9 1990 by The American Society for Biochemistry and Molecular Biology, Inc. THE JOURNAL OF BIOLOGICAL CHEMISTRY Val. 265, No. 9. Issue of March 25, pp. 5081-5085,1990

Printed in U.S.A.

Occurrence of Chondroitin Sulfate E in Glycosaminoglycan Isolated from the Body Wall of Sea Cucumber Stichopus juponicus*

(Received for publication, November 11, 1989)

Yutaka KariyaS, Shugo Watabes, Kanehisa Hashimoto!l(I, and Keiichi Yoshida** From the 11 Laboratory of Marine Biochemistry, Faculty of Agriculture, The University of Tokyo, Bunkyo, Tokyo 113 and the **Tokyo Research Institute, Seikagaku Kogyo Co. Ltd., Higashiyamato, Tokyo 189, Japan

Glycosaminoglycan was isolated from the body wall of sea cucumber Stichopus japonicus by a method con- sisting of enzymatic digestion, gel filtration, and ion- exchange chromatography. One gram of sea cucumber glycosaminoglycan was composed of 2.50 mmol of sul- fate, 0.47 mmol of N-acetylgalactosamine (GalNAc), 0.53 mmol of glucuronic acid (GlcA), 1.73 mmol of fucose, and a small amount of peptide.

When mildly hydrolyzed with 0.1 N HzS04, this gly- cosaminoglycan released two products, one consisting of fucose plus sulfate and the other of fucose only. Partially hydrolyzed glycosaminoglycan thus obtained was composed of sulfate, GalNAc, GlcA, and fucose at a molar ratio of 3:2:2:1. Partially hydrolyzed glycos- aminoglycan was easily digested with chondroitinase AC 11. In ion-exchange chromatography, the digest exhibited four sharp peaks whose retention times agreed with those of unsaturated 0-(ADi-OS), mono- (ADi-4S and ADi-GS), and di-(ADi-SE) sulfated disac- charide, respectively. The disaccharide unit of sea cuc- umber glycosaminoglycan was composed of 22.4% chondroitin sulfate E, 11.2% chondroitin, 10.4% chon- droitin 4-sulfate, and 56.0% chondroitin 6-sulfate.

Glycosaminoglycans (GAGs)’ are widely distributed in an- imals. GAGs have so far been isolated and examined for disaccharide unit composition by paper electrophoresis (l), paper chromatography (2-4), and high performance liquid chromatography ( 5 , 6) after enzymatic digestion. GAGs are differentiated into two major families, i.e. chondroitin sulfate derivatives and heparan sulfate ones, according to sulfate

* This work was supported by a grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Present address: Institute of Research and Development, Taiyo Fisheries Co. Ltd., Chuo, Tokyo 104, Japan.

§ To whom correspondence should be addressed. V Present address: Food Science Laboratory, Faculty of Education,

Ibaraki University, Mito, Ibaraki 310, Japan. ’ The abbreviations used are: GAG, glycosaminoglycan; HPLC,

high performance liquid chromatography; GlcA, glucuronic acid; ADi- OS, 2-acetamido-2-deoxy-3-0-(4-deoxy-~-~-threo-hex-~-enopyrano- syluronic acid)-D-galactose; ADi-6S, 2-acetamido-2-deoxy-3-0-(4- deoxy-a-~-threo-hex-4-enopyranosyluronic acid)-6-sulfo-~-galactose; ADi-4S, 2-acetamide-2-deoxy-3-0-(4-deoxy-cu-~-threo-hex-4-enopyr- anosyluronic acid)-4-O-sulfo-~-galactose; ADi-SD, 2-acetamide-2- de0xy-3-0-(4-deoxy-2-O-sulfo-a-~-threo-hex-4-enopyranosyluronic acid)-6-~-sulfo-~-galactose; ADi-SE, 2-acetamido-2-deoxy-3-0-(4- deoxy-a-~-threo-hex-4-enopyranosyluronic acid)-4,6-bis-O-sulfo-~- galactose; ADi-TriS, 2-acetamido-2-deoxy-3-0-(4-deoxy-2-~-s~l~~-~- ~-threo-hex-4-enopyranosyluronic acid)-4,6-bis-O-sulfo-~-galactose.

position and hexosamine species (7). In order to determine the structure of GAGs belonging to the chondroitin sulfate family, chondroitinases ABC and AC have been used, releas- ing unsaturated 0-(ADi-OS), mono-(ADi-4S and ADi-GS), di- (ADi-S, and ADi-SE), and/or tri-(ADi-TriS) sulfated disac- charides.

GAGs from echinodermata contain glucuronic acid (GlcA), N-acetylgalactosamine (GalNAc) and sulfate, suggesting them to be typical chondroitin sulfates (8). Most GAGs in the chondroitin sulfate family are easily digested by chondroiti- nases ABC and AC. However, the structures of GAGs isolated from echinodermata had remained unknown for a long time, because those GAGs were not digested at all by both these chondroitinases (9). Very recently, Vieira and Mouriio (10) have succeeded in enzymatic digestion of GAG from the sea cucumber Ludwigothurea grisea with chondroitinases after mild acid treatment, and found unsaturated 0- and 6-sulfated disaccharides in the digests. They examined the reducing sugars released from the GAG at several stages of hydrolysis, and found that a branching fucan sulfate was separated from the oligosaccharide rich in GlcA and hexosamine. In spite of their breakthrough work, exclusive existence of unsaturated 0- and 6-sulfated dissacharides seemed not reasonable for sea cucumber GAGs, since these GAGs generally are highly sul- fated (8-11).

The purpose of the present study was to characterize GAG which was isolated from the body wall of a representative Japanese sea cucumber Stichopus japonicus, by adopting a mild acid treatment followed by subsequent digestion with chondroitinases and sulfatases. As expected, we detected a.1 unsaturated 4,6-disulfated disaccharide (ADi-SE), along with mono- and 0-sulfated ones.

MATERIALS AND METHODS AND RESULTS~

DISCUSSION

The composition of the sea cucumber GAG demonstrated the presence of a large amount of fucose (3.68 mol/mol GalNAc) together with a high sulfate content (5.32 mol/mol GalNAc). The presence of fucose could be responsible for indigestibility of our GAG with chondroitinases. Vieira and Mouriio (10) also reported a similar indigestibility with chon- droitinases of a fucose-containing GAG from another sea cucumber L. grisea. Accordingly, we mildly treated the GAG with acid. A 6-min hydrolysis with 0.1 N H,SO, at 100 “C gave a partially hydrolyzed GAG and low molecular weight products, although the former was hardly susceptible to chon-

’ Portions of this paper (including “Materials and Methods,” “Re- sults,” Figs. 1-9, and Tables 1-111) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

5081

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5082 Chondroitin Sulfate E in Sea Cucumber Glycosaminoglycan

droitinase ABC digestion. When hydrolyzed with 0.1 N H2S04 a t 80 "C for 6 h, GAG was converted into another partially hydrolyzed form, which was susceptible to chondroitinases AC I and AC I1 digestion. This difference seemed to be related to no production of H-P2 (free fucose) fraction on 100 "C hydrolysis (see Fig. 3). Such fucoses bound to GAG could inhibit chondroitinase digestion. Then, GAG was hydrolyzed with 0.1 N H2S04 a t 80 "C for 6 h. Such partial hydrolysate of GAG gave three fractions: fraction A containing sulfate, GaINAc, GlcA, and fucose; H-P1 containing sulfate and fu- cose; and H-P2 containing fucose only (Fig. 3). During hy- drolysis, the amount of H-P1 fraction remained roughly un- changed, while H-P2 increased. This suggested that the hy- drolytic reaction was composed of two steps, a rapid one releasing sulfate plus fucose and a slow one releasing fucose only.

Purified GAG and fraction A (Table 11) were similar to each other in GalNAc or GlcA contents, suggesting that glycosidic bonds between GalNAc and GlcA were hardly de- stroyed by such mild acid treatment. On the other hand, sulfate and fucose contents were markedly lower in fraction A, indicating that mild acid hydrolysis released both sub- stances from the chondroitin sulfate core of intact GAG. This, along with the above two-step reaction, suggested that the fucose moiety binds to the chondroitin sulfate core much more strongly than does the sulfate/fucose moiety. In other words, part of fucose could bind directly to the chondroitin sulfate core.

The disaccharide unit of our GAG was composed of 1 GalNAc and 1 GlcA along with 5 sulfates and 3-4 (3.7) fucose, on the assumption that the distribution of sulfates and fucoses in disaccharide units were homogeneous. Analysis of unsatu- rated disaccharide revealed that the digestible chondroitin sulfate core was accounted for by 11.2% ADi-OS, 56.0% ADi- 6S, 10.4% ADi-4S, and 22.4% ADi-SE. The average sulfate content in dissacharide units was calculated to be 1.1 mol/ mol, assuming that the remaining indigestive chondroitin sulfate core had the same unsaturated dissacharide composi- tion. Therefore, the difference in sulfate content between intact GAG and chondroitin sulfate core (or fraction A) was 4 mol/mol. These 4 sulfates could indirectly be bound to the dissacharide unit through fucoses. H-P1 which was released from GAG rapidly on hydrolysis (Fig. 6) contained approxi- mately 2 mol of sulfate/mol of fucose. The content of fucose released rapidly (H-PI) was almost twice as high as that released slowly (Table 11). From all these results, the two moieties were assumed to have the structure shown in Fig. 9 which consists of 2 fucoses with 4 sulfates and 1 fucose. In addition, chondroitin sulfate was estimated to account for 35% (w/w) of intact GAG.

Razin et ai. (18) reported for the first time that chondroitin sulfate E is localized inside mammalian mast cells, forming

intracellular proteoglycan. Later, they (19) demonstrated in- terleukin 3 to be a differentiation/growth factor for the mouse mast cell containing chondroitin sulfate E proteoglycan, sug- gesting the involvement of chondroitin sulfate E in the im- munological function of the cell.

On the other hand, chondroitin sulfate E accounted for 61.5% of squid cartilage GAG (7, 16), 12.1% of bovine kidney GAG (7), and 7.0% of shark fin GAG (7). Therefore, the content of chondroitin sulfate E in fraction A (22.4%) is lower than that in squid cartilage GAG, but higher than those in other GAGS.

Sea cucumber body wall becomes considerably tough by a nervous stimulation (20, 21). This phenomenon, called "con- nective tissue catch" (22, 23), may be associated with the unique chondroitin sulfate structure as demonstrated here.

Further studies are now in progress to elucidate the struc- ture of sea cucumber GAG.

REFERENCES 1. Harada, T., Murata, K., Fujiwara, T., and Furuhashi, T. (1969)

2. Habuchi, O., Sugiura, K., Kawai, N., and Suzuki, S. (1977) J.

3. Murata, K., and Bjelle, A. 0. (1980) Connect. Tissue Res. 7 , 143-

4. Murata, K. (1981) J. Mol. Cell. Cardiol. 13, 281-292 5. Seldin, D. C., Seno, N., Austen, K. F., and Stevens, R. L. (1984)

6. Murata, K., and Yokoyama, Y. (1987) J. Chromatogr. 423, 51-

7. Yoshida, K., Miyauchi, S., Kikuchi, H., Tawada, A., and Toku-

8. Motohiro, T. (1960) Nippon Suisan Gakkaishi 26, 1171-1174 9. Cassaro, C. M. F., and Dietrich, C. P. (1977) J. Biol. Chem. 252,

10. Vieira, R. P., and Mourlo, P. A. S. (1988) J. Biol. Chem. 263,

11. Kariya, Y., Watabe, S., Ochiai, Y., Srikantha, S., Hashimoto, K.,

12. Bitter, T., and Muir, H. (1962) Anal. Biochem. 4, 330-334 13. Dimler, R. L., Schaefer, W. C., Wise, C . S., and Rist, C. E. (1952)

14. Nauto, V. (1963) Acta Chem. Scand. 17,857 15. Seno, N., Meyer, K., Anderson, B., and Hoffman, P. (1965) J.

16. Kawai, Y., Seno, N., and Anno, K. (1966) J. Biochem. (Tokyo)

17. Yamagata, T., Saito, H., Habuchi, O., and Suzuki, S. (1968) J. Biol. Chem. 243,1523-1535

18. Razin, E., Stevens, R. L., Akiyama, F., Schmid, K., and Austen, K. F. (1982) J. Biol. Chem. 257,7229-7236

19. Razin, E., Ihle, J. N., Seldin, D., Mencia-Huerta, J. M., Katz, H. R., Leblanc, P. A., Hein, A., Caulfield, J. P., Austen, K. F., and Stevens, R. L. (1984) J . Immunol. 132, 1479-1486

20. Uexkull, J. (1926) Pfliigers Arch. Eur. J . Physiol. 213, 1-14 21. Buddenbrock, S. E. (1963) Vie Milieu 14, 55-70 22. Motokawa, T. (1981) Comp. Biochem. Physiol. 70C, 41-48 23. Motokawa, T. (1982) J. Exp. Biol. 99, 29-41

Biochim. Biophys Acta 177 , 676-679

Biol. Chem. 252,4570-4576

156

Anal. Biochem. 141 , 291-300

61

yasu, K. (1989) Anal. Biochem. 177,327-332

2254-2261

18176-18183

and Murata, K. (1986) Connect. Tissue 18,312-313

Anal. Chem. 24, 1411-1414

Biol. Chem. 240,1005-1010

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Chondroitin Sulfate E in Sea Cucumber Glycosaminoglycan SUPPLEYEITAL MATERIAL TO:

5083

Occurr-ence of chondroitin sulfate E in Glycosam~noqlycan Isolated from Body w a l l of Sea Cucumber SeichopuS iaoonlcus

by Y. K d c i y a , S . Wdtabe, K. Ha$himoto and K . Yoshlda

Preparation g Partially Hvdrolvzed E

In the flrst step to determine the Optimum hydrolytic condLtiona, purlfled GAG ( 3 nql was dissolved in 300 p l of 0.1 N H SO and the mixture was incubated at 80 or 10O.c for 8 h. AlIqUOts I 50 pl l wey2:aten out at due Intervals durinq hydcolyala, and nFutCdllzFd rlth 2.4 x 10- P: 8aIOHI After CFntrlfvqlnq a : 900 x q for 20 mrn. the supernatants were evrporaqed to dryness. To each residue was added 200 pl of dlstllled rater and an aliquot I30 p l ) of each solution was applied to a TSK-gel G-14.000~3.000+2,500I PW 187.5 x 300 srnl column. HPLC run was performed on a Tosoh CCPI liquid chrdkoqraph using 0.2 I NaCl at 40'C and a flow rate of 0.6 mlfmin, under mOnitOrLnq by refractometry and spectrophotonetry at 210 om. Peak areas rere measured by a Shinadru C-RqI

Preparation g Partially Hvdrolvzed E

In the flrst step to determine the Optimum hydrolytic condLtiona, purlfled GAG ( 3 nql was dissolved in 300 p l of 0.1 N H SO and the mixture was incubated at 80 or 10O.c for 8 h. AlIqUOts I 50 pl l wey2:aten out at due Intervals durinq hydcolyala, and nFutCdllzFd rlth 2.4 x 10- P: 8aIOHI After CFntrlfvqlnq a : 900 x q for 20 mrn. the supernatants were evrporaqed to dryness. To each residue was added 200 pl of dlstllled rater and an aliquot I30 p l ) of each solution was applied to a TSK-gel G-14.000~3.000+2,500I PW 187.5 x 300 srnl column. HPLC run was performed on a Tosoh CCPI liquid chrdkoqraph using 0.2 I NaCl at 40'C and a flow rate of 0.6 mlfmin, under mOnitOrLnq by refractometry and spectrophotonetry at 210 om. Peak areas rere measured by a Shinadru C-RqI tnteqrator.

In the next place, the purlfled GAG 180 m q l was partially hydrolyzed with 0.1 N H so under the Optimum hydrolytic CondltlOnS 180-C and 6 hl. After neutral?rar?on n t h 0.1 N NIOH, the hydrolyzate was evaporated to dryness. The r e s u l t ~ n q s o l i d was s o l u b l l ~ z e d ~n 5 m l of 0.2 I NdCl and applied Onto D Cellvloftne GCL-90 1Selkaqaku KoqyOl column 1 6 3.0 x 95 C D I equlllbrated vlth 0.2 ?I NaCl. Elvtlon was ~erformed wlth 0.2 I NaCl at a f lw rate Of 66 mllh.

tnteqrator. In the next place, the purlfled GAG 180 m q l was partially hydrolyzed with

0.1 N H so under the Optimum hydrolytic CondltlOnS 180-C and 6 hl. After neutral?rar?on n t h 0.1 N NIOH, the hydrolyzate was evaporated to dryness. The r e s u l t ~ n q s o l i d was s o l u b l l ~ z e d ~n 5 m l of 0.2 I NdCl and applied Onto D Cellvloftne GCL-90 1Selkaqaku KoqyOl column 1 6 3.0 x 95 C D I equlllbrated vlth 0.2 ?I NaCl. Elvtlon was ~erformed wlth 0.2 I NaCl at a f lw rate Of 66 mllh.

anthrone methods. respect~vely. Tvo peaks A and 8 appeared, A belnq rich l n under monitoring for uronlc acld and neutral suqar by the Carbazole and

rhrch were found to contain the partially hydrolyzed GAG IPH-GAG1 by the uronic .acid and B In neutral sugar lrefer to F l q . 51. Fractions Of peak A

carbazole IeaCtlOn were comblned and applied onto a CellulOfrne GCL-25 columr. ( # 1.8 x 3 3 cml equilibrated with dlstilled water for desaltlnq. Elutlon vas

~ r d c t ~ o n s of PH-GAG were comblned and dried under reduced pressure. The yield erformed with diatllled water, under monltorinq for PH-GAG at 210 nm.

w a s 20 mq on an average. On the other hand, fractions of peak 8 r1Ch in neutral suqar were combined, desalted and concentrated a s venttoned above. The

Toyopearl HW-40 superfine ITosohI column 18 1.6 x 91 csl cqulllbrated with 0.2 resultlnq s o l ~ d was s o l u b ~ i i r e d In 3 m l of 0.2 M NaCl and applied Onto d

X SAC1 to 09Cimdlr the chernlcal ~onposxtion. Elution w a s performed with 0.2 V NaCl at a flow ra te of 36 m l l h . under mon~torlnq for neutral Suqac at 210 nm.

Enzymatic OIqPStlo"

PH-GAG 1150 pql was digested vlth 0.1 U of Chondroltlndse AC I ~n 80 pl 0 1 50 mll TrLs-HCl IpH 7.31 or with 0.1 U of ChondrOLtLnaS* AC I1 10 80 pl of 50 IT' sodxum acetate IpH 6.01. at 37'C for 2 h. In order to determlnc the p o ~ ~ t i o n o! sulfate qroup ~n the molecule, the chondroiC;nase AC I1 diqegt was dLvlder Into two halves, and one was further dlqested vlth 0.1 U of ~hondro-4-sulfatas~ and the other with ChondrO-6-Sulfatd?le, ~n 30 pl Of 200 m* TrLS-HCl IpH 7 . 5 1 37.C for 2 h.

Determlnatlon Of Unsaturated OisaCCharides

The digests obtained a s above were analyzed for unsaturated disaccharide by D Hltachi 638-30 llquid chromatograph equipped with a Hitachi 635I Y V

monitor 1 7 1 . usinq a column I+ 2.6 x 250 m m l packed rlth LiChrosorb NH

over 60 min at a flow rate O f 1.5 ml/min and 40'C. Unsaturated disaccharide2rat Incrckl. HPLC run -as performed by a l lnear qrsdient from 16 to 800 mM N a x Po2

wonltored at 230 nm. Peak areas were measured by a Herlett-Packard 3390A inteqratoc.

Cellulose-Acetate Strlp Electrophoresis

electrophoresis vas performed on Scparax cellulose-acetate strips ( F u j i Film CO. Ltd., Tokyo1 in 0.1 I calciuln acetate (pH7.71 at 2.5 Vfsm width for 3 h. After the run. the Strips vere stained with 0.25 s A l c l a n blue in 0.5 8 a c e t i c a c i d , a n d d e s t a l n e d w i t h 0.5 * a c e t i c a c i d 1 1 4 . 1 5 1 .

Identlfisation of Neutral S w a m

to dryness. To the residue v a s added 100 pl of distilled water and it was Purlfled GAG vas hydrolyzed vlth 3 N HC1 at 100Dc for 15 h and evaporated

eppllad to TLC on a DC-Fcrtiqatten Cellulose F TLC plstc IIcrck, lsycr-

distlllcd vater 16 : 4 : 3, v l v l . Neutral sugers were detected vlth AqN02. thlCknFc3s 0.1 m m l usinq a s o l v e n t S ~ n s l s t i n q of ?-butanol, pyridine and

Determination for Uoleculac Yeiqht of GAG

HPLC on 0 TSK-gel G-~4,000+3,000t2,500I PWxL column. The rnol=Cular weight of Purified GAG, along with authentic chondroitin sulfates. was appllcd to

the GAG was dctermlnsd by intrspolstlnq the retention time.

Chemical Analyses

Sulfatc - The aanplc was subjected to a 7 h-hydrolysis with 2 N HC1 at 11O.c.

added 50 p l of dlstilled rater and a n aliquot of the solution was applied t o a followed by removal of HC1 under reduced P ~ C I S Y C C at 50'C. To the resldue l a 3

T S K - g e l I C - A n i o n P w column I p 4.6 x 50 l n m l . HPLC run w a s p c r f o r ~ e d

conductivity monitor, u s ~ n q TSK IC-A a s eluent at 40-C and a f low rate of 0.6 ISOCratLCa11y on a Tosoh CCPI liquid chromatograph equipped with a CM-8.000

mlfmin. Peak areas rere measured by a Shimadru C-RIA inteqrstor. Uronic Acld - Uronlc acid was determined by the carbazole method 1121. Sulfurlc acid COntaLnlnq 0.25 I Na 8 0 w a s added to a sample solUt10n under Ice-COOliW and shdkinq. The mixture $:='heated ~n bolling rater for 10 min. After adding the carbazole reaqent, the mixture -8s shaken Viqorously. After heating I n boilinq water for 15 inin, the r n i x ~ v r e was mcasurcd for absorbance at 530 nl. Hexosamine and Amino Acld - To a sample SOIutLOn was added Zml Of 3 N HC1 and t'le ~ L X ~ U I C w a s h y d r o l y z e d a t IOO'C for 15 h. After removing HC1, the hydrolyzate was mixed with 800 pl of 0.01 N Hcl and applled to a Hltachl 83'

RESULTS

~" Purification of GAG - Fig. la shows a Sephadex G-100 gel flltration pattern of

peak I G l l , as monitored by both the carbazole and anthrone method=. A s m l l the crude sea c u c u ~ b e c GAG. Just after the Yo voluse appeared a sharp. larqe

oeak vas located at the lame oosition when monitored bv absorbance at 200 nm.

suqgeatinq it to be iiqhly ncgativc-char&i under the p&ent conditions.

I" "

b1;arbonate 1pH 8.01. and eluted vith the ; m e buffer at a flow rate of 20 mlfh. Five-ml fractions were collected and monitored for uronlc acid at 530 nm after reacting with the carbazole reaqent lbl, for neutral sugar at 620 nm after reectinq with the anthrone reaqent ( D l , and for peptide at 280 nm 1 0 1 .

dqdinst distilled rater and lyophilized. Ibl About 200 mq of the lyophilized G' Indrcsted fractions from two resultinq peska G1 and G2 were comblned, dialyzed

was d l s s d w d In 40 m l of 100 mM s o d ~ u m acetate IpH 5.01. applltd to a OEAE- cellulose column 16 1.8 x 18 cml eaull~brated with 100 mI sodium acetate IPH

~n the follorinq experment.

Ch-85

Ch--4s

D S

Crude GAG

01

Purified GAG

Ch/Me

G 2

H A

H S

ne0

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5084 Chondroitin Sulfate E in Sea Cucumber Glycosaminoglycan

The purlfled GAG Contalned sulfate. CalEi&c. GlcA and neutral ~ u g a i wlth 2 . 5 0 , 0.41, 0.53 and 1.13 mmal per gram GAG, respectlvely. Because of a n almost equlmolar ratlo of GalNAC and GlcA, it seemed reasonable to assume that the

structure vhLch 1s typlcal to chondroitln sulfate. The sulfur to nltrogen purlfled G A G has a dlsaccharlde unlt structure of G a l N A c plus GlcA, the

malar ratio I S I N I v a s calculated to be 5.32. Such a hlgh content of sulfur has never been reported far any G G 5 . In thL5 connectiont the S I N molar rdtro of

far reported. A l a r t e amount of neutral sugar ( 3 . 6 8 mol/mol GalNAcl could 'guld cartilage Ch-S was 1 . 5 5 1 1 6 ) . whlch was the highest sulfur content so

with the relative moblllty vhlch was consistent wlth fucose and xylose under partly be involved in the hlgh sulfur content I" our GAG. TLC gave one spat

the condltlona adopted (data not shown]. Slnce Sea cucumber GAGS s o far reported contalned fucose dominantlv $8. 1 0 ) . the neutral s ~ a a r I" the oresent study was assumed to be fucose.

A small amount of peptlde Of 1 8 . 1 mg/g GAG was detected in our GAG (Table I). Elght amlno acids were detected. ser showed the hlghest content 11.05 mol/mal GAG) followed by G1U 1 0 . 8 8 1 , Thr (0.741, et=. Other amlno acrds disclased laver Contents. 0.33 - 0.68 mollmol. These results Suggested that our G I G was not homogeneous in respect of peptlde molety, and that thls w a s

Actlnase dlqestmn. resulted from random rembval of C- or N-termrnal reglon Of the peptide during

. -

a"alyZer.

Amlno acid pmol/g GAG mg/g GILG mollmol GAG

ASP 21.1 2.50 0 . 6 5

Thr 24.8 2.51 0.74

ser 35.2 3.70 1.05

Glu 29.2 3.16 0.88

Gly 22.6 1.29 0 . 6 8

&la 11.1 0.19 0.33

11e 13.3 1 .51 0.40

Le" 17.7 2.00 0 . 4 3

Total 175.6 18.10 5.26

;nd to become m01e indeciinite than that of intact GAG. Instead, the slaw sharp peak (retention tlme. 43 minl became dominant. The hydrolyzate Shoved essentially the Same HPLC profile when treated with Chondroitlnase ABC (data not shorn). When hydrolyzed with 0.1 N H SO at lO0'C for 5 4 min, sea cucumber GAG Showed a drastic decrease of thz f:St peak a r e a and a simvltaneoua ~ n c r e a r e of low-molecular-weight peaks ldata not ehovnl. 1t was judged from

partially hydrolyzed GAG susceptible to ohondraitinaae digestion. these results that lower tempreratures were preferable to prepare the

. . ~ " " .~ ~

A cd ) n

Flg. 3 - Changes in HPLC pattern of sea C Y C Y m b e r GAG during mild acid- hydrolysis. Purified GAG was partially hydrolyzed vlth 0.1 N H SO at 80'C far 1 h ( a l l 3 h (bl, 4 h r c l , 6 h Id1 and B h ( e l . and applled tzH$Lc an a TSX-

rate Of 0.6 mlfmin and 4O.C. under monitarlng by refzdctometry. Peak areal were gel G-I4 ,000+3,000+2,5001 PWxL column equilibrated with 0.2 M NaCl at a flaw

measured by a Shmadru GR4A integrator. Two well-deflned peaks IH-PI and H- P21, and a dull peak IH-GI whose retentLon time tended to increase, appeared.

X SO at 8 0 - C for 8h. Durlng hydrolysir, portions were taken out at due The parifxed GliG vas then subjected to milder acld-hydrolysis with 0.1 P1

~!?te:vals and analyzed by HPLc. As shown in Fig. 3 , the hydrolyzates gave similar patterns comprising three peaks. These peaks were designated H-G, H- P 1 and H-P2 i n the order of elution. H-Pl Fxhlblted essentially the same retention time a s the low-molecular-weight peak with the hydrolyzate a t 1OD'c far 6 mi". In adaxtion. the area of H-P1 was not greatly changed during 80°c. hydrolysis. suggesting that the release Of this fraction from the intact GAG proceeded in a rapid reaction. On the Other hand the area of H-P2 tended to

changes in the retention tlme of H-G peak durlng hydrolysis. The retention increase gradually dvrlng hydrolysis. In Flg. 4'are Shawn the tlme-dependent

time Increased Sharply during the first 4 h but did not change so vldely thereafter. The area ratio of H-P2 to the sum of H-G and H-P2 also shoved a

remained roughly constant thereafter (Fig. 4). It was. found from these re:nlts time-dependent change : The ratio increased gradually dvrxng the fliIt 4 h and

hydrolyze partially. that the 6 h-heatlng ~n 0.1 N H2S01 at 80-C 1s optimum for sea cucumber GAG to

F1g. 4 - Time-dependent changes in retention time of H-G Peak 1 0 1 and In the peak area ratio IH-PZ/H-G plus H-P2, I of sea cucumber GAG during mild acid- hydrolysis. Refer to the legend Of Flg. 3 for H-G peak and the Condltions of acid-hydrolysis.

GAG w a s partl~lly hydrolyzed u n d e r x e s e condltlons and applied onto d FTdCtlonation of P d r t l a l l y HVdrOlvred GAG - A portlo" ( 8 0 mg) of the purlfled

Cellulafine column. AI Shawn in Flg. 5, the pdrtlal hydrolyzate of GAG gave

anthrone reagent. FrDCtIOns of each peak. along wlth the fractions a s rise to one ma3or peak each when detected either by the carbazale OT the

lndicated In Flg. 5, were combined, designated FrS. A, B and A', and subjedted

consisted Of H-PI and H-P2. Therefore, Fr. B w d l further frdctlonated on d to g e l permeation HPLC. Frs. A and A' corresponded to H-G, whereas FT. B

Toyapearl HW-40 ~uperfine column, resulting ~n separdt~on ~ n t a two ne* fractmns. 8-1 and B-2. As shown ln F14. 6 , 8-1 and 8 - 2 corresponded to x-P1 and H-P2. respectlvely.

0.t

0 (0 20 30 40 50 60 70

Flg. 5 - Gel filtration of a partial acid-hydrolyzate of sea Cucumber GAG. Purlfled GAG I 8 0 mgI was partially hydrolyzed ~n 0.1 N H SO at 80'C for 6 hr and applled Onto a Cellulofine GCL-90 column. Elutlon wa: P2rfarmed with 0.2 M NaCl at a flow rate of 6 6 ml/h. Ten-ml feactlons were callected and monitored for uronic acld at 530 nm by the carbazole method (AI and for neutcal sugar a t 6 2 0 nm by the anthrone method (01. Fraction5 were Combined a s lndlcated, and three newly-provlded fractions (A, A' and B I were used ~n the fallawlng experment.

;I F..B-t

Fr.B-2

25 30 35 4a 45 R e t e n l i o n t i m i n

Flg. 6 - APtC of Fr+. B, 8-1 and 8-2. Fr. B (Fig. 7 ) . and F r s . 8 - 1 and 8-2 (Fig. 91, were sublected to HPLC on a TSK-gel G-~4,000i3,000+2,5001 PWxL column using 0.2 M NaCl at a flow rate of 0.6 mllmin and 40-C, under monltorlnq by refractometry.

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Chondroitin Sulfate E in Sea Cucumber Glycosaminoglycan 5085

lrnmollq GAG1

Sulfate CdlNAc GlcA FYCOSF

PurlfLed GAG 2.50 0.47 0.53 1 .11

Fr. A 0.68 0.43 0.44 0.21

A' 0.26 0.07 0.15 0.12

8 1.79 0.00 0.03 1.06

Totd 1 2.71 0.50 0.59 1.19

F:. R - 1 1.19 0.00 0.00 0.46

8-2 0.02 0.00 0.00 0.26

To ta 1 1.21 0.00 0.00 0.72 ......"""""""."""""""""""""~.."""""""

moblllty. which vas bnrermedldte between those of ch-sE and Ch-6s. suqqestlnq As shown I n F l q . 7, either Fr. A or A' exhibited a broad band with the same

r r L h Alclan blue, demonatrating the absence of acxdlc polysaccharide. that both A and A' were composed of these Substances. Fr. 8 was nor SLained

Origin

I -(+)

Ch-6s 0 Ch"4S 8 O S

PurifiedGAG 0 d

0

e A'

B

Ch-SE

H A

ns

Q b 0

Hep 0 F l q . 7 - Cellulose-acetate strlp electrophoresis of Frs. A, A ' and 8 II'Iq. ? I , s l onq with purifed sea cuctmber GAG and sotme authentic samples. Electrophoresls w a s carr~ed out on a ~ e l l ~ l o ~ e - d ~ e t d t c strip ISepdraxI ~n 0.1 I4 calclurn acetate IpH 7 . 7 1 a t 2.5 V l c m for I h, and Stained utt'l 0.l5 % Alclan blue ~n 0 . 5 % D C ~ ~ L C acid.

Chase AC lIa 17.2 56.0 10.4 22.4

Chaac AC I1 25.1 54.4 - 20.6 1 4sa.e'

Chase A: 11 67.8 - 12.2 - + 6Saae

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Y Kariya, S Watabe, K Hashimoto and K Yoshidawall of sea cucumber Stichopus japonicus.

Occurrence of chondroitin sulfate E in glycosaminoglycan isolated from the body

1990, 265:5081-5085.J. Biol. Chem. 

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