Structure of chondroitin sulfates analyses of the products formed from chondroitin sulfates A and C by the action of the chondroitinases C and AC from Flavobacterium heparinum

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Biochimica et Biophysica Acta, 451 (1976) 436--443 © Elsevier/North-Holland Biomedical Press

BBA 28095

STRUCTURE OF CHONDROITIN SULFATES

ANALYSES OF THE PRODUCTS FORMED FROM CHONDROITIN SULFATES A AND C BY THE ACTION OF THE CHONDROITINASES C AND AC FROM F L A V O B A C T E R I U M H E P A R I N U M

YARA M. MICHELACCI and CARL P. DIETRICH Departarnento de Bioqulmica e Farmacologia, Escola Paulista de Medicina, C P. 20372, S~o Paulo SP (Brazil)

(Received May 31st, 1976)

Summary

The structures of chondroit in sulfate A from whale cartilage and chondroit in sulfate C from shark cartilage have been examined with the aid of the chondroitinases A t and C from F l a v o b a c t e r i u m h e p a r i n u m . The analyses of the products formed from the chondroit in sulfates by the action of the chondroit inases have shown that three types of oligosaccharides compose the structure of chondroit in sulfate A, namely, a dodeca-, hexa- and a tetra-saccharide, containing five, two and one 4-sulfated disaccharides per 6-sulfated disaccharide residue, respectively. The polymer contains an average of 3 mol of each oligosaccharide per mol of chondroit in sulfate A. Each mol of chondroit in sulfate C contains an average of 5 mol of 4-sulfated disaccharide units. A tetra-saccharide containing one 4-sulfated disaccharide and one 6-sulfated disaccharide was isolated from this mucopolysaccharide by the action of the chondroitinase C, indicating that the 4-sulfated disaccharides are not linked together in one specific region but spaced in the molecule.

Int roduct ion

It is well established that chondroit in sulfate B from pig skin is a hybrid structure made of copolymers containing regions akin to chondroit in sulfate A

A b b r e v i a t i o n s : C h o n d r o i t i n su l fa te A a n d C, e h o n d r o i t i n 4 -su l fa te a n d e h o n d r o i t i n 6 -su l fa te , re- s p e c t i v e l y ; e h o n d r o i t i n su l fa te B, d e r m a t a n su l fa te ; A Di-4S, 2 - ace t amido -2 -deoxy-3 -O- ( f l -D-g luco - 4 - e n e p y r a n o s y l u r o n i c ac id ) -4 -O-su l fo -D-ga lae tose ; A Dl-fiS, 2 - a e e t a m i d o - 2 - d e o x y - 3 - O - ( ~ - D - g l u c o - 4 - e n e p y r a n o s y l u r o n i c ac id ) -6 -O-su l fo -D-ga lac tose ; A Di-OS, 2 - a c e t a m i d o - 2 - d e o x y - 3 - O - ( ~ - D - g l u c o - 4 - c n e p y r a n o s y l u r o n t c ac id ) -D-ga l ac tose : A Di -Glu-OS, 2 - ace t a rn ido -2 -deoxy-3 -O- (~ -D-g luco -4 - e n e p y r a n o s y l u r o n i c ac id ) -D-g lucose .

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(glucuronic acid-containing disaccharides) and regions made of iduronic acid- containing disaccharides [ 1--3]. Chondroitin sulfate B obtained from umbilical cord and horse aorta were shown to contain chondroitin sulfate C regions besides the above mentioned copolymers [4]. It is also becoming apparent that human fibrous cartilage contains copolymers made of chondroitin sulfates B and C [5]. Furthermore, the chondroitin sulfate molecules from cartilages seem to be also hybrid molecules composed of 4- and 6-sulfated disaccharides in different proportions depending upon the type of cartilage examined [6--8]. These results were largely obtained with the use of enzymes such as testicular hyaluronidase, chondroitinase AC and chondroitinase B that cleave selectively the different uronic acid-containing copolymers.

The present paper describes new structural details of the chondroitin sulfates A and C by the use of a recently described chondroitinase [9] that cleaves only 6-sulfated or non-sulfated hexosaminido-glucuronic acid bonds.

A preliminary communication of these results has been presented [10].

Experimental procedure

Materials and enzymes. Chondroitin sulfate A (from whale cartilage) chondroitin sulfate C (from shark cartilage), chondroitinase AC (E.C. 4.2.2.5) and chondroitinase ABC (E.C. 4.2.2.4) were purchased from Miles Laboratories, Elkhart, Ind. Chondroitinase C was prepared by a method previously described [9].

Preparation of degradation products. For the isolation of di- and oligo-saccharides, 200 mg of chondroitin sulfates A and C were incubated with 1 to 3 units of chondroitinase C in 0.05 M ethylenediamine-acetate buffer pH 8.0 at 20°C in a total volume of 20 ml. After 24 h incubation the mixtures were concentrated in a rotary evaporator to about 5 ml and applied as 40 cm bands in 2 sheets of Whatmann 3 MM filter paper and subjected to descending chromatography in isobutyric acid/1 M NH3, 5 : 3, v/v (solvent A) for 72 h. The di- and oligo-saccharides were eluted from the paper with water after visual obser- vation with the aid of an ultraviolet lamp or after silver nitrate staining of guide strips prepared from the chromatograms. Usually these eluted products were re- subjected to descending chromatography in the same solvent or in 1-butanol/ acetic acid/1 M NH3, 2 : 3 : 1, v/v (solvent B) or in 1-butanol/acetic acid/water, 10 : 3 : 7, v/v (solvent C) for 48 h. After chromatography and elution, the products were concentrated to 50 gl and further purified by precipitation with the addition of 50 ~l of methanol and 10 volumes acetone. The precipitate formed was then dried under vacuum and resuspended in water to a final solution of 10 mg per ml.

Analytical methods. The methods used for hexosamine, uronic acid and sulfate content as well as reducing power and reducing end were the ones previously refered to [9,11,12]. The degradation products obtained from the chondroitin sulfates A and C by the action of the chondroitinase C were incubated with the chondroitinases AC and C as follows: about 100/ag of degradation products were incubated with chondroitinase AC at 37°C and chondroitinase C at 20°C in 0.05 M ethylenediamineacetate buffer, pH 8.0 in a final volume of 20 pl. After 4 h incubation the mixtures were chromatographed

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in solvent A and the degradation products quantitated by densi tometry after silver nitrate staining [11,12]. Alternatively, the products formed were visu- alised with the aid of an ultraviolet lamp, eluted from the paper and quantitated by absorption at 232 nm or hexosamine content after acid hydrolysis.

The molecular weight of the degradation products were determined by polyacrylamide electrophoresis in gel slabs [11]. The molecular weight markers used were chondroitin sulfate B (average mol.wt. 19 000), heparitin sulfate C (mol.wt. 9300), and hexa- (mol.wt. 1800), tetra- (mol.wt. 1100) and trisulfated disaccharide (mot.wt. 600) prepared from heparin by methods previously described [ 11,14].

Results

Enzymatic degradation of chondroitin sulfate A Chondroitin sulfate A is totally degraded by the chondroitinase AC produc-

ing 80% of A Di-4S and 20% of A Di-6S (Fig. 1). When this mucopolysaccharide is incubated with chondroitinase C it produces 20% of a tetrasaccharide, 21% of an hexasaccharide and 57% of an oligosaccharide besides a small amount of A Di-6S (Table I). These products were prepared in large scale and analysed. Table I shows that the ratios of uronic acid, hexosamine and sulfate were approximately the same for all the fragments. The reducing power and hexosamine remaining after borohydride reduction of the compounds varied as a function of their chain lengths. The disappearance of hexosamine after borohyd.ride reduction also indicates that hexosamine is at the reducing end of the fragments. These compounds were again incubated with chondroitinase C and also with chondroitinase AC. None of the fragments were fur-

C ~ AC Ch~e C

C A C A

~ ,- AWi4S

Fig . 1. P a p e r e h r o m a t o g r a m o f t h e e h o n d r o i t i n a s e s A C a n d C d i g e s t s o f t h e c h o n d r o i t i n s u l f a t e s . 3 • 10 -3 u n i t s o f t h e c h o n d r o i t i n a s e A C ( C h a s e A C ) a n d 2 .8 • 10 -3 u n i t s o f t h e c h o n d r o i t i n a s e C ( C h a s e C) w e r e i n c u b a t e d w i t h 1 0 0 # g o f c h o n d r o i t i n s u l f a t e s A a n d C ( A a n d C, r e s p e c t i v e l y ) in 0 . 0 5 M e t h y l e n e d i a m i n e - a c e t a t e b u f f e r p H 8 .0 in a f i na l v o l u m e o f 2 0 #tl. A f t e r 4 h i n c u b a t i o n a t 3 7 ° C f o r t h e c h o n d r o l t i n a s e A C

a n d 2 0 ° C f o r t h e c h o n d r o i t i n a s e C, t h e m i x t u r e s w e r e s p o t t e d o n W h a t m a n N o . 1 P a p e r a n d c h r o m a t o - g r a p h e d in s o l v e n t A f o r 48 h. A f t e r t h e r u n , t h e r e d u c i n g p r o d u c t s w e r e l o c a t e d b y s i l ve r n i t r a t e s t a i n i n g .

4 3 9

TABLE I

ANALYTICAL DATA FOR CHONDROITIN S U L F A T E A DEGRADATION PRODUCTS

C h o n d r o i t i n a s e C m o l / m o l o f h e x o s a m i n e Reduc ing % o f remain- Average prod uc t sugars ing h e x o s - m o l e c u -

Uronic T o t a l t o o l / t o o l a mine af ter lar w e i g h t acid su l fate o f h e x o s - bo ro hy dr ide

am in e reduc t io n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C h o n d r o i t i n

su lphate A 1 . 0 3 1 . 0 5 0 . 0 3 1 0 0 1 5 , 0 0 0 - -

Oligosaccharides 0 . 9 6 0 . 9 8 0 . 1 6 85 3 , 0 0 0 57

Hexasacchar ides 1 .04 1 . 0 2 0 . 2 7 7 5 - - 21

Tctrasaccharides 1 . 3 4 1 . 0 4 0 . 4 0 56 - - 20

A Di -6S 1 .17 1 .2 0 . 8 0 5 - - 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A m o u n t

f o r m e d b y

chondro- i t inase C (%)

ther degraded by the chondroitinase C but they were all degraded by chondroitinase AC (Fig. 2). The oligo-, hexa- and tetrasaccharides formed A Di-6S and A Di-4S in different proportions by the action of chondroitinase AC, as shown in Table II. The results permit to conclude that: the tetrasaccharide is composed of one 4-sulfated disaccharide and one 6-sulfated disaccharide; the hexasaccharide contains two 4-sulfated disaccharides and one 6-sulfated disaccharide; the oligosaccharide contains five 4-sulfated disaccharides and one 6- sulfated disaccharide. The oligosaccharide has the same molecular weight as the oligosaccharide obtained from chondroitin sulfate B after the action of the chondroitinase B [3] but differs from it by the presence of one 6-sulfated disaccharide in its structure.

One of the possible structures of the chondroitin sulfate A and the mode of

Clam AC

2 3

@ * i

I ChmmC I -- 4 1 2 3 4 1 2 3 4

0 # - - -

- t t ~ - e S

~L.. ..

f

Fig. 2. Paper c h r o m a t o g r a m o f the c h on d r o i t in ase s AC and C digests o f the p r o d u c t s prepared by degrada- t ion o f c h o n d r o i t i n sul fate A wi th chondro i t inase C. I 0 0 ~g o f the di- (1 ) , tetra- (2 ) , hexa- (3 ) and o l igo -

s a c c h a r i d e s (4) f o r m e d f r o m c h on d r o i t in sul fate A b y the ac t ion o f the chondro i t inase C were incuba ted and analysed as descr ibed in Fig. 1 : Chase, chondro i t inase .

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T A B L E II

A M O U N T O F D I S A C C H A R I D E S F O R M E D F R O M C H O N D R O I T I N S U L F A T E A P R O D U C T S BY T H E

A C T I O N OF T H E C H O N D R O I T I N A S E AC

Subs t r a t e s *

C h o n d r o i t i n su l fa te A

Ol igosacchar ides

H e x a s a c c h a r i d e s

Te t r a saccha r ide s A Di-6S

Produc t s f o r m e d (%)

A Di-4S ~ Di-6S

79 21

82 18 65 35

50 50 -- 100

* P r o d u c t s f o r m e d f r o m c h o n d r o i t i n su l fa te A by the ac t ion of the c h o n d r o i t i n a s e C.

action of the chondroitinase C and chondroitinase AC upon this mucopolysaccharide are shown in Fig. 3.

Enzymatic degradation of chondroitin sulfate C The products formed by the action of chondroitinase AC upon chondroitin

sulfate C are shown in Fig. 1. About 95% of A Di-6S and 5% of A Di-4S are formed from chondroitin sulfate C {Table III). When this mucopolysaccharide is incubated with the chondroitinase C, A Di.6S, tetra-, hexa- and oligosaccharides are formed. These fragments were prepared in large scale and incubated with chondroitinase AC and chondroitinase C. The result of this experi- ment is sh'own in Fig. 4. With the exception of the tetrasaccharide the other fragments of higher molecular weight were still slowly degraded by the chondroitinase C to A Di-6S. The tetrasaccharide was degraded only by the chondroitinase AC producing A Di-6S and A Di-4S. The hexa- and oligosaccharides were also degraded by the chondroitinase AC producing mainly A Di-6S and small amounts of another product (not analysed) with a chromatographic migration

C~A- CtA-A-A-A -A- C.*A-A-C:A-C'~A-A-A-A-A-C'~A-A-C ~A-CIA-A-A-A-A-C~A-A- C

,lChase C}

A. tA ' ;A-A-A '~C + A~A~C + A'~C

:]Chase ACI

A • C

Fig. 3, P roposed s t r u c t u r e of e h o n d r o i t i n su l fa te A and the m o d e of ae t ton of the chondro i t inases AC and

C in the degradation of choncLroitin sulfate A to disaccharides. The abreviations used are: A, 2-acetamido-

2-deoxy-3-O-(glucopyranosyluronic acid)-4-O-sulfo-D-galactose; C, 2-acetamido-2-deoxy-3-O-(glucopyranosyluronic acid)-6-O-sulfo-D-galactose. Chase, chondroitinase.

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T A B L E Ill

A N A L Y T I C A L D A T A FOR C H O N D R O I T I N S U L F A T E C D E G R A D A T I O N P R O D U C T S

Chondro i t inase C m o l / m o l of h e x o s a m i n e Reduc ing % of r e m a i n - A m o u n t of di- Average p r o d u c t sugars ing hexos- sacchar ides mo lecu la r

Uronic Tota l tool / tool amin e a f t e r f o r m e d by chon- weight acid sulfa te of hexos- b o r o h y d r i d e droi t inase AC (%)

a mine r ed u c t i o n A Di-4S A Di-6S

Ch ondro i t in sulfate C 0 .96 0 .98 0.01 100 5 95 55 000

Te t ra saccha r ide 1.50 1.06 0 .42 58 50 50 -- A Di-6S 1.07 1.22 0 .80 10 -- 100 --

similar to the disulfated disaccharide obtained from chondroitin sulfate D by Suzuki et al. [15]. The chemical analyses of the fragments are shown in Table III. Except for the high value of uronic acid found in the tetrasaccharide the proport ions of hexosamine, uronic acid and sulfate of the fragments were the same. The reducing power and hexosamine remaining after borohydride reduction indicate that the tetrasaccharide is composed of two disaccharides and that the hexosamine is at the reducing end of the fragment. About 50% of A Di-4S and 50% of A Di-6S are formed by the action of the chondroitinase AC upon the tetrasaccharide {Table III). Considering that the average molecular weight of the chondroitin sulfate C is around 55000 and that 5% of it contains 4-sulfated disaccharides, it can be concluded that there are 5 mol of 4- sulfated disaccharides per mol of chondroitin sulfate C. Fig. 5 shows one of the

Chase AC Chew C - .

1 2 3 4 1 2 3 4 1 2 3 4

, , , , + + e . ; o O . +

X :~ ~MsL ~-~,~ .

Fig. 4. Paper c h r o m a t o g r a m o f the chondro i t i nases AC and C digests o f the d eg rad a t i o n p r o d u c t s f o r m e d by the ac t ion o f the chondro i t inase C on c hond ro i t i n sulfa te C. The e x p e r i m e n t was p e r f o r m e d as described in Fig. 3, e x c e p t tha t the subs t ra tes were degrada t ion p r o d u c t s o b t a i n e d f rom c h o n d r o i t i n sulfate

C by the ac t i on of the chondro i t i na se C. 1, di-; 2, te t ra- ; 3+ hexa- ; 4, o l lgosacchar ide; Chase, chondroi t in - ase.

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C. C-(C)17- C-C ;A-C;C ; (C) I /C;C;A-CiC- (C)1.~ C -C-A-C-C- (C)17 C-C .A-C- C- (C)17-C- C-a

,J Chase c l

C + A:C

H =1 Chase AC I

A + C

Fig. 5. P r o p o s e d s t ruc ture o f c h o n d r o i t i n su l fa te C and m o d e of ac t i on of the c h o n d r o i t i n a s e s AC and C

in t h e d e g r a d a t i o n o f c h o n d r o i t i n su l fa te C to disacehaxides . T h e abbrev ia t i ons u s e d axe the s a m e as the o n e s s h o w n in Fig. 3.

possible arrangements of 4-sulfated disaccharides within the chondroitin sulfate C structure as well as the mode of action of the chondroitinases. Other analyses of A Di-4S and A Di-6S produced by the action of the chondroitinases upon chondroitin sulfates A and C have been previously reported [9].

Discussion

Three types of oligosaccharides seem to compose the structure of chondroitin sulfate A from whale cartilage (Fig. 3). These oligosaccharides contain five, two and one 4-sulfated disaccharides per 6-sulfated disaccharide residue and occur in the ratio of approximately 1 : 1 : 1 in the polymer. Considering the average molecular weight of chondroitin sulfate A which is around 15000, an average of 3 mol of each oligosaccharide is present for each mol of the polymer. We were not able to detect other oligosaccharides containing different proportions of the disaccharides than the ones mentioned above, using the polyacrylamide method to visualize the compounds. It could be argued that the dodecasaccharide would not be separated from a hypothetical deca- or octa- saccharide by the methods employed. This seems unlikely since the dodecasaccharide has a ratio of 5 to 1 of the two types of disaccharides and a molecular weight of 3000.

The sequence of these three types of oligosaccharides in the chondroitin sulfate A molecule shown in Fig. 3 is arbitrary. No evidence for this or any other arrangement was obtained with the methods presently employed.

The degradation of chondroitin sulfate C with the chondroitinase C provided evidence that this polymer is also composed of small amounts of 4- sulfated disaccharide units. The i~olation of a tetrasaccharide containing 4- and 6-sulfated disaccharides rules out the possibility that this mucopolysaccharide is contaminated with chondroitin sulfate A. Fig. 5 shows one of the

443

possible arrangements of the 4-sulfated disaccharides within the structure of chondroitin sulfate C. There is no evidence, however, that they are evenly spaced as shown in the figure.

The proposed structure of the chondroitin sulfates presented in this paper are based in the generally accepted assumption that they are linear polymers. All the evidences provided so far by different methods, indicate absence of branches in these mucopolysaccharides.

It is clear from the results presented in this paper that the chondroitin sulfates A and C are also copolymers as chondroitin sulfate B isolated from different tissues [1--5].

Acknowledgements

We wish to express our gratitude to Dr. S.M.C. Dietrich for help in the preparation of this manuscript. Aided by grants from Funda~io de Amparo Pesquisa do Estado de S~o Paulo (FAPESP) and Conselho Nacional de Pes- quisas (CNPq), Brazil.

References

1 Fransson, L.A. and Roddn, L. (1967) J. Biol. Chem. 242, 4170--4175 2 Fransson, L.A., Coster, L., Malmstr~m, A. and Sj6berg, I. (1974) Biochem. J. 143 ,369- -378 3 Michelacci, Y.M. and Dietrich, C.P. (1975) Biochem. J. 151 ,121- -129 4 Fransson, L.A. and Havsmark0 B. (1970) J. Biol. Chem. 245, 4770---4783 5 Habuchl, H., Yamagata, T., lwata, H. and Suzuki, S. (1973) J. Biol. Chem. 249, 6019--6028 6 Seno, N., Anno, K., Yaegashi, Y. and Okuyama, T. (1975) Connect. Tiss. Res. 3, 87---96 7 Mour~o, P.A.S., Rosenfeld, S., Laredo, J. and Dietrich, C.P. (1976) Biochim. Biophys. Acta 428,

19--26 8 Mottriio, P.A.S. and Dietrich, C.P. (1973) Biochim. Blophys. Acta. 320, 210--213 9 Michelacci. Y.M. and Dietrich, C.P. (1976) J. Biol. Chem. 251, 1154--1158

10 Michelacci, Y.M. and Dietrich, C.P. (1975) Fourth Meeting of the Brazilian Biochemical Society, April 27th, 1975

11 Dietrich, C.P. and Nader, H.B. (1974) Bioehim. Biophys. Acta 343, 34--44 12 Silva, M.E. and Dietrich, C.P. (1975) J. Biol. Chem. 250, 6841--6846 13 Dietrich, C.P. (1968) Biochem. J. 108 ,647- -654 14 Dietrich. C.P., Silva, M.E. and Michelacci, Y.M. (1973) J. Biol. Chem. 248, 6408--6415 15 Suzuki. S., Saito, H.. Yamagata, T., Anno, K., Seno. N., Kawal. Y. and Furuhashl, T. (1968) J. Biol.

Chem. 243, 1543--1550

Documents

Structure of chondroitin sulfates analyses of the products formed from chondroitin sulfates A and C by the action of the chondroitinases C and AC from Flavobacterium heparinum