/ . Biochem., SO, 513-524 (1976)
Dermatan Sulfate-Chondroitin Sulfate Copolymers from Umbilical Cord Isolation and Characterization Sadako INOUE and Mariko IWASAKI School of Pharmaceutical Sciences, Showa University, Hatanodai, Shinagawa-ku, Tokyo 142 Received for publication, March 19, 1976
Dermatan sulfate-chondroitin sulfate copolymers have been isolated from human umbilical cord as a major galactosaminoglycan component of this tissue. The galactosaminoglycan fraction was obtained from this tissue by papain [EC 3.4. 22.2] digestion followed by precipitation with cetylpyridinium chloride in a yield of 700 mg per 100 g of dry tissue. Ethanol fractionation resolved 4-5 subfractions differing in relative content of L-iduronic acid and D-glucuronic acid. No galactosaminoglycan containing either solely L-iduronic acid or r>glucuronic acid was obtained. The copolymeric structure of the material in each subfraction was demonstrated by analysis of oligosaccharide fragments obtained by chondroitinase-AC [EC 4.2.2.5] digestion. All the polymers contained repeating disaccharide units, D-glucuronosyl-N-acetylgalactosamine, D-glucuronosyl-N-acetylgalactosamine 4-sulfate, D-glucuronosyl-N-acetylgalactosamine 6-sulfate, and L-iduronosyl-N-acetylgalactosamine 4-sulfate, of which D-glucuronosyl-N-acetylgalactosamine 6-sulfate and L-iduronosyl-N-acetylgalactosamine 4-sulfate were predominant. Both iduronic acid- and glucuronic acid-containing units were arranged in clusters. The presence of a considerable amount of nonsulfated disaccharide units was noted. The copolymers show extensive polydispersity in electrophoresis on cellulose acetate and gel chromatography on Sephadex G-200.
The isolation of dermatan sulfate in a yield about half that of chondroitin sulfate C from human umbilical cord has been reported by Danishefsky and Bella (1). Fransson has shown that umbilical cord dermatan sulfate is a hybrid containing both chondroitin sulfate C-
type disaccharide units and L-iduronic acidcontaining disaccharide units (2). In the present study we reexamined the nature of galactosaminoglycans from human umbilical cord and found that the major components have copolymeric structures composed
The following abbreviations are used: GlcUA, D-glucuronic acid; IdUA, L-iduronic acid; GalNAc, Nacetylgalactosamine; GalNAc(4S), N-acetylgalactosamine 4-sulfate; GalNAc(6S), N-acetylgalactosamine 6-sulfate ; JUA, 44-uronic acid; JDi-OS or JGlcUA-GalNAc, 3-O-zf-glucuronosyl N-acetylgalactosamine ; JDi-4S or 4GlcUA-GalNAc(4S), 3-O-44-glucuronosyl-N-acetylgalactosamine 4-sulfate; JDi-6S or JGlcUAGalNAc(6S), 3-O-J4-glucuronosyl-N-acetylgalactosamine 6-sulfate. Vol. 80, No. 3, 1976
513
S. INOUE and M. IWASAKI
514
of various proportions of disaccharide units, Dglucuronosyl - N - acetylgalactosamine, D - glu curonosyl-N-acetylgalactosamine 4-sulfate, r> glucuronosyl-N-acetylgalactosamine 6-sulfate, and L-iduronosyl-N-acetylgalactosamine 4-sulfate. The material isolated from umbilical cord shares its structural complexity and extremely disperse nature with the dermatan sulfatechondroitin sulfate copolymers from the meniscus of the human knee joint (3). EXPERIMENTAL Materials—Chondroitinase-AC [EC 4.2.2.5] from Arthrobacter aurescens and chondroitinaseABC [EC 4. 2.2.4] from Proteus vulgaris were purchased from Seikagaku Kogyo, Co., Tokyo, and Pronase-P was purchased from Kaken Kagaku Co., Tokyo. The following materials (used as references) were products of Seikagaku Kogyo Co.: chondroitin sulfate A, chondroitin sulfate B (dermatan sulfate), chondroitin sulfate C, 4Di-0S, ^Di-4S, and 4Di-6S. DEAESephadex A-25 and Sephadexes were products of Pharmacia, Uppsala, and Separax (cellulose acetate film) was purchased from Jok5 Sangyo Co., Tokyo. Chemical Analyses—The uronic acid content was determined by the method of Bitter and Muir (4). Carbazole-to-orcinol ratios were calculated according to Hoffman et al. (5) from the color yields of the carbazole (6) and the orcinol ( 7 ) reactions, the latter being performed with a boiling time of 30 min. Hexosamine was determined after hydrolysis in 3 N HC1 at 100° for 18 hr with a Hitachi amino acid analyzer. Amino acids were analyzed on the amino acid analyzer after hydrolysis in 6 N HC1 at 110° for 22 hr. Sulfate was determined by the turbidimetric method -. of Dodgson (8). The amounts of oligosaccharides obtained by chondroitinase digestion were estimated from the galactosamine contents. Reduction with sodium borohydride was performed as described by Spiro (9). Galactosaminitol was determined on the amino acid analyzer equipped with a 0.9x20 cm column, which was equilibrated and eluted with 0.35 M sodium citrate buffer, pH 5.2, containing boric acid at a final concentration of 0.1 M. This procedure,
a slight modification of that recommended by Weber and Winzler (10), gave an adequate separation of galactosaminitol from galactosamine. The ninhydrin color yield with this buffer was about half that with citrate buffer alone. N-acetylgalactosamine at reducing terminals of oligosaccharides was estimated by the method of Reissig et al. (11). Galactose and xylose were determined after paper chromatographic separation of the hydrolysates (1 N HC1, 100°, 18 hr) by the method of Park and Johnson (12). Values were corrected for losses during hydrolysis and chromatography based on authentic sugars treated similarly. Physical Analyses — Viscosity - average molecular weights (MT) were determined by the procedure of Mathews (13). Infrared spectra were obtained on a JASCO IRA-2 spectrophotometer as KBr pellets. Optical rotation was determined with a JASCO ORD/ UV-5 spectropolarimeter. Electrophoresis and Chromatography—Electrophoresis of glycosaminoglycans was carried out on cellulose acetate at a constant current of 1 mA per cm. Buffers used were 0.15 M pyridine-formic acid, pH 3.0(74) and 0.2 M calcium acetate (15). After electrophoresis the strips were stained and washed as recommended by Seno et al. (15). Oligosaccharides produced by chondroitinase digestion were separated by descending chromatography on Toyo No. 51A or Whatman No. 3MM filter paper in Solvent A, butyric acid-0.5 N ammonia (5 : 3, by volume), at room temperature. Solvent B, 1-butanol-ethanol-water (13 : 8 : 4, by volume), was used for desalting the digestion products on paper and for washing chromatograms after development in Solvent A. Compounds were detected by viewing under ultraviolet light, eluted with water from the paper and subjected to chemical analyses. L-Iduronic acid and D-glucuronic acid in galactosaminoglycans were determined by gas chromatography as described previously (16), with the following modifications: (i) Samples were deacetylated with 2 N trifluoroacetic acid instead of 2 N HC1: the recovery of L-iduronic acid was improved, and (ii) 1.5% OV-1 on Shimalite W (80-100 mesh, Shimadzu Seisakusho, Kyoto) was used as a column packing instead of 3.5%
/ . Biochtm.
S. INOUE and M. IWASAKI
516
TABLE I. Analyses of galactosaminoglycan fractions from umbilical cord. Fraction' UC25 Yield (mg/lOOg dry tissue) Galactosamineb (%) Uronic acid (%) Carbazole-to-orcinol ratio Sulfur (%) Amino acid (%) MT (X10-1) [ tions from UC40 and UC50. (a) Gel chromatography . glucuronic acid and nonsulfated galactosamine on Sephadex G-10 (fine, 1x180 cm) of the oligosacis associated with both L-iduronic acid and Dcharide fraction from UC40. (b) Gel chromatography glucuronic acid. Examination of the oligosacon Sephadex G-25 (superfine, 1x190 cm) of Fraction charide fractions by paper chromatography UC40-I from the Sephadex G-10 column, (c) Gel revealed that gel chromatography on Sephadex chromatography on Sephadex G-25 of the oligosacG-25 columns resulted in partial fractionation charide fraction from UC50. (d) Rechromatography of Fraction 50-1 on the same column as (c). Elutions of unsaturated disaccharide isomers. For instance, Fractions UC50-Ib, -Ic, and -II were were with 10% ethanol. An aliquot from each 1 ml composed of JGlcUA-GalNAc(4S)-MGlcUAfraction was analyzed for uronic acid. Vo, void GalNAc(6S), JGlcUA-GalNAc(6S), and volume.
V
x.
Vol. 80, No. 3, 1976
. A
S. INOUE and M. IWASAKI
520
TABLE IV. Analyses of oligosaccharide fractions separated by Sephadex columns.
Fraction
Yield
Degree of polymerization 1
Identification by paper chromatography
(% of the sum)
Unsaturated disaccharides produced by chondroitinase-ABC (mole/100 moles)" Di-OS
Di-4S
Di-6S
c
4
-Ilia -Illb
16 33 17
10 2.6
10 5 9
88 87 88 65
8 3 7 26
-IIIc
11
1.2
18
43
39
-Hid
9
1.2
7
16
77
4 6 9 18
93 91 81 51
3 3 10 31
7
22
71
8 10 4
85 77 70
•7 13 26
0 12
46 12
54
12
46
42
1 0 39
39 0 1
60 100 60
UC25-I
-n
UC30-I
14 —
-Ilia
-nib
15
24 6.1 1.5
-IIIc
12
1.4
UC40-Ia -Ib
6
tetrasaccharides JDi-6S
—
13 16 44
-II
higher oligomers, tetrasaccharides tetrasaccharides JDWS, 4Di-6S
higher oligomers, tetrasaccharides JDi-lS tetrasaccharides JDi^S, 4Di-6S
12
-Ic
10 8
7.0 3.6
-Id -II
14 61
2.2
UC50-Ia
12
—
-Ib •Ic -II
25 19 44
— —
1.1
—
higher oligomers, tetrasaccharides tetrasaccharide ^Di-OS, JDi-4S JDi-6S higher oligomers, tetrasaccharides JDi^tS, JDi-6S JDi^tS, JDi-6S JDi-6S JDi-OS, 4Di-6S
76
a
Expressed as the ratio of total galactosamine to galactosaminitol formed by reduction with NaBH 4 . b No other product was detected on paper chromatograms. The sum of the 3 disaccharide isomers eluted from paper accounted for more than 70% of each undigested sample on the basis of galactosamine analysis. Digestion with chondroitinase-ABC was omitted for fractions UC40-II, UC50-Ib, UC50-Ic, and UC50-II which contained only disaccharides. c Not determined.
GalNAc + JGlcUA - GalNAc(6S), respectively. Thus, unsaturated disaccharides were eluted in the order ^GlcUA - GalNAc(4S), JGlcUAGalNAc(6S), and ^GlcUA-GalNAc from the Sephadex column. Since the elution sequence was apparently affected by sulfate substitution, gel chromatography afforded only incomplete fractionation based on degree of polymerization.
Analyses of Di- and Tetrasaccharide Components Liberated by Chondroitinase-AC and -ABC—In order to compare the amounts of di- and tetrasaccharides liberated by chondroitinase-AC from various polymer fractions, the digestion products were directly separated by paper chromatography and each component was quantified by hexosamine analysis. The results are shown in Table V. Although dif/ . Biochem.
522
S. INOUE and M. IWASAKI
TABLE VI. Characterization of some copolymeric oligosaccharide3 from UC40.
Fraction
Yield
Degree of polymerization6
Id
9.2
2.0
Ic-1 Ic-2 Ic-3
1.6
1.9
1.3
3.0
4.4
3.0
Ic^
0.9
2.5
Morgan-Elson reaction Negative Negative C
Negative —
Unsaturated disaccharides produced by chondroitinase-ABC (mole/100 moles) Di-OS
Di-4S
Di-6S
0
46
54
Q
§0
40
19 0 0
70 81
11 19
79
21
1
Expressed as % of the total oligosaccharide fractions obtained with chondroitinase-AC. b The ratio of total galactosamine to galactosamine destroyed by reduction with NaBH 4 . c Not determined.
ADi6S UC40-1C
cm o
to
15
Fig. 7. Tracing Of a paper saccharide fraction UC40-Ic. rated by gel chromatography chromatography in Solvent temperature.
chromatogram of oligoFraction UC40-Ic sepawas subjected to paper A for 3 days at room
at the expense of nonsulfated units in L-iduronic acid-rich polymers as compared to D-glucuronic acid-rich polymers. Isolation and Characterization of Some Copolymeric Oligosaccharides—O\igosacchande fractions UC40-Ic and -Id were subjected to preparative paper chromatography for further purification. One major component, which migrated in the position expected for terrasaccharide, was obtained from UC40-Id. UC40Ic was separated into 4 components by prolonged development (Fig. 7). Analytical data for each component eluted from the paper are
given in Table VI. Based on these results, UC40-H appears to be a tetrasaccharide, JGlcUA - GalNAc(6S) - IdUA - GalNAc(4S) and UC40-Ic-l is a mixture of two different tetrasaccharides, JGlcUA - GalNAc(6S) - IdUA GalNAc(4S) and JGlcUA - GalNAc(4S) - IdUA GalNAc(4S). UC40-Ic-2, UC40-Ic-3, and UC40Ic-4 are composed of hexasaccharides, as indicated by the apparent degrees of polymerization, but analyses of the disaccharide units produced by chondroitinase-ABC suggested that these fractions are still heterogeneous. Since unpurified UC40-Ic gave a negative MorganElson reaction, the reducing terminal galactosamine should be 4-sulfated in these oligosaccharides. DISCUSSION A copolymeric structure containing both Liduronic acid and D-glucuronic acid has been proposed for many dermatan sulfate fractions isolated from various tissues (2, 3, 20—22). When D-glucuronic acid occurs as a minor component, the polymer is easily distinguishable from chondroitin sulfates in many physical and chemical properties. Dermatan sulfatechondroitin sulfate copolymers from the meniscus of the human knee joint ( 4 ) and horse aorta (20), which contained a large proportion of D-glucuronic acid, showed properties intermediate between those of "dermatan sulfate" and chondroitin sulfate, like the material from umbilical cord. / . Biochem.
DERMATAN SULFATE-CHONDROITIN SULFATE FROM UMBILICAL CORD TABLE V.
521
Relative amounts of digestion products formed by chondroitinases. Relative amounts of the products (mole/100 moles)b
Fraction"
Chondroitinase Di-OS
UC18
UC25
UC30
ABC
4
AC
1
ABC
6
AC
2
ABC
6
AC UC40
UC50
3
ABC
11
AC
12
ABC
16
AC
16
Di-4S 67
( 8)e
3
4
(19)
56 ( 8)
6
(18)
(15)
12 14
(17)
11
0
0 84
27
0
0
14
(71)
4
76 0
(74)
0 5
62
0
0
4
12
0
0
3
3
25 65
(15)
59
(70)
70 (12)
a
Higher oligomer
4
8
37
23
Tetrasaccharide
(69)
30 (23)
67 (10)
Di-6S
67
(71)
Preparation 2. b Expressed relative to the total disaccharides produced by chondroitinase-ABC. in parentheses are relative to the total disaccharides liberated by chondroitinase-AC.
ferent preparation of the original polymers were used, the results obtained here support the con- ' elusion based on analyses of fragments separated by gel chromatography: the amounts of higher oligosaccharides decreased with decreasing content of L-iduronic acid in the original polymers, and the yields of tetrasaccharides were small, being similar for all polymer fractions. The amounts of three isomers of unsaturated disaccharide liberated from each polymer fraction by chondroitinase-AC and by chondroitinase-ABC are also given in Table V. It is clear that, although a major portion of 4-sulfated galactosamine is associated with Liduronic acid-containing segments and is liberated by chondroitinase-ABC, a small portion is also found in D-glucuronic acid-containing segments and is liberated by chondroitinase-AC. A large amount of 4GlcUA-GalNAc(6S) was liberated by chondroitinase-AC digestion, indicating that 6-sulfated galactosamine residues occur in consecutive glucuronic acid-containing units to a large extent. At the same time, a considerable portion of JGlcUA-GalNAc(6S) was recovered only after chondroitinase-ABC digestion from L-iduronic acid-rich polymers (UC18, UC25, and UC30). Apparently, some of the 6-sulfated units originate from -GlcUAVol. 80, No. 3, 1976
c
Values
GalNAc(6S)-units which occur singly and are liberated only after chondroitinase-ABC digestion. However, judging from the chain lengths of the higher oligosaccharides resistant to chondroitinase-AC digestion, single -GlcUAGalNAc(6S)- units could not account for all the 6-sulfated units liberated by chondroitinaseABC and some GalNAc(6S) residues must be present internally in consecutive L-iduronic acid-containing units in the iduronic acid-rich polymer fractions. Finally, nonsulfated galactosamine residues were largely associated with D-glucuronic acid in glucuronic acid-rich polymers (UC40 and UC50). In iduronic acidrich polymers, a considerable portion of nonsulfated galactosamine residues was located adjacent to L-iduronic acid. In order to see whether there was any difference in sulfate substitution in glucuronic acid-containing segments between L-iduronic acid-rich and D-glucuronic acid-rich polymers, we examined the amount of each disaccharide liberated by chondroitinase-AC by expressing it relative to the total amount liberated by the same enzyme (parenthesized values in Table V). While the proportion of the 6-sulfated units was similar in all fractions, the content of 4sulfated units showed a tendency to increase
DERMATAN SULFATE-CHONDROITIN SULFATE FROM UMBILICAL CORD
Ethanol fractionation resulted in partial resolution of umbilical cord galactosaminoglycans into a series of variants with a gradual increase in D-glucuronic acid content. Although each fraction must still contain a large number of molecular species, some of the structural features of the copolymeric chains have been determined in the present study. First, the disaccharide units unequivocally identified as structural elements are D-glucuronosyl-N-acetylgalactosamine, D - glucuronosyl - N - acetylga lactosamine 4-sulfate, D-glucuronosyl-N-acetylgalactosamine 6-sulfate, and L-iduronosyl-Nacetylgalactosamine 4-sulfate. The 6-sulfated isomer was the major D-glucuronic acid-containing unit. The presence of L-iduronosyl-Nacetylgalactosamine and L-iduronosyl-N-acetylgalactosamine 6-sulfate also seems likely, but further evidence is required. In this context, Fransson has demonstrated the presence of Liduronosyl - N - acetylgalactosamine 6 - sulfate units in a dermatan sulfate fraction from umbilical cord (2). Second, both D-glucuronic acid-containing units and L-iduronic acid-containing units occur in clusters. The yield of a tetrasaccharide fraction in which a D-glucuronic acid-containing unit alternates with an L-iduronic acid-containing unit was small from each polymer fraction. The mechanism by which the cell synthesizes L-iduronic acid-containing glycosaminoglycans has been elucidated partially. Thus, Hook et al. (23) have shown that L-iduronic acid in heparin is formed by the epimerization of D-glucuronic acid at the polymer level, and the reaction is somehow dependent on concomitant sulfation of hydroxyl groups. Recently, Malstrom et al. (24), using a fibroblast particulate fraction, demonstrated that Liduronic acid in dermatan sulfate is also formed by epimerization of D-glucuronic acid at the polymer level, and the process is promoted by 4-sulfation of neighboring N-acetylgalactosamine moieties. However, it is not known whether sulfation occurs before or after epimerization in either case. In the case of galactosaminoglycans, the position of sulfation of N-acetylgalactosamine appears to be related to the epimerization of D-glucuronic acid, since in all dermatan sulfate-chondroitin sulfate co.Vol. 80, No. 3, 1976
523
polymers so far reported, L-iduronic acid-containing regions are mainly 4-sulfated, while Dglucuronic acid-containing regions are either 4- or 6-sulfated. In this connection, it would be interesting to know which side (reducing or nonreducing) of a D-glucuronic acid residue is sulfated in relation to its epimerization. In all oligosaccharides obtained from the umbilical cord copolymers by chondroitinase-AC digestion and examined so far, the reducing terminal galactosamine residues were 4-sulfated. To determine the position of sulfate on the nonreducing terminal disaccharide unit, we attempted to remove the nonreducing terminal •4UA by partial acid hydrolysis (25). However, analyses of the hydrolysates revealed partial cleavage of glycosidic linkages as well as partial desulfation, especially of 4-sulfate. Moreover, authentic JGIcUA - GalNAc(6S) showed considerable resistance to acid hydrolysis under these conditions. Removal of nonreducing terminal 4UA with a glucuronidase from Fiavobacterium heparinum (18) may be a better approach. The significance of nonsulfated galactosamine residues is also of interest in relation to sulfation and epimerization. Studies on the macromolecular properties of dermatan sulfate-chondroitin sulfate copolymers alone or interacting with other biopolymers will aid an understanding of the function of galactosaminoglycans in connective tissues. We are grateful to Prof. G. Matsumura for his interest and for helpful suggestions. We thank Mrs. Kazuko Uehara for help in the first stage of preparation of umbilical cord galactosaminoglycans. REFERENCES 1. Danishefski, I. & Bella, A. Jr. (1966) / . Biol. Chem. 241, 143-146 2. Fransson, L.-A. (1968) / . Biol. Chem. 243, 15041510 3. Habuchi, H., Yamagata, T., Iwata, H., & Suzuki, S. (1973) / . Biol. Chem. 248, 6019-6028 4. Bitter, T. & Muir, H.M. (1962) Anal. Biochem. 4, 330-334 5. Hoffman, P., Linker, A., & Meyer, K. (1957) Arch. Biochem. Biophys. 69, 435-440 6. Dische, Z. (1947) / . Biol. Chem. 167, 189-198 7. Brown, A.H. (1946) Arch. Biochem. 11, 269-278
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8. Dodgson, K.S. (1961) Biochem. J. 78, 312-319 9. Spiro, R.G. (1972) in Methods in Enzymology (Ginsburg, V., ed.) Vol.28, part B, pp.3-43, Academic Press, New York 10. Weber, P. & Winzler, R.J. (1969) Arch. Biochem. Biophys. 129, 534-538 11. Reissig, J.L., Strominger, J.L., & Leloir, L.F. (1955) / . Biol. Chem. 217, 959-966 12. Park, J.T. & Johnson, M.J. (1949) / . Biol. Chem. 181, 149-151 13. Mathews, M.B. (1956) Arch. Biochem. Biophys. 61, 367-377 14. Fransson, L.-A. & Rcxten, L. (1967) / . Biol. Chem. 242, 4161-4169 15. Seno, N., Anno, K., Kondo, K., Nagase, S., & Saito, S. (1970) Anal. Biochim. 37, 197-202 16. Inoue, S. & Miyawaki, M. (1975) Anal. Biochem. 65, 164-174 17. Rod6n, L., Baker, J.R., Cifonelli, J.A., & Mathews,
18. 19. 20. 21. 22. 23.
24. 25.
M.B. (1972) in Methods in Enzymology (Ginsburg, V., ed.) Vol.28, part B, pp.73-140, Academic Press, New York Yamagata.T., Saito, H., Habuchi, 0., & Suzuki, S. (1968) / . Biol. Chem. 243, 1523-1535 Stern, E.L., Lindahl, B., & RodSn, L. (1971) / . Biol. Chem. 246, 5707-5715 Fransson, L.-A. & Havsmark, B. (1970) / . Biol. Chem. 245, 4770-4783 Fransson, L.-A. & Rod6n, L. (1967) / . Biol. Chem. 242, 4161-4169 Fransson, L.-A., Antonopoulos, C.A., & Gardell, S. (1970) Carbohyd. Res. 15, 73-89 Hook, M., Lindahl, U., Backstrom, G., Malstrom, A., & Fransson, L.-A. (1974) / . Bid. Chem. 249, 3908-3915 Malstrom, A., Fransson, L.-A., Hook, M., & Lindahl, U. (1975) / . Bid. Chem. 250, 3419-3425 Suzuki, S. (1960) / . Bid. Chem. 235, 3580-3588
/ . Biochem.
Dermatan Sulfate-Chondroitin Sulfate Copolymers from Umbilical Cord Isolation and Characterization Sadako INOUE and Mariko IWASAKI School of Pharmaceutical Sciences, Showa University, Hatanodai, Shinagawa-ku, Tokyo 142 Received for publication, March 19, 1976
Dermatan sulfate-chondroitin sulfate copolymers have been isolated from human umbilical cord as a major galactosaminoglycan component of this tissue. The galactosaminoglycan fraction was obtained from this tissue by papain [EC 3.4. 22.2] digestion followed by precipitation with cetylpyridinium chloride in a yield of 700 mg per 100 g of dry tissue. Ethanol fractionation resolved 4-5 subfractions differing in relative content of L-iduronic acid and D-glucuronic acid. No galactosaminoglycan containing either solely L-iduronic acid or r>glucuronic acid was obtained. The copolymeric structure of the material in each subfraction was demonstrated by analysis of oligosaccharide fragments obtained by chondroitinase-AC [EC 4.2.2.5] digestion. All the polymers contained repeating disaccharide units, D-glucuronosyl-N-acetylgalactosamine, D-glucuronosyl-N-acetylgalactosamine 4-sulfate, D-glucuronosyl-N-acetylgalactosamine 6-sulfate, and L-iduronosyl-N-acetylgalactosamine 4-sulfate, of which D-glucuronosyl-N-acetylgalactosamine 6-sulfate and L-iduronosyl-N-acetylgalactosamine 4-sulfate were predominant. Both iduronic acid- and glucuronic acid-containing units were arranged in clusters. The presence of a considerable amount of nonsulfated disaccharide units was noted. The copolymers show extensive polydispersity in electrophoresis on cellulose acetate and gel chromatography on Sephadex G-200.
The isolation of dermatan sulfate in a yield about half that of chondroitin sulfate C from human umbilical cord has been reported by Danishefsky and Bella (1). Fransson has shown that umbilical cord dermatan sulfate is a hybrid containing both chondroitin sulfate C-
type disaccharide units and L-iduronic acidcontaining disaccharide units (2). In the present study we reexamined the nature of galactosaminoglycans from human umbilical cord and found that the major components have copolymeric structures composed
The following abbreviations are used: GlcUA, D-glucuronic acid; IdUA, L-iduronic acid; GalNAc, Nacetylgalactosamine; GalNAc(4S), N-acetylgalactosamine 4-sulfate; GalNAc(6S), N-acetylgalactosamine 6-sulfate ; JUA, 44-uronic acid; JDi-OS or JGlcUA-GalNAc, 3-O-zf-glucuronosyl N-acetylgalactosamine ; JDi-4S or 4GlcUA-GalNAc(4S), 3-O-44-glucuronosyl-N-acetylgalactosamine 4-sulfate; JDi-6S or JGlcUAGalNAc(6S), 3-O-J4-glucuronosyl-N-acetylgalactosamine 6-sulfate. Vol. 80, No. 3, 1976
513
S. INOUE and M. IWASAKI
514
of various proportions of disaccharide units, Dglucuronosyl - N - acetylgalactosamine, D - glu curonosyl-N-acetylgalactosamine 4-sulfate, r> glucuronosyl-N-acetylgalactosamine 6-sulfate, and L-iduronosyl-N-acetylgalactosamine 4-sulfate. The material isolated from umbilical cord shares its structural complexity and extremely disperse nature with the dermatan sulfatechondroitin sulfate copolymers from the meniscus of the human knee joint (3). EXPERIMENTAL Materials—Chondroitinase-AC [EC 4.2.2.5] from Arthrobacter aurescens and chondroitinaseABC [EC 4. 2.2.4] from Proteus vulgaris were purchased from Seikagaku Kogyo, Co., Tokyo, and Pronase-P was purchased from Kaken Kagaku Co., Tokyo. The following materials (used as references) were products of Seikagaku Kogyo Co.: chondroitin sulfate A, chondroitin sulfate B (dermatan sulfate), chondroitin sulfate C, 4Di-0S, ^Di-4S, and 4Di-6S. DEAESephadex A-25 and Sephadexes were products of Pharmacia, Uppsala, and Separax (cellulose acetate film) was purchased from Jok5 Sangyo Co., Tokyo. Chemical Analyses—The uronic acid content was determined by the method of Bitter and Muir (4). Carbazole-to-orcinol ratios were calculated according to Hoffman et al. (5) from the color yields of the carbazole (6) and the orcinol ( 7 ) reactions, the latter being performed with a boiling time of 30 min. Hexosamine was determined after hydrolysis in 3 N HC1 at 100° for 18 hr with a Hitachi amino acid analyzer. Amino acids were analyzed on the amino acid analyzer after hydrolysis in 6 N HC1 at 110° for 22 hr. Sulfate was determined by the turbidimetric method -. of Dodgson (8). The amounts of oligosaccharides obtained by chondroitinase digestion were estimated from the galactosamine contents. Reduction with sodium borohydride was performed as described by Spiro (9). Galactosaminitol was determined on the amino acid analyzer equipped with a 0.9x20 cm column, which was equilibrated and eluted with 0.35 M sodium citrate buffer, pH 5.2, containing boric acid at a final concentration of 0.1 M. This procedure,
a slight modification of that recommended by Weber and Winzler (10), gave an adequate separation of galactosaminitol from galactosamine. The ninhydrin color yield with this buffer was about half that with citrate buffer alone. N-acetylgalactosamine at reducing terminals of oligosaccharides was estimated by the method of Reissig et al. (11). Galactose and xylose were determined after paper chromatographic separation of the hydrolysates (1 N HC1, 100°, 18 hr) by the method of Park and Johnson (12). Values were corrected for losses during hydrolysis and chromatography based on authentic sugars treated similarly. Physical Analyses — Viscosity - average molecular weights (MT) were determined by the procedure of Mathews (13). Infrared spectra were obtained on a JASCO IRA-2 spectrophotometer as KBr pellets. Optical rotation was determined with a JASCO ORD/ UV-5 spectropolarimeter. Electrophoresis and Chromatography—Electrophoresis of glycosaminoglycans was carried out on cellulose acetate at a constant current of 1 mA per cm. Buffers used were 0.15 M pyridine-formic acid, pH 3.0(74) and 0.2 M calcium acetate (15). After electrophoresis the strips were stained and washed as recommended by Seno et al. (15). Oligosaccharides produced by chondroitinase digestion were separated by descending chromatography on Toyo No. 51A or Whatman No. 3MM filter paper in Solvent A, butyric acid-0.5 N ammonia (5 : 3, by volume), at room temperature. Solvent B, 1-butanol-ethanol-water (13 : 8 : 4, by volume), was used for desalting the digestion products on paper and for washing chromatograms after development in Solvent A. Compounds were detected by viewing under ultraviolet light, eluted with water from the paper and subjected to chemical analyses. L-Iduronic acid and D-glucuronic acid in galactosaminoglycans were determined by gas chromatography as described previously (16), with the following modifications: (i) Samples were deacetylated with 2 N trifluoroacetic acid instead of 2 N HC1: the recovery of L-iduronic acid was improved, and (ii) 1.5% OV-1 on Shimalite W (80-100 mesh, Shimadzu Seisakusho, Kyoto) was used as a column packing instead of 3.5%
/ . Biochtm.
S. INOUE and M. IWASAKI
516
TABLE I. Analyses of galactosaminoglycan fractions from umbilical cord. Fraction' UC25 Yield (mg/lOOg dry tissue) Galactosamineb (%) Uronic acid (%) Carbazole-to-orcinol ratio Sulfur (%) Amino acid (%) MT (X10-1) [ tions from UC40 and UC50. (a) Gel chromatography . glucuronic acid and nonsulfated galactosamine on Sephadex G-10 (fine, 1x180 cm) of the oligosacis associated with both L-iduronic acid and Dcharide fraction from UC40. (b) Gel chromatography glucuronic acid. Examination of the oligosacon Sephadex G-25 (superfine, 1x190 cm) of Fraction charide fractions by paper chromatography UC40-I from the Sephadex G-10 column, (c) Gel revealed that gel chromatography on Sephadex chromatography on Sephadex G-25 of the oligosacG-25 columns resulted in partial fractionation charide fraction from UC50. (d) Rechromatography of Fraction 50-1 on the same column as (c). Elutions of unsaturated disaccharide isomers. For instance, Fractions UC50-Ib, -Ic, and -II were were with 10% ethanol. An aliquot from each 1 ml composed of JGlcUA-GalNAc(4S)-MGlcUAfraction was analyzed for uronic acid. Vo, void GalNAc(6S), JGlcUA-GalNAc(6S), and volume.
V
x.
Vol. 80, No. 3, 1976
. A
S. INOUE and M. IWASAKI
520
TABLE IV. Analyses of oligosaccharide fractions separated by Sephadex columns.
Fraction
Yield
Degree of polymerization 1
Identification by paper chromatography
(% of the sum)
Unsaturated disaccharides produced by chondroitinase-ABC (mole/100 moles)" Di-OS
Di-4S
Di-6S
c
4
-Ilia -Illb
16 33 17
10 2.6
10 5 9
88 87 88 65
8 3 7 26
-IIIc
11
1.2
18
43
39
-Hid
9
1.2
7
16
77
4 6 9 18
93 91 81 51
3 3 10 31
7
22
71
8 10 4
85 77 70
•7 13 26
0 12
46 12
54
12
46
42
1 0 39
39 0 1
60 100 60
UC25-I
-n
UC30-I
14 —
-Ilia
-nib
15
24 6.1 1.5
-IIIc
12
1.4
UC40-Ia -Ib
6
tetrasaccharides JDi-6S
—
13 16 44
-II
higher oligomers, tetrasaccharides tetrasaccharides JDWS, 4Di-6S
higher oligomers, tetrasaccharides JDi-lS tetrasaccharides JDi^S, 4Di-6S
12
-Ic
10 8
7.0 3.6
-Id -II
14 61
2.2
UC50-Ia
12
—
-Ib •Ic -II
25 19 44
— —
1.1
—
higher oligomers, tetrasaccharides tetrasaccharide ^Di-OS, JDi-4S JDi-6S higher oligomers, tetrasaccharides JDi^tS, JDi-6S JDi^tS, JDi-6S JDi-6S JDi-OS, 4Di-6S
76
a
Expressed as the ratio of total galactosamine to galactosaminitol formed by reduction with NaBH 4 . b No other product was detected on paper chromatograms. The sum of the 3 disaccharide isomers eluted from paper accounted for more than 70% of each undigested sample on the basis of galactosamine analysis. Digestion with chondroitinase-ABC was omitted for fractions UC40-II, UC50-Ib, UC50-Ic, and UC50-II which contained only disaccharides. c Not determined.
GalNAc + JGlcUA - GalNAc(6S), respectively. Thus, unsaturated disaccharides were eluted in the order ^GlcUA - GalNAc(4S), JGlcUAGalNAc(6S), and ^GlcUA-GalNAc from the Sephadex column. Since the elution sequence was apparently affected by sulfate substitution, gel chromatography afforded only incomplete fractionation based on degree of polymerization.
Analyses of Di- and Tetrasaccharide Components Liberated by Chondroitinase-AC and -ABC—In order to compare the amounts of di- and tetrasaccharides liberated by chondroitinase-AC from various polymer fractions, the digestion products were directly separated by paper chromatography and each component was quantified by hexosamine analysis. The results are shown in Table V. Although dif/ . Biochem.
522
S. INOUE and M. IWASAKI
TABLE VI. Characterization of some copolymeric oligosaccharide3 from UC40.
Fraction
Yield
Degree of polymerization6
Id
9.2
2.0
Ic-1 Ic-2 Ic-3
1.6
1.9
1.3
3.0
4.4
3.0
Ic^
0.9
2.5
Morgan-Elson reaction Negative Negative C
Negative —
Unsaturated disaccharides produced by chondroitinase-ABC (mole/100 moles) Di-OS
Di-4S
Di-6S
0
46
54
Q
§0
40
19 0 0
70 81
11 19
79
21
1
Expressed as % of the total oligosaccharide fractions obtained with chondroitinase-AC. b The ratio of total galactosamine to galactosamine destroyed by reduction with NaBH 4 . c Not determined.
ADi6S UC40-1C
cm o
to
15
Fig. 7. Tracing Of a paper saccharide fraction UC40-Ic. rated by gel chromatography chromatography in Solvent temperature.
chromatogram of oligoFraction UC40-Ic sepawas subjected to paper A for 3 days at room
at the expense of nonsulfated units in L-iduronic acid-rich polymers as compared to D-glucuronic acid-rich polymers. Isolation and Characterization of Some Copolymeric Oligosaccharides—O\igosacchande fractions UC40-Ic and -Id were subjected to preparative paper chromatography for further purification. One major component, which migrated in the position expected for terrasaccharide, was obtained from UC40-Id. UC40Ic was separated into 4 components by prolonged development (Fig. 7). Analytical data for each component eluted from the paper are
given in Table VI. Based on these results, UC40-H appears to be a tetrasaccharide, JGlcUA - GalNAc(6S) - IdUA - GalNAc(4S) and UC40-Ic-l is a mixture of two different tetrasaccharides, JGlcUA - GalNAc(6S) - IdUA GalNAc(4S) and JGlcUA - GalNAc(4S) - IdUA GalNAc(4S). UC40-Ic-2, UC40-Ic-3, and UC40Ic-4 are composed of hexasaccharides, as indicated by the apparent degrees of polymerization, but analyses of the disaccharide units produced by chondroitinase-ABC suggested that these fractions are still heterogeneous. Since unpurified UC40-Ic gave a negative MorganElson reaction, the reducing terminal galactosamine should be 4-sulfated in these oligosaccharides. DISCUSSION A copolymeric structure containing both Liduronic acid and D-glucuronic acid has been proposed for many dermatan sulfate fractions isolated from various tissues (2, 3, 20—22). When D-glucuronic acid occurs as a minor component, the polymer is easily distinguishable from chondroitin sulfates in many physical and chemical properties. Dermatan sulfatechondroitin sulfate copolymers from the meniscus of the human knee joint ( 4 ) and horse aorta (20), which contained a large proportion of D-glucuronic acid, showed properties intermediate between those of "dermatan sulfate" and chondroitin sulfate, like the material from umbilical cord. / . Biochem.
DERMATAN SULFATE-CHONDROITIN SULFATE FROM UMBILICAL CORD TABLE V.
521
Relative amounts of digestion products formed by chondroitinases. Relative amounts of the products (mole/100 moles)b
Fraction"
Chondroitinase Di-OS
UC18
UC25
UC30
ABC
4
AC
1
ABC
6
AC
2
ABC
6
AC UC40
UC50
3
ABC
11
AC
12
ABC
16
AC
16
Di-4S 67
( 8)e
3
4
(19)
56 ( 8)
6
(18)
(15)
12 14
(17)
11
0
0 84
27
0
0
14
(71)
4
76 0
(74)
0 5
62
0
0
4
12
0
0
3
3
25 65
(15)
59
(70)
70 (12)
a
Higher oligomer
4
8
37
23
Tetrasaccharide
(69)
30 (23)
67 (10)
Di-6S
67
(71)
Preparation 2. b Expressed relative to the total disaccharides produced by chondroitinase-ABC. in parentheses are relative to the total disaccharides liberated by chondroitinase-AC.
ferent preparation of the original polymers were used, the results obtained here support the con- ' elusion based on analyses of fragments separated by gel chromatography: the amounts of higher oligosaccharides decreased with decreasing content of L-iduronic acid in the original polymers, and the yields of tetrasaccharides were small, being similar for all polymer fractions. The amounts of three isomers of unsaturated disaccharide liberated from each polymer fraction by chondroitinase-AC and by chondroitinase-ABC are also given in Table V. It is clear that, although a major portion of 4-sulfated galactosamine is associated with Liduronic acid-containing segments and is liberated by chondroitinase-ABC, a small portion is also found in D-glucuronic acid-containing segments and is liberated by chondroitinase-AC. A large amount of 4GlcUA-GalNAc(6S) was liberated by chondroitinase-AC digestion, indicating that 6-sulfated galactosamine residues occur in consecutive glucuronic acid-containing units to a large extent. At the same time, a considerable portion of JGlcUA-GalNAc(6S) was recovered only after chondroitinase-ABC digestion from L-iduronic acid-rich polymers (UC18, UC25, and UC30). Apparently, some of the 6-sulfated units originate from -GlcUAVol. 80, No. 3, 1976
c
Values
GalNAc(6S)-units which occur singly and are liberated only after chondroitinase-ABC digestion. However, judging from the chain lengths of the higher oligosaccharides resistant to chondroitinase-AC digestion, single -GlcUAGalNAc(6S)- units could not account for all the 6-sulfated units liberated by chondroitinaseABC and some GalNAc(6S) residues must be present internally in consecutive L-iduronic acid-containing units in the iduronic acid-rich polymer fractions. Finally, nonsulfated galactosamine residues were largely associated with D-glucuronic acid in glucuronic acid-rich polymers (UC40 and UC50). In iduronic acidrich polymers, a considerable portion of nonsulfated galactosamine residues was located adjacent to L-iduronic acid. In order to see whether there was any difference in sulfate substitution in glucuronic acid-containing segments between L-iduronic acid-rich and D-glucuronic acid-rich polymers, we examined the amount of each disaccharide liberated by chondroitinase-AC by expressing it relative to the total amount liberated by the same enzyme (parenthesized values in Table V). While the proportion of the 6-sulfated units was similar in all fractions, the content of 4sulfated units showed a tendency to increase
DERMATAN SULFATE-CHONDROITIN SULFATE FROM UMBILICAL CORD
Ethanol fractionation resulted in partial resolution of umbilical cord galactosaminoglycans into a series of variants with a gradual increase in D-glucuronic acid content. Although each fraction must still contain a large number of molecular species, some of the structural features of the copolymeric chains have been determined in the present study. First, the disaccharide units unequivocally identified as structural elements are D-glucuronosyl-N-acetylgalactosamine, D - glucuronosyl - N - acetylga lactosamine 4-sulfate, D-glucuronosyl-N-acetylgalactosamine 6-sulfate, and L-iduronosyl-Nacetylgalactosamine 4-sulfate. The 6-sulfated isomer was the major D-glucuronic acid-containing unit. The presence of L-iduronosyl-Nacetylgalactosamine and L-iduronosyl-N-acetylgalactosamine 6-sulfate also seems likely, but further evidence is required. In this context, Fransson has demonstrated the presence of Liduronosyl - N - acetylgalactosamine 6 - sulfate units in a dermatan sulfate fraction from umbilical cord (2). Second, both D-glucuronic acid-containing units and L-iduronic acid-containing units occur in clusters. The yield of a tetrasaccharide fraction in which a D-glucuronic acid-containing unit alternates with an L-iduronic acid-containing unit was small from each polymer fraction. The mechanism by which the cell synthesizes L-iduronic acid-containing glycosaminoglycans has been elucidated partially. Thus, Hook et al. (23) have shown that L-iduronic acid in heparin is formed by the epimerization of D-glucuronic acid at the polymer level, and the reaction is somehow dependent on concomitant sulfation of hydroxyl groups. Recently, Malstrom et al. (24), using a fibroblast particulate fraction, demonstrated that Liduronic acid in dermatan sulfate is also formed by epimerization of D-glucuronic acid at the polymer level, and the process is promoted by 4-sulfation of neighboring N-acetylgalactosamine moieties. However, it is not known whether sulfation occurs before or after epimerization in either case. In the case of galactosaminoglycans, the position of sulfation of N-acetylgalactosamine appears to be related to the epimerization of D-glucuronic acid, since in all dermatan sulfate-chondroitin sulfate co.Vol. 80, No. 3, 1976
523
polymers so far reported, L-iduronic acid-containing regions are mainly 4-sulfated, while Dglucuronic acid-containing regions are either 4- or 6-sulfated. In this connection, it would be interesting to know which side (reducing or nonreducing) of a D-glucuronic acid residue is sulfated in relation to its epimerization. In all oligosaccharides obtained from the umbilical cord copolymers by chondroitinase-AC digestion and examined so far, the reducing terminal galactosamine residues were 4-sulfated. To determine the position of sulfate on the nonreducing terminal disaccharide unit, we attempted to remove the nonreducing terminal •4UA by partial acid hydrolysis (25). However, analyses of the hydrolysates revealed partial cleavage of glycosidic linkages as well as partial desulfation, especially of 4-sulfate. Moreover, authentic JGIcUA - GalNAc(6S) showed considerable resistance to acid hydrolysis under these conditions. Removal of nonreducing terminal 4UA with a glucuronidase from Fiavobacterium heparinum (18) may be a better approach. The significance of nonsulfated galactosamine residues is also of interest in relation to sulfation and epimerization. Studies on the macromolecular properties of dermatan sulfate-chondroitin sulfate copolymers alone or interacting with other biopolymers will aid an understanding of the function of galactosaminoglycans in connective tissues. We are grateful to Prof. G. Matsumura for his interest and for helpful suggestions. We thank Mrs. Kazuko Uehara for help in the first stage of preparation of umbilical cord galactosaminoglycans. REFERENCES 1. Danishefski, I. & Bella, A. Jr. (1966) / . Biol. Chem. 241, 143-146 2. Fransson, L.-A. (1968) / . Biol. Chem. 243, 15041510 3. Habuchi, H., Yamagata, T., Iwata, H., & Suzuki, S. (1973) / . Biol. Chem. 248, 6019-6028 4. Bitter, T. & Muir, H.M. (1962) Anal. Biochem. 4, 330-334 5. Hoffman, P., Linker, A., & Meyer, K. (1957) Arch. Biochem. Biophys. 69, 435-440 6. Dische, Z. (1947) / . Biol. Chem. 167, 189-198 7. Brown, A.H. (1946) Arch. Biochem. 11, 269-278
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8. Dodgson, K.S. (1961) Biochem. J. 78, 312-319 9. Spiro, R.G. (1972) in Methods in Enzymology (Ginsburg, V., ed.) Vol.28, part B, pp.3-43, Academic Press, New York 10. Weber, P. & Winzler, R.J. (1969) Arch. Biochem. Biophys. 129, 534-538 11. Reissig, J.L., Strominger, J.L., & Leloir, L.F. (1955) / . Biol. Chem. 217, 959-966 12. Park, J.T. & Johnson, M.J. (1949) / . Biol. Chem. 181, 149-151 13. Mathews, M.B. (1956) Arch. Biochem. Biophys. 61, 367-377 14. Fransson, L.-A. & Rcxten, L. (1967) / . Biol. Chem. 242, 4161-4169 15. Seno, N., Anno, K., Kondo, K., Nagase, S., & Saito, S. (1970) Anal. Biochim. 37, 197-202 16. Inoue, S. & Miyawaki, M. (1975) Anal. Biochem. 65, 164-174 17. Rod6n, L., Baker, J.R., Cifonelli, J.A., & Mathews,
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M.B. (1972) in Methods in Enzymology (Ginsburg, V., ed.) Vol.28, part B, pp.73-140, Academic Press, New York Yamagata.T., Saito, H., Habuchi, 0., & Suzuki, S. (1968) / . Biol. Chem. 243, 1523-1535 Stern, E.L., Lindahl, B., & RodSn, L. (1971) / . Biol. Chem. 246, 5707-5715 Fransson, L.-A. & Havsmark, B. (1970) / . Biol. Chem. 245, 4770-4783 Fransson, L.-A. & Rod6n, L. (1967) / . Biol. Chem. 242, 4161-4169 Fransson, L.-A., Antonopoulos, C.A., & Gardell, S. (1970) Carbohyd. Res. 15, 73-89 Hook, M., Lindahl, U., Backstrom, G., Malstrom, A., & Fransson, L.-A. (1974) / . Bid. Chem. 249, 3908-3915 Malstrom, A., Fransson, L.-A., Hook, M., & Lindahl, U. (1975) / . Bid. Chem. 250, 3419-3425 Suzuki, S. (1960) / . Bid. Chem. 235, 3580-3588
/ . Biochem.
