Hyaluronic Acid Conformations and Interactions

7 Hyaluronic Acid Conformations and Interactions

Downloaded by UNIV OF PITTSBURGH on May 4, 2015 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0048.ch007

W. T. WINTER, J. J. CAEL, P. J. C. SMITH, and STRUTHER ARNOTT Department of Biological Sciences, Purdue University, West Lafayette, IN 47907

F o r some t i m e now we h a v e b e e n i n t e r e s t e d i n glycosaminoglycan conformations, thefactors control l i n g s e l e c t i o n o f p a r t i c u l a r c o n f o r m a t i o n a l modes, a n d the p o s s i b l e r e l a t i o n s h i p s o f s t r u c t u r e and s t r u c t u r a l changes t o b i o l o g i c a l f u n c t i o n . I n t h i s p a p e r we w o u l d l i k e t o p r e s e n t o u r r e s u l t s w i t h t h e l e a s t complex o f the g l y c o s a m i n o g l y c a n s , h y a l u r o n i c acid. H y a l u r o n i c a c i d i s found i n most c o n n e c t i v e t i s s u e s w i t h t h e l a r g e s t amounts i n t h e s o f t e r t i s s u e s e.g. W h a r t o n s j e l l y , v i t r e o u s a n d s y n o v i a l f l u i d s . The p r i n c i p l e a c t i v i t i e s i n w h i c h h y a l u r o n i c a c i d i s suspected t o play a s i g n i f i c a n t r o l e a r e morphogenetic r e g u l a t i o n (1J a n d s h o c k a b s o r b a n c y (2Γ) . Chemically, hyaluronic acid i sa polydisaccharide - ( A - B - ) where A i s^-glucuronic a c i d and Β i s 2-acetamido-2-deoxy-D glucose. T h e g l y c o s i d i c l i n k a g e s A-B a n d B-A a r e β (1+3) a n d β(1->4) r e s p e c t i v e l y a s s h o w n i n F i g u r e 1. Linkages i ndermatan, c h o n d r o i t i n and k e r a t a n s u l f a t e s are g e o m e t r i c a l l y e q u i v a l e n t t o t h e s e a n d we t h e r e f o r e e x p e c t many o f o u r o b s e r v a t i o n s w i t h h y a l u r o n i c acid w i l l obtain t o these s u l f a t e d polysaccharides. Light s c a t t e r i n g r e s u l t s o f M a t h e w s a s r e p o r t e d b y Swann ( 3 ) , v i s c o s i t y (3) a n d m a g n e t i c r e s o n a n c e (£) a l l s u g g e s t the e x i s t e n c e , i n d i l u t e s o l u t i o n , o f s t i f f segments, i n v o l v i n g more t h a n 50% o f t h e r e s i d u e s , s t a b i l i z e d by cooperative long range i n t e r a c t i o n s extending over several residues. The e x i s t e n c e o f s u c h segments implies that considerable conformational r e g u l a r i t y persists i nsolution. Since hyaluronic acid concentra tions i nsoft connective tissue ares u f f i c i e n t l y large t h a t t h ec a l c u l a t e d excluded volume exceeds t h e t o t a l v o l u m e o f s o l u t i o n ( 5 ) , i t seems r e a s o n a b l e t h a t m o r e c o m p a c t a n d o r d e r l y s t r u c t u r e s a l s o p e r t a i n in vivo. We h a v e u s e d X - r a y d i f f r a c t i o n f r o m o r i e n t e d f i l m s t o i n v e s t i g a t e t h enature o f such ordered conformations, 1

n

91

In Cellulose Chemistry and Technology; Arthur, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.


CELLULOSE CHEMISTRY AND TECHNOLOGY

Figure 1. Chemical structure of the repeating unit in hyaluronates. A is D glucuronate and Β is 2-acetamido-2-deoxy-O glucose.


7.

WINTER ET AL.

Hyaluronic

Acid

93

t h e f o r c e s w h i c h s t a b i l i z e them a n d t h e v a r i o u s f a c t o r s influencing conformation and s p a t i a l o r g a n i z a t i o n .


Hyaluronate

Geometries

S t u d i e s i n o u r l a b o r a t o r y and i n B r i s t o l by E.D.T. A t k i n s a n d c o - w o r k e r s s u g g e s t t h a t hyaluronates exhibit considerable conformational versatility ( F i g u r e 2 ) . The d i f f e r e n t c o n f o r m e r s a r e m o s t s i m p l y c l a s s i f i e d i n terms o f t h e i r h e l i x symmetry (n) and a x i a l p e r i o d i c i t y p e r d i s a c c h a r i d e u n i t {h). These parameters a r e r e a d i l y o b t a i n a b l e from t h e observed i n t e n s i t y d i s t r i b u t i o n and l a y e r l i n e s e p a r a t i o n ( F i g u r e 2 ) . Table I summarizes these parameters f o r t h e known h y a l u r o n a t e conformations. TABLE I .

Helix Symmetry

a

Summary o f O b s e r v e d H y a l u r o n i c Conformations

4

6

Conformational Effector

Reference

0.98

free

13

0.95

divalent cation

7,8,13,18

0.85

monovalent cation

6,20,21

0. 93

chondroitin-6s u l f a t e (*10%)

21,22

0.91

hemiprotonated

b

Disaccharide p i t c h (nm)

2l

3

4

Acid

acid

H e l i x symmetries a r e assigned assuming a r e g u l a r helix of disaccharides. I n some i n s t a n c e s s u c h a s c a l c i u m h y a l u r o n a t e o r sodium h y a l u r o n a t e , a t h i g h h u m i d i t i e s t h e c r y s t a l symmetry i n d i c a t e s t h a t successive disaccharides are not exactly equivalent. This

paper.

Conformational

Features

We h a v e e x a m i n e d s e v e r a l p a c k i n g v a r i a n t s o f e a c h o f t h e two p r i n c i p a l h y a l u r o n a t e c o n f o r m a t i o n a l motifs, t h e s i n u o u s 4 - f o l d f o r m s w i t h h = 0.8 5nm (6) a n d t h e e x t e n d e d 3 - f o l d c o n f o r m e r s w i t h h = 0.95nm (7_,3) l i n k a g e s . S i d e c h a i n o r i e n t a t i o n s are s i m i l a r to those observed i n r e l a t e d carbohydrate m a t e r i a l s w i t h hydroxymethyl groups o r i e n t e d e i t h e r gt o r gg a n d t h e a c e t a m i d o s i d e c h a i n r o t a t e d 20 t o 40° f r o m e c l i p s i n g H ( 2 ) . This arrange ment p l a c e s t h e p l a n e o f t h e a c e t a m i d o g r o u p n e a r l y p e r p e n d i c u l a r t o t h e f i b e r a x i s b u t does n o t p e r m i t the k i n d o f continuous sequence o f B


Hyaluronic

B

A

B

- Ν - H---0

= C Ν - H---0

= C -

h y d r o g e n bonds found i n c h i t i n (9) and N - a c e t y l - a - D glucosamine (10,11). A s e c o n d v i e w o f t h e two c o n f o r m a t i o n a l v a r i a n t s ( F i g u r e 4) r e v e a l s s e v e r a l i n t e r e s t i n g d i f f e r e n c e s . F i r s t , t h e c h a i n s o f t h e compact 4 - f o l d form a r e h i g h l y i n t e r d i g i t a t e d and t h e r e a r e o p p o r t u n i t i e s f o r d i r e c t i n t e r m o l e c u l a r hydrogen bonding i n v o l v i n g each c h a i n w i t h i t s f o u r n e a r e s t n e i g h b o r s and even w i t h i t s f o u r next nearest neighbors a t the adjacent u n i t c e l l corners. I n t h e 3 - f o l d forms the i n d i v i d u a l c h a i n s a r e t i g h t l y wound a b o u t t h e i r r e s p e c t i v e h e l i x a x e s a n d each s u c c e s s i v e d i s a c c h a r i d e a c t s as a donor i n o n l y one d i r e c t i n t e r m o l e c u l a r h y d r o g e n b o n d . A l l of the h y d r o p h i l i c groups are d i s t r i b u t e d near the molecular p e r i p h e r y and under h i g h h u m i d i t y c o n d i t i o n s (ambient r e l a t i v e h u m i d i t y g r e a t e r t h a n 70%) h y d r o g e n b o n d i n g between chains proceeds l a r g e l y through water b r i d g e s . The s e c o n d m a j o r d i f f e r e n c e b e t w e e n t h e s e two s t r u c t u r e s i n v o l v e s t h e p o s i t i o n and o r i e n t a t i o n o f t h e c a r b o x y l a t e groups together w i t h t h e i r a s s o c i a t e d counter-cation. I n t h e 4 - f o l d f o r m (6) t h e c h a r g e d g r o u p s a r e a l l l o c a t e d l e s s t h a n 0.15nm f r o m t h e h e l i x axis. T h i s g e o m e t r y , i n a d d i t i o n t o p r o v i d i n g maximum s e p a r a t i o n o f s i m i l a r l y charged groups, a l s o r e s u l t s i n the f o r m a t i o n o f charge stacks along the h e l i x a x i s . With the 3 - f o l d forms the r a d i a l c o o r d i n a t e s o f



Figure

S.

The (a.) regular

three-fold

and (b) compressed regular four-fold tion normal to the helix axis

hyaluronates


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Hyaluronic Acid Conformations and Interactions

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