Fungal Polysaccharides - American Chemical Society

12

Texture and Crystal Structure of Fungal Polysaccharides

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on April 15, 2016 | http://pubs.acs.org Publication Date: June 10, 1980 | doi: 10.1021/bk-1980-0126.ch012

ROBERT H. MARCHESSAULT and YVES DESLANDES Xerox Research Centre of Canada, 2480 Dunwin Drive, Mississauga, Ontario L5L 1J9

The study of crystalline conformation and cell wall morphology of polysaccharides is helpful in the search for structure-function relationships. For example, in the materials system composing fungal cell walls one finds polysaccharides such as: chitin, (1->3)-β-D-glucan, nigeran and (1->3)-α-D-glucan, whose known physical properties are quite different. The first two are only slightly crystalline in fungi, but fibrous and water insoluble; chitin(1) is a microfibrillar entity, while (1->3)-β-D-glucan(2) is a triple helix "network forming" skeletal substance. Nigeran(3) on the other hand, is highly crystalline, but it is soluble in warm water and is considered to play a space-filling role in the cell wall. Its propensity to "chainfold" when crystallizing from dilute solution and its high level of crystallinity can provide it with resistance to enzymatic attack (3). The water insoluble (1->3)-α-D-glucan has only recently been studied in the cell walls of different fungi (4). This polysaccharide seems to have a structural function in those organisms. Extracellular fungal polysaccharides are also produced by some fungi(5), however, very little information is available about the crystal structure of that class of polysaccharides (6). The crystalline conformations and cell wall morphologies of t y p i c a l fungal p o l y s a c c h a r i d e s have been derived from x - r a y fiber diffraction studies and m i c r o d i f f r a c t i o n a n a l y s i s in the e l e c t r o n m i c r o s c o p e . The r e c o r d i n g of s u i t a b l e d i f f r a c t i o n data r e q u i r e s d e i n c r u s t r a t i o n and c r y s t a l l i z a t i o n inducing t r e a t ments (7_). From the r e c o r d e d data i t i s p o s s i b l e to gain some idea of the i n t i m a t e r e l a t i o n of v a r i o u s c r y s t a l l i n e polysaccharides in the cell wall, although it is sometimes necessary to complement these s t u d i e s with

0-8412-0555-8/80/47-126-221$07.50/0 ©

1980 A m e r i c a n C h e m i c a l Society

Sandford and Matsuda; Fungal Polysaccharides ACS Symposium Series; American Chemical Society: Washington, DC, 1980.


222

FUNGAL POLYSACCHARIDES

i n - d e p t h a n a l y s i s of a m a t e r i a l from a pure n o n - f u n g a l source for a precise determination of the crystal structure. Almost a l l the l i n k a g e types are e n c o u n t e r e d i n the fungal p o l y s a c c h a r i d e s and s e v e r a l combinations of d i f f e r e n t l i n k a g e s a r e p o s s i b l e l e a d i n g t o a l a r g e number of p o l y s a c c h a r i d e s with d i f f e r e n t p r o p e r t i e s . Figure 1 shows two g l u c o s e r e s i d u e s w h i c h c a n be l i n k e d i n e i g h t d i f f e r e n t w a y s . The a n o m e r i c o x y g e n , 0 ( 1 ) , c a n be 1 i n k e d to f o u r different o x y g e n atoms l o c a t e d on t h e n e x t residue. When one c o n s i d e r s t h a t e a c h d i s a c c h a r i d e c a n be l i n k e d t o t h e n e x t g l u c o s e w i t h a g a i n e i g h t p o s s i b l e l i n k a g e s and t h a t d i f f e r e n t d i s a c c h a r i d e s c a n be u s e d , t h e number o f p o s s i b l e p o l y s a c c h a r i d e s i s v e r y g r e a t . T h i s r e p o r t i s a g e n e r a l s u r v e y of the p r i n c i p a l s t r u c t u r a l and m o r p h o l o g i c a l c h a r a c t e r i s t i c s o f t h e most common f u n g a l p o l y s a c c h a r i d e s . The f o u r p o l y s a c c h a r i d e s a l r e a d y m e n t i o n e d g e n e r a l l y a r e t h e most a b u n d a n t and extensive information is generally available. Some o t h e r l e s s c h a r a c t e r i z e d p o l y s a c c h a r i d e s which however h a v e i n t e r e s t i n g p r o p e r t i e s , w i l l a l s o be d e s c r i b e d . The focus of our review will be the correlation of c r y s t a l l i n e s t r u c t u r e and m o l e c u l a r c o n f o r m a t i o n w i t h m o r p h o l o g i c a l and f u n c t i o n a l features. The

X-Ray

Diffraction

Approach

X - r a y d i f f r a c t i o n t e c h n i q u e s a r e t h e o n l y way o f determining the crystal structure of natural and synthetic polymers, although the x-ray data itself o b t a i n e d f r o m a c r y s t a l l i n e p o l y m e r i c f i b e r or f i l m i s not s u f f i c i e n t to a l l o w c o m p l e t e r e f i n e m e n t of the structure. Conformational analysis and electron d i f f r a c t i o n r e p r e s e n t complementary methods which w i l l f a c i l i t a t e the d e t e r m i n a t i o n of the s t r u c t u r e . The necessary requirements for the x - r a y approach are: c r y s t a l 1 i η i t y and o r i e n t a t i o n . X - r a y d a t a c a n n o t be o b t a i n e d f r o m an a m o r p h o u s s a m p l e w h i c h means t h a t a n o n c r y s t a l l i n e p o l y m e r i c m a t e r i a l must be t r e a t e d i n o r d e r t o i n d u c e o r i m p r o v e c r y s t a l 1 i n i t y . Some p o l y m e r s , s u c h as c e l l u l o s e and c h i t i n , a r e c r y s t a l l i n e and o r i e n t e d i n t h e n a t i v e s t a t e . ( 1) In o r d e r t o c r y s t a l l i z e , a p o l y m e r must p o s s e s s r e g u l a r i t y in the c h e m i c a l s t r u c t u r e along the c h a i n . The m o n o m e r i c u n i t c a n be v e r y s i m p l e as i n p o l y e t h y l e n e or very complex as it is in the pneumococcal polysaccharides^), b u t as l o n g as i t is regularly repeated, the polymer will be suitable for crystallization. An irregularly branched chain



12.

MARCHESSAULT AND DESLANDES

Texture

ir Crystal

Structure

223

crystallizes with difficulty, whereas a randomly occurring single sugar substituent on a homop o l y s a c c h a r i d e backbone o n l y p r e v e n t s crystallization if the s u b s t i t u t i o n i s too f r e q u e n t . Wood x y l a n s , w i t h their 4-0-methyl-3 -D-glucuronic acid moieties are examples of the l a t t e r . ( 3 0 ^ ) If the s u b s t i t u e n t r e s i d u e or o t h e r f u n c t i o n a l group o c c u r s w i t h r e g u l a r or near r e g u l a r p e r i o d i c i t y , c r y s t a l l i n i t y s h o u l d be f o u n d a t a l l degrees of s u b s t i t u t i o n ; gal actomannans h a v i n g a - D g a l a c t o s e r e s i d u e s a p p e n d e d t o a D-mannose i n t h e m a i n chain are an e x a m p l e . {9) Another example is the secondary c e l l u l o s e a c e t a t e which c r y s t a l l i z e s even though a l l h y d r o x y l s are not acetylated.(10) O r i e n t a t i o n of the c h a i n s a l o n g the f i b e r a x i s i s required for x-ray structure determination. As c o n t r a s t e d w i t h small m o l e c u l e s which y i e l d perfect s i n g l e c r y s t a l s , a f i l m or a f i b e r o b t a i n e d f r o m a ρ ο lymer material consist of local areas of order called c r y s t a l l i t e s embedded i n a m o r p h o u s d o m a i n s . Once t h e c h a i n s a r e o r i e n t e d , t h e c r y s t a l l i t e s have a common o r i e n t a t i o n f o r one u n i t c e l l a x i s , n a m e l y t h e " f i b e r a x i s " and t h e o t h e r two unit c e l l dimensions have c y l i n d r i c a l s y m m e t r y a b o u t t h i s a x i s . F i g u r e 2 shows t h e different levels of o r g a n i z a t i o n from the oriented c h a i n , to the a n i s o t r o p i c c r y s t a l l i t e w i t h i t s unique o r i e n t a t i o n i n s i d e the f i b e r : the c h a i n a x i s p a r a l l e l to the f i b e r axis, but a l l possible rotations of the crystallite about the chain axis are present. A c c o r d i n g l y , an o r i e n t e d f i b e r y i e l d s a s i n g l e c r y s t a l r o t a t i o n p a t t e r n without r o t a t i n g the f i b e r , s i n c e the xr a y beam " s e e s " a l a r g e number o f c r y s t a l l i t e s a t o n c e , each r o t a t e d d i f f e r e n t l y about the f i b e r a x i s . F i g u r e 3 shows t h e d i f f e r e n t s t e p s i n v o l v e d i n t h e crystal structure determination of a p o l y s a c c h a r i d e starting from the natural sources. Once the polysaccharide has been isolated and i t s chemical structure is well defined, crystal structure determination can proceed provided that the p o l y s a c c h a r i d e c a n be made t o c r y s t a l l i z e . When a n a t u r a l p o l y s a c c h a r i d e i s a m o r p h o u s , a f i l m o r a f i b e r must be c a s t , s t a r t i n g from a s o l u t i o n ; subsequent annealing and stretching provides a c r y s t a l l i n e and o r i e n t e d s a m p l e . The t r e a m e n t s e q u e n c e u s u a l l y i n v o l v e s s t r e t c h i n g the sample at h i g h h u m i d i t y (or r e l a t i v e l y high t e m p e r a t u r e f o r a t h e r m o p l a s t i c ) , f o l l o w e d by a n n e a l i n g t r e a t m e n t w i t h t h e s a m p l e k e p t under t e n s i o n to m a i n t a i n o r i e n t a t i o n . The x - r a y d i f f r a c t i o n p a t t e r n i s t h e n r e c o r d e d f r o m w h i c h i n f o r m a t i o n on t h e u n i t c e l l o f t h e p o l y s a c c h a r i d e



224


ΑΝΟΜΕRIC OXYGEN

( α - or β - )

OXYGEN CARBON

Figure 1. Two glucose residues can be linked in eight different ways leading to different polysaccharides with different properties. Since it is possible to have different types of linkages and different sugar residues, the number of possible polysaccharides is very large.

Figure 2. Hierarchy of structural or ganization: (a) single chain in crystalline conformation; (b) chain in crystallite; (c) crystallites oriented inside fiber

(a)



MARCHESSAULT A N D DESLANDES

Texture

b- Crystal

Structure

225

STEPS FOR X-RAY CRYSTAL STRUCTURE ANALYSIS

RAW MATERIALS

STANDARD STRUCTURAL PARAMETERS MOLECULAR MODEL

PURIFICATION

PACKING REFINEMENT

CHEMICAL STRUCTURE

|Fo(hkl)| CALCULATION

ORIENTED FIBERS (ANNEALING) I FIBER DIAGRAM

SINGLE CRYSTALS

COMPARISON OF |F |and |F | 0

C

ELECTRON DIFFRACTION DENSITY H UNIT CELL SPACE GROUP HELIX PITCH HELIX SYMMETRY

GOOD ^AGREEMENT? YES FINAL MODEL

Figure 3. Flowchart of operations involved in evaluation of x-ray fiber diagram for polysaccharide crystal structure analysis: left, experimental procedure and evaluation of fiber diagram; right, computational refinement



226


as w e l l as on t h e s y m m e t r y and p i t c h o f a s i n g l e c h a i n helix are o b t a i n e d . A fortunate fact is that the m o l e c u l a r a x i s of symmetry, t h a t i s , the long c h a i n a x i s g e n e r a l l y c o i n c i d e s w i t h t h a t u n i t c e l l a x i s w h i c h has a common o r i e n t a t i o n a l o n g t h e f i b e r d i r e c t i o n , n a m e l y t h e fiber axis. Unit c e l l p e r i o d i c i t y in t h i s d i r e c t i o n , o f t e n r e f e r r e d t o as t h e " f i b e r r e p e a t " , i s d i r e c t l y r e l a t e d t o t h e c o n f o r m a t i o n and s y m m e t r y o f t h e monomer r e s i d u e s i n the d i r e c t i o n of the c h a i n . Experimentally, the f i b e r r e p e a t i s d i r e c t l y d e r i v e d from the l a y e r l i n e s p a c i n g of a f i b e r di agram.(11) The p a r a m e t e r s o f t h e u n i t c e l l and t h e s p a c e g r o u p can be d e t e r m i n e d s o l e l y f r o m t h e x - r a y f i b e r d i a g r a m although electron diffraction recorded from single c r y s t a l s of the p o l y s a c c h a r i d e are very h e l p f u l s i n c e t h e y g i v e much more p r e c i s e i n f o r m a t i o n a b o u t t h e b a s e plane.(1_2) Once t h e u n i t c e l l d i m e n s i o n s a r e k n o w n , dens i t y m e a s u r e m e n t s w i l l t e l l how many a s y m m e t r i c u n i t s are p r e s e n t i n the u n i t c e l l . M o r e o v e r , once the symmetry and t h e a d v a n c e p e r monomer o f t h e h e l i x ( o b t a i n e d f r o m the f i b e r diagram) a r e k n o w n , t h e number o f c h a i n s p r e s e n t i n t h e u n i t c e l l c a n be d e d u c e d . In a f i r s t s t e p , t h e t h r e e d i m e n s i o n a l s t r u c t u r e o f a single helix is generated s t a r t i n g with the known s t r u c t u r e of a s i n g l e sugar r e s i d u e f o r which the c r y s t a l s t r u c t u r e i s w e l l k n o w n . The h e l i x s y m m e t r y and a d v a n c e p e r monomer, d e r i v e d f r o m t h e f i b e r d i a g r a m , a r e t h e basic helix determinants. I f no s t e r i c h i n d r a n c e o r s h o r t c o n t a c t s are p r e s e n t , the "packing" of the h e l i c e s can be p e r f o r m e d w i t h t h e c h a i n s d i s p o s e d a c c o r d i n g t o the space group r e q u i r e m e n t s . The b e s t " p a c k i n g " m o d e l ( t h e one w i t h t h e minimum e n e r g y ) w i l l be u s e d f o r comparison of t h e o r e t i c a l l y p r e d i c t e d x - r a y d i f f r a c t i o n i n t e n s i t i e s with those observed. If the structure p r e s u m e d i s c o r r e c t , t h e r e w i l l be good a g r e e m e n t b e t w e e n t h e two s e t s o f i n t e n s i t i e s . On t h e o t h e r h a n d , i f t h e m o d e l p o s t u l a t e d i s w r o n g , t h e w h o l e p r o c e d u r e w i l l have t o be r e p e a t e d a f t e r a d j u s t m e n t s have been made i n t h e o r i g i n a l model . H i g h - s p e e d c o m p u t e r s and v e r s a t i l e p r o g r a m s a l l o w one t o r e f i n e t h e s t r u c t u r e by s i m u l t a n e o u s l y v a r y i n g many p a r a m e t e r s . T h i s l e a d s t o a model i n a g r e e m e n t w i t h the e x p e r i m e n t a l data. Unfortunately, in a f i b e r d i a g r a m , t h e r e are not enough x - r a y r e f l e c t i o n s t o p e r m i t t h e use o f F o u r i e r s y n t h e s i s m e t h o d s . However, p r i o r knowledge of the chemical structure coupled with c o n f o r m a t i o n a l and p a c k i n g a p p r o a c h e s , b a s e d on minimum energy c o n s i d e r a t i o n s , a l l o w a r e a s o n a b l e approach to f i n d i n g a very probably c o r r e c t c r y s t a l structure.


12.


Texture

b- Crystal

Structure

227

In the next s e c t i o n , the d e t e r m i n a t i o n of the s t r u c t u r e o f ( 1+3 ) -3-D - g l u c a n w i l l be d e s c r i b e d i n d e t a i l s i n c e i t r e p r e s e n t s â complex s t r u c t u r e s o l v e d w i t h the help of x-ray diffraction and computer-based c o n f o r m a t i o n a l and p a c k i n g a n a l y s i s .


(l->3)-3-D-G1ucan A l t h o u g h e n c o u n t e r e d i n many y e a s t s and f u n g i , t h i s p o l y s a c c h a r i d e i s u b i q u i t o u s in the b i o l o g i c a l w o r l d . It f i l l s a v a r i e t y o f f u n c t i o n s ( T a b l e I) and i s known u n d e r s e v e r a l d i f f e r e n t names. For e x a m p l e , paramylon i s a h i g h l y c r y s t a l 1 i ne g r a n u l a r ( l->3 ) - 3 - D - g l u c a n w h i c h i s a reserve polysaccharide in Euglena graci1i s.(13) C u r d l a n ( 2 ) , a l s o a p u r e ( 1+3 ) - 3 - D - g l u c a n , i t s s y n t h e s i z e d e x t r a c e l T u l a r l y by a b a c t e r i a ( A l c a l i g e n e s f a e c a l i s v a r . m y x o g e n e s ) and i s p o o r l y c r y s t a l 1 i ne . ( 14") I t i s f o u n d i n o t h e r a l g a e ( 1 5 , 1 6 ) and a l s o i n more a d v a n c e d o r g a n i s m s s u c h as c o t t o n f i b e r s ( l _ 7 ) and i n t h e p o l l e n t u b e s o f L i 1 iurn longif1orum(18) where it is fibrillar and associated with c e l l u l o s e . T h i s g l u c a n i s a l m o s t a l w a y s p r e s e n t i n y e a s t s and f u n g a l s p e c i e s and t h e most w e l l known a r e l i s t e d i n Table I. In those or gan i sms ( 7 , 1 9 ^ 20 ,21_, 22 ) the polysaccharide is believed to play a structural f u n c t i o n in the c e l l w a l 1 . ( 2 0 ) C r y s t a l S t r u c t u r e . C u r d l a n powder was t h e s o u r c e o f (1+ 3 ) - 3 - D - g l u c a n ù~sed in our study since it is c o m m e r c i a î l y a v a i l a b l e and has been d e m o n s t r a t e d t o be a l i n e a r c h a i n c o n s t i t u t e d almost e x c l u s i v e l y of (l+3)-3-Dglucose residues. The p o l y s a c c h a r i d e was o b t a i n e d f r o m T a k e d a C h e m i c a l s Company i n J a p a n and i t s m o r p h o l o g y has been p r e v i o u s l y d e s c r i b e d . ( Z I t i s r e c e i v e d as a s p r a y - d r i e d powder w h i c h i s v e r y p o o r l y crystalline. Fibers were obtained by extrusion of a 10% dimethylsulfoxyde (DMSO) s o l u t i o n o f c u r d l a n i n t o a m e t h a n o l b a t h f o l l o w e d by w a s h i n g o f t h e g e l f i b e r s i n w a t e r and d r y i n g a t c o n s t a n t l e n g t h . The x - r a y f i b e r patterns r e c o r d e d (2_3) for these fibers show poor c r y s t a l 1 i η i t y b u t good o r i e n t a t i o n . O n l y a few b r o a d d i f f r a c t i o n s p o t s can be m e a s u r e d , l e a d i n g t o a u n i t c e l l determination (Table II). The d i f f r a c t i o n d a t a i s n o t e x t e n s i v e enough t o a l l o w one t o p r o p o s e a d e f i n i t e structure for this polymorph. However, Takeda, e t à1.{2A_) h a v e been a b l e t o i m p r o v e t h i s x - r a y p a t t e r n and~~Îhey proposed a s t r u c t u r e c o n s i s t i n g of a s i n g l e helix with a 7 symmetry. 9

9

1


228



TABLE and

STORAGE

Natural F u n c t i o n s of

I

Sources (1+3)-β-D-Glucans

Unicellular Algae

Euglena

crocilis(13)

Brown A l g a e

Cladophona

Green A l g a e

L a m i n a r i a( 16)

EXOCELLULAR

Bacteria

Alcaligenes faecalis myxogenes(2)

STRUCTURAL

Plant

Lilium

rupestris(15)

var,

Longif1orum(18)

Gossypiumarboneum ( c o t t o n ) (17) Yeast

Saccharomyces cerevi s i ae(19,20) Schi zosaccharomyces octosporus(21)

Fungi

Armi1laria Pénicillium Lentinus

mel1ea(7) notatum(22)

elodes(7)


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230


In o r d e r t o i m p r o v e t h e c r y s t a l 1 i n i t y , t h e f i b e r s w e r e a n n e a l e d u n d e r t e n s i o n i n a s e a l e d bomb i n t h e p r e s e n c e of water at 1 4 5 ° C . This treatment greatly i m p r o v e d t h e c r y s t a l 1 i η i t y and t h e d i f f r a c t i o n d a t a so o b t a i n e d and p r o v i d e d enough i n f o r m a t i o n t o a l l o w a complete s t r u c t u r e d e t e r m i n a t i o n . D e p e n d i n g on t h e r e l a t i v e humidity (R.H.), two d i f f e r e n t x - r a y d i a g r a m s a r e o b t a i n e d , f r o m w h i c h two d i f f e r e n t u n i t c e l l s can be d e r i v e d . T a b l e II shows t h a t b o t h u n i t c e l l s a r e h e x a g o n a l w i t h t h e v a l u e s o f

Fungal Polysaccharides - American Chemical Society

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