Carbohydrate-Protein Interaction - ACS Publications - American

14 Carbohydrate-Protein Interactions in Proteoglycans

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L. ROSENBERG, H . CHOI, S. PAL, and L. TANG Montefiore Hospital and Medical Center, Orthopedic and Connective Tissue Research, 111 East 210th Street, Bronx, NY 10467

P r o t e o g l y c a n s are major structural components o f the intercellular matrix o f connective t i s s u e s . In t h e formation of proteoglycans, a proteoglycan b a s i c unit is first formed, called p r o t e o g l y c a n monomer. P r o t e o g l y c a n monomer c o n s i s t s o f g l y c o s a m i n o g l y c a n c h a i n s c o v a l e n t l y bound t o a p r o t e i n c o r e . Several classes of p r o t e o g l y c a n s have been isolated from different connective t i s s u e s . These different classes o f proteoglycans are d e f i n e d in terms o f the k i n d s o f g l y c o s a m i n o g l y c a n c h a i n s which a r e bound t o the p r o t e i n c o r e . In cartil a g e s , p r o t e o g l y c a n monomer c o n s i s t s o f c h o n d r o i t i n sulfate and k e r a t a n s u l f a t e bound t o t h e same p r o t e i n c o r e . In t h e intercellular m a t r i x o f b l o o d v e s s e l wall, p r o t e o g l y c a n monomer c o n s i s t s o f dermatan sulfate and c h o n d r o i t i n s u l f a t e bound t o t h e same p r o t e i n c o r e . In the plasma membranes o f some cells, heparan s u l f a t e is bound t o t h e p r o t e i n c o r e . In t h e intercellular matrix o f cartilage, and p e r haps o t h e r t i s s u e s , most o f the p r o t e o g l y c a n e x i s t s in the form o f p r o t e o g l y c a n a g g r e g a t e s , formed by t h e nonc o v a l e n t a s s o c i a t i o n o f p r o t e o g l y c a n monomers w i t h hya l u r o n i c a c i d and link p r o t e i n s . C a r b o h y d r a t e - p r o t e i n i n t e r a c t i o n s a r e i n v o l v e d in t h e b i n d i n g o f p r o t e o g l y can monomer t o h y a l u r o n a t e , and in the b i n d i n g o f link protein t o hyaluronate. Carbohydrate-protein interact i o n s a r e a l s o i n v o l v e d in t h e n o n - c o v a l e n t a s s o c i a t i o n o f p r o t e o g l y c a n s w i t h c o l l a g e n fibers i n intercellular matrix. The purpose o f this r e v i e w is t o d e s c r i b e t h e s t r u c t u r e o f p r o t e o g l y c a n s , and t o summarize t h e results of recent s t u d i e s of carbohydrate-protein i n t e r a c t i o n s between p r o t e o g l y c a n monomer and h y a l u r o n a t e , link p r o t e i n and h y a l u r o n a t e , and between p r o t e o g l y c a n s and collagen.

0-8412-0466-7/79/47-088-186$07.50/0 © 1979 American Chemical Society

Goldstein; Carbohydrate-Protein Interaction ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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Cartilage

E T A L .

Interactions in Proteoglycans

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Proteoglycans

C a r t i l a g e s are highly s p e c i a l i z e d connective t i s sues composed o f r e l a t i v e l y few c e l l s d i s t r i b u t e d throughout an abundant i n t e r c e l l u l a r m a t r i x . The i n t e r c e l l u l a r m a t r i x g i v e s c a r t i l a g e unusual m e c h a n i c a l p r o p e r t i e s e s s e n t i a l f o r t h e normal f u n c t i o n o f d i a r t h r o dial joints. F o r example, because o f t h e p r o p e r t i e s o f the i n t e r c e l l u l a r m a t r i x , a r t i c u l a r c a r t i l a g e i s a r e l a t i v e l y hard, y e t e l a s t i c t i s s u e . Articular cartilage p r o v i d e s a smooth c o v e r i n g f o r t h e bony elements o f d i a r t h r o d i a l j o i n t s and c o n t r i b u t e s t o t h e almost f r i c t i o n l e s s g l i d i n g o f o p p o s i n g j o i n t s u r f a c e s . The i n t e r c e l l u l a r m a t r i x i s composed m a i n l y o f c o l l a g e n , p r o t e o g l y c a n s and water. C o l l a g e n i s an i n s o l u b l e f i b r o u s p r o t e i n with t e n s i l e strength. Proteoglycans are e l a s t i c m o l e c u l e s which t e n d t o expand i n s o l u t i o n and r e s i s t compression i n t o a s m a l l e r volume o f s o l u t i o n . The remarkable m e c h a n i c a l p r o p e r t i e s o f a r t i c u l a r c a r t i l a g e r e s u l t from t h e s t r u c t u r e o f c o l l a g e n and p r o t e o g l y c a n s , and from t h e p r o p e r t i e s o f t h e f i b r o u s compo s i t e formed by t h e i n t e r a c t i o n s o f c o l l a g e n and p r o t e o glycans i n i n t e r c e l l u l a r matrix. A diagrammatic model o f c a r t i l a g e p r o t e o g l y a n monomer i s shown i n F i g u r e 1. C a r t i l a g e p r o t e o g l y c a n monomer c o n s i s t s o f c h o n d r o i t i n s u l f a t e and k e r a t a n s u l f a t e c h a i n s c o v a l e n t l y bound t o s e r i n e and t h r e o n i n e residues w i t h i n the p r o t e i n core. Chondroitin sulfate and k e r a t a n s u l f a t e a r e members o f t h e group o f p o l y s a c c h a r i d e s termed g l y c o s a m i n o g l y c a n s . Glycosaminoglycans a r e composed o f two d i f f e r e n t sugar r e s i d u e s which a l t e r n a t e r e g u l a r l y i n t h e p o l y s a c c h a r i d e c h a i n . One sugar r e s i d u e i s u s u a l l y N - a c e t y l g a l a c t o s a m i n e o r Nacetylglucosamine. The o t h e r sugar r e s i d u e i s u s u a l l y glucuronic acid or iduronic acid. Thus g l y c o s a m i n o g l y cans a r e composed o f d i s a c c h a r i d e r e p e a t i n g u n i t s . The structures of the disaccharide repeating u n i t s of the g l y c o s a m i n o g l y c a n s , and o f t h e l i n k a g e r e g i o n o f t h e g l y c o s a m i n o g l y c a n c h a i n s t o p r o t e o g l y c a n monomer c o r e p r o t e i n , a r e shown i n F i g u r e 2. C h o n d r o i t i n s u l f a t e i s c o v a l e n t l y bound t o s e r i n e r e s i d u e s v i a t h e n e u t r a l sugar t r i s a c c h a r i d e , g a l a c t o s e - g a l a c t o s e - x y l o s e ( 1-5^ ). K e r a t a n s u l f a t e i s c o v a l e n t l y bound m a i n l y t o t h r e o n i n e and s e r i n e r e s i d u e s v i a ϋ-acetylgalactosamine, t o which a s i a l y l g a l a c t o s y l d i s a c c h a r i d e i s a l s o a t t a c h e d (6-10 ). P r o t e o g l y c a n monomers from d i f f e r e n t c a r t i l a g e s v a r y i n m o l e c u l a r weight and c h e m i c a l c o m p o s i t i o n , p a r t i c u l a r l y i n t h e r e l a t i v e amounts o f c h o n d r o i t i n sulfate and k e r a t a n s u l f a t e . Indeed, p r o t e o g l y c a n monomers from the same t i s s u e a r e p o l y d i s p e r s e and v a r y i n s i z e and


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-Chondroitin sulfate Hyaluronic acid binding region

-Protein core

TT

-Keratan sulfate

PROTEOGLYCAN MONOMER

Figure 1.

Diagram of the cartilage monomer


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STRUCTURES OF THE GLYCOSAMINOGLYCANS AND THEIR LINKAGE REGIONS TO PROTEIN

OSO3-

OSO " Ψ 3

4·

GalNAc( 31+4)GlcUA( 31+3)GalNAc( 31+4 )GlcUA( 31+3)Gal( 31+3)Gal(31+4)Xyl+Ser

CHONDROITIN 4-SULFATE

GlcNAc(31+4)GlcUA(31+3)GlcNAc(31+4)GlcUA(31+3)Gal(31+3)Gal(31+4)Xyl+Ser

HYALURONATE

OSO3""

OS0 ~ Ψ 3

4·

GalNAc( 31+4) IdUA(al+3)GalNAc( 31+4)GlcUA( 31+3)Gal( 31+3)Gal( 31+4)Xy1+Ser

DERMATAN

SULFATE

OSO 3 " OSO " Ψ 46 6 GlcNAc(al->4)IdUA(al->4)GlcNAc(al->4)GlcUA(31+3)Gal(31+3)Gal(31+4)Xyl-^Ser HEPARAN SULFATE 3

OS0 ~

OSO3

3

OSO3

GlcNS0 "(al-»»4)IdUA(al^4)GlcNAc(al^4)GlcUA(31+3)Gal(31+3)Gal(.31+4)Xyl^Ser 3

HEPARIN

OSO 3 OSO 3 " 4 - 4 -

OSO " Ψ 3

OSO 4-

Gal(31+4)GlcNAc(31+3)Gal(31+4)GlcNAc(31+6)GalNAc-»»Thr 3+

ι

Gal(4+-la)NeuAc

KERATAN SULFATE Figure 2. Structures of the glycosaminoglycans and their linkage regions to pro tein. Two disaccharide repeating units are shown to emphasize the microheterogeneity that exists in some cases. Heparan sulfate and heparin show many struc tural simihrities. However, heparan sulfate contains more GlcNAc(a-l-*4)GhUA repeating units; fewer glucosamine residues are Ν'-sulfated, and few iduronic acid residues are sulfated at C2.


190


INTERACTION

composition. However, a r e p r e s e n t a t i v e p r o t e o g l y c a n monomer from b o v i n e a r t i c u l a r c a r t i l a g e would have a p r o t e i n c o r e a p p r o x i m a t e l y 200,000 i n m o l e c u l a r weight, measuring 3000 A l o n g . To t h i s would be a t t a c h e d about 100 c h o n d r o i t i n s u l f a t e c h a i n s v a r y i n g from 2 χ 10 * t o 3 χ 10 * i n m o l e c u l a r w e i g h t . Keratan s u l f a t e chains 3 χ 1 0 t o 7 χ 1 0 i n m o l e c u l a r weight would a l s o be a t t a c h e d t o t h e p r o t e i n c o r e . The e n t i r e p r o t e o g l y c a n monomer would be a p p r o x i m a t e l y 2 χ 1 0 t o 3 χ 10* i n m o l e c u l a r weight. 1

1

3

3

6

S t r u c t u r e o f P r o t e o g l y c a n Aggregates. In t h e i n t e r c e l l u l a r m a t r i x o f c a r t i l a g e , most o f t h e p r o t e o g l y can e x i s t s i n t h e form o f aggregates o f h i g h m o l e c u l a r weight. The m o l e c u l a r a r c h i t e c t u r e o f t h e c a r t i l a g e p r o t e o g l y c a n aggregate i s shown i n F i g u r e 3. H y a l u r o n i c a c i d forms t h e f i l a m e n t o u s backbone o f t h e aggregate (11-19). The aggregate i s formed by t h e n o n - c o v a l e n t a s s o c i a t i o n o f many p r o t e o g l y c a n monomers w i t h h y a l u r o nate. As i n d i c a t e d i n t h e model o f t h e p r o t e o g l y c a n aggregate shown i n F i g u r e 3, p r o t e o g l y c a n monomer c o r e p r o t e i n c o n s i s t s o f t h r e e major r e g i o n s which d i f f e r i n s t r u c t u r e and f u n c t i o n . One end o f p r o t e o g l y c a n monomer where p r o t e o g l y c a n monomer b i n d s t o h y a l u r o n a t e , con t a i n s l i t t l e o r no c h o n d r o i t i n s u l f a t e o r k e r a t a n s u l fate. I t c o n s i s t s o f a p o l y p e p t i d e about 60,000 i n m o l e c u l a r weight w i t h a g l o b u l a r c o n f o r m a t i o n . I t con t a i n s t h e b i n d i n g s i t e o f p r o t e o g l y c a n monomer c o r e pro tein for hyaluronic acid. This region i s c a l l e d the h y a l u r o n i c a c i d b i n d i n g r e g i o n o f p r o t e o g l y c a n monomer core p r o t e i n . Most o f t h e l e n g t h o f c o r e p r o t e i n , which extends towards t h e o t h e r t e r m i n u s o f t h e m o l e c u l e , i s composed m a i n l y o f a number o f p o s s i b l y homologous, r e p e a t i n g , s h o r t p e p t i d e s t o each o f which a c h o n d r o i t i n s u l f a t e c h a i n i s a t t a c h e d ( F i g u r e s 1 and 3 ) . L o c a t e d between c l u s t e r s o f t h e s e c h o n d r o i t i n s u l f a t e c o n t a i n i n g pep t i d e s a r e s h o r t p e p t i d e s t o which k e r a t a n s u l f a t e c h a i n s are a t t a c h e d . T h i s r e g i o n which c o n t a i n s most o f t h e c h o n d r o i t i n s u l f a t e and some o f t h e k e r a t a n s u l f a t e c h a i n s i s c a l l e d t h e p o l y s a c c h a r i d e attachment region. Between t h e h y a l u r o n i c a c i d b i n d i n g r e g i o n and t h e po l y s a c c h a r i d e attachment r e g i o n i s a r e g i o n c o n t a i n i n g m a i n l y k e r a t a n s u l f a t e bound t o p e p t i d e , c a l l e d the k e r atan s u l f a t e - r i c h r e g i o n . A low m o l e c u l a r weight p r o t e i n , c a l l e d l i n k protein is a l s o a component o f p r o t e o g l y c a n a g g r e g a t e s . Link p r o t e i n appears t o b i n d s i m u l t a n e o u s l y t o h y a l u r o n a t e and t o t h e h y a l u r o n i c a c i d b i n d i n g r e g i o n o f c o r e p r o t e i n , and s t a b i l i z e s t h e bond between p r o t e o g l y c a n


ROSENBERG

E T A L .


PROTEOGLYCAN Figure 3.

AGGREGATE

Diagram of a cartilage proteoglycan aggregate


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INTERACTION

monomer and h y a l u r o n a t e . I s o l a t i o n of Proteoglycan Species. Proof o f the c o n c e p t s d e s c r i b e d above r e q u i r e d t h a t p r o t e o g l y c a n agg r e g a t e , p r o t e o g l y c a n monomer and l i n k p r o t e i n be i s o l a t e d and c h a r a c t e r i z e d , and t h a t t h e i n t e r a c t i o n s o f p r o t e o g l y c a n monomer and l i n k p r o t e i n w i t h h y a l u r o n a t e be s t u d i e d by a v a r i e t y o f methods. The p r o c e d u r e now g e n e r a l l y used f o r t h e i s o l a t i o n o f p r o t e o g l y c a n aggreg a t e s and p r o t e o g l y c a n monomer from c a r t i l a g e s i n v o l v e s four steps c a l l e d 1) d i s s o c i a t i v e e x t r a c t i o n ; 2) r e a s s o c i a t i o n ; 3) e q u i l i b r i u m d e n s i t y g r a d i e n t c e n t r i f u g a t i o n under a s s o c i a t i v e c o n d i t i o n s ; and 4) e q u i l i b r i u m d e n s i t y g r a d i e n t c e n t r i f u g a t i o n under d i s s o c i a t i v e conditions. In s t e p 1, d i s s o c i a t i v e e x t r a c t i o n , f r e s h wet t i s s u e i s s l o w l y s t i r r e d a t 4° i n 4 M g u a n i d i n e hydroc h l o r i d e (GnHCl), pH 5.8 t o 6.3. The non-covalent bonds between p r o t e o g l y c a n monomers, h y a l u r o n a t e and l i n k p r o t e i n a r e broken i n c o n c e n t r a t e d s o l u t i o n s o f GnHCl o r d i v a l e n t c a t i o n s (20-26). P r o t e o g l y c a n monomer, h y a l u r o n a t e , and l i n k p r o t e i n d i f f u s e out o f t h e i n s o l u b l e c o l l a g e n network a t a r e l a t i v e l y r a p i d r a t e , i n t o t h e e x t r a c t i o n s o l v e n t . The e x t r a c t i s s e p a r a t e d from t h e i n s o l u b l e c o l l a g e n o u s c a r t i l a g e r e s i d u e by filtration. The f i l t e r e d e x t r a c t c o n t a i n s extraneous p r o t e i n s i n c l u d i n g p r o t e a s e s which must be s e p a r a t e d from t h e p r o t e o g l y c a n s . In s t e p 2, r e a s s o c i a t i o n , p r o t e o g l y c a n monomer, l i n k p r o t e i n and h y a l u r o n a t e a r e r e a s s o c i a t e d i n t o p r o t e o g l y c a n aggregates by d i a l y z i n g o f f t h e GnHCl. In s t e p 3, extraneous m a t r i x p r o t e i n s and p r o t e a s e s a r e s e p a r a t e d from t h e p r o t e o g l y c a n agg r e g a t e s by an e q u i l i b r i u m d e n s i t y g r a d i e n t c e n t r i f u g a t i o n i n 3.5 M C s C l , under a s s o c i a t i v e c o n d i t i o n s . The gradient i s f r e q u e n t l y d i v i d e d i n t o s i x equal fractions. The f r a c t i o n s from t h e t o p o f t h i s a s s o c i a t i v e g r a d i e n t a r e c a l l e d A l through A6 (16,24,27). P r o t e o g l y c a n agg r e g a t e s a r e o f h i g h buoyant d e n s i t y and a r e c o n c e n t r a t e d i n f r a c t i o n A l i n t h e bottom o n e - s i x t h ( p ^ l . 6 g/ml) of the g r a d i e n t . Proteoglycan f r a c t i o n A l i s the prep a r a t i o n used f o r t h e p h y s i c a l c h a r a c t e r i z a t i o n o f p r o t e o g l y c a n aggregates by s e d i m e n t a t i o n v e l o c i t y s t u d i e s (26-28), by e l e c t r o n microscopy (19), and as t h e s t a r t i n g p o i n t f o r t h e p r e p a r a t i o n o f l i n k p r o t e i n (29-34) or the h y a l u r o n i c a c i d - b i n d i n g region o f proteoglycan monomer c o r e p r o t e i n (18,28). In s t e p 4, e q u i l i b r i u m d e n s i t y g r a d i e n t c e n t r i f u g a t i o n under d i s s o c i a t i v e cond i t i o n s , t h e p r o t e o g l y c a n aggregate i s s e p a r a t e d i n t o i t s component s p e c i e s . F r a c t i o n A l from an a s s o c i a t i v e g r a d i e n t , which c o n t a i n s p r o t e o g l y c a n aggregate, i s d i s s o l v e d i n guanidine hydrochloride. The aggregate i s d i s -


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E T A L .


193

s o c i a t e d i n t o p r o t e o g l y c a n monomer, h y a l u r o n a t e and l i n k p r o t e i n . Cesium c h l o r i d e i s added and a d i s s o c i a t i v e g r a d i e n t i s c a r r i e d out i n 4 M GnHCl-3 M C s C l . The g r a d i e n t i s d i v i d e d i n t o s i x f r a c t i o n s c a l l e d A l - D l (bottom) through A1-D6 ( t o p ) . Link p r o t e i n i s separat e d i n t o A1-D6 a t t h e t o p o f t h e g r a d i e n t . Hyaluronate d i s t r i b u t e s i n t h e middle o f t h e g r a d i e n t . Most o f t h e p r o t e o g l y c a n monomer, o f h i g h m o l e c u l a r weight and h i g h c h o n d r o i t i n s u l f a t e - p r o t e i n r a t i o , i s concentrated at the bottom o f t h e g r a d i e n t ( A l - D l ) , f r e e o f h y a l u r o n i c a c i d and l i n k p r o t e i n ( 35-37 ) . However s m a l l e r amounts of p r o t e o g l y c a n monomers o f lower m o l e c u l a r weight d i s t r i b u t e throughout t h e d i s s o c i a t i v e g r a d i e n t , i n d i v i d u a l members o f t h e p o l y d i s p e r s e p o p u l a t i o n o f p r o t e o g l y c a n s u b u n i t s banding at buoyant d e n s i t i e s determined m a i n l y by t h e i r c h o n d r o i t i n s u l f a t e t o p r o t e i n r a t i o s . S t r u c t u r a l Basis f o r the P o l y d i s p e r s i t y of Proteog l y c a n Monomer. P o l y d i s p e r s e p r o t e o g l y c a n monomers from b o v i n e a r t i c u l a r c a r t i l a g e (36 ) and from b o v i n e n a s a l c a r t i l a g e (3.7 ) have been s e p a r a t e d i n t o a s e r i e s o f r e l a t i v e l y monodisperse f r a c t i o n s by d i s s o c i a t i v e e q u i l i b r i u m d e n s i t y g r a d i e n t c e n t r i f u g a t i o n , and t h e s e f r a c t i o n s have been c h e m i c a l l y and p h y s i c a l l y characterized. Columns 2 t h r o u g h 9 o f T a b l e I show t h e c h e m i c a l c o m p o s i t i o n and s e d i m e n t a t i o n c o e f f i c i e n t s o f e i g h t r e l a t i v e l y monodisperse p r o t e o g l y c a n monomer f r a c t i o n s from bovine a r t i c u l a r c a r t i l a g e ( 36). Column 1 o f T a b l e I g i v e s t h e amino a c i d c o m p o s i t i o n o f t h e h y a l u r o n i c a c i d - b i n d i n g r e g i o n o f p r o t e o g l y c a n monomer i s o l a t e d from b o v i n e n a s a l c a r t i l a g e by H e i n e g a r d and H a s c a l l (18 ). The m o l e c u l a r weight o f t h e p r o t e o g l y can monomer i n c r e a s e s i n p r o p o r t i o n t o i t s c h o n d r o i t i n s u l f a t e c o n t e n t , as i n d i c a t e d by t h e i n c r e a s e i n uronate or galactosamine values with i n c r e a s i n g s i z e . The chond r o i t i n s u l f a t e - t o - p r o t e i n r a t i o also increases with size. T h i s r e l a t i o n s h i p suggests that proteoglycan monomers might c o n t a i n p r o t e i n c o r e s i d e n t i c a l i n molec u l a r weight and c o m p o s i t i o n , t o which c h o n d r o i t i n s u l fate chains of d i f f e r e n t chain lengths are attached. However, s e v e r a l o b s e r v a t i o n s r u l e out t h i s p o s s i b i l i t y . F i r s t , e l e c t r o n m i c r o s c o p i c s t u d i e s show t h a t p r o t e o g l y can monomer c o r e p r o t e i n i s o f v a r i a b l e l e n g t h (19 ). Second, t h e amino a c i d c o m p o s i t i o n o f p r o t e o g l y c a n monomer v a r i e s i n a c h a r a c t e r i s t i c f a s h i o n w i t h m o l e c u l a r weight. P r o t e o g l y c a n monomer o f t h e lowest m o l e c u l a r weight (Column 2, T a b l e I ) c o n t a i n s l i t t l e c h o n d r o i t i n s u l f a t e , and an amino a c i d c o m p o s i t i o n r e l a t i v e l y low i n s e r i n e and g l y c i n e , and r e l a t i v e l y h i g h i n c y s t e i n e , methionine and a s p a r t i c a c i d . As t h e m o l e c u l a r weight


194


INTERACTION

TABLE I

Chemical c o m p o s i t i o n and s e d i m e n t a t i o n c o e f f i c i e n t s (s° ) p r o t e o g l y c a n monomer f r a c t i o n s from b o v i n e a r t i c u l a r c a r t i l a g e ( 3 6 ) . Column 1 shows t h e amino a c i d composition of the h y a l u r o n i c a c i d - b i n d i n g r e g i o n o f p r o t e o g l y c a n monomer c o r e p r o t e i n , i s o l a t e d by H e i n e g a r d and H a s c a l l (18 ). o

f

0

Column

1

FRACTIONS

HA-PGS*

2 ,

3

4

Al-D3,4,5 — ι

5

6

1

7 ι

8 Al-Dl

9 1

Yield, g/g

.019

.039

.036

.045

.074

.053

.209

.451

Uronate, X

9.7

10.3

11.5

15.3

16.1

17.1

19.0

20.1

6.6

8.1

12.7

14.3

15.4

14.8

17.5

18.7

Hexose

Galactosamine

12.5

13.5

12.9

14.3

13.3

11.7

11.8

12.2

Glucosamine

6.0

10.4

11.1

10.0

9.1

8.6

5.6

6.5

Sialate

3.0

3.1

2.9

2.4

2.8

1.8

1.8

1.4

Protein

30.7

23.9

17.3

13.0

14.9

10.3

11.1

9.9

Density, g/ml

1.41

1.46

1.52

1.61

1.57

1.57

1.65

1.62

5.7

7.8

8.8

9.7

10.3

10.8

12.7 . 14.3

18.8

32.1

β* , subunit 0

8

2o» aggregate

Amino Acid Composition residues/1000 60

Aspartic acid

98

96

92

71

68

70

62

65

Threonine

60

61

65

68

63

65

62

62

61

Serine

72

69

77

90

105

103

115

123

125

122

139

138

149

147

141

150

146

150

75

84

96

101

111

° 110

104

105

101

87

93

102

102

117

114

118

Glutamic acid Proline Glycine

80

81

Alanine

85

75

76

77

74

76

71

73

70

Half-cystine

21

20

21

17

14

17

12

13

12

Valine

60

60

56

56

59

56

59

59

57

6

8

6

7

5

32

31

32

33

40

Methionine'

14

12

10

10

Isoleucine

48

35

34

33

Leucine

70

81

78

74

73

74

74

78

78

Tyrosine

48

42

27

33

29

20

27

25

24

Phenylalanine

33

40

45

41

43

41

39

38

38

Lysine

24

32

28

24

19

19

15

15

13

Histidine

14

14

17

12

11

11

12

13

13

Arginine

58

58

55

51

44

47

42

41

37

*HA-PGS: Hyaluronic acid binding region of PGS core protein, isolated by Heinegard and Hascall


14.

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E T

A L .


195

and c h o n d r o i t i n s u l f a t e content o f t h e p r o t e o g l y c a n monomer i n c r e a s e s , t h e r e i s a p a r a l l e l i n c r e a s e i n s e r i n e and g l y c i n e c o n t e n t s , and a d e c r e a s e i n c y s t e i n e , m e t h i o n i n e and a s p a r t i c a c i d c o n t e n t s o f t h e p r o t e o g l y c a n monomer c o r e p r o t e i n . An i n t e r p r e t a t i o n o f t h e s i g n i f i c a n c e o f t h e s e changes was made p o s s i b l e when H e i n e g a r d and H a s c a l l i s o l a t e d and c h a r a c t e r i z e d t h e h y a l u r o n i c a c i d - b i n d i n g r e g i o n o f c o r e p r o t e i n ( 1 8 ). H e i n e g a r d and H a s c a l l made a remarkable o b s e r v a tion. They found t h a t i n p r o t e o g l y c a n a g g r e g a t e s , t h e p o l y s a c c h a r i d e attachment r e g i o n o f c o r e p r o t e i n was r e a d i l y and s e l e c t i v e l y degraded by t r y p s i n ( 18 ). As shown i n F i g u r e 4, when p r o t e o g l y c a n aggregate was t r e a t e d w i t h t r y p s i n and c h o n d r o i t i n a s e , t h e p o l y s a c c h a r i d e attachment r e g i o n was s h a t t e r e d i n t o s m a l l fragments. However, t h e c e n t r a l p o r t i o n o f t h e p r o t e o g l y c a n aggregate remained r e l a t i v e l y u n a l t e r e d . The c e n t r a l p o r t i o n o f t h e p r o t e o g l y c a n aggregate c o n s i s t e d of the h y a l u r o n i c a c i d - b i n d i n g region o f core p r o t e i n , n o n - c o v a l e n t l y a s s o c i a t e d w i t h l i n k p r o t e i n and h y a l uronate. As shown i n F i g u r e 4, t h i s complex, c o n s i s t ing of the hyaluronic acid-binding region, l i n k protein and h y a l u r o n a t e , was s e p a r a t e d from p o l y s a c c h a r i d e a t tachment r e g i o n fragments by Sepharose 6B chromatography. The h y a l u r o n i c a c i d - b i n d i n g r e g i o n was then i s o l a t e d from t h e complex by chromatography on Sephadex G-200 i n 4 M GnHCl. On sodium d o d e c y l s u l f a t e - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s , w i t h o r w i t h o u t mercaptoethanol, the hyaluronic acid-binding region consisted o f a s i n g l e p o l y p e p t i d e fragment a p p r o x i m a t e l y 90,000 i n m o l e c u l a r weight. As shown i n T a b l e I, p r o t e o g l y c a n monomer o f t h e lowest m o l e c u l a r weight (Column 2) c o n t a i n s l i t t l e chondroitin s u l f a t e , i s r e l a t i v e l y r i c h i n keratan s u l f a t e , and has an amino a c i d c o m p o s i t i o n low i n s e r i n e and g l y c i n e , and h i g h i n c y s t e i n e , m e t h i o n i n e and a s p a r t i c a c i d , almost i d e n t i c a l t o t h a t o f t h e h y a l u r o n i c a c i d - b i n d i n g r e g i o n (Column 1, T a b l e I ) . Proteoglycan monomer o f t h e lowest m o l e c u l a r weight appears t o cons i s t m a i n l y o f t h e h y a l u r o n i c a c i d - b i n d i n g r e g i o n and the k e r a t a n s u l f a t e - r i c h r e g i o n ; i t c o n t a i n s a s h o r t p o l y s a c c h a r i d e attachment r e g i o n composed o f few S e r G l y c o n t a i n i n g p e p t i d e s t o which c h o n d r o i t i n s u l f a t e c h a i n s a r e a t t a c h e d (36,38,39). As t h e m o l e c u l a r weight o f p r o t e o g l y c a n monomer i n c r e a s e s , t h e p o l y s a c c h a r i d e attachment r e g i o n appears t o p r o g r e s s i v e l y i n c r e a s e i n l e n g t h , w i t h a concomitant i n c r e a s e i n t h e s e r i n e and g l y c i n e c o n t e n t s o f c o r e p r o t e i n , and i n t h e c h o n d r o i t i n s u l f a t e c o n t e n t o f t h e monomer. T h i s i n t e r p r e t a t i o n i s i n accord with e l e c t r o n microscopic



TRYPSIN

CHONDROITINASE

SEPHAROSE 6B

6-200

4M GuHCI

SEPHADEX

J

Figure 4. Diagramamtic representation of the procedure used by Heinegard and Hascall (IS) for the isofotion of the hyaluronic acid-binding region of proteoglycan monomer core protein

********

ISOLATION OF THE HYALURONIC ACID BINDING REGION OF PGS CORE PROTEIN

14.

ROSENBERG

ET

197


AL.

s t u d i e s o f p r o t e o g l y c a n a g g r e g a t e s , which show p r o t e o g l y c a n monomers v a r y i n g i n l e n g t h from 1000 t o 4000 Â bound t o h y a l u r o n i c a c i d at one terminus ( 1 9 ) . In p r o t e o g l y c a n a g g r e g a t e s , a l l p r o t e o g l y c a n monomers c o n t a i n f u n c t i o n a l h y a l u r o n i c a c i d - b i n d i n g r e g i o n s , but p o s s e s s p o l y s a c c h a r i d e attachment r e g i o n s o f v a r i a b l e l e n g t h . P r o t e o g l y c a n monomers appear t o c o n t a i n a h y a l u r o n i c a c i d - b i n d i n g r e g i o n o f c o n s t a n t s i z e and c o m p o s i t i o n l o c a t e d at one terminus o f the m o l e c u l e and a p o l y s a c c h a r i d e attachment r e g i o n o f v a r i a b l e l e n g t h e x t e n d i n g towards the o t h e r t e r m i n u s o f the m o l e c u l e . The p o l y d i s p e r s i t y o f p r o t e o g l y c a n monomers appears t o be det e r m i n e d by the v a r i a b l e l e n g t h o f the p o l y s a c c h a r i d e attachment r e g i o n of c o r e p r o t e i n . E v i d e n c e t h a t the H y a l u r o n i c A c i d - B i n d i n g Region i s L o c a t e d at the N H - t e r m i n u s o f P r o t e o g l y c a n Monomer Core P r o t e i n . P r o t e o g l y c a n monomer ( A l - D l ) c o r e p r o t e i n i s a p p r o x i m a t e l y 200,000 i n m o l e c u l a r weight and c o n s i s t s o f t h r e e r e g i o n s which d i f f e r i n s t r u c t u r e and f u n c t i o n . P r o t e o g l y c a n workers have t h e r e f o r e examined the p o s s i b i l i t y t h a t c o r e p r o t e i n c o n s i s t s of more than one p o l y p e p t i d e c h a i n . However, once s e c r e t e d e x t r a c e l l u l a r l y , p r o t e o g l y c a n monomer c o r e p r o t e i n appears t o be a c o v a l e n t l y - l i n k e d s t r u c t u r e , not d i s sociable into smaller units. Further, proteoglycan monomer c o r e p r o t e i n appears to c o n t a i n a s i n g l e NH t e r m i n a l amino a c i d . C h o i , £t al.. (40) have r e c e n t l y determined the N H - t e r m i n a l amino a c i d s o f p r o t e o g l y c a n monomers (A1-D1-D1) from b o v i n e n a s a l c a r t i l a g e , b o v i n e a r t i c u l a r c a r t i l a g e , and b o v i n e f e t a l e p i p h y s e a l c a r t i lage. P r o t e o g l y c a n monomers were d a n s y l a t e d , t r e a t e d w i t h c h o n d r o i t i n a s e ABC, and h y d r o l y z e d ; the d a n s y l a t e d amino a c i d s were then s e p a r a t e d by chromatography on polyamide s h e e t s ( F i g u r e 5 ). V a l i n e was t h e o n l y NH t e r m i n a l amino a c i d demonstrated i n each o f t h e p r o t e o g l y c a n monomers examined. V a l i n e was a l s o the o n l y N H - t e r m i n a l amino a c i d demonstrated when the h y a l u r o n i c a c i d - b i n d i n g r e g i o n o f p r o t e o g l y c a n monomer c o r e p r o t e i n ( k i n d l y p r o v i d e d by Dr. V i n c e n t H a s c a l l ) was examined. These o b s e r v a t i o n s suggest t h a t the h y a l u r o n i c a c i d - b i n d i n g r e g i o n i s l o c a t e d at the N H - t e r m i nus o f p r o t e o g l y c a n monomer c o r e p r o t e i n . Proteoglycan monomers o f s m a l l e r s i z e from b o v i n e n a s a l c a r t i l a g e (A1-D2 through A1-D4) were a l s o examined. These cons i s t e n t l y showed v a l i n e as a s i n g l e N H - t e r m i n a l amino acid. These r e s u l t s s u p p o r t the concept o f a h y a l u r o n i c a c i d - b i n d i n g r e g i o n o f c o n s t a n t s i z e and composition l o c a t e d at the N H - t e r m i n u s , and a p o l y s a c c h a r i d e a t tachment r e g i o n o f v a r i a b l e l e n g t h , e x t e n d i n g towards 2

2

2

2

2

2

2

2



198

INTERACTION

Figure 5. Photograph of a micropolyamide plate showing that valine is the NH -terminal amino acid of proteoglycan monomer. Identical results were obtained with the hyaluronic acid-binding region of proteoglycan monomer core protein. 2

Journal of Biological Chemistry

Figure 6. Dark field electron micrograph of a Uirge proteoglycan aggregate from bovine articular cartilage. Proteoglycan monomers of varying length arise laterally from the opposite sides of an elongated central filament (hyaluronate) approximately 42,000 À in length (X71,000). From Rosenberg, et al, J. Biol. Chem. (1975) 250,1887-1883.


14.

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E T

A L .


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the COOH-terminus o f p r o t e o g l y c a n monomer c o r e p r o t e i n . The P r o t e o g l y c a n Monomer-Hyaluronate I n t e r a c t i o n . E v i d e n c e t h a t H y a l u r o n i c A c i d i s t h e F i l a m e n t o u s Backbone o f P r o t e o g l y c a n Aggregates. Even i n t h e absence of l i n k p r o t e i n , p r o t e o g l y c a n monomers b i n d a v i d l y t o h y a l u r o n a t e t o form h i g h m o l e c u l a r weight complexes. E l e c t r o n micrographs o f p r o t e o g l y c a n aggregates show t h a t p r o t e o g l y c a n monomers a r i s e from an e l o n g a t e d cent r a l f i l a m e n t . Taken t o g e t h e r , t h e r e s u l t s o f c h e m i c a l b i n d i n g s t u d i e s and e l e c t r o n m i c r o s c o p i c s t u d i e s have f i r m l y e s t a b l i s h e d t h a t h y a l u r o n a t e forms a f i l a m e n t o u s backbone ( F i g u r e 3) t o which monomers a r e n o n - c o v a l e n t l y bound i n p r o t e o g l y c a n a g g r e g a t e s . Hardingham and Muir f i r s t demonstrated t h a t t h e a d d i t i o n o f s m a l l amounts o f h y a l u r o n i c a c i d t o p r o t e o g l y c a n monomer r e s u l t e d i n t h e f o r m a t i o n o f h i g h molec u l a r weight complexes, demonstrable by g e l chromatography o r v i s c o m e t r y ( 1 1 ) . These r e s u l t s were s u r p r i s i n g , s i n c e i t was thought t h a t no h y a l u r o n i c a c i d was present i n c a r t i l a g e . Hardingham and Muir q u i c k l y r e s o l v e d t h e m a t t e r by i s o l a t i n g h y a l u r o n a t e from p i g l a r y n g e a l c a r t i l a g e , and from p r o t e o g l y c a n a g g r e g a t e s p r e p a r e d from t h i s t i s s u e ( 1 2 ) . H a s c a l l and H e i n e g a r d (16) s u b s e q u e n t l y i s o l a t e d h y a l u r o n a t e from p r o t e o g l y can a g g r e g a t e s p r e p a r e d from b o v i n e n a s a l c a r t i l a g e . H a s c a l l and H e i n e g a r d a l s o showed t h a t p r o t e o g l y c a n monomer from b o v i n e n a s a l c a r t i l a g e , t r e a t e d w i t h c h o n d r o i t i n a s e ABC t o remove c h o n d r o i t i n s u l f a t e , i n t e r a c t e d w i t h h y a l u r o n a t e t o form complexes o f h i g h e r m o l e c u l a r weight ( 1 7 ) . The m o l e c u l a r a r c h i t e c t u r e o f p r o t e o g l y c a n aggreg a t e s has been demonstrated by e l e c t r o n m i c r o s c o p y o f proteoglycan-cytochrome c monolayers ( 1 9 ) . F i g u r e 6 shows a dark f i e l d e l e c t r o n micrograph o f a p r o t e o g l y can aggregate from b o v i n e a r t i c u l a r c a r t i l a g e i n which p r o t e o g l y c a n monomers a r i s e at f a i r l y r e g u l a r i n t e r v a l s from t h e o p p o s i t e s i d e s o f a c e n t r a l f i l a m e n t a p p r o x i mately 42,000 Â i n l e n g t h . Measurements o f e l e c t r o n m i c r o g r a p h s o f p r o t e o g l y c a n aggregates i n d i c a t e t h a t the s p a c i n g between p r o t e o g l y c a n monomers a l o n g t h e c e n t r a l f i l a m e n t i s 200 t o 300 A ( 1 9 ) . C a l c u l a t i o n s made from t h e r e s u l t s o f c h e m i c a l b i n d i n g s t u d i e s i n d i cate t h a t t h e s p a c i n g between native proteoglycan monomers on h y a l u r o n a t e i s ^240 1 ( 1 3 ) . T h i s c o r r e s pondence between t h e s p a c i n g o f monomers c a l c u l a t e d from c h e m i c a l b i n d i n g s t u d i e s , and t h a t demonstrated by e l e c t r o n microscopy, s u p p o r t s t h e concept t h a t t h e f i l a m e n t o u s backbone o f t h e aggregate seen i n e l e c t r o n micrographs i s hyaluronate ( 1 9 ) .


200


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C o m p e t i t i v e I n h i b i t i o n o f the P r o t e o g l y c a n MonomerH y a l u r o n a t e I n t e r a c t i o n by H y a l u r o n a t e O l i g o s a c c h a r i d e s P r o t e o g l y c a n monomers b i n d préfèrentially t o h y a l u r o n a t e m o l e c u l e s o f low m o l e c u l a r weight i n the p r e s e n c e o f h y a l u r o n a t e m o l e c u l e s o f b o t h h i g h and low m o l e c u l a r weights. Because o f t h i s , h i g h m o l e c u l a r weight comp l e x e s formed by the b i n d i n g o f p r o t e o g l y c a n monomers t o h i g h m o l e c u l a r weight h y a l u r o n a t e a r e d i s s o c i a t e d by h y a l u r o n a t e o l i g o s a c c h a r i d e s (1^,JL7)« competitive i n h i b i t i o n o f the p r o t e o g l y c a n monomer-hyaluronate i n t e r a c t i o n by h y a l u r o n a t e o l i g o s a c c h a r i d e s has been s t u d i e d by H a s c a l l and H e i n e g a r d (17) and by Hardingham and Muir (13) w i t h i n t e r e s t i n g r e s u l t s . H a s c a l l and H e i n e g a r d (17) p r e p a r e d a p r o t e o g l y c a n monomer c o r e p r e p a r a t i o n of m o l e c u l a r weight 450,000 by c h o n d r o i t i n a s e d i g e s t i o n o f p r o t e o g l y c a n monomer. The c o r e p r e p a r a t i o n chromatographed as a s i n g l e r e t a r d e d peak on Sepharose 2B. M i x t u r e s o f the c o r e p r e p a r a t i o n and h y a l u r o n a t e o f m o l e c u l a r weight 230,000 were p r e p a r e d and chromatographed on Sepharose 2B. When a mixt u r e o f c o r e and 20% h y a l u r o n a t e were chromatographed, t h e r e was l i t t l e change i n the e l u t i o n volume o f the core. Under t h e s e c o n d i t i o n s o f e x c e s s h y a l u r o n a t e , o n l y a few c o r e m o l e c u l e s bound t o each h y a l u r o n a t e m o l e c u l e , and the complexes formed were r e l a t i v e l y small. M i x t u r e s o f c o r e p l u s 4.1%, 1.7% and 0.8%hyaluronate were chromatographed; t h e r e was a p r o g r e s s i v e i n c r e a s e i n the amount o f c o r e e l u t e d i n the v o i d volume, u n t i l at 0.6% h y a l u r o n a t e a p p r o x i m a t e l y 70% o f the c o r e m o l e c u l e s e l u t e d near the v o i d volume. Under t h e s e cond i t i o n s , each h y a l u r o n a t e m o l e c u l e had been s a t u r a t e d w i t h c o r e m o l e c u l e s , and e l u t e d near t h e v o i d volume as a h i g h m o l e c u l a r weight complex. H a s c a l l and H e i n e g a r d c a l c u l a t e d t h a t each c o r e molecule, from which c h o n d r o i t i n s u l f a t e c h a i n s have been removed w i t h c h o n d r o i t i n ase, o c c u p i e d a l e n g t h o f about 8-10 h y a l u r o n i c a c i d d i s a c c h a r i d e s (80-100 A) along the h y a l u r o n i c a c i d c h a i n . As noted above, i n the i n t a c t aggregate ( 1 9 ) , o r i n complexes formed between h y a l u r o n a t e and p r o t e o g l y c a n monomers w i t h i n t a c t c h o n d r o i t i n s u l f a t e c h a i n s , the minimum s p a c i n g between p r o t e o g l y c a n monomers i s 250 A. These o b s e r v a t i o n s i n d i c a t e t h a t the s p a c i n g o f p r o t e o g l y c a n monomers on h y a l u r o n a t e i s determined by the lengths of c h o n d r o i t i n s u l f a t e chains. O l i g o s a c c h a r i d e s r a n g i n g from two t o s i x r e p e a t i n g u n i t s were p r e p a r e d by t e s t i c u l a r h y a l u r o n i d a s e d i g e s t i o n o f h y a l u r o n a t e , and i s o l a t e d by Sephadex G-50 chromatography. Experiments were c a r r i e d out t o determine the minimum c h a i n l e n g t h o f h y a l u r o n a t e t o which c o r e m o l e c u l e s would b i n d . When c o r e m o l e c u l e s were mixed T

n

e


14.

ROSENBERG

E T

A L .


201

w i t h h i g h m o l e c u l a r weight h y a l u r o n a t e , complexes were formed which e l u t e d i n t h e v o i d volume o f Sepharose 2B columns. When c o r e m o l e c u l e s were mixed f i r s t w i t h h y a l u r o n a t e o c t a s a c c h a r i d e , then w i t h h i g h m o l e c u l a r weight h y a l u r o n a t e , h i g h m o l e c u l a r weight complexes were s t i l l formed which e l u t e d i n t h e v o i d volume. However, when c o r e m o l e c u l e s were mixed f i r s t w i t h h y a l u r onate d e c a s a c c h a r i d e s , then w i t h h i g h m o l e c u l a r weight h y a l u r o n a t e , no h i g h m o l e c u l a r weight complexes were formed and t h e c o r e m o l e c u l e s were r e t a r d e d . Moreover, the h y a l u r o n a t e d e c a s a c c h a r i d e s , which e l u t e d i n t h e column t o t a l volume when chromatographed a l o n e , now e l u t e d together with the core molecules. Similar res u l t s were o b t a i n e d when t h e c o r e m o l e c u l e s and h i g h m o l e c u l a r weight h y a l u r o n a t e were mixed f i r s t , then h y a l u r o n a t e d e c a s a c c h a r i d e s were added. Thus, p r o t e o g l y c a n monomer w i l l b i n d t o h y a l u r o n a t e w i t h a c h a i n l e n g t h o f f i v e r e p e a t i n g u n i t s but not t o h y a l u r o n a t e of f o u r r e p e a t i n g u n i t s . F i v e r e p e a t i n g u n i t s may be r e q u i r e d f o r h y a l u r o n a t e t o assume a c o n f o r m a t i o n e s s e n t i a l f o r p r o t e o g l y c a n monomer-hyaluronate i n t e r a c tion. Core m o l e c u l e s d i d not b i n d t o c h o n d r o i t i n o l i g o s a c c h a r i d e s , which c o n t a i n g a l a c t o s a m i n e , and d i f f e r from h y a l u r o n a t e o n l y i n t h a t t h e 4 - h y d r o x y l o f t h e amino sugar i s i n an a x i a l r a t h e r t h a n an e q u a t o r i a l position. Hardingham and Muir (13) have a l s o s t u d i e d t h e binding of o l i g o s a c c h a r i d e s of hyaluronate t o proteog l y c a n monomer from p i g l a r y n g e a l c a r t i l a g e , u s i n g viscometry. When o l i g o s a c c h a r i d e s o f a p p r o p r i a t e c h a i n l e n g t h were added t o a p r o t e o g l y c a n monomer-hyaluronate m i x t u r e , t h e r e was a d e c r e a s e i n v i s c o s i t y t h a t was p r o p o r t i o n a l t o t h e amount o f o l i g o s a c c h a r i d e added. R e l a t i v e l y l i t t l e e f f e c t was o b s e r v e d w i t h h y a l u r o n a t e t e t r a s a c c h a r i d e , hexasaccharide o r octasaccharide, i n d i c a t i n g t h a t p r o t e o g l y c a n monomer does not b i n d t o t h e s e o l i g o s a c c h a r i d e s . D e c a s a c c h a r i d e s and duodecasaccharides strongly i n h i b i t e d the binding of proteog l y c a n t o h i g h m o l e c u l a r weight h y a l u r o n i c a c i d , r e s u l t i n g i n a sharp d e c r e a s e i n v i s c o s i t y . Chemical M o d i f i c a t i o n s o f H y a l u r o n i c A c i d . Native p r o t e o g l y c a n a g g r e g a t e s , and complexes formed between p r o t e o g l y c a n monomer and h y a l u r o n a t e , a r e d i s s o c i a t e d at pH 3 t o 4, a t which t h e c a r b o x y l groups o f h y a l u r o nate a r e p r o t o n a t e d . T h i s o b s e r v a t i o n suggested that the c a r b o x y l groups o f h y a l u r o n a t e might be i n v o l v e d i n t h e b i n d i n g o f p r o t e o g l y c a n monomer t o h y a l u r o n a t e . C h r i s t n e r , Brown and D z i e w i a t k o w s k i have r e c e n t l y s t u died the e f f e c t of chemical m o d i f i c a t i o n of the c a r -


202


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b o x y l groups o f h y a l u r o n a t e on t h e p r o t e o g l y c a n monomerh y a l u o n a t e i n t e r a c t i o n ( 4 1 ) . T h e i r r e s u l t s show t h a t , f o r p r o t e o g l y c a n monomer t o b i n d t o h y a l u r o n a t e , h y a l u r o n a t e c a r b o x y l groups must be p r e s e n t and i n a s p e c i f i c spatial orientation. Hyaluronate o l i g o s a c c h a r i d e s of 5 t o 15 r e p e a t i n g u n i t s were p r e p a r e d . These i n h i b i t e d t h e b i n d i n g o f p r o t e o g l y c a n monomer t o h i g h molec u l a r weight h y a l u r o n a t e . The h y a l u r o n a t e c a r b o x y l groups were then c h e m i c a l l y m o d i f i e d i n s e v e r a l ways. F i r s t , t h e c a r b o x y l groups were t r e a t e d w i t h diazomethane t o form t h e carboxymethyl e s t e r s . The o l i g o s a c c h a r i d e s no l o n g e r i n h i b i t e d t h e b i n d i n g o f p r o t e o g l y c a n monomer t o h i g h m o l e c u l a r weight h y a l u r o n a t e . When t h e methyl groups were removed by s a p o n i f i c a t i o n , t h e h y a l uronate o l i g o s a c c h a r i d e s again i n h i b i t e d the b i n d i n g o f p r o t e o g l y c a n monomer t o h i g h m o l e c u l a r weight h y a l ronate. I f t h e carboxymethyl e s t e r was reduced w i t h NaBHi*, so t h a t t h e g l u c u r o n i c a c i d r e s i d u e o f h y a l u r o nate was t r a n s f o r m e d t o g l u c o s e , t h e o l i g o s a c c h a r i d e also lost i t s inhibitory capacity. In o t h e r experiments, t h e amide was formed between g l y c i n e methyl e s t e r and t h e c a r b o x y l groups o f g l u c u r o n i c a c i d r e s i d u e s . The i n h i b i t o r y c a p a c i t y o f t h e o l i g o s a c c h a r i d e s was l o s t . Ttje methyl group was removed by s a p o n i f i c a t i o n , y i e l d i n g t h e g l y c i n e amide o f g l u c u r o n i c a c i d , i n which a f r e e c a r b o x y l a t e group a r i s e s from g l u c u r o n i c a c i d , but i s d i s p l a c e d by the i n t e r p o s i t i o n o f a g l y c i n e r e s i d u e . The i n h i b i t o r y c a p a c i t y o f t h e o l i g o s a c c h a r i d e was not r e s t o r e d . The c o n f o r m a t i o n o f t h e g l u c u r o n i c a c i d c a r b o x y l a t e group i s e s s e n t i a l f o r p r o t e o g l y c a n monomer t o b i n d t o h y a l uronate. S t r u c t u r e and F u n c t i o n o f L i n k P r o t e i n . Cartilage p r o t e o g l y c a n aggregates a r e formed by t h e non-covalent a s s o c i a t i o n o f p r o t e o g l y c a n monomer, h y a l u r o n a t e and l i n k p r o t e i n . L i n k p r o t e i n can be i s o l a t e d from p r o t e o g l y c a n aggregate by e q u i l i b r i u m d e n s i t y g r a d i e n t c e n t r i f u g a t i o n under d i s s o c i a t i v e c o n d i t i o n s , f o l l o w e d by g e l chromatography under d i s s o c i a t i v e c o n d i t i o n s . L i n k p r o t e i n i s f i r s t p a r t i a l l y p u r i f i e d by e q u i l i b r i u m dens i t y gradient c e n t r i f u g a t i o n of proteoglycan f r a c t i o n A l i n 4 M GnHCl - 3 M C s C l . L i n k p r o t e i n i s r e c o v e r e d at low buoyant d e n s i t i e s from t h e t o p o n e - t h i r d o f 4 M GnHCl - 3 M C s C l g r a d i e n t s , mixed w i t h some h y a l u r o n a t e and low m o l e c u l a r weight, p r o t e i n - r i c h p r o t e o g l y c a n monomer. Most o f t h e h y a l u r o n a t e can be s e p a r a t e d from l i n k p r o t e i n by a s e q u e n t i a l 4 M GnHCl - 2 M C s C l d i s s o c i a t i v e g r a d i e n t . L i n k p r o t e i n may then be s e p a r a t e d from low m o l e c u l a r weight, p r o t e i n - r i c h p r o t e o g l y c a n


14.

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ET

AL.


203

monomer by chromatography on Sephadex G-200 i n 4 M GnHCl ( 1 8 ) , o r on S e p h a c r y l S-200 i n 4 M GnHCl, o r on U l t r a g e l 34 i n 1% SDS (30) o r on Sepharose CL-6B i n 4 M GnHCl ( 3 3 ) . In our hands, chromatography on Sephac r y l S-200 i n 4 M GnHCl y i e l d s f u n c t i o n a l l y a c t i v e , pure l i n k p r o t e i n , f r e e o f p r o t e i n - r i c h p r o t e o g l y c a n monomer, based on immunodiffusion: s t u d i e s ( 3 4 ) . L i n k p r o t e i n p r e p a r a t i o n s from most c a r t i l a g e s , i s o l a t e d as d e s c r i b e d above, c o n t a i n two p r o t e i n s a p p r o x i m a t e l y 44,000 and 48,000 i n m o l e c u l a r weight, on sodium d o d e c y l s u l f a t e p o l y a c r y l a m i d e g e l e l e c t r o p h o resis. These two p r o t e i n s have been c a l l e d l i n k p r o t e i n s 1 and 2. M o l e c u l a r weights o f l i n k p r o t e i n s 1 and 2 from s e v e r a l c a r t i l a g e s are g i v e n i n T a b l e I I . TABLE I I 3

M o l e c u l a r Weights ( x l O ) o f L i n k P r o t e i n s from S e v e r a l C a r t i l a g e s , Based on Sodium Dodecyl S u l f a t e P o l y a c r y l amide G e l E l e c t r o p h o r e s i s . CARTILAGE

Bovine N a s a l Bovine N a s a l Bovine N a s a l Human A r t i c u l a r Human Chondrosarcoma Swarm Rat Chondrosarcoma

LINK PROTEIN 1 2 45 48 51 49 49

REFERENCE

40 44 47 40 40

Oegema, e t a l . (28) Tang, et""aTT"(34) Baker and Caterson (30,33) P a l , e t j i l . (27) P a l , e t al_. (27)

40

Oegema, e t a l . (29)

Baker and C a t e r s o n have s e p a r a t e d l i n k p r o t e i n s 1 and 2 by p r e p a r a t i v e g e l e l e c t r o p h o r e s i s , and have p r e s e n t e d e v i d e n c e t h a t l i n k p r o t e i n s 1 and 2 a r e g l y c o p r o t e i n s which d i f f e r i n t h e i r o l i g o s a c c h a r i d e components, but not i n t h e i r amino a c i d c o m p o s i t i o n ( 3 3 ) . The compos i t i o n s o f l i n k p r o t e i n s 1 and 2 from b o v i n e n a s a l c a r t i l a g e ( k i n d l y p r o v i d e d by Dr. John B a k e r ) a r e shown in Table III (22). C l o s e e x a m i n a t i o n o f the model o f the p r o t e o g l y c a n aggregate d e p i c t e d i n F i g u r e 3 s u g g e s t s t h a t l i n k p r o t e i n b i n d s s i m u l t a n e o u s l y t o h y a l u r o n a t e , and t o t h e h y a l u r o n i c a c i d - b i n d i n g r e g i o n o f p r o t e o g l y c a n monomer c o r e p r o t e i n , and may s e r v e t o s t r e n g t h e n o r s t a b i l i z e the b i n d i n g o f p r o t e o g l y c a n monomer t o h y a l u r o n a t e . The s t u d i e s o f H e i n e g a r d and H a s c a l l p r o v i d e e v i d e n c e t h a t l i n k p r o t e i n i s l o c a t e d i n the r e g i o n where the h y a l u r o n i c a c i d - b i n d i n g r e g i o n o f p r o t e o g l y c a n monomer


204


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TABLE I I I

C h e m i c a l C o m p o s i t i o n s o f L i n k P r o t e i n s 1 and 2 Link Protein 1 Link Protein 2 AMINO ACID COMPOSITION ( r e s i d u e s p e r 1000 r e s i d u e s ) L-aspartic acid L-threonine L-serine L-glutamic acid L-proline Glycine L-alanine L-valine L-methionine L-isoleucine L-leucine L-tyrosine L-phenylalanine L-histidine L-lysine L-arginine

135 52 62 76 48 104 80 61 3 29 80 66 53 29 58 64

133 52 63 84 54 103 77 62 2 28 82 61 52 26 59 62

CARBOHYDRATE COMPOSITION (moles p e r 1 0 g p r o t e i n ) 5

L-fucose D-mannose D-galactose N-acetyl-D-glucosamine u-acetyl-D-galactosamine S i a l i c acid Total T o t a l (% by w e i g h t )

1.8 16.6 5.7 16.5 5.6 0.9 47.2 9.5

2.3 5.7 1.1 5.1 1.3 tr 15.6 3.0

(FROM: B a k e r , J.R. and C a t e r s o n , B. : The I s o l a t i o n and C h a r a c t e r i z a t i o n o f t h e Link P r o t e i n s from P r o t e o g l y c a n Aggregates o f Bovine Nasal C a r t i l a g e . Submitted f o r p u b l i c a t i o n i n J . B i o l . Chem.)


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b i n d s t o h y a l u r o n a t e ; as noted above ( F i g u r e 4 ) , deg r a d a t i o n o f proteoglycan aggregates with t r y p s i n r e moves t h e p o l y s a c c h a r i d e attachment r e g i o n o f p r o t e o g l y c a n monomers, y i e l d i n g a complex composed o f h y a l u ronate, hyaluronic a c i d - b i n d i n g region of proteoglycan monomer c o r e p r o t e i n , and l i n k p r o t e i n , r e p r e s e n t i n g the c e n t r a l p o r t i o n o f p r o t e o g l y c a n aggregate. C a t e r s o n and Baker (_31) have shown t h a t l i n k p r o t e i n b i n d s t o p r o t e o g l y c a n monomer i n t h e absence o f hyaluronate. L i n k p r o t e i n from b o v i n e n a s a l c a r t i l a g e was i s o l a t e d by chromatography on Sepharose CL 6B i n 4 M GnHCl. The l i n k p r o t e i n was mixed w i t h p r o t e o g l y can monomer. The m i x t u r e was s u b j e c t e d t o e q u i l i b r i u m d e n s i t y g r a d i e n t c e n t r i f u g a t i o n under a s s o c i a t i v e conditions. The d i s t r i b u t i o n o f l i n k p r o t e i n i n t h e g r a d i e n t f o l l o w e d t h a t o f p r o t e o g l y c a n monomer, which d i s t r i b u t e d a t h i g h buoyant d e n s i t i e s . When t h e l i n k p r o t e i n - p r o t e o g l y c a n monomer m i x t u r e was chromatographed on Sepharose CL 2B under a s s o c i a t i v e c o n d i t i o n s , l i n k p r o t e i n was e l u t e d w i t h p r o t e o g l y c a n monomer. These observations indicate that l i n k protein non-covalently a s s o c i a t e s w i t h p r o t e o g l y c a n monomer i n t h e absence o f hyaluronate. Tang, e t a l . (34) have p r o v i d e d e v i d e n c e t h a t l i n k p r o t e i n s t a b i l i z e s t h e b i n d i n g o f p r o t e o g l y c a n monomer to hyaluronate. L i n k p r o t e i n p r e s e n t i n low d e n s i t y f r a c t i o n s from t h e t o p o f 4 M GnHCl-2 M C s C l g r a d i e n t s was s e p a r a t e d from p r o t e i n - r i c h p r o t e o g l y c a n monomer and h y a l u r o n a t e by chromatography on S e p h a c r y l S-200 i n 4 M GnHCl. L i n k p r o t e i n p r e p a r e d by t h i s p r o c e d u r e was i m m u n o l o g i c a l l y pure. Since l i n k protein i s i n s o l uble i n most a s s o c i a t i v e s o l v e n t s , a study was c a r r i e d out t o i d e n t i f y a s s o c i a t i v e s o l v e n t s i n which l i n k p r o tein i s soluble. S e v e r a l a s s o c i a t i v e s o l v e n t s were i d e n t i f i e d i n which l i n k p r o t e i n i s s o l u b l e . In t h e s e s o l v e n t s , l i n k p r o t e i n was p r e s e n t as an 8 S s p e c i e s i n sedimentation v e l o c i t y studies, suggesting that l i n k p r o t e i n e x i s t s as an o l i g o m e r under a s s o c i a t i v e c o n d i tions. P r o t e o g l y c a n monomer was p r e p a r e d from b o v i n e n a s a l c a r t i l a g e , which i n t e r a c t e d w i t h h y a l u r o n a t e i n the absence o f l i n k p r o t e i n , t o form a h i g h m o l e c u l a r weight complex ( s J o = 68 S) demonstrable i n sedimentat i o n v e l o c i t y s t u d i e s . The a d d i t i o n o f l i n k p r o t e i n t o the monomer-hyaluronate m i x t u r e r e s u l t e d i n an i n c r e a s e i n t h e s e d i m e n t a t i o n c o e f f i c i e n t o f t h e complex from 68 t o 81 S. The complex formed between p r o t e o g l y c a n monomer and h y a l u r o n a t e i n t h e absence o f l i n k p r o t e i n was uns t a b l e a t a c i d pH. A p p r o x i m a t e l y o n e - h a l f o f t h e comp l e x was d i s s o c i a t e d a t pH 5; a l l o f t h e complex was


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d i s s o c i a t e d a t pH 4 o r 3. The a d d i t i o n o f l i n k p r o t e i n g r e a t l y i n c r e a s e d t h e s t a b i l i t y o f t h e complex a g a i n s t d i s s o c i a t i o n a t a c i d pH. In t h e presence o f l i n k p r o t e i n , t h e r e was no d e t e c t a b l e d i s s o c i a t i o n o f t h e comp l e x a t pH 5, and t h e complex was o n l y 50% d i s s o c i a t e d at pH 4.0. Even a t a pH of 3, some complex remained und i s s o c i a t e d i n the presence of l i n k p r o t e i n . These o b s e r v a t i o n s i n d i c a t e t h a t one b i o l o g i c r o l e o f l i n k p r o t e i n i s t o s t a b i l i z e t h e i n t e r a c t i o n between p r o t e o g l y c a n monomers and h y a l u r o n a t e i n p r o t e o g l y c a n aggregates. Blood Vessel Proteoglycans C a r t i l a g e c o n t a i n s one c l a s s o f p r o t e o g l y c a n monomer i n which c h o n d r o i t i n s u l f a t e and k e r a t a n s u l f a t e a r e c o v a l e n t l y bound t o the same p r o t e i n c o r e . Blood v e s s e l , k i d n e y and l u n g c o n t a i n s e v e r a l g l y c o s a m i n o g l y cans, i n c l u d i n g c h o n d r o i t i n 4 - s u l f a t e , c h o n d r o i t i n 6s u l f a t e , dermatan s u l f a t e , heparan s u l f a t e and h e p a r i n (42-53). Compared t o c a r t i l a g e , l i t t l e i s known about the p r o t e o g l y c a n s o f b l o o d v e s s e l , kidney and l u n g . However, b l o o d v e s s e l and l u n g parenchyma c o n t a i n a t l e a s t two c l a s s e s o f p r o t e o g l y c a n monomers, d i f f e r e n t from t h a t found i n c a r t i l a g e . One c l a s s c o n s i s t s o f dermatan s u l f a t e and c h o n d r o i t i n s u l f a t e c h a i n s bound t o t h e same p r o t e i n c o r e . The o t h e r c l a s s c o n s i s t s o f heparan s u l f a t e bound t o a p r o t e i n c o r e . The study o f p r o t e o g l y c a n s has r e c e n t l y e n t e r e d an e x c i t i n g new phase, i n which i n t e r e s t i s b e i n g f o c u s e d on t h e i s o l a t i o n and c h a r a c t e r i z a t i o n o f each o f t h e s e c l a s s e s o f p r o t e o g l y c a n monomers, t h e i r l o c a l i z a t i o n i n t h e i n t e r c e l l u l a r m a t r i x , basement membranes and plasma membranes o f c e l l s i n d i f f e r e n t t i s s u e s , and t h e e l u c i dation of t h e i r b i o l o g i c functions i n blood vessel, k i d n e y and l u n g . Dermatan S u l f a t e - C o n t a i n i n g P r o t e o g l y c a n s . The d i s a c c h a r i d e r e p e a t i n g u n i t o f dermatan s u l f a t e ( F i g u r e 2) c o n s i s t s o f L - i d u r o n i c a c i d and ^ - a c e t y l g a l a c t o s a mine (5ji). L - i 3 u r o n i c a c i d i s t h e C5 epimer o f D - g l u c u r o n i c a c i d , i n which t h e c a r b o x y l group i s i n an a x i a l r a t h e r than i n an e q u a t o r i a l p o s i t i o n . The N - a c e t y l g a l a c t o s a m i n e r e s i d u e o f dermatan s u l f a t e c a r r i e s an e s t e r s u l f a t e group u s u a l l y on carbon number 4, but sometimes on carbon number 6. Dermatan s u l f a t e c h a i n s c o n t a i n r e p e a t i n g u n i t s composed o f g l u c u r o n i c a c i d and N - a c e t y l g a l a c t o s a m i n e ( F i g u r e 2) as w e l l as i d u r o n i c a c i d and N - a c e t y l g a l a c tosamine. Thus, t h e dermatan s u l f a t e c h a i n i s a c o -


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polymer composed of dermatan s u l f a t e and c h o n d r o i t i n s u l f a t e r e p e a t i n g u n i t s . The h y b r i d s t r u c t u r e o f d e r matan s u l f a t e has been e x t e n s i v e l y s t u d i e d by F r a n s s o n (54-58). Dermatan s u l f a t e was i s o l a t e d from d i f f e r e n t s o u r c e s by methods c a p a b l e of removing c h o n d r o i t i n s u l fate. The dermatan s u l f a t e f r a c t i o n s c o n t a i n e d g l u c u r o n i c a c i d as w e l l as i d u r o n i c a c i d . The dermatan s u l f a t e f r a c t i o n s were degraded w i t h t e s t i c u l a r h y a l uronidase. T h i s enzyme c l e a v e s GalNAc(βl+4)GlcUA l i n kages i n c h o n d r o i t i n s u l f a t e r e p e a t i n g u n i t s , but not GalNAc(β1+4)IdUA l i n k a g e s i n dermatan s u l f a t e r e p e a t i n g units. V i s c o s i t y measurements showed t h a t dermatan s u l f a t e c h a i n s were degraded by t e s t i c u l a r h y a l u r o n i dase. Fragments were formed w i t h g l u c u r o n i c a c i d r e s i d u e s at newly formed n o n - r e d u c i n g t e r m i n i . Following t e s t i c u l a r hyaluronidase degradation of u m b i l i c a l cord dermatan s u l f a t e , F r a n s s o n i s o l a t e d and c h a r a c t e r i z e d a h y b r i d o c t a s a c c h a r i d e w i t h the f o l l o w i n g s t r u c t u r e : GlcUA-^GalNAc->IdUA-^GalNAc^IdUA->GalNAc->GlcUA->GalNAc Γ ι ι ι o s o

3

OS0

3

OS0

3

OS0

3

When dermatan s u l f a t e c h a i n s were degraded w i t h t e s t i c u l a r h y a l u r o n i d a s e , the fragments formed were m a i n l y o f h i g h m o l e c u l a r weight and composed o f dermatan s u l f a t e r e p e a t i n g u n i t s , o r were low m o l e c u l a r weight o l i g o s a c c h a r i d e s , c o n t a i n i n g most o f the g l u c u r o n i c acid. Fragments o f i n t e r m e d i a t e m o l e c u l a r weight were scarce. F r a n s s o n s u g g e s t e d t h a t i n the n a t i v e dermatan s u l f a t e c h a i n s , l o n g segments composed e n t i r e l y o f i d u r o n i c a c i d - c o n t a i n i n g r e p e a t i n g u n i t s are s e p a r a t e d by c l u s t e r s o f g l u c u r o n i c a c i d - c o n t a i n i n g r e p e a t i n g u n i t s , l o c a t e d a d j a c e n t t o one another. Fransson*s o b s e r v a t i o n s were based on a study o f dermatan s u l f a t e i s o l a t e d from p i g s k i n and human u m b i l i c a l c o r e . Lit t l e i s known about the c o - p o l y m e r i c s t r u c t u r e o f d e r matan s u l f a t e from b l o o d v e s s e l , k i d n e y and l u n g . B l o o d v e s s e l , k i d n e y and l u n g c o n t a i n p r o t e o g l y c a n monomers c o n s i s t i n g o f dermatan s u l f a t e and c h o n d r o i t i n s u l f a t e c h a i n s c o v a l e n t l y bound t o the same p r o t e i n core. The l i n k a g e r e g i o n o f dermatan s u l f a t e t o p r o t e i n i s i d e n t i c a l to that of c h o n d r o i t i n s u l f a t e ( F i g ure 2) ( 58, 59). Only a few attempts have been made t o i s o l a t e and c h a r a c t e r i z e dermatan s u l f a t e c o n t a i n i n g p r o t e o g l y c a n monomers. In 1971, K r e s s e , H e i d e l and Buddecke (60, 61) e x t r a c t e d p r o t e o g l y c a n s from b o v i n e a o r t a by h i g h speed homogenization i n 0.15 M phosphate, 0.05 M EDTA, pH 7. Dermatan s u l f a t e - c o n t a i n i n g p r o t e o g l y c a n was p u r i f i e d by r e p e a t e d c e t y l p y r i d i n i u m c h l o r i d e p r e c i p i t a t e s with MgCl . A dermatan s u l f a t e - c o n 2


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t a i n i n g p r o t e o g l y c a n was o b t a i n e d which c o n t a i n e d 20% p r o t e i n and 80% g l y c o s a m i n o g l y c a n . The g l y c o s a m i n o g l y c a n c o n s i s t e d o f 75% c h o n d r o i t i n s u l f a t e and 25% dermatan s u l f a t e . The a u t h o r s s t a t e d t h a t the p r o t e o g l y c a n behaved as a s i n g l e component i n the a n a l y t i c a l u l t r a c e n t r i f u g e , a l t h o u g h no d a t a from s e d i m e n t a t i o n v e l o c i t y experiments o r the c o n d i t i o n s o f the sedimen t a t i o n v e l o c i t y s t u d i e s were p r e s e n t e d . The weight average m o l e c u l a r weight o f the dermatan s u l f a t e con t a i n i n g p r o t e o g l y c a n from l i g h t s c a t t e r i n g was 2 χ 1 0 . F o l l o w i n g d e g r a d a t i o n o f the dermatan s u l f a t e - c o n t a i n i n g p r o t e o g l y c a n w i t h pronase and t e s t i c u l a r h y a l u r o n i dase, o l i g o s a c c h a r i d e s r a n g i n g from t e t r a s a c c h a r i d e s t o o c t a s a c c h a r i d e s were i s o l a t e d , whose i d u r o n i c acid/glucu r o n i c a c i d r a t i o i n c r e a s e d w i t h i n c r e a s i n g c h a i n length. S t u d i e s o f b l o o d v e s s e l s and o t h e r t i s s u e s , and o f c e l l s i n c u l t u r e i n d i c a t e t h a t dermatan s u l f a t e c o n t a i n i n g p r o t e o g l y c a n s are d i s t r i b u t e d throughout the i n t e r c e l l u l a r substance i n t e r c o n n e c t i n g c o l l a g e n f i b e r s e l a s t i n and c e l l s , w h i l e heparan s u l f a t e - c o n t a i n i n g p r o t e o g l y c a n s are found i n the plasma membranes o f cells. Wight and Ross r e c e n t l y s t u d i e d the u l t r a s t r u c t u r a l l o c a l i z a t i o n o f p r o t e o g l y c a n s i n the i n t i m a o f non-human p r i m a t e a r t e r i e s ( 6 2 ) . Numerous 200-500 Â d i a m e t e r p o l y g o n a l g r a n u l e s w i t h a marked a f f i n i t y f o r ruthenium r e d were d i s t r i b u t e d throughout the i n t e r c e l l u l a r s u b s t a n c e ( F i g u r e 7 ) . The g r a n u l e s p o s s e s s e d 30-60 Â t h i c k f i l a m e n t o u s p r o j e c t i o n s which appeared t o interconnect adjacent granules. The g r a n u l e s and t h e i r f i l a m e n t s i n t e r c o n n e c t e d c o l l a g e n f i b e r s at r e g u l a r i n t e r v a l s i n r e g i s t e r w i t h the p e r i o d i c i t y of the c o l l a gen f i b e r s , and e l a s t i c f i b e r s , and appeared t o form c o n n e c t i o n s between the plasma membranes o f smooth musc l e c e l l s and i n t e r c e l l u l a r f i b e r s ( F i g u r e 7 ) . Most o f the i n t e r c e l l u l a r g r a n u l e s and f i l a m e n t s were r e moved w i t h c h o n d r o i t i n a s e ABC. Wight and Ross (63) a l s o found t h a t 60-80% o f the g l y c o s a m i n o g l y c a n s y n t h e s i z e d and s e c r e t e d i n t o t h e medium by a r t e r i a l smooth muscle c e l l s i n c u l t u r e was dermatan s u l f a t e , w h i l e o n l y 10-20% was c h o n d r o i t i n 4- o r 6 - s u l f a t e . Taken t o g e t h e r , the r e s u l t s i n d i c a t e d t h a t most o f the i n t e r c e l l u l a r m a t r i x g r a n u l e s are p r o b a b l y dermatan s u l f a t e containing proteoglycans. Wight and Ross suggested t h a t the i n t e r c e l l u l a r p r o t e o g l y c a n might f u n c t i o n t o h o l d c o l l a g e n f i b e r s , e l a s t i n and c e l l s t o g e t h e r , and at the same time m a i n t a i n t i s s u e t u r g o r as a r e s u l t o f t h e i r e l a s t i c p r o p e r t i e s . They suggested t h a t the p r o t e o g l y c a n s might f u n c t i o n as a t y p e o f p l a s t i c i n t e r s t i t i a l s u b s t a n c e , important i n a b s o r b i n g and/or d i s s i pating stress. 6


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Journal of Cell Biology

Figure 7. Portion of an intimai smooth muscle cell and the adjacent intercellular matrix from a primate artery (Macaca nemestrina) stained with ruthenium red. The ruthenium red stains numerous polygonal granules associated with each other through 30-60 A diameter fihmentous projections which, based on studies with chondroitinases, must represent dermatan sulfate-containing proteoglycans (X40,000). As shown in the insert, the 30-60 A filaments interconnect granules, collagen fibers, elastin, and the surfaces of cells (X140,000). Reproduced from Proteoglycans in Primate Arteries. I. Ultrastructural Localization and Distribution in the Intima, by Thomas N. Wight and Russell Ross, J. Cell Biol. (1975) 67, 660-674.


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INTERACTION

E i s e n s t e i n and K u e t t n e r r e p o r t e d s i m i l a r o b s e r v a t i o n s i n an e l e c t r o n m i c r o s c o p i c study o f the u l t r a s t r u c t u r e o f p r o t e o g l y c a n s i n b o v i n e a o r t a (64,65). Ruthenium r e d s t a i n e d g r a n u l e s , 300-400 A i n diameter, i n t e r c o n n e c t e d by s l e n d e r f i l a m e n t s , were found i n the i n t e r c e l l u l a r m a t r i x , on the s u r f a c e s o f c o l l a g e n f i b e r s , at the edges o f e l a s t i c f i b e r s and near the p l a s ma membranes o f smooth muscle c e l l s . The g r a n u l e s d i s appeared a f t e r d i g e s t i o n w i t h c h o n d r o i t i n a s e ABC, o r a f t e r e x t r a c t i o n w i t h 4 M g u a n i d i n e h y d r o c h l o r i d e . The p r e s e n c e o f dermatan s u l f a t e - c o n t a i n i n g p r o t e o g l y c a n s on t h e s u r f a c e s o f c o l l a g e n f i b e r s was a l s o i n d i c a t e d by the b i n d i n g o f p e r o x i d a s e - l a b e l e d a n t i b o d i e s r a i s e d a g a i n s t dermatan s u l f a t e - c o n t a i n i n g p r o t e o g l y c a n s t o the s u r f a c e s o f c o l l a g e n f i b e r s . In b o t h the s t u d i e s p f Wight and Ross, and o f E i s e n s t e i n and K u e t t n e r , ruthenium r e d s t a i n i n g m a t e r i a l was a l s o found i n t h e plasma membranes o f endothel i a l c e l l s and smooth muscle c e l l s which was not r e moved w i t h c h o n d r o i t i n a s e ABC and i s t h e r e f o r e not chond r o i t i n s u l f a t e o r dermatan s u l f a t e . O b s e r v a t i o n s by s e v e r a l workers suggest t h a t at l e a s t some o f the ruthenium r e d s t a i n i n g m a t e r i a l found i n t h e plasma membranes o f c e l l s i s heparan s u l f a t e c o n t a i n i n g p r o t e o g l y c a n . C e l l s i n c u l t u r e appear t o synthesize three d i s c r e e t pools of glycosaminoglycans; 1) an e x t r a c e l l u l a r p o o l , s e c r e t e d i n t o t h e c u l t u r e medium, p r o b a b l y r e p r e s e n t a t i v e o f the p r o t e o g l y c a n s s e c r e t e d i n t o the i n t e r c e l l u l a r m a t r i x in v i v o ; 2) a cell-membrane a s s o c i a t e d o r p e r i c e l l u l a r p o o l ; and 3) an i n t r a c e l l u l a r p o o l . F o r a p a r t i c u l a r c e l l t y p e , each p o o l f r e q u e n t l y shows a c h a r a c t e r i s t i c d i s t r i b u t i o n i n terms o f the p e r c e n t a g e o f the t o t a l g l y c o s a m i n o g l y c a n t h a t i s c h o n d r o i t i n s u l f a t e , dermatan s u l f a t e , heparan s u l f a t e , h e p a r i n or h y a l u r o n a t e . Kraemer has shown t h a t heparan s u l f a t e i s p r e s e n t as a p r o t e o g l y c a n i n the plasma membrane o f a v a r i e t y o f c e l l s (66-70). Treatment o f c u l t u r e d c e l l s w i t h t r y p s i n r e l e a s e d T r a g ments o f plasma membrane heparan s u l f a t e - c o n t a i n i n g p r o t e o g l y c a n w i t h a m o l e c u l a r weight o f 135,000 ( 7 0 ) . The fragments were e x c l u d e d on B i o - G e l A 0.5 m. When the e x c u l d e d m a t e r i a l was t r e a t e d w i t h n i t r o u s a c i d , o l i g o s a c c h a r i d e s c h a r a c t e r i s t i c o f heparan s u l f a t e were formed, which were r e t a r d e d on B i o - G e l P-10. After a l k a l i n e b o r o h y d r i d e treatment o f the t r y p t i c fragments, r e i s o l a t e d heparan s u l f a t e c h a i n s had a m o l e c u l a r weight o f 44,000, s u g g e s t i n g at l e a s t 3 c h a i n s p e r t r y p t i c fragment. S i l b e r t has s t u d i e d s i m i l a r heparan s u l f a t e c o n t a i n i n g t r y p t i c fragments d e r i v e d from the plasma membranes o f c u l t u r e d normal s k i n f i b r o b l a s t s (71,72).


14.

ROSENBERG

E T

A L .


211

U s i n g h e p a r i n a s e and h e p a r i t i n a s e from Flavobacterium heparinum, B u o n a s s i s i has p r o v i d e d e v i d e n c e f o r t h e p r e s e n c e o f heparan s u l f a t e - c o n t a i n i n g p r o t e o g l y c a n s i n t h e plasma membranes o f e n d o t h e l i a l c e l l s from r a b b i t a o r t a (73,74). The o b s e r v a t i o n s d e s c r i b e d above r a i s e q u e s t i o n s about t h e f u n c t i o n s o f dermatan s u l f a t e - c o n t a i n i n g proteoglycans i n the i n t e r c e l l u l a r matrix of a r t e r i a l w a l l , and about t h e f u n c t i o n s o f heparan s u l f a t e - c o n t a i n i n g p r o t e o g l y c a n s i n t h e plasma membranes o f endo t h e l i a l c e l l s and smooth muscle c e l l s . Essentially n o t h i n g i s known about t h e b i o l o g i c a l f u n c t i o n o f he p a r a n s u l f a t e - c o n t a i n i n g p r o t e o g l y c a n s i n t h e plasma membranes o f c e l l s . Methods f o r t h e e x t r a c t i o n and i s o l a t i o n o f n a t i v e heparan s u l f a t e - c o n t a i n i n g p r o t e o g l y c a n s have y e t t o be d e v e l o p e d . However, some f u n c t i o n s o f dermatan s u l f a t e - c o n t a i n i n g p r o t e o g l y c a n s a r e g r a d u a l l y becoming a p p a r e n t . As i n d i c a t e d above, t h e u l t r a s t r u c t u r a l s t u d i e s o f Wight and Ross and o f E i s e n s t e i n and K u e t t n e r suggest t h a t dermatan s u l f a t e - c o n t a i n i n g p r o t e o g l y c a n s i n t e r connect c o l l a g e n , e l a s t i n and c e l l s , and c o n t r i b u t e t o the e l a s t i c and m e c h a n i c a l p r o p e r t i e s o f a r t e r i a l w a l l . Implicit i n t h i s concept i s t h e i d e a t h a t dermatan s u l f a t e - c o n t a i n i n g p r o t e o g l y c a n s may b i n d t o and noncovalently associate with collagen. Recent s t u d i e s show t h a t dermatan s u l f a t e - c o n t a i n i n g p r o t e o g l y c a n s n o n - c o v a l e n t l y b i n d t o c o l l a g e n , and i n f l u e n c e b o t h the c o n f o r m a t i o n a l s t a b i l i t y o f c o l l a g e n monomer, and the f o r m a t i o n o f c o l l a g e n f i b r i l s from c o l l a g e n mono mer. T o o l e and Lowther (_75) i s o l a t e d a dermatan s u l fate containing proteoglycan, following e x t r a c t i o n of b o v i n e h e a r t v a l v e s w i t h 6 M u r e a a t 6 0 ° . When i t was mixed w i t h c o l l a g e n monomer, c o l l a g e n f i b r i l s w i t h t y p i c a l p e r i o d i c i t y were formed immediately. No f i b r i l s were formed when t h e c o l l a g e n monomer was mixed w i t h hyaluronate or chondroitin s u l f a t e - c o n t a i n i n g proteo glycan. T o o l e and Lowther s u g g e s t e d t h a t t h e p r i m a r y b i o l o g i c r o l e o f dermatan s u l f a t e - c o n t a i n i n g p r o t e o g l y c a n s might be i n t h e f o r m a t i o n and o r i e n t a t i o n o f c o l l a g e n f i b r i l s from c o l l a g e n monomer. B l a c k w e l l and h i s co-workers have used c i r c u l a r d i c h r o i s m s p e c t r o s c o p y t o study t h e i n t e r a c t i o n s o f g l y c o s a m i n o g l y c a n s w i t h c o l l a g e n , and w i t h s y n t h e t i c cationic polypeptides. In t h e absence o f g l y c o s a m i n o g l y c a n s , p o l y - L - l y s i n e and p o l y - L - a r g i n i n e exist i n an extended charged c o i l c o n f o r m a t i o n . Glycosaminogly cans b i n d t o t h e s e c a t i o n i c p o l y p e p t i d e s and cause them t o assume an α-helical c o n f o r m a t i o n . In a s e r i e s o f s y s t e m a t i c s t u d i e s (76-83). B l a c k w e l l and h i s co-workers


212


INTERACTION

have e x t e n s i v e l y examined t h e c o n f o r m a t i o n - d i r e c t i n g e f f e c t o f g l y c o s a m i n o g l y c a n s on c a t i o n i c p o l y p e p t i d e s . The e x t e n t o f α-helix f o r m a t i o n was a s s e s s e d from t h e n e g a t i v e e l l i p t i c i t y found on c i r c u l a r d i c h r o i s m spec t r o s c o p y i n d i l u t e aqueous s o l u t i o n a t n e u t r a l pH. F o r each m i x t u r e , maximum α-helix c o n t e n t was o b t a i n e d at a c h a r a c t e r i s t i c r a t i o o f amino a c i d r e s i d u e s t o disaccharide repeating u n i t s (Table IV). TABLE IV I n t e r a c t i o n s o f Glycosaminoglycans peptides . *

with C a t i o n i c Poly

Comparison o f C o n f o r m a t i o n - D i r e c t i n g E f f e c t s , Residue R a t i o s , and M e l t i n g Temperature a t Maximum I n t e r a c t i o n f o r t h e Seven G l y c o s a m i n o g l y c a n s (76,81,83). HA

Ratio 1:1 Effect α t (°C) 35.0 m

C4S

HS C6S KS1 Poly-L-arginine

2:1 α 54.5

1:1 α 65.0

2:1 α 76.0

1.2:1 α >90

DS

HEP

1.4:1 α >90

3.3: 1 α >90

1.4:1 α 74.5

2.3:1 α >90

Poly-L-lysine Ratio 1:1 Effect R t (°C) -

1:1 α 25.0

m

2:1 R -

1:1 α 47.0

1.2: 1 R -

* A b b r e v i a t i o n s used i n TABLE IV HA - h y a l u r o n i c a c i d C4S - c h o n d r o i t i n 4 - s u l f a t e HS - heparan s u l f a t e C6S - c h o n d r o i t i n 6 - s u l f a t e KS1 - k e r a t a n s u l f a t e DS - dermatan s u l f a t e HEP - h e p a r i n G l y c o s a m i n o g l y c a n s were compared i n terms o f t h e r e s i due r a t i o a t which maximum α-helix f o r m a t i o n was ob t a i n e d (TABLE I V ) . G l y c o s a m i n o g l y c a n - p o l y p e p t i d e i n t e r a c t i o n s were weakened and f i n a l l y a b o l i s h e d , as t h e temperature was i n c r e a s e d . The α-helical c o n f o r m a t i o n of t h e p o l y p e p t i d e was l o s t , and t h e p o l y p e p t i d e r e v e r t e d t o an extended charged c o i l c o n f o r m a t i o n . The m e l t i n g temperature f o r each g l y c o s a m i n o g l y c a n - p o l y p e p t i d e m i x t u r e was d e f i n e d as t h e m i d p o i n t o f t h e t r a n s i t i o n from α-helix t o extended c o i l o f t h e p o l y peptide. The m e l t i n g temperature o f a p a r t i c u l a r p o l y p e p t i d e was d i f f e r e n t f o r each g l y c o s a m i n o g l y c a n ( T a ble IV). Therefore, the strength o f the i n t e r a c t i o n between g l y c aminoglycans and c a t i o n i c p o l y p e p t i d e s c o u l d a l s o be e v a l u a t e d i n terms o f t h e m e l t i n g tem p e r a t u r e ( t , °c) o f t h e m i x t u r e . As i n d i c a t e d by t h e dermatan s u l f a t e - p o l y - L - a r g i n i n e o r p o l y - L - l y s i n e m e l t m


14.

ROSENBERG

E TA L .


213

i n g temperature shown i n T a b l e IV, t h e i n t e r a c t i o n be tween dermatan s u l f a t e and c a t i o n i c p o l y p e p t i d e s i s p a r t i c u l a r l y strong. In c o n n e c t i v e t i s s u e s such as b l o o d v e s s e l , k i d n e y and l u n g , dermatan s u l f a t e - c o n t a i n i n g p r o t e o g l y c a n s must i n t e r a c t s t r o n g l y w i t h colrl a g e n f i b e r s , and i n c r e a s e t h e s t a b i l i t y o f t h e c o l l a gen monomer t r i p l e h e l i x ( 8 4 ) . In doing so, dermatan s u l f a t e - c o n t a i n i n g p r o t e o g l y c a n s must a l s o envelop and s h i e l d c o l l a g e n f i b e r s , thereby r e g u l a t i n g t h e i n t e r a c t i o n s o f c o l l a g e n w i t h p l a t e l e t s and o t h e r c e l l s .

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Supported by g r a n t s AM HD 21498 and CA AM 23945 from the N a t i o n a l I n s t i t u t e s o f H e a l t h RECEIVED

September 8, 1978.


Carbohydrate-Protein Interaction - ACS Publications - American

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