Occurrence of a dermatan sulfate isomer in sea urchin larvae

469 Biochimica et Biophysica A cta, 372 (1974) 469--473 © Elsevier Scientific Publishing Company Amsterdam -- Printed in The Netherlands

BBA Report BBA 21398 OCCURRENCE OF A D E R M A T A N S U L F A T E ISOMER IN SEA URCHIN L A RVAE

TATSUYA YAMAGATA and KAYO OKAZAKI Mitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Machidashi, Tokyo 194 and Department of Biology, Faculty of Science, Tokyo Metropolitan University, Tokyo 158 (Japan) (Received August 5th, 1974)

Summary T h e d er matan sulfate isomer was isolated f r o m the larvae of a sea urchin, P s e u d o c e n t r o t u s depressus. Its repeating unit was characterized as 2-acetamide2-deoxy-3-O-(~-D-idopyranosyluronic acid)-4,6-O-disulfo-galactose. This r e p o r t is the first one to show t he occur r ence of a de rm at an sulfate in invertebrates.

It has been shown t hat larval spicules in the sea urchin are f o r m e d in matrices p r o d u c e d by m e s enchym a l cells which show metachromasia by toluidine blue [ 1 ] . An a t t e m p t was made to characterize mucopolysaccharides in t h e matrix of a sea urchin larva. Evidence is obtained which shows the occurrence o f a d e r m a t a n polysulfate, repeating unit of which consists of 2-acetamide-2-deoxy-3-O-(~-D-idopyranosyluronic acid)-4,6-O-disulfo-galactose. Artificially fertilized sea urchin eggs ( P s e u d o c e n t r o t u s depressus) were plated in culture dishes containing artificial sea water. T he eggs were allowed to develop up to the early pluteus stage, harvested with a sheet of cheese cloth, washed with acet one and dried. 400 mg of the dried larvae were exhaustively digested with pronase and t he n treated with trichloroacetic acid. The supernatant was dialyzed and t he mucopolysaccharides were precipitated with an addition o f 2 vol. o f ethanol. T h e yield was 260 mg. F u r t h e r purification was achieved by isolation of der m at an sulfate as a copper complex. 0.1 vol. o f 70% NaOH and 0.75 vol. of qualitative Benedict solution were added to a 1% solution of the crude mucopolysaccharide preparation. T he solution was k e p t for 10 min with occasional shaking. After centrifugation, the precipitate was washed with 10 ml of alkaline copper solution ( w a t e r - N a O H - - B e n e d i c t

470

solution, 10:1:2, by vol.). The precipitate was dissolved in 3 ml of 3 M acetic acid, followed b y the addition of 2 vol. of ethanol. The precipitate thus formed was dissolved in distilled water and passed through a small column of Dowex-50, H ÷. After the effluent was neutralized and concentrated, the mucopolysaccharide (27 mg) was precipitated with an addition of ethanol. The mucopolysaccharide sample thus obtained was dissolved in 1 ml of 0.02 M Tris--HC1 (pH 7.2) and applied to a column (0.5 × 5 cm) of DEAEcellulose. The column was washed with the buffer and then the mucopolysaccharide fraction was eluted with 6 ml of 3 M NaC1. By adding ethanol to the effluent, the final preparation of mucopolysaccharide was obtained, yielding 5 mg. Analytical data on the purified sample are given in Table I. 97% of hexosamine was found to be galactosamine and the rest glucosamine, when analyzed b y the m e t h o d of Gardell [2]. The ratio of carbazole to orcinol was 0.23, suggesting that uronic acid of the sample consists solely of L-iduronic acid. The hydrolysate of the sample with 1 M H2 SO4 for 2 h at 100°C gave only a single peak on Dowex-1 chromatography, when analyzed following the method of Fransson et al. [3] ; the peak corresponded to that of standard L-iduronic acid. No evidence was found for the occurrence of glucuronic acid in this preparation. The sulfate/hexosamine ratio of 2.27 indicates that the mucopolysaccharide is over-sulfated. When subjected to electrophoresis on a cellulose acetate strip, using 0.37 M formic acid--0.1 M pyridine at pH 3.0, the sample was detected as a single spot occupying a position between the spots of standard chondroitin sulfate and dermatan sulfate. 1

2

3

ORIGIN

I (.9 z 7 hi (J U~ hi £3

o;

~ADi-diSo ~ADi-diS B ~ADi-diS E

I

~ADi-6S

i

~AOi-hS

Ii

~ADi-OS

Fig.1. T r a c i n g of a p a p e r c h r o m a t o g r a m of digestion p r o d u c t s o f the m u c o p o l y s a c c h a r i d e w i t h c h o n dxoitinase A C (1) a n d c h o n d r o i t i n a s e ABC (2) a l o n g w i t h s t a n d a r d s (3), visualized w i t h a M i n e r a l i g h t , M o d e l S - 2 5 3 7 . E n z y m a t i c digests were applied o n a W h a t m a n No. 1 paper and subjected t o d e s c e n d i n g paper c h r o m a t o g r a p h y d e v e l o p e d w i t h b u t y r i c acid--2 M N H 4 O H ( 5 : 3 , b y vol.) f o r 48 h, N o t e o n l y o n e s p o t ( A D i - d i S x ) w a s o b s e r v e d in c h o n d r o i t i n a s e ABC digest.

471

The sample was not digested with chondroitinase AC [4], whereas a treatment with chondroitinase ABC [4] resulted in its complete degradation, as revealed b y an increase in absorption at 232 nm measured by the m e t h o d previously described [4]. The digested product, when run on paper chromatography, yielded only one ultraviolet-absorbing material (Fig. 1), the position of which corresponded to that of an unsaturated, disulfated disaccharidebearing sulfates on C-4 and C-6 of the N-acetylgalactosamine residue, ADi-diSE [2-acetamide-2-deoxy-3-O-(~-D-gluco-4-enepyranosyluronic acid)D-galactose 4,6-disulfate]. This material will be referred as ADi-diSx, hereafter. An additional confirmation was obtained, which demonstrated that ADi-diSx was the only product obtained, when the sample was digested with chondroitinase ABC. From a paper chromatogram of chondroitinase ABC digests, the origin and the areas corresponding to the spots represented by individual standards of unsaturated disaccharides were cut out and eluted for a hexosamine analysis. Hexosamine was found only at the spot of 5Di-diSx. In the case of the digestion with chondroitinase AC, hexosamine was completely recovered from the origin.

1

2

3

4

5

ililp el lip

qp o

®

~ADi-OS

ADi-4S, ~ADi-6S

-ADi-diSE

® Fig.2. E f f e c t of c h o n d x o s u l f a t a s e s o n t h e u n s a t u r a t e d d i s a c c h a r i d e ( A D i - d i S x ) o b t a i n e d b y the d i g e s t i o n o f t h e d e r m a t a n s u l f a t e w i t h c h o n d r o i t i n a s e ABC. T h e s a m p l e was e l u t e d w i t h distilled w a t e r f r o m t h e paper c h r o m a t o g r a m s h o w n in Fig. 1. I n c u b a t i o n m i x t u r e c o n t a i n e d , in a f i n a l v o l u m e o f 50/~1: A D i - d i S x , 0.1 # m o l e ; T r i s - - a e e t a t e ( p H 8 . 0 ) , 2.5 /~moles; a n d e n z y m e s . (1) N o e n z y m e a d d e d . (2) 0 . 0 5 u n i t of c h o n d r o - 6 - s u l f a t a s e . (3) 0 . 0 5 u n i t of c h o n d r o - 4 - s u l f a t a s e . (4) 0 . 0 5 u n i t o f c h o n d r o - 6 - s u l f a t a s e plus 0 . 0 5 u n i t of c h o n d r o - 4 - s u l f a t a s e . A f t e r i n c u b a t i o n f o r 1 h a t 37 ° C, t h e e n t i r e r e a c t i o n m i x t u r e w a s a p p l i e d o n a strip of W h a t m a n No. 1 p a p e r . E l e c t r o p h o r e s i s was c a r r i e d o u t in 0 . 0 5 M a m m o n i u m a c e t a t e b u f f e r ( p H 5.0) a t a p o t e n t i a l g r a d i e n t of 3 0 V p e r c m for 4 5 rain. A t r a c i n g of a n u l t r a v i o l e t a b s o r p t i o n p r i n t is p r e s e n t e d .

Further characterization of A Di-diS x was performed with the following results. As clearly shown in Fig. 2, ADi-diSx, when treated with chondro-6sulfatase [4] or chondro-4-sulfatase [4], gave a single spot which electro-

472 TABLE I A N A L Y S E S OF D E R M A T A N S U L F A T E FROM SEA U R C H I N L A R V A E Molar ratio Hexosamine Galactosamine Glucosamine U r o n i c acid (as G I c U A ) Carbazole Orcinol L-Iduronic acid D - G l u c u r o n i c acid Sulfate

%

1.00 97 3 0.41 1.80 100 0 2.27

phoretically coincided with an unsaturated, monosulfated disaccharide, ADi-4S or ADi-6S [2-acetamide-2-deoxy-3-O-(~-D-gluco-4-enepyranosyluronic acid)-4, or -6 sulfo-D-galactose]. Simultaneous digestion of ADi-diSx with both chondrosulfatases gave a single spot corresponding to the electrophoretic position of an unsaturated, nonsulfated disaccharide, ADi-OS [2-acetamide-2deoxy-3-O-(/~-D-gluco-4-enepyranosyluronic acid)-D-galactose]. The above results confirm the identity of ADi-diSx with ADi-diSE. From the results shown above, it is concluded that the dermatan polysulfate obtained from sea urchin larvae is solely composed of (1-~4)~-L-idopyranuronosyl-( 1-* 3)-2-acetamide-2-deoxy-~-D-galactopyranosyl 4,6-disulfate units. It has been reported by one of the authors and his colleagues [5] that a dermatan polysulfate from hagfish notochord contains the same repeating unit as just described. In the case of hagfish, however, this repeating unit accounts for a b o u t 68% 6f the total repeating units. Chondroitin sulfate isomers, including dermatan sulfate isomers, are c o m m o n constituents of the connective tissues of vertebrates occurring as proteochondroitin sulfates (or proteodermatan sulfates in case of dermatan sulfates). In invertebrates, on the other hand, the occurrence of cartilage or of chondroitin sulfate isomers has been reported only in three species. Chondroitin sulfate E was found in the endocranial cartilage of the squid [6] and chondroitin sulfate K in the gill cartilage of king crab [7]. A marine polychaete was reported to have cartilage in its tentacles [8]. The detailed study, however, has n o t y e t been presented on the nature of the mucopolysaccharide in the cartilage of this species, except that it contains mainly glucuronic acid as a uronic acid c o m p o n e n t and is oversulfated. The present report is the first one to show the occurrence of a dermatan sulfate isomer in an invertebrate. The function of the dermatan sulfate presented in this report is unknown. It may be worth mentioning that, in sea urchin larvae, the metachromatic matrix is formed prior to the initiation of spicule formation [1]. An autoradiographic study using radioactive inorganic sulfates showed that the matrix seemed to contain the dermatan sulfate isomer reported in this paper, since silver grains on the matrix decreased after the treatment with chondroitinase ABC.

473

The authors wish to express their sincere gratitude to Drs L. R o d i n , J.A. Cifonelli and Y. Kato for constructive criticism to accomplish this paper. They also thank S. Suzuki for his technical assistance.

References 1 0 k a z a k i , K. (1967) Jap. J. Exp. Morph. 21, 12--18 2 Gardell, S. (1953) Acta Chem. Scand. 7, 2 0 7 - - 2 1 5 3 Fransson, L.-A., R o d i n , L. and Spach, M.L. (1968) Anal. Biochem. 23, 317--330 4 Yarnagata, T., Saito, H., Habuchi, O. and Suzuki, S. (1968) J. Biol. Chem. 243, 1523---1535 5 Anno, K., Seno, N., Mathews, M.B., Yamagata, T. and Suzuki, S. (1971) Bioehim. Biophys. Acta 237, 173--177 6 Suzuki, S., Saito, H., Yamagata, T., Anno, K., Seno, N., Kawai, Y. and Furuhashi, T. (1968) J. Biol. Chem. 243, 1 5 4 3 - - 1 5 5 0 7 Seno, N., Yamashiro, S. and Anno, K. (1974) Biochim. Biophys. Aeta 343, 423--426 8 Person, P. and Mathews, M.B. (1967) Biol. Bull. 132, 244--252