Structural Heterogeneity among Unique Sulfated L-Galactans from Different Species of Ascidians (Tunicates)*

The sulfated polysaccharides that occur in the tunic of ascidians differ markedly in molecular weight and chemical composition. A high molecular weight fraction (F-l), which has a high galactose content and a strong negative optical rotation, is present in all species. Several structural differences were observed among the F-1 fractions obtained from three species of ascidians that were studied in detail. Large numbers of a-L-galactopyranose residues sulfated at position 3 and linked glycosidically through position 1+4 are present in F-1 from all three ascidians. However, a - ~ - galactopyranose units, 1+3-linked and partially sulfated at position 4, comprise about half of the sugar units in the central core of F-1 from Ascidian nigra. In addition, L-galactopyranose nonreducing end units occur in F-1 from Styela plicata and A. nigra, but comprise only a minor fraction of F-1 from Clavelina sp. The combination of these various component units gives a complex structure for F-1 from S. plicata and A. nigra, whereas F-1 from Clavelina sp. possesses a simpler structure. The structures of these ascidian glycans

The sulfated polysaccharides that occur in the tunic of ascidians differ markedly in molecular weight and chemical composition. A high molecular weight fraction (F-l), which has a high galactose content and a strong negative optical rotation, is present in all species. Several structural differences were observed among the F-1 fractions obtained from three species of ascidians that were studied in detail. Large numbers of a-L-galactopyranose residues sulfated at position 3 and linked glycosidically through position 1+4 are present in F-1 from all three ascidians. However, a -~galactopyranose units, 1+3-linked and partially sulfated at position 4, comprise about half of the sugar units in the central core of F-1 from Ascidian nigra. In addition, L-galactopyranose nonreducing end units occur in F-1 from Styela plicata and A. nigra, but comprise only a minor fraction of F-1 from Clavelina sp. The combination of these various component units gives a complex structure for F-1 from S. plicata and A. nigra, whereas F-1 from Clavelina sp. possesses a simpler structure. The structures of these ascidian glycans are unique among all previously described sulfated polysaccharides, since they are highly branched (except that from Clauelina sp), sulfated at position 3, and contain large amounts of L-galactose without its D-enantiomorph. These data show unusual examples of polyanionic glycans with structural function in animal tissues.
Sulfated polysaccharides are widespread in nature, occurring in a great variety of organisms. In marine algae, for example, there are the carrageenans and fucoidan, which are composed mainly of sulfated galactose and fucose, respectively (1). In the animal kingdom, sulfated glycosaminoglycans abound in vertebrate connective tissue (2) and, to a lesser extent, are also present in invertebrates (2,3).
The glycosaminoglycans display many biological functions and in connective tissues they exert a crucial role in the maintenance of the structural integrity, mainly through interactions with other molecules of the extracellular matrix (4,

) .
* This work was supported by grants from Financiadora de Estudos e Projetos (FINEP), Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), and International Foundation for Science (IFS) and by support from The British Council to P. A. S. M. 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.
ll To whom correspondence should be addressed.
In previous studies we have reported the isolation of novel sulfated polysaccharides from invertebrate tissues, namely the tunic of ascidians (6)(7)(8) and the body wall of sea cucumber (6,9,10). We speculated that the occurrence of high amounts of sulfated polysaccharides in these tissues indicated that they were playing important structural roles, perhaps like those of glycosaminoglycans in vertebrate connective tissue.
In this paper is described the purification and chemical analysis of the sulfated polysaccharides extracted from the tunics of various species of ascidians. The main polysaccharide found in all species was a high molecular weight sulfated L-galactan, which differs from all previously described galactose-rich glycans of marine algae and also from the animal glycosaminoglycans. Interestingly, marked structural variations were observed among the sulfated L-galactans from different species of ascidians. In this class of compounds there were differences in the proportion of nonreducing end units, in the content of 1+3-and 1 4 -l i n k e d units and in the degree and position of sulfation. The sulfated polysaccharides extracted from the tunics of five species of ascidians were analyzed by agarose and polyacrylamide gel electrophoresis (Fig. 1).
The electrophoretic mobilities of the sulfated polysaccharides on agarose gel were characteristic for each species of ascidian (Fig. 1A). Two metachromatic bands with electrophoretic mobilities different from those of standard glycosaminoglycans are observed in the agarose gel electrophoretograms of the sulfated polysaccharides from Ascidian nigra, Botryllus sp., and Styelu plicata. The glycans extracted from Cluvelina sp. and Herdmania monus show a single widespread band.
Polyacrylamide gel electrophoresis of the ascidian polysaccharides (Fig. 1B) indicates that all the species have at least two fractions: one of high molecular weight that stays at the origin and another of low molecular weight that migrates into the gel. The glycans from A. nigra, Clavelina sp., and H. monus show a single low molecular weight fraction, while those from Botryllus sp. and S. plicata have two distinct bands of low molecular weight.
Chemical analysis of the polysaccharides from five ascidi- The "Experimental Procedures" 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. Electrophoresis of the sulfated polysaccharmes mom different species of ascidians. A, agarose gel. About 25 pg of the polysaccharides extracted from the various species of ascidians and a mixture of glycosaminoglycans containing 10 pg each of standard chondroitin 4-sulfate (CS), dermatan sulfate (DS), and heparan sulfate ( H 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% N-cetyl-N,N,Ntrimethylammonium bromide solution. After 8 h, the gel was dried and stained with 0.1% toluidine blue in acetic acidethanokwater (0.1:5:5, v/v). B, polyacrylamide gel. About 25 pg of the polysaccharides from the various species of ascidians were subjected to 6% polyacrylamide gel electrophoresis in 0.02 M sodium barbital buffer (pH 8.6) for 30  ans (Table I) shows the presence of sulfate ester, a high galactose content, and small amounts of glucose and hexosamine. Small amounts of mannose and fucose are also present in the glycans from A. nigra and Clavelina sp., respectively. The sulfated polysaccharides from all species have a strong negative optical rotation.
The polysaccharides from S. plicata were also analyzed after purification on DEAE-cellulose chromatography. The molar proportions of hexoses, hexosamine, and sulfate in this purified polysaccharide (Table I) show no significant changes when compared with the nonpurified glycan. This result excludes the possibility of neutral polysaccharide contamination in the ascidian sulfated glycans.
Fractionation and Chemical Analysis of the Ascidian Polysaccharides Gel Chromatography on Sepharose CL-4B and Sephadex G-200-The sulfated glycans from three species of ascidians were fractionated by gel filtration on Sepharose CL-4B and Sephadex G-200 (Fig. 2). A high molecular weight fraction, designated F-1, is observed in all species and accounts for a t least 50% of the total polysaccharides. This fraction does not enter the polyacrylamide gel due to its high molecular weight (Fig. 3B) and on agarose gels shows a single band (Fig. 3A).
A low molecular weight fraction, designated F-2, is eluted near the total volume of the Sepharose CL-4B column (Fig.  2). In S. plicata, this fraction has two components, which were further purified by gel chromatography on Sephadex G-200 (fractions F-2-A and F-2-B in the insert to Fig. 2C). Each fraction F-2 (or F-2-A and F-2-B) migrates as a single band on the polyacrylamide gel, but the average molecular weight differs for each species (Fig. 3B). Electrophoresis on an agarose gel confirms the homogeneity of the F-2 fractions (Fig.  3A).
Chemical Composition of the Fractions- Table I1 shows the chemical analysis and the specific rotation of the purified polysaccharides from the three species of ascidians. Fraction F-1 from all species is composed mainly of galactose and sulfate. Fraction F-2 shows a more heterogeneous chemical composition and a wide variation among the various species of ascidians. However, it always has a higher hexosamine content than F-1, and its predominant amino sugar is glucosamine, while galactosamine predominates in F-1. The sulfate content of both fractions increases from A. nigra to S. plicata and Clavelina sp. (Table 11).
Sulfated polysaccharides from the other two species (Botryllus sp. and H. monus) were also purified by gel filtration and checked for purity by agarose and polyacrylamide gel electrophoresis (results not shown). The high molecular weight fraction (F-1) of these two species is a sulfated galactan, with minor amounts of glucose and amino sugar, while the F-2 fractions have always a higher hexosamine content than F-1, as already reported for the other ascidians in Table  11.
In the ascidian glycans, the presence of amino sugars, which are especially prominent in F-2 fractions, resembles the glycosaminoglycans from animal tissues. In fact, F-2 from A. nigra and Clnuelina sp. possess equimolar proportions of galactose and glucosamine, as do keratan sulfate from mammalian cartilages and corneas (19). However, the specific optical rotation (Table 11) and the resistance to the mucopolysaccharidases (6,7) indicate that the ascidian glycans differ from all previously described glycosaminoglycans.
Fraction F-1 from the various species of ascidians forms broad peaks on Sepharose CL-4B columns (Fig. 2). In order to determine whether this fraction has a homogeneous chemical composition, F-1 from S. plicata was subdivided into F-l-A and F-1-B (Fig. 2C, see also Ref. 7). The chemical analysis of these two subfractions shows comparable chemical compositions with respect to hexoses, hexosamine, and sulfate, confirming previous data (7).' This indicates that the high molecular weight fraction encompasses a broad range of molecular weights but is structurally homogeneous.
L-Galactose in the Ascidian Polysaccharides-The strongly negative specific rotations of the ascidian polysaccharides (Tables I and 11), especially of the F-1 fractions (-100 to In the present study we also methylated F-I-A and F-1-B and obtained similar proportions of methylated derivatives for both of them (data not shown). ' These analyses were carried out after purification of the polysaccharides on a DEAE-cellulose column. -132"), is compatible with residues of a-L-galactopyranose, since the specific rotation of methyl a-L-galactopyranoside is -179". Furthermore, the presence of a-L-galactopyranosyl units in the ascidian polysaccharides was demonstrated unequivocally by the finding that galactose in these glycans occurs only in the L-enantiomeric form and that its D-enantiomorph is lacking (7,20). This finding indicates that the ascidian polysaccharides are unique among known galactose-rich polysaccharides (1,(21)(22)(23)(24), in that their major constituent is Lgalactose, the D-enantiomorph being entirely absent.
Main Structural Features of Fraction F-1 In spite of their chemical similarities (Table 11) the very different electrophoretic mobilities of the various F-1 fractions (Fig. 3A) suggest important structural differences. These were examined in detail for A. nigra, Clavelina sp., and S. plicata, using periodate oxidation, methylation, 'H NMR, and I 3 C NMR, as described below.
Periodate Oxidation-The products obtained by acid hydrolysis of the periodate-oxidated F-1 fractions after borohy-  dride reduction are shown in Table 111. Fractions F-1 from S. plicata and A. nigra yield large amounts of glycerol and some threitol and erythritol, while F-1 from Clavelinu sp. forms only small amounts of threitol. Since glycerol is produced only from nonreducing end or 1 4 glycosidically linked units, these data indicate that these two features are virtually absent from the Clavelina polysaccharide in contrast to the other two species.
About 76% of the galactose units in F-1 from S. plicata and A. nigra are resistant to periodate oxidation (Table  111). However, detectable amounts of threitol and erythritol indicate additional complexity in the structure of these two poly-

Sulfated
Polysaccharides from Ascidians 9975 mers. Regarding the amino sugars, it is possible that they occur at nonreducing ends or are 1 -6 glycosidically linked units, since most of them disappear from F-1 of S. plicata and A. nigra after periodate oxidation.
Methylation Studies-The methylation studies of fraction F-1 from Clavelinu sp. indicate that this polysaccharide is constituted mainly of a core of galactose-linked glycosidically through position 1 4 and sulfated at position 3. That is, 2,3,6-tri-O-methylgalactose as alditol acetate derivative is the main methyl ether obtained from desulfated F-1, whereas 2,6di-0-methylgalactose is the predominant methyl ether derived from sulfated F-1 (Table IV). Small amounts of 2,3,4,6-tetra-0-methylgalactose indicate the presence of some galactose at nonreducing ends.
Another interesting feature of the Clauelina F-1 polysaccharide is the presence of fucose, as already reported in Table  11. The only fucose methyl derivative obtained from sulfated F-1 is 2-mono-O-methylfucose, which after desulfation is entirely converted into 2,3-di-O-rnethylfucose (Table IV). Therefore, fucose occurs as intrachain residues glycosidically linked through position 1 4 and sulfated at position 3.
Methylation studies of fraction F-1 from S. plicata indicate that this polymer has a carbohydrate core similar to that reported for F-1 from Clauelina sp. It is constituted mainly of galactose units linked glycosidically through position 1 4 and sulfated at position 3. Therefore, the main methyl ether derivative obtained from the sulfated polymer is 2,6-di-0methylgalactose, whereas after desulfation the 2,6-di-O-methylgalactose almost disappears and the 2,3,6-tri-O-methylgalactose increases almost 4-fold (Table IV).
However, fraction F-1 from S. plicata has several distinctive features. Interestingly, it possesses large amounts of nonreducing end sugars. Thus, the proportion of 2,3,4,6-tetra-Omethyl derivative from galactose is very high compared with F-1 from Clavelina sp. This agrees with the results obtained by analysis of the products formed from the periodate-oxidized F-1 fractions (Table 111), showing that no detectable glycerol is produced from F-1 of Clavelina sp., while high yields of glycerol are obtained from F-1 of S. plicata and A. nigra.
The higher amounts of 2,3,4,6-tetra-O-methylgalactose obtained from desulfated F-1 of S. plicata suggest that some galactose residues at nonreducing ends are also sulfated in this species.
Based on the methylation studies, it is difficult to determine the branching point of this polymer, although the 0 -2 position is a good candidate. Thus, small amounts of 6-mono-0-methylgalactose were obtained from the intact F-1 and 3,6-di-0methylgalactose from desulfated F-1. The sum of these derivatives does not amount to the proportion of tetra-0-methyl derivatives. This underestimate could be due to a low recovery of branching residues under our hydrolysis conditions. However, even when the methylated F-l was hydrolyzed under milder conditions (90% formic acid, 1 h, 100 "C, followed by 0.15 M H2S04, 16 h, 100 "C), the same proportion of methylated sugars as that reported in Table IV was obtained. Furthermore, we methylated and hydrolyzed glycogen under the same experimental conditions used for ascidian polysaccharides. The formation of equivalent proportions of 2,3,4,6tetra-0-methylglucose and 2,3-di-O-methylglucose from glycogen indicates that the underestimation of the branching residues in the ascidian polysaccharides cannot be attributed to our experimental protocol.
The presence of 2,4,6-tri-O-methylgalactose in the methylation of desulfated F-1 from s. plicata (Table IV) indicates the presence of small but reproducible amounts of galactose glycosidically linked through position 1-3, and sulfated at position 4, whereas this type of unit is absent in F-1 from Clavelinu sp. Methylation of F-1 from A. nigra produces essentially the same products as reported for F-1 from S. plicata, but the relative proportions of the methylated derivatives differ considerably between these two species. The distribution among the various methylated derivatives, without the preponderance of any type of linkage, suggests that this fraction in A. nigra may have a more heterogeneous structure. However, some distinctive features are observed. More 2,4,6-tri-O-methylgalactose is obtained from intact F-1 of A. nigra, and the level increases 2-fold after desulfation. Therefore, F-1 from A. nigra includes large amounts of 1-3-linked galactose residues that are partially sulfated at position 4.
For A. nigra a better recovery of the branching galactose units may account for the higher yields of 6-mono-0-methylgalactose, which after desulfation yields 3,6-di-O-methyl-  galactose. This result also confirms the presence of sulfate ester at position 3 of the galactose residues and indicates that the branching occurs only in the 14-linked units. As already mentioned for F-1 from S. plicata, these branching derivatives are probably underestimated.
The formation of large amounts of 2,3,4,6-tetra-O-methyl derivatives of galactose, glucose and/or mannose, which did not increase after desulfation, indicates that many nonsulfated end groups are present in F-1 from A. nigra.
'H NMR-The 'H NMR spectra of F-1 fractions from the three species of ascidians (Figs. 4, A, 0, and F ) show signals in the vicinity of 6 = 4.7 and 5.3 ppm, which are consistent with the anomeric protons of a-galactopyranosides (25,26). Several other signals that resonate in the range expected for a @-galactopyranoside (4.3-4.6 ppm) (25,27) are also found; however, they strongly decrease in relative intensity (Fig. 4C) or totally disappear (Fig. 4E) after chemical desulfation. Since protons from carbons bearing a sulfate ester are shifted downfield by about 0.42-0.74 ppm (25,26), these signals in the region of the p-anomeric proton are probably attributable to protons of the sulfate-substituted carbons. Overall, the ' H NMR analysis confirms the preponderance of a-anomeric protons in all F-1 fractions. These observations agree with the proposal that the F-1 fractions are composed mainly of a-L-galactopyranosyl units, as already suggested by their strong negative optical rotations (Table 11).
The 'H NMR spectra also show a signal at approximately 6 = 2.0 ppm in the F-1 fraction from S. plicata (Fig. 4A) and A. nigra (Fig. 4F), but it is absent in F-1 from Cluvelina sp. (Fig. 40). Since the chemical analyses show amino sugars only in F-1 from S. plicata and A. nigra (Table 11), and since these polymers resist deaminative cleavage by nitrous acid (6, 7), we attribute this signal at 6 = 2.0 ppm to acetamido methyl. Another peak at approximately 6 = 1.3 ppm, which is attributed to deoxymethyl, occurs only in F-1 from Clauelina sp. This signal is rather large in the 'H spectrum, more consistent with about 20% of fucose than the 9-10% that we found by chemical analysis of this fraction (Tables I1 and IV). However, we have no explanation for this discrepancy. Interestingly, F-1 from S. plicata after Smith degradation shows a 'H NMR spectrum with much sharper peaks (Fig.  4B), which strongly resembles the spectrum of F-1 from Clauelinu sp. (Fig. 40). In fact, the chemical shifts of the protons in the anomeric region are very similar for both polysaccharides, except for the peak at 6 = 4.78, which is absent in the 'H NMR spectrum of F-1 from Cluuelinu sp. 13C NMR-The 13C NMR spectra obtained from F-1 of the three species of ascidians (Fig. 5, A, C, and 0 ) show a major signal in the region of anomeric carbon, which resonates at 6 = 101.3 ppm. This result shows that the major anomeric carbon of F-1 is more strongly shielded than is the corresponding carbon of @-galactopyranoside, which resonates at 6 = 103.4-105.2 ppm (28,29). In addition, the anomeric carbon of F-1 resonates in the range expected for an a-galactopyranoside (97.7-101.5 ppm) (28,29). The 13C NMR spectra show also an intense signal attributed to nonsubstituted carbon 6, which resonates at 6 = 62.4-60.7 ppm. This result agrees with the methylation studies in showing the absence of substitution at carbon 6.
Fraction F-1 from Cluvelina sp. affords a well resolved I3C spectrum (Fig. 50). Six signals are clearly distinguished. From the shapes of these signals and their relative intensities, it is possible to conclude that this is a polysaccharide containing mainly six nonequivalent carbons. Because of their distinctive  (Table  IV), it is possible to conclude that most of carbons 2 and 5 in F-1 from Clavelina sp. are neither sulfated nor substituted by glycosidic linkage. Therefore, signals at 6 = 68.6 and 72.6 ppm, which resonate in the region of nonsubstituted secondary carbons, may be attributed tentatively to carbons 2 and 5, respectively (29). Two other signals ascribed to substituted secondary carbons resonate at 6 = 77.7 and 77.1 ppm. From the methylation data (Table IV), it is possible to attribute these signals to carbons 3 and 4, which are sulfated and substituted by glycosidic linkage, respectively. However, the specific assignment of these two peaks is not possible because a similar downfield displacement with respect to the parent sugars is observed for the shifts of carbon atoms carrying a sulfate group (30)(31)(32), or substituted by a glycosidic linkage (29).
For fractions F-1 from S. plicata (Fig. 5A) and A. nigra ( Fig. 5C), more complex 13C NMR spectra were obtained. The 13C nuclei of these polysaccharides resonate at 6 = 72.4-68.2 ppm and a large variety of signals attributable to glycosidically linked or sulfated secondary carbons resonate at 6 = 80.2-74.3 ppm. The complexity of the spectra in the region of substituted secondary carbons does not permit the identification of the main glycosidically or sulfate-substituted carbons. However, the spectra demonstrate that the 13C nuclei of both F-1 fractions are more strongly shielded than are the corresponding nuclei of a-or @-galactofuranosides, which resonate at 84.7-71.7 ppm (34). The 13C nuclei of both F-1 fractions resonate in the range expected for an a-or @galactopyranoside (71.6-69.2 ppm) (35,36). The 13C spectrum of A. nigra (Fig. 5C) shows small signals of acetamido sugar, as suggested by the chemical analysis (Table 11). However, the spectrum of S. plicata (Fig. 5A) has only a minor signal for acetamido methyl and the expected signal corresponding to carbon 2 of the 2-deoxy-2-acetamido hexose at about 6 = 55 ppm appears to be lost in the noise.
The methylation studies (Table IV) suggest that the major heterogeneity of F-1 from S. plicata occurs in the sugar residues at the nonreducing end, while the carbohydrate core of this fraction is similar to that of F-1 from Clauelina sp. Therefore, it is expected that the Smith degradation, which oxidizes the periodate-sensitive sites and removes the polyalcohols formed by mild acid hydrolysis, would produce from F-1 of S. plicata a polysaccharide similar to F-1 of Clavelina sp. In fact, F-1 from S. plicata after Smith degradation ex- hibits a 13C NMR spectrum (Fig. 5B) that closely resembles the 13C spectrum of intact F-1 from Clavelina sp. (Fig. 5 0 ) . Furthermore, F-1 from S. plicata after Smith degradation produces a simpler 'H NMR spectrum, with much sharper peaks and a lower acetamido methyl signal (Fig. 4B), which resembles the 'H NMR spectrum of intact F-1 from Clavelina sp. (Fig. 40). In addition, the chemical analysis of F-1 from S. plicata after Smith degradation shows an increase in the relative proportions of L-galactose and sulfate, lower hexosamine and glucose contents, and a stronger negative optical rotation ( Table V). These results indicate that the Smith degradation removes mainly the nonreducing end groups, which are mostly nonsulfated, and the intrachain glucose residues, which are linked through position 1 4 , increasing the relative proportion of 3-sulfated a-L-galactopyranosyl units in the polymer. CONCLUSION In the present work sulfated polysaccharides were extracted from the tunics of several species of ascidians and fractionated by gel filtration (Fig. 2). A high molecular weight fraction (Fl ) , which contains high amounts of L-galactose, is obtained from all species. The other fraction (F-2 or F-2-A and F-2-B) has a molecular weight and a chemical composition that vary greatly among the different species.
It is interesting that F-1 from Clavelina sp. differs from F-1 of the other two ascidians in the amounts of branching units. Clavelina sp. is considered to be a more primitive tunicate, as compared to S. plicata and A. nigra (36). Therefore, we can speculate that the addition of nonreducing end units, possibly by a different biosynthetic enzyme, occurred only in the later evolution of the tunicates. This comparison is based on analysis of the main structural features of fraction F-1 from the three species of ascidians. Fig. 6 summarizes the main galactose units found in these polysaccharides. Large amounts of a-L-galactopyranose residues, sulfated at position 3 and linked glycosidically through positions 1 and 4 (I, Fig. 6) are present in all F-1 fractions. However, a-L-galactopyranose units, 1+3-linked and partially sulfated at position 4, are present in large amounts only in F-1 from A. nigra (3, Fig. 6C). They occur in small amounts in F-1 from S. plicata (Fig. 6A) and are absent in F-1 from Clavelina sp. (Fig. 6B). The methylation studies (Table IV) show high amounts of L-galactopyranose nonreducing end units in F-1 from A. nigra and S. plicutu, indicating that both F-1 fractions are highly branched polymers. However, methylation (Table IV) and 13C NMR (Fig. 5 0 ) show that F-1 from Clavelina sp. is distinguished by a paucity of nonreducing end units.
Fraction F-1 from S. plicata subjected to Smith degradation, which oxidizes the periodate-sensitive sites and removes the polyalcohols formed by mild acid hydrolysis, gives a 13C NMR spectrum (Fig. 5B) similar to that of intact F-1 from Clavelina sp. (Fig. 5 0 ) . Both 13C NMR spectra contain mainly six Our studies did not determine the branching point of these polymers, although the 0 -2 position is a good candidate. The methylation studies (Table IV)  nonequivalent carbons. Two of them resonate in the region of the substituted secondary carbons, as expected for a linear polysaccharide whose units are sulfated. Such results confirm that the major heterogeneity of F-1 from S . plicutu is the presence of nonreducing end units.
The F-1 fractions from the various ascidians are unique among previously described sulfated glycans. The main galactose-rich sulfated polysaccharides described in living tissues are keratan sulfate and carrageenans. Keratan sulfate, which occurs mainly in mammalian cartilages and corneas, is composed of P-D-galaCtOpyranOSe units 1 4 -l i n k e d glycosidically to N-acetyl-D-glucosamine 6-sulfate (19). The algal carrageenans present a more heterogeneous structure (1). They are linear chains of @-D-gak3CtOpyranOSe residues linked glycosidically through position 1+3 to a-galactopyranose. The agalactopyranose can occur in either D or L form or can be wholly or partly converted to 3,6-anhydro forms. The sulfate ester may occur at positions 2,4, or 6 of the galactose residues in the carrageenans.
Fraction F-1 from the ascidians differs from these previ-ously described sulfated polysaccharides not only in the types of linkages and position of sulfation, but also in the extensive branching (except F-1 from Cluuelinu sp.), while both keratan sulfate and carrageenans are linear polysaccharides. Furthermore, the ascidian glycans are the first group of polysaccharides that contain large amounts of L-galactose and not its Denantiomorph. The presence of extensive branching and the abundance of sulfate ester in these polysaccharides may increase their water binding capacity. As in the cartilaginous proteoglycans of vertebrates, this property would contribute to the resilience of the tissue.