116 Plenary lectures STRUCTURE-FUNCTION RELATIONSHIPS FOR HEPARIN-LIKE POLYSACCHARIDES M. H66k (Uppsala, Sweden) The d r a m a t i c e f f e c t s o f h e p a r i n - 1 i k e p o l y s a c c h a r i d e s i n v a r i o u s b i o l o g i c a l systems a r e p r o b a b l y o f t e n due t o i n t e r a c t i o n s between t h e p o l y s a c c h a r i d e and a t a r g e t p r o t e i n . For example, t h e a c t i o n o f h e p a r i n as an a n t i c o a g u l a n t can be e x p l a i n e d by t h e s p e c i f i c i n t e r a c t i o n o f t h e p o l y s a c c h a r i d e w i t h a n t i t h r o m b i n I l l , a p r o t e a s e i n h i b i t o r a c t i n g on s e v e r a l o f t h e s e r i n e p r o - t e a s e s i n t h e c o a g u l a t i o n cascade. B i n d i n g o f h e p a r i n t o t h e p r o t e i n g r e a t l y p o t e n t i a t e s t h e a c t i v i t y o f a n t i t h r o m b i n I l l , t h u s l e a d i n g t o an e f f e c t i v e b l o c k a g e o f t h e s e l e c t i v e p r o t e o l y t i c t r a n s f o r m a t i o n o f zymogens t o a c t i v e coagu 1 a t i o n f a c t o r s . i n j e c t i o n o f h e p a r i n i n t o t h e b l o o d o f an animal a f f e c t s n o t o n l y t h e co- a g u l a t i o n system b u t a l s o t h e plasma c o n c e n t r a t i o n o f t r i g l y c e r i d e s . T h i s l i p o l y t i c e f f e c t o f h e p a r i n i s p r o b a b l y caused by t h e r e l e a s e o f l i p o p r o t e i n 1 ipase, a t r i g l y c e r i d e d e g r a d i n g enzyme, f r o m t i s s u e s i t e s t o t h e c i r c u - l a t i n g b l o o d . L i p o p r o t e i n l i p a s e r e a d i l y b i n d s t o c e r t a i n p o l y a n i o n s and i s p r o b a b l y r e l e a s e d as a complex w i t h h e p a r i n . Heparan s u l f a t e , a p o l y s a c c h a r i d e s t r u c t u r a l l y r e l a t e d t o h e p a r i n , o c c u r s a s s o c i a t e d w i t h t h e c e l 1 s u r f a c e o f s e v e r a l mammal i a n c e l l t y p e s . Recent work i n o u r l a b o r a t o r y has shown t h a t p a r t o f t h e c e l l s u r f a c e heparan s u l - f a t e i s bound t o s p e c i f i c b i n d i n g s i t e s i n t h e plasma membrane. I n t h e p r e s - e n t communication t h e s t r u c t u r a l r e q u i r e m e n t s for b i n d i n g o f h e p a r i n - 1 i k e p o l y s a c c h a r i d e s t o a n t i t h r o m b i n I I I , 1 i p o p r o t e i n l i p a s e and s p e c i f i c s i t e s o n t h e s u r f a c e o f r a t l i v e r c e l l s w i l l be d i s c u s s e d . B i n d i n g t o a n t i t h r o m b i n I l l . H e p a r i n i s t h e o n l y g l y c o s a m i n o g l y c a n t h a t b i n d s w i t h h i g h a f f i n i t y t o a n t i t h r m b i n I I I . Furthermore, n o t a l l h e p a r i n m o l e c u l e s c o n i a i n t h e s t r u c t u r a l f e a t u r e s r e q u i r e d f o r h i g h a f f i n i t y b i n d i n g t o t h e p r o t e i n ; and h e p a r i n can i n f a c t be s e p a r a t e d i n t o two d i s t i n c t f r a c - t i o n s h a v i n g h i g h and low a f f i n i t y f o r a n t i t h r o m b i n , r e s p e c t i v e l y . A n a l y s i s o f c h e m i c a l l y m o d i f i e d h e p a r i n s suggests t h a t t h e a n t j t h r o m b i n - s t i m u l a t i n g a c t i v i t y i s l o s t when N - s u l f a t e groups a r e removed from t h e polymer and r e - s t o r e d on r e - N - s u l f a t i o n b u t n o t on N - a c e t y l a t i o n o f t h e N - d e s u l f a t e d p o l y - s a c c h a r i d e . A h e p a r i n fragment w i t h a m o l e c u l a r w e i g h t o f about 5 0 0 0 con- t a i n i n g t h e a n t i t h r o m b i n - b i n d i n g s i t e and p o s s e s s i n g a n t i t h r o m b i n - e n h a n c i n g a c t i v i t y c o u l d be i s o l a t e d a f t e r d i g e s t i o n o f t h e h e p a r i n - a n t i t h r o m b i n complex w i t h a b a c t e r i a l h e p a r i n a s e . The s t r u c t u r e o f t h i s fragment w i l l be d i scussed. B i n d i n g t o 1 i p o p r o t e i n I ipase. Beside h e p a r i n a l s o heparan s u l f a t e and dermatan s u l f a t e a r e c a p a b l e o f b i n d i n g t o 1 i p o p r o t e i n 1 ipase. The enzyme appears to b i n d much s t r o n g e r t o h e p a r i n t h a n t o t h e l o w - s u l f a t e d g l y c o s - aminoglycans; however, t h e a f f i n i t y o f t h e l i p a s e f o r t h e p o l y s a c c h a r i d e s does n o t depend s o l e l y on charge d e n s i t y o f t h e g l y c o s a m i n o g l y c a n as chon- d r o i t i n s u l f a t e o n l y b i n d s s m a l l amount o f enzyme a l t h o u g h i t s c h a r g e den- s i t y i s s i m i l a r t o t h a t o f dermatan [ s u l f a t e and heparan s u l f a t e . A n a l y s i s o f c h e m i c a l l y m o d i f i e d h e p a r i n s suggests t h a t N - a c e t y l groups can be sub- s t i t u t e d f o r N - s u l f a t e groups w i t h o u t s i g n i f i c a n t l y a f f e c t i n g t h e a f f i n i t y f o r 1 i p o p r o t e i n 1 ipase. Upshla J Med Sci 82 Plenary lectures 117 A f t e r p a r t i a l d e g r a d a t i o n o f h e p a r i n , a fragment w i t h a m o l e c u l a r w e i g h t o f about 3 5 0 0 w i t h a p p r e c i a b l e a f f i n i t y f o r 1 i p o p r o t e i n 1 i p a s e c o u l d be i s o l a t e d . These r e s u l t s i n d i c a t e t h a t t h e b i n d i n g s i t e s f o r l i p o - p r o t e i n 1 i p a s e and a n t i t h r o m b i n I I I i n t h e h e p a r i n m o l e c u l e a r e d i f f e r e n t . I n s u p p o r t o f t h i s c o n c l u s i o n t h e two h e p a r i n s p e c i e s separated by a f f i n i t y chromatography on a n t i t h r o m b i n - S e p h a r o s e gave e s s e n t i a l l y i d e n t i c a l e l u t i o n p r o f i l e s when chromatographed on i m n o b i l ized 1 i p o p r o t e i n 1 i p a s e . B i n d i n g t o r a t l i v e r c e l l s . The heparan s u l f a t e s y n t h e t i s e d i n r a t l i v e r c e l l s u l t i m a t e l y appears a t t h e c e l l s u r f a c e where p a r t o f t h e m o l e c u l e s o c c u r s a s s o c i a t e d w i t h a " r e c e p t o r " . T h i s p o l y s a c c h a r i d e can be r e l e a s e d by h e p a r i n and by some heparan s u l f a t e s . P o l y s a c c h a r i d e s c a p a b l e o f r e - l e a s i n g endogenous r a t l i v e r heparan s u l f a t e f r o m t h e c e l l s a l s o b i n d t o t h e c e l l s . R e s u l t s w i l l be p r e s e n t e d s u g g e s t i n g t h a t b i n d i n g o f heparan s u l f a t e t o c e l l s depends on t h e c o n t e n t o f e s t e r s u l f a t e b u t n o t on t h e presence o f N - s u l f a t e groups i n t h e polymer. Conclusions: The p r e s e n t e d r e s u l t s i n d i c a t e d i f f e r e n t s t r u c t u r a l r e q u i r - ments f o r b i n d i n g of h e p a r i n - r e l a t e d p o l y s a c c h a r i d e s t o a n t i t h r o m b i n I I I , 1 i p o p r o t e i n 1 i p a s e and r a t 1 i v e r c e l I s , r e s p e c t i v e l y . -- Upsala J Med Sci 82