e s u l t s ( p r i v a t e communication). Larsen, B., unpublished r e s u l t s ( p r i v a t e communication). Mangel-Din Hussein, M., Phytochem., (1975), 14, 1866. Medcalf, D.G., T.L. Schneider and R.W. Barnett, Carbohydr. Res., in p r e s s . Quatrano, R.S. and M.A. Crayton, Dev. Biol., (1973), 30, 29. J a f f e , L.F., Advan. Morphog., (1968), 7, 295. Crayton, M.A., E. Wilson, and R.S. Quatrano, Dev. Biol., (1974), 39, 164. Hogsett, W.E. and R.S. Quatrano, Plant P h y s i o l . , (1975), 55, 25. Hogsett, W.E. and R.S. Quatrano, unpublished r e s u l t s . F u l c h e r , R.G. and M.E. McCully, Can. J. Bot., (1971), 49, 161. Otness, J.S. and D.G. Medcalf, Compar, Biochem. P h y s i o l . , (1972), 43B, 443.

RECEIVED February 6, 1978.


Sulfated Fucose-Containing Polysaccharides from Brown Algae

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