Eur. J. Biochem. 166,639-645 (1987) 0 FEBS 1987

Highly acidic glycans from sea cucumbers Isolation and fractionation of fucose-rich sulfated polysaccharides from the body wall of Ludwigothurea grisea

Paulo A. S. MOURAO and Izaias G. BASTOS Departamento de Bioquimica, Centro de CiCncias da Saide, Universidade Federal do Rio de Janeiro

(Received February 16,1987) - EJB 87 0177

The body wall of the sea cucumber contains high amounts of sulfated glycans, which differ in structure from glycosaminoglycans of animal tissues and also from the fucose-rich sulfated polysaccharides isolated from marine algae and from the jelly coat of sea urchin eggs. In Ludwigothurea grisea, glycans can be separated into three fractions which differ in molecular mass and chemical composition. The fraction containing a high-molecular- mass component has a high proportion of fucose and small amounts of amino sugars, whereas another fraction contains primarily a sulfated fucan. The third fraction, which represents the major portion of the sea cucumber polysaccharides, contains besides fucose, approximately equimolar proportions of glucuronic acid and amino sugars, and has a sulfate content higher than that in the other two fractions. Both D and L-isomers of fucose are found in these polysaccharides, and the sulfate is linked to the 0-3 position of the fucose residues. The attachment position of the sulfate groups to the glucuronic acid units and amino sugars is still undetermined. It is possible that these compounds are involved in maintaining the integrity of the sea cucumber’s body wall, in analogy with the role of other macromolecules in the vertebrate connective tissue.

Connective tissues of invertebrates and of vertebrates have been compared in relation to function and chemical composi- tion [l]. In our laboratory, we have searched for structures in invertebrates that showed the presence of sulfated glycans with functions presumably similar to those of connective tissue glycosaminoglycans. Interestingly, we isolated sulfated polysaccharides from the tunic of various species of ascidians, which contain galactose, glucose and amino sugars [2, 31.

In this work we describe the presence of unusually high amounts of sulfated glycans in the body wall of sea cucumbers (Echinodermata-Holothuroidea), which are different from all the sulfated polysaccharides described so far in animal and algal tissues. A more detailed study was carried out on the polymers from Ludwigothurea grisea. Sulfated poly- saccharides occur in the body wall of this species of sea cucumber which can be separated into three fractions that differ markedly in molecular mass and chemical composition. Two fractions contain high amounts of fucose, whereas the thrd fraction has a high proportion of amino sugars and also glucuronic acid.

MATERIALS AND METHODS

Materials Sea cucumbers were collected in Guanabara Bay, Rio de

Janeiro. Chondroitin 4-sulfate, chondroition 6-sulfate, dextran sulfate (average M, 500000), dextran sulfate (average M, 10 000), pork liver L-fucose dehydrogenase and Sepharose CL-4B were purchased from Sigma Chemical Company (St Louis, MO, USA). Dermatan sulfate, chondroitinase AC and chondroitinase ABC were from Miles Laboratories Company

Correspondence to P. A. S. Mouriio, Departamento de Bio- quimica, Centro de Ci2ncias da Saude, Universidade Federal do Rio de Janeiro, Caixa Postal 68041, Rio de Janeiro, BR-21910, Brazil

(Elkhart, IN, USA), papain form E. Merck A.G. (Darmstadt, FRG), toluidine blue from Fisher Scientific Company (New Jersey, USA) and agarose from Bio-Rad Laboratories (Richmond, CA, USA). Heparan sulfate was a gift from Dr J. A. Cifonelli (Department of Pediatrics, University of Chicago, Chicago, IL, USA). Keratan sulfate from ox cornea [4], crude extracts from heparan-sulfate-induced Flavobacterium hepa- rinum [S] and crude extracts from keratan-sulfate-induced Pseudomonas sp. (IFO-13 309 [6] were prepared by methods previously described.

Isolation of acidic glycans f rom the body wall of sea cucumbers

The body wall of sea cucumbers was carefully separated from other tissues, immediately immersed in acetone, and kept for 24 h at 4°C. Acidic glycans were extracted from the dry tissue by papain as previously described for another type of tissue [3].

Fractionation of acidic glycans

Fractionation on Sepharose CL-4B. About 40 mg of acidic glycans from the body wall of L. grisea dissolved in 1.5 ml of 0.3 M pyridinelacetate buffer, pH 6.0, was chromatographed on a Sepharose CL-4B column (115 x 1.5 cm), eluted with the same buffer and with 4.0 M guanidine hydrochloride in 0.3 M pyridinelacetate buffer, pH 6.0. Columns were eluted at a flow rate of 6 ml/h and aliquots of approximately 1.0 ml were collected. The presence of sulfated glycans in each fraction was detected by the DuBois reaction [7], and by the metachromatic property [3], as previously described. Columns were calibrated using blue dextran as a marker for V,, and cresol red as a marker for V,.

DEAE-cellulose chromatography. About 40 mg of the sulfated glycans purified on Sepharose CL-4B were applied

640

to a DEAE-cellulose column (3.5 x 2 cm) equilibrated with 0.1 M sodium acetate buffer (pH 6.0) and washed with 100 ml of the same buffer. The column was developed by a linear gradient prepared by mixing 35 ml of 0.1 M sodium acetate buffer (pH 6.0) with 35 ml of 1.0 M NaCl and 35 ml of 2.0 M NaCl in the same buffer. The flow rate of the column was 10 ml/h, and fractions of 2.5 ml were collected. They were checked by the DuBois reaction [ 71, ultraviolet absorbing material and conductivity. Fractions were pooled, dialyzed against distilled water and lyophilized.

Preparative agarose gel electrophoresis. About 15 mg of the acidic glycans purified on Sepharose CL4B and DEAE- cellulose was mixed with 50 pg cresol red (dye indicator) and applied on a 1 .0-cm-thick agarose gel (0.5% agarose in 0.05 M 1,3-diaminopropane/acetate buffer, pH 9.0). The agarose slide was then electrophoresed at 5°C and 150 V (100 mA) in a chamber similar to that described by Wieme [8]. The electrophoresis was carried out for about 3 h or until the cresol red migrated 3.0 cm from the origin. After the run, the agarose gel was cut in 0.2-cm-wide strips which were frozen. Upon thawing, the agarose lost its gel structure and was removed by centrifugation (5000 x g for 15 min at 5°C). The clear supernatants were precipitated with 3 vol. 95% ethanol and maintained at - 10 'C for 24 h. The precipitates which formed were collected by centrifugation (2000 x g for 15 min at room temperature), washed with 80% ethanol, dried in vacuum and dissolved in 0.5 ml distilled water. Sulfated glycans of each fraction were analyzed by analytical agarose gel electrophoresis (see below), and the fractions containing polysaccharides with the same electrophoretic migration were pooled.

Isolation oj:fucose from the sea cucumber polysaccharides

A solution of the sea cucumber polysaccharides (10 mg) in 2 ml6.0 M trifluoroacetic acid was heated at 100°C for 4 h. After evaporation of the acid in a rotary evaporator, the mixture was applied to Whatman no. 1 paper and chromato- graphed in ethyl acetate/pyridine/water (8 : 2: 1, v/v) for 24 h. A strip guide of the chromatogram was developed with silver nitrate, and the region corresponding to fucose cut and eluted with distilled water. The eluate was concentrated to about 1 ml, and fucose estimated by the Dische and Shettles reaction [91.

Isolat ion of sulfated, fucose from the seu cucumber po1ysaccharidt.s

About 200 mg of the unfractionated polysaccharides from the body wall of L. grisea was submitted to partial acid hy- drolysis in 10 ml of 75 mM H2S04 at 100°C for 60 min. Saturated solution of Ba(OH)2 was added to give a pH of 6.5, and the BaS04 precipitate was removed by centrifugation (2000 x g for 15 min at room temperature). The supernatant was passed through a column (15 x 1 cm) of Dowex 1-X2 (50- 100 mesh, OH- form). The column was washed with 60 ml water and developed by a gradient formed with 30 ml water mixed with 30 m10.3 M HCl and 30 m10.6 M HCI. The flow rate of the column was 10 ml/h, and fractions of 2.5 ml were collected, measured by the DuBois reaction [7], and checked for conductivity. The sugar-positive fractions were pooled, neutralized with pyridine and concentrated by rotatory evaporation. This material was further purified by preparative paper electrophoresis in 0.3 M pyridine/acetate buffer (pH 4.5) at 500 V. A strip guide of the paper electropho-

resis was developed with silver nitrate, and the region cor- responding to the sulfated fucose was cut and eluted with distilled water. The eluate was concentrated to about 1 ml, and was further purified by preparative chromatography on Whatman no. 1 paper for 12 h in isobutyric acid/l.O M NH40H (5:3, v/v). A yield of about 2.5 mg sulfated fucose was obtained by this procedure.

Periodate oxidation

The periodate consumption was determined by the spectrophotometric method [lo, 111. The course of the oxida- tion was followed by measuring consumption of periodate with time at room temperature. The unreactive periodate which was not involved in the oxidation reaction was de- stroyed with arsenite and the amounts of formaldehyde [12], acetaldehyde [13] and formic acid [14] were measured by meth- ods described previously.

Chemical analyses

Total methylpentose was measured by the method of Dische and Shettles [9], total hexose by the phenol/sulfuric acid method of DuBois et al. [7] and hexuronic acid by the carbazole reaction [15]. After acid hydrolysis of the polysaccharides (6.0 M HC1, at 100°C for 8 h), total hexosamine was measured by a modified Elson-Morgan reac- tion [16], and sulfate by the BaC12/gelatin method [17]. Stan- dard curves for hexosamine were constructed with glucos- amine subjected to exactly the same hydrolytic conditions as the biological samples. The percentages of hexoses and methylpentoses in acid hydrolyzates were estimated by gas- liquid chromatography of the correspondent alditol acetates [18], and by paper chromatography in ethyl acetate/pyridine/ water (8: 2: 1, v/v) for 24 h [19]. Sugars were revealed by silver nitrate staining. Methylpentoses were also identified in the chromatograms by staining with p-anisidine hydrochloride [19]. The relative proportions of glucosamine and galactos- amine were determined by densitometry of chromatograms run on Whatman no. 1 paper for 48 h in ethyl acetate/ pyridineiwater (8 : 2: 1, v/v), stained with silver nitrate. Identi- fication of hexuronic acid was performed by paper chroma- tography for 48h in isobutyric acid/l.OM NH40H (5:3, v/v). Sialic acid was determined by the procedure of Warren (201 and by the resorcinol method [21].

Analytical agarose and polyacrylamide gel electrophoresis

Sulfated polysaccharides were analyzed by agarose gel electrophoresis, as previously described [22]. About 100 pg of sulfated glycans were applied to 0.5% agarose gel in 0.05 M 1,3-diaminopropane/acetate buffer (pH 9.0) and after electro- phoresis, the glycans in the gel were fixed with N-cetyl- N,N,N-trimethylammonium bromide in water and stained with 0.1 % toluidine blue in acetic acid/ethanol/water (0.1 : 5 : 5, v/v). The molecular mass of glycans were determined by polyacrylamide gel electrophoresis [23]. About 50 pg of the sulfated glycans were applied to a 6% polyacrylamide slab gel (1 mm), and after electrophoresis the gel was stained with 0.1 % toluidine blue in 1 YO acetic acid. After staining, the gel was washed for about 12 h in 1 YO acetic acid.

Enzymatic degradation

Incubation with mucopolysaccharidases. About 100 pg of the sea cucumber polysaccharides or of the purified fraction

A

F-l- F-2-

B

-cs - DS -HS

64 1

1 'p * x

4 - 5 f 10

- 1 2 3 S t 1 2 3 S,S,S,S,

Fig. 1. Electrophoresis ojpolysaccharides extractedfrom the body wall of three species of sea cucumbers. (A) Agarose gel. About 100 pg of the sulfated glycans obtained from Zsosticopus badionotus (l), Ludwigothurea grisea (2), and Brandtothurea arenicola (3), and a mixture of glycosaminoglycans containing 20 pg each of chondroitin 4/6-sulfate (CS), dennatan sulfate (DS) and heparan sulfate (HS) (S,) were applied to a 0.5% agarose gel and run for 1 h at 120 V in 0.05 M 1,3-diaminopropane/acetate buffer, pH 9.0. The polysaccharides in the gel were fixed with 0.1 YO N-cetyl-N,N,N-trimethylammonium bromide solution. After 8 h, the gel was dried and stained with 0.1% toluidine blue in acetic acid/ethanol/water (0.1 : 5: 5, v/v). (B) Polyacrylamide gel. About 50 pg of the sulfated polysaccharides from I . badionotus (I), L. grisea (2) and B. arenicola (3) were submitted to 6% polyacrylamide gel electrophoresis in 0.02 M sodium barbital buffer, pH 8.6, for 30 min at 100 V. Glycans in the gel were stained with 0.1 YO toluidine blue in 1 YO acetic acid. After staining, the gel was washed for about 12 h in 1 % acetic acid solution. The M , markers used were dextran sulfate, average M , = 500000 (Sl); chondroitin 6-sulfate, average M , =40000 (S2); dermatan sulfate, average M , = 27000 (S,) and dextran sulfate, average M, = 10000 (S,)

Table 1. Chemical analysis of the polysaccharides extracted from the body wall of three species of sea cucumber

Organism Proportion of extracted material

fucose galactosamine glucosamine glucuronic acid sulfate

% (mol/mol)

Ludwigothurea grisea 13.9 (1.00) 1 .O (0.05) 0.5 (0.03) 2.3 (0.14) 13.9 (1.15)

Brandtothurea arenicola 27.7 (1.00) 2.0 (0.06) 1.3 (0.03) 3.6 (0.11) 18.2 (0.76) lsosticopus badionotus 23.0 (1.00) 1.2 (0.04) 0.8 (0.03) 3.0 (0.11) 22.1 (1.11)

F-2-d were incubated with: (a) 0.01 unit chondroitinase AC or with 0.01 unit chondroitinase ABC [17] in 0.5 M Tris/HCl buffer (pH 8.0) at 37°C for 8 h; (b) 30 pg (as protein) of crude extract from heparan-sulfate-induced Flavobacterium heparinum in 0.05 M ethylenediamine/acetate buffer pH 8.0, at 30°C for 8 h [ 5 ] ; or (c) 30 pg (as protein) of crude extracts from keratan-sulfate-induced Pseudomonas sp. (IFO-13 309) in 0.05 M Tris/HCl buffer, pH 7.2, at 37 "C for 8 h [6]. Agarose and polyacrylamide gel electrophoresis of control and enzyme-incubated glycans was used to assess the enzymatic activity.

Incubation with I;-fucose dehydrogenase. Varying amounts of fucose isolated from the sea cucumber polysaccharides, and authentic samples of D- or L-fucose, were incubated with 0.2 units porcine liver L-fucose dehydrogenase [24] and 2.5 pmol NAD' in 2 ml of 0.01 M glycine/NaOH buffer (pH 8.0). The reaction mixtures were incubated at 30 "C for different times and the formation of NADH was followed by measuring the absorbance at 340 nm.

Other methods

Deamination by nitrous acid at pH 4.0 and at pH 2.0 was performed as described by Shively and Conrad [25].

Densitometry was performed using a Quick Scan densi- tometer (Helena Laboratories, Beaumont, TX, USA). Con- ductivity was measured with a Konduktometer model E-527 (Metrohm Herisau, Switzerland) and optical rotations with a digital polarimeter Perkin-Elmer model 243-B. Infrared spectra were recorded with a Perkin-Elmer infrared spectrophotometer model 298.

RESULTS

Sulfated glycans f rom the body wall of three species of sea cucumbers

The papain-extracted sulfated glycans from the body wall of three species of sea cucumbers were subjected to agarose and polyacrylamide gel electrophoresis (Fig. 1). The sulfated glycans obtained from the sea cucumbers showed metachro- matic bands in agarose gel electrophoresis which are not ex- actly the same as those of standard glycosaminoglycans. The main band designated as F-2 has a slower electrophoretic migration when compared with that of standard glycos- aminoglycans, while a more diffuse band, designated as F-I, migrated among the standard glycosaminoglycans (Fig. 1 A). Polyacrylamide gel electrophoresis of the sea cucumber polysaccharides (Fig. 1 B) showed two fractions of different molecular mass: one fraction had a high M , and therefore remained at the origin of the gel, whereas the other fraction migrated into the gel. Analysis of the polysaccharides from the three species of sea cucumbers indicated the presence of fucose, amino sugars, glucuronic acid and sulfate (Table 1).

To exclude the presence of well-known glycosamino- glycans in the sea cucumber polysaccharides, the solution of papain-extracted glycans from the body wall of the three species were incubated with chondroitinase AC, chondroitinase ABC, crude extracts from heparan-sulfate- induced F1. heparinum, or crude extracts from keratan-sulfate- induced Pseudomonas sp. (IFO-13 309). In addition, these glycans were deaminated by nitrous acid (pH 4.0 and pH 2.0). The agarose and polyacrylamide gel electrophoresis of the control glycans and those treated with enzyme or nitrous acid

642 -

rF-*l t A E .5- c

4

' .1- cd 2 0-

I I I 30 50 70 90

Fraction number

0 0.5 1.0 K av

I I I I I

B

Origin T F.1 F.2

Fig. 2. Fractionation of su!fiatedglycans cxtractedfrom the body wall of L. grisea on Sepharose CL-4B column. (A) About 40 mg of the sulfated glycans from the body wall of L. grisea were chromatographed on a Sepharose CL4B column (115 x 1.5 cm), eluted with 0.3 M pyridine/ acetate buffer, pH 6.0, and the fractions measured by the DuBois reaction (0) and by the metachromatic property ( O ) , T.B., toluidine blue. (B) About 100 pg of the sulfated glycans from L. grisea (T), and 100 pg each of fractions F-1 and F-2 obtained from the Scpharose CL-4B column were submitted to agarose gel electrophoresis, as described in the legend of Fig. 1

1 1 1 1 1 1 1 1 1 1 1

10 30 50 70 90 110 Fraction Number

B C D

Fig. 3. Subfractionation of F-1 on DEAE-cellulose chromatography and on preparative agarose gel ekctrophoresis. (A) About 40 mg of fraction F-1 obtained by Sepharose CL-4B gel chromatography was applied to a DEAE-cellulose column (2.0 x 3.5 cm). The column was washed with 100 ml of 0.1 M sodium acetate buffer (pH 6.0), and at the point indicated (arrow), the column was eluted by a linear gradient of NaC1. as described in Methods. The fractions were measured by the DuBois reaction (O) , and checked for ultraviolet-absorbing material (0), and for NaCl concentration (----). Fractions were pooled as indicated by the horizontal bars in the figure, dialyzed against distilled water and lyophilized. (€3) About 100 pg of fraction F-1 obtained by Sepharose CL-4B gel chromatography, and 100 pg each of subfractions F-1-a (a), F-I-b (b), F-I-c (c) and F-1-d (d) obtained from the DEAE-cellulose column were submitted to agarose gel electrophoresis, as described in Fig. 1. (C) The fraction F-I-d obtained by Sepharose CL-4B and DEAE-cellulose columns was further purified by preparative agarose gel electrophoresis, as described under Methods. About 100 pg of the two fractions obtained (F-1-d-x and F-1 -d-y) werc analyzed by agarose gel electrophoresis. (D) 50 kg each of fractions F-1-d-x-, F-I-d-y and F-1 -d were analyzed by polyacrylamide gel electrophoresis as described in the legend of Fig. 1. The M , markers used in this experiment are the same as those described in Fig. 1

(not shown) indicated that these compounds are resistant to both the mucopolysaccharidases and to deamination by nitrous acid, thus differing from all previously described glycosaminoglycans.

Fractionation and chemical analysis of the polysaccharides ,from L. grisea

Since the sulfated polysaccharides from the body wall of the three species of sea cucumbers showed comparable migrations on electrophoresis in agarose and polyacrylamide

gels (Fig. l), and had also a similar composition (Table l), we decided to concentrate our studies on the fractionation of the glycans obtained from one species, L. grisea.

The papain-extracted polysaccharides from L. grisea were fractionated in columns of Sepharose CL-4B and DEAE- cellulose and by preparative agarose gel electrophoresis. Gel filtration chromatography on Sepharose CL4B (Fig. 2 A) separated the sulfated polysaccharides into two peaks: a broad one designated F-1 which eluted near the void volume, and another one with a K,, between 0.5 and 0.75, designated F-2. Most hexoses eluted as a sharp peak separated from the

643

I ,a,

I l l l l l l l l l l l l 70 90 110

lo 30Frac% Number

B C

1 L,

F-2 a b c d S, S2 S3 Origin - F-2 a b c d

Fig. 4. Fractionation of F-2 on DEA E-cellulose chromatography. (A) About 40 mg of fraction F-1 obtained by Sepharose CL-4B gel chro- matography (see Fig. 2) were applied to a DEAE-cellulose column (2.0 x 3.5 cm). The column was washed with 100 ml of 0.1 M sodium acetate buffer (pH 6.0), and at the point indicated (arrow), the column was eluted by a linear gradient of NaCI, as described in Methods. The fractions were assessed by the DuBois reaction (O) , and checked for ultraviolet-absorbing material (0), and for NaCl concentration (----). The fractions were pooled as indicated by the horizontal bars in the figure, dialyzed against distilled water and lyophilized. (B) About 100 pg of fraction F-3 obtained by Sepharose CL4B gel chro- matography, and 100 pg each of subfractions F-2-a (a), F-2-b (b), F-2-c (c) and F-2-d (d) obtained from the DEAE-cellulose column were submitted to agarose gel electrophoresis, as described in the legend of Fig. 1. (C) About 50 pg each of the same polysaccharides described in B were submitted to polyacrylamide gel electrophoresis, as described in the legend of Fig. 1. The M , markers used were chondroitin 6-sulfate, average M, = 40000 (Sl); dermatan sulfate, average M , = 27000 (&); and dextran sulfate, average M , = 10000

sulfated polysaccharides, probably consisting of short oligosaccharides. On agarose electrophoresis (Fig. 2 B), F-1 showed a predominant component with high mobility along with two less intense bands; F-2 provided a major band with lower electrophoretic mobility.

When fraction F-I purified on Sepharose CL4B was chro- matographed on DEAE-cellulose (Fig. 3 A), ultraviolet-ab- sorbing materials were eluted before or at the beginning of the salt gradient while the sugars were eluted as a single peak at higher NaCl concentrations. The sulfated polysaccharides were detected only in the subfraction F-1-d and showed a similar electrophoretic migration on agarose gel before and after purification on DEAE-cellulose (Fig. 3 B).

Fractionation on preparative agarose gel electrophoresis (Fig. 3C) separated F-1-d into a subfraction of higher electrophoretic mobility (F-1-d-y) and another fraction of lower mobility (F-I-d-x). F-1-d-x did not enter the polyacryl- amide gel due to its high Mr (Fig. 3 D), whereas F-1-d-y moved as a diffuse metachromatic band with an apparent M, ranging over 30000 - 5000 (compared with glycosaminoglycan standards).

Elution of F-1 near the void volume of the Sepharose CL- 4B column (Fig. 2A), together with the presence of low-M, polysaccharides detected on polyacrylamide gel electrophore-

Table 2. Analysis of fractions of sulfated glycans f rom the body wall of L. grisea The fractions F-1-d and F-2-d were obtained by Sepharose CL-4B and DEAE-cellulose columns (Figs 2A, 3 A and 5 A), and fractions F-1-d-x and F-1 -d-y were obtained by preparative agarose gel electro- phoresis (Fig. 3 C, D)

Fraction Molar ratios of

fucose hexos- glucu- sulfate/ amine ronic total

acid sugar

F-1-d 1 .oo 0.03 0 0.85 -26" F-1 -d-x 1 .oo 0.11 0 0.42 + 2" F-1-d-y 1.00 0 0 0.60 -21" F-2-d 1 .oo 0.46" 0.54 1.10 -19"

a Galactosamine is the predominant aminosugar in this fraction.

sis of the purified F-1-d-y (Fig. 3D), suggested that this sub- fraction may occur as an aggregate of glycoconjugates. To test this hypothesis, the Sepharose-CL-4B-purified fraction F-1 was re-chromatographed in the same gel but eluted with 4.0 M guanidine hydrochloride (dissociative conditions). Under these conditions, fraction F-I-d-y was eluted near the total volume (Kay 0.5-0.9; result not shown), a result that is consistent with the hypothesis of aggregation.

The DEAE-cellulose chromatography of the Sepharose- CL-4B-purified fraction F-2 (Fig. 4A) showed ultraviolet absorbing materials eluted before the salt gradient (peak a), and three peaks containing sugars eluted with increasing salt (peaks b, c, and d). Only peak d contained sulfated polysaccharides, as shown by the agarose gel electrophoresis (Fig. 4B). Peaks b and c corresponded to non-sulfated polysaccharides or oligosaccharides.

Chemical analyses of the purified polysaccharides (Table 2) showed that fraction F-1-d contained unusually high amounts of fucose while glucuronic acid and hexosamines predominated in F-2-d. Subfraction F-1-d-y- consisted of a single sulfated fucan while small amounts of hexosamine were found in F-1-d-x. Besides the sugars listed in Table 2, no other sugars were detected in these polysaccharides at a sensitivity of 0.02 mg/mg polysaccharide. Infrared spectra of fractions F-1-d and F-2-d showed absorption bands at 1240 cm-', 1550 cm-' and 1680 cm-l, which can be attributed to S=O, C = 0 and N - H groups, respectively [26].

The presence of an approximately equimolar proportion of glucuronic acid and hexosamine in fraction F-2-d (Table 2) could suggest the presence of glycosaminoglycan-like struc- tures in this sea cucumber polysaccharide. However, fraction F-2-d is resistant to enzymatic degradation by chondroitinase AC, chondroitinase ABC, crude extracts from heparan- sulfate-induced F1. heparinurn and crude extracts from keratan-sulfate-induced Pseudomonas sp., thus differing from all previously described glycosaminoglycans.

Analysis of the fucose and sulfated fucose obtained from the polysaccharides of L. grisea

Both D- and L-isomers of fucose were obtained by strong acid hydrolysis of the sea cucumber polysaccharides (Table 3) . However, while the D-isomer is preponderant in the purified fractions (F-1-d and F-2-d), the L-isomer is obtained in a higher proportion from the non-purified sea cucumber

644

Table 3. Determination of fucose enantiomeric forms in the poly- saccharides of L. grisea 'Total' polysaccharides are the non-purified polysaccharides extracted from the sea cucumber body wall by papain. n.d., not determined

Pol ysaccharide [ago 'c Oxidation with L-fucose dehydrogenase

Yo total fucose

'Total' poiysaccharides - 40 66 Fraction F-I-d n.d. 32 Fraction F-2-d + 25 19

Table 4. C'haructerlzution of sulfated fucose obtained by partial acid hydrolysis from sea cucumber polysaccharides

Method of characterization Results obtained

Chemical composition (molar ratios): fucose glucuronic acid hexosamine sulfate

[cI]BO""

Relative proportion of reducing sugar" Periodate oxidation:

periodate uptake (as mol/mol sugar) products formed (as mol/mol sugar):

acetaldehyde formaldehyde formic acid

Formaldehyde obtained from sulfated fucositol by periodate oxidation (as mol/mol sugar)

1 .oo <0.01 <0.01

0.90

-51" 89

2.34

1.18 < 0.01

1.30

1.34

a The relative proportion of reducing sugar was determined by the decrease of the DuBois reaction after sodium borohydride reduc- tion, as previously described [28].

polysaccharides. This apparent controversial result can be explained by the observation that other non-sulfated oligo- saccharides or polysaccharides were also removed from the sea cucumber body wall by papain. However, these components, which might contain L-fucose, were separated from the sulfated polysaccharides by Sepharose CL4B and DEAE-cellulose chromatography (Figs 2 - 4).

Since the fucose glycosidic linkages are very sensitive to acid in contrast to the sulfate esters, attempts were made to isolate sulfated fucose after partial acid hydrolysis of the sea cucumber polysaccharides. The sulfated fucose was purified by Dowex 1-X2 column and preparative paper electrophoresis and chromatography (see Methods). The chemical composi- tion of the product obtained was compatible with sulfated fucose (Table 4), and the strong negative optical rotation suggested a preponderance of L-fucose. Several oligo- saccharides were identified close to the origin and well separated from sulfated fucose in the paper chromatography. These oligosaccharides are rich in glucuronic acid and hexosamines, which form glycosidic linkages that are more acid-resistant [27] than that of fucose.

The periodate oxidation was employed to determine the location of the sulfate groups in the fucose. Formation of 1 mol acetaldehyde and 1 mol formic acid/mol sugar should

occur if the sulfate groups were linked at positions 2 or 3, whereas 2 mol formic acid but no acetaldehyde should form in the case of a linkage at position 4. The results of Table 4 suggest that the sulfate groups are attached at positions 2 or 3 of fucose.

In order to distinguish between these possibilities, the sulfated fucose was reduced with NaBH4, and the sulfated fucositol obtained was oxidized with periodate. The produc- tion of formaldehyde after these treatments (Table 4) demonstrates that the sulfate is linked at position 3. Further- more, the sulfated fucose was reduced with [3H]NaBH4 at pH 9.0 and oxidized with sodium metaperiodate. The prod- ucts obtained were submitted to strong acid hydrolysis and then reduced with non-radioactive NaBH4. By using this tech- nique [3H]glycerol should form if the sulfate groups were linked at position 2 and non-labeled glycerol should be detected in the case of a linkage at position 3. The absence of [3H]glycerol in the products obtained after periodate oxida- tion of the sulfated [3H]fucositol is another indication that the sulfate is linked at position 3 of the sulfated fucose pre- pared from the sea cucumber polysaccharides.

DISCUSSION

In the present work we report the presence of unusually high amounts of sulfated polysaccharides in the body wall of sea cucumbers. In L. griseu these compounds are distributed in three fractions which differ in molecular mass and chemical composition. The high-molecular-mass polysaccharide (F-l- d-x in Fig. 3C, D) has a high fucose content and small amounts of amino sugars. Fraction F-1-d-y is primarily a sulfated fucan. Fraction F-2-d contains, besides fucose, a high proportion of amino sugars and glucuronic acid.

The chemical analysis in Table 2 and infrared spectra indi- cate the presence of high amounts of sulfate esters in the holothurian polysaccharides. The sulfate groups are linked to positions 3 of the fucose residues. However, we cannot exclude the possibility that part of the sulfated fucose residues in the polysaccharides may have been lost during the mild acid hydrolysis or subsequent purification procedures. Thus, the strongly negative optical rotation of the sulfated fucose units (Table 4) may be an indication that the L-isomers are more sulfated than the D-isomers, or that some sulfate-bearing D-fucose has been lost. The site ofattachment of sulfate groups to the glucuronic acid as well as to the hexosamine units is still unknown.

Although F-2-d is more negatively charged than F-1-d due to its higher sulfate content and presence of glucuronic acid units (Table 2), more NaCl was necessary to elute F-1-d from the DEAE-cellulose column than F-2-d (Figs 3 and 4). This apparent discrepancy may be due to stronger hydrophobic interaction between F-1 -d and the cellulose column.

The presence of fucose-rich sulfated polysaccharides has already been shown in marine algae and in the jelly coat of sea urchin eggs. Structural studies of sulfated fucans from several species of marine algae (named fucoidans) have in- dicated the presence of (1 + 2)-linked cc-L-fucopyranose units sulfated at position 4 [29, 301. Small amounts of D-galactose, D-mannose, D-xylose and D-glucuronic acid have also been reported in fucoidan preparations [29, 301. The structure of the fucose-rich glycans from the jelly coat of sea urchin eggs is not known with certainty [31- 331. Linkages between the carbohydrate residues and attachment sites of the sulfate groups remain undetermined. Sialic acid, hexoses, amino

sugars and high amounts of amino acids occur in sulfated glycans from the jelly coat of the sea urchm [32, 331. Other sulfated glycoproteins containing small amounts of fucose were isolated from the liver and lung of chicken embryos [34], and from the hog gastric mucosa [35].

As shown in the present work the sulfated polysaccharides from the body wall of sea cucumber differ from previously described glycosaminoglycans and also from the sea urchin fucose-rich-glycans. The absence of sulfate substituted at posi- tion 4 of the fucose residues and the preponderance of the D-isomer of fucose are the main features that distinguish the holothurian sulfated polysaccharides from the fucoidans of marine algea. Furthermore, the chemical composition of the major fraction of the sea cucumber polysaccharides (F-2-d), rich in fucose, amino sugars and glucuronic acid, is unique among other previously described sulfated glycans.

The function of the holothurian polysaccharides is a matter of speculation. The structure of the body wall of the sea cucumber is formed by a network of thick collagen fibers, embedded in an amorphous substance. Small irregular microfibrils, which resemble the proteoglycans of vertebrate cartilages, form bridges among collagen fibers [36]. In- terestingly, it is known that several species of sea cucumber, when stimulated or disturbed undergo a process of quick degradation of the body wall called autotomy [37]. Although no morphological evidence of collagen fiber digestion could be seen in the stimulated animals, the microfibrils described above were not observed in this material [36]. The above- mentioned phenomenon was thought to be linked to the diges- tion of proteoglycans [36]. These results and the presence of the holothurian polysaccharides in concentrations which resemble the amount of glycosaminoglycans which are charac- teristic of cartilages 138, 391, suggest that these sulfated polysaccharides are essential for maintaining the structural integrity of the sea cucumber’s body wall, thus resembling the structural function of glycosaminoglycans in connective tissues.

This work was supported by grants from Fundo de Zncentivo a Pesquisa Tkcnico-Cientifica (FIPEC), Financiadora de Estudos e Projetos (FINEP) and Conselho Nacional de Desenvolvimento Cientifico e Tecnolbgico (CNPq). The authors wish to express their appreciation to Dr Luiz R. Travassos for his help in the preparation of this manuscript and to Rui N.L.T.R. Costa for assistance in some experiments.

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