THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 241, No. 1, Issue of January 10, 1966 Printed in U.S.A. Structure of the Chondroitin 4-Sulfate-Protein Linkage Region ISOLATION AND CHARACTERIZATION OF THE DISACCHARIDE ~-O-~-D-GLUCURO~OSYL-D- GALACTOSE* (Receieved for publication, June 21, 19651 LENNART RoDtiNt AND GI~RARD ARMAND$ From the La Rabida-University of Chicago Institute and the Department of Biochemistry, University of Chicago, Chicago, Illinois 60649 SUMMARY A disaccharide composed of glucuronic acid and D-gal- actose has been isolated from chondroitin 4-sulfate and chondroitin 4-sulfate glycopeptides. Jts structure has been determined in the following way. (a) Reduction with boro- hydride demonstrated that galactose constitutes the reduc- ing end of the disaccharide; (b) digestion with /3-glucuroni- dase cleaved the disaccharide into glucuronic acid and galactose, demonstrating a /I linkage; (c) periodate oxidation of the disaccharide methylglycoside resulted in no loss of galactose; and (d) oxidation of the disaccharide with lead tetraacetate degraded the galactose moiety to lyxose. This establishes the structure of the disaccharide as 3-O-p-~- glucuronosyl-n-galactose. A glycosidic linkage between xylose and the hydroxyl group o serine constitutes the carbohydrate-protein linkage in the chon- droitin 4-sulfate-protein complex (2, 3). The presence of galac- tose in glycopeptides from the complex (4) indicates that this sugar is also part of a “bridge” structure between the chondroitin sulfate chains proper and the protein moiety of the complex. On the basis of the composition of the chondroitin sulfate glyco- peptides, it was postulated (4) that the chondroitin sulfate chains are linked to galactose via glucuronic acid residues. Evidence for such a linkage is presented in this paper, which describes the isolation and characterization of the disaccharide 3-O-p-n- glucuronosyl-n-galactose. MATERIALS Chondroitin 4-sulfate glycopeptides were isolated by enzymatic degradation of the chondroitin 4-sulfate-protein complex of bovine nasal septum, as described previously (4). The tetrasac- * A preliminary report has appeared (1). This work was sup- norted bv Grant AM-05996 from the National Institutes of Health. and by grants from The National Foundation and The Chicago Heart Association. f This work was done during the tenure of an Established In- vestigatorship of the American Heart Association. $ Present address, Retina Foundation, Boston 14, Massachu- setts. charide of chondroitin 4-sulfate was prepared by hyaluronidase digestion of chondroitin 4-sulfate and separated by gel filtration (5). Chondroitin 4-sulfate was prepared from bovine nasal septa by digestion with papain, essentially as described by Scott (6). Acetone-dried septa, 500 g, were digested at 65” with 240 mg of crystalline papain (Worthington) in 3 liters of a buffer containing 0.01 M EDTA, 0.01 M cysteine-HCI, and 0.5 M NaCl, pH 6.5. After 24 hours, the solution was clarified by filtration through gauze and subsequently through a pad of Celite. The polysac- charide was precipitated from the combined digest and t.he washings (4 liters) by the addition of 5 liters of absolute ethanol. After standing overnight, the chondroitin 4-sulfate was again dissolved in 4 liters of water and reprecipitated with 2 volumes of ethanol in the presence of 0.5 y0 sodium acetate. The product, dried with ethanol and ether, weighed 155 g. Analyses (see below for analytical methods) were: uranic acid, 29.9%; hexosa- mine, 24.3%; and sulfur, 4.501,. Hexosamine analysis with the Technicon automatic amino acid analyzer showed that glucosa- mine constituted 0.7% or less of the total hexosamine. Alternatively, the chondroitin 4-sulfate was purified from the digestion mixture by precipitation with cetylpyridinium chloride, essentially according to Scott (6) ; an excess of detergent was used in order to avoid precipitation of keratosulfate. Further purifi cation was achieved by reprecipitation with cetylpyridinium chloride from 0.5 M NaCl. The product had a glucosamine con- tent of 1.0% of the total hexosamine. 6-o-P-u-Glucuronosyl-n-galactose was a gift from Dr. Michael Heidelberger. P-Glucuronidase was purchased from Sigma. n-Galactose dehydrogenase was prepared from Pseudomonas saccharophila (7) and purified through the first ammonium sul- fate precipitation. METHODS Glucuronic acid was determined by the method of Dische (8). Galactose analyses were performed with the anthrone method (9) or a modification1 of more specific cysteine-sulfuric acid method (10). Galactose was also assayed enzymatically with n-galactose dehydrogenase, as previously described (11). Hex- osamine was determined after hydrolysis in 4 M HCl for 14 hours, by a modification of the method of Boas (12) with omission of the resin treatment. The glucosamine content of chondroitin 1 Z. Dische, personal communication. 65 by guest on December 29, 2019 http://www.jbc.org/ Downloaded from
Transcript
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 241, No. 1, Issue of January 10, 1966
Printed in U.S.A.
Structure of the Chondroitin 4-Sulfate-Protein Linkage Region
ISOLATION AND CHARACTERIZATION OF THE DISACCHARIDE ~-O-~-D-GLUCURO~OSYL-D-
GALACTOSE*
(Receieved for publication, June 21, 19651
LENNART RoDtiNt AND GI~RARD ARMAND$ From the La Rabida-University of Chicago Institute and the Department of Biochemistry, University of Chicago, Chicago, Illinois 60649
SUMMARY
A disaccharide composed of glucuronic acid and D-gal- actose has been isolated from chondroitin 4-sulfate and chondroitin 4-sulfate glycopeptides. Jts structure has been determined in the following way. (a) Reduction with boro- hydride demonstrated that galactose constitutes the reduc- ing end of the disaccharide; (b) digestion with /3-glucuroni- dase cleaved the disaccharide into glucuronic acid and galactose, demonstrating a /I linkage; (c) periodate oxidation of the disaccharide methylglycoside resulted in no loss of galactose; and (d) oxidation of the disaccharide with lead tetraacetate degraded the galactose moiety to lyxose. This establishes the structure of the disaccharide as 3-O-p-~-
glucuronosyl-n-galactose.
A glycosidic linkage between xylose and the hydroxyl group o serine constitutes the carbohydrate-protein linkage in the chon- droitin 4-sulfate-protein complex (2, 3). The presence of galac- tose in glycopeptides from the complex (4) indicates that this sugar is also part of a “bridge” structure between the chondroitin sulfate chains proper and the protein moiety of the complex. On the basis of the composition of the chondroitin sulfate glyco- peptides, it was postulated (4) that the chondroitin sulfate chains are linked to galactose via glucuronic acid residues. Evidence for such a linkage is presented in this paper, which describes the isolation and characterization of the disaccharide 3-O-p-n- glucuronosyl-n-galactose.
MATERIALS
Chondroitin 4-sulfate glycopeptides were isolated by enzymatic degradation of the chondroitin 4-sulfate-protein complex of bovine nasal septum, as described previously (4). The tetrasac-
* A preliminary report has appeared (1). This work was sup- norted bv Grant AM-05996 from the National Institutes of Health. and by grants from The National Foundation and The Chicago Heart Association.
f This work was done during the tenure of an Established In- vestigatorship of the American Heart Association.
$ Present address, Retina Foundation, Boston 14, Massachu- setts.
charide of chondroitin 4-sulfate was prepared by hyaluronidase digestion of chondroitin 4-sulfate and separated by gel filtration
(5). Chondroitin 4-sulfate was prepared from bovine nasal septa
by digestion with papain, essentially as described by Scott (6). Acetone-dried septa, 500 g, were digested at 65” with 240 mg of crystalline papain (Worthington) in 3 liters of a buffer containing 0.01 M EDTA, 0.01 M cysteine-HCI, and 0.5 M NaCl, pH 6.5. After 24 hours, the solution was clarified by filtration through gauze and subsequently through a pad of Celite. The polysac- charide was precipitated from the combined digest and t.he washings (4 liters) by the addition of 5 liters of absolute ethanol. After standing overnight, the chondroitin 4-sulfate was again dissolved in 4 liters of water and reprecipitated with 2 volumes of ethanol in the presence of 0.5 y0 sodium acetate. The product, dried with ethanol and ether, weighed 155 g. Analyses (see below for analytical methods) were: uranic acid, 29.9%; hexosa- mine, 24.3%; and sulfur, 4.501,. Hexosamine analysis with the Technicon automatic amino acid analyzer showed that glucosa- mine constituted 0.7% or less of the total hexosamine.
Alternatively, the chondroitin 4-sulfate was purified from the digestion mixture by precipitation with cetylpyridinium chloride, essentially according to Scott (6) ; an excess of detergent was used in order to avoid precipitation of keratosulfate. Further purifi cation was achieved by reprecipitation with cetylpyridinium chloride from 0.5 M NaCl. The product had a glucosamine con- tent of 1.0% of the total hexosamine.
6-o-P-u-Glucuronosyl-n-galactose was a gift from Dr. Michael Heidelberger. P-Glucuronidase was purchased from Sigma. n-Galactose dehydrogenase was prepared from Pseudomonas saccharophila (7) and purified through the first ammonium sul- fate precipitation.
METHODS
Glucuronic acid was determined by the method of Dische (8). Galactose analyses were performed with the anthrone method (9) or a modification1 of more specific cysteine-sulfuric acid method (10). Galactose was also assayed enzymatically with n-galactose dehydrogenase, as previously described (11). Hex- osamine was determined after hydrolysis in 4 M HCl for 14 hours, by a modification of the method of Boas (12) with omission of the resin treatment. The glucosamine content of chondroitin
66 Glucuronosylgalactose from Chondroitin 4-Sulfate Vol. 241, No. 1
4-sulfate preparations was obtained in the course of amino acid analysis with the Technicon automatic amino acid analyzer after hydrolysis in 6 M HCl at 100” for 20 hours. Since this procedure results in more pronounced destruction of galactosamine than glucosamine (e.g. Reference 13), the content of glucosamine relative to that of galactosamine is probably lower than in- dicated by the actual analyses. Sulfate was estimated accord- ing to Muir’s modification (14) of the method of Dodgson and Spencer (15). Reducing power was determined by t,he method of Park and Johnson (16).
Paper chromatography was carried out on Whatman No. 1 paper with ethyl acetate-acetic acid-formic acid-water, 18 : 3 : 1:4 (Solvent A), or butanol-ethanol-water, 10 : 3 : 5 (Solvent B). Papers were stained with aniline-hydrogen phthalate (17) or a silver dip reagent (18).
High voltage electrophoresis was carried out on strips (27 X 112 cm) of Whatman No. 3MM paper at 4600 volts for 3 hours in 0.75 M formic acid-l.0 M acetic acid buffer, pH 1.9, or pyridine- acetic acid, pH 6.3 (19). The papers were stained wibh a silver dip reagent (18) or ninhydrin (20). For preparative elec- trophoresis at pH 2, washed papers were used and the time was extended to 7 hours.
Isolation of Glucuronosylgalactose from Chondroitin ..-Suljate- A solution of 20 g of chondroitin 4-sulfate in 2 liters of 1 M HCl was heated at 100” for 4 hours. After cooling, the solution was adjusted to pH 2.0 with Dowex 3, COs=, and passed through a column (10 x 48 cm) of Dowex 50-X8 (H+ form, 200 to 400 mesh). The combined effluent and washings were concentrated to 75 ml with repeated adjustments to pH 2.0 with Dowex 3, COa=. This solution was brought to pH 6.3 by the addition of NaOH. After lyophilization, 900 mg of crude material were recovered which contained principally glucuronic acid, galactose, xylose, and glucuronosylgalactose. This crude mixture was dissolved in approximately 8 ml of water and applied to a column (2.5 x 200 cm) of Sephadex G-25 (fine grade, bead-polymerized), which was eluted with distilled water. Fractions of 12 to 13 ml were collected at a rate not exceeding 25 ml per hour and aliquots were analyzed by the carbazole (8) and anthrone (9) reactions. The disaccharide fraction (Peak I, Fig. 2) was concentrated to a small volume and lyophilized. After drying in a desiccator over P205, the material weighed 120 mg.
Peak 1 was occasionally contaminated with monosaccharides. Paper electrophoresis of the various fractions in pyridine-acetic acid, pH 6.3, provided a simple means for determining the extent of the admixture. The impure fractions were chromatographed on Dowex 1 by a modification of the method described by Weiss- man et al. (21) for the fractionation of hyaluronic acid oligosac- charides. Samples containing up to 100 mg of uranic acid were separated on a column (1 x 20 cm) of Dowex l-Xl0 (formate form, 200 to 400 mesh), which was eluted with 0.03 M formic acid at a rate of approximately 25 ml per hour. After the elution of the disaccharide fraction (carbazole and anthrone analysis) glucuronic acid was eluted by increasing the acid concentration to 0.25 M. The disaccharide-containing eluate was neutralized with NaOH, concentrated to a small volume, and desalted on a column (1.8 x 140 cm) of Sephadex G-25, medium grade. A small amount of NaCl was added to the sample in order to facilitate t,he localization of the salt peak with silver nitrate. The desalted solution was lyophilized.
Isolation of Glucuronosylgalactose from Chondroitin 4-Sulfate Glycopeptides-Crude glycopeptide B (4), 5 mg, was dissolved in
0.5 ml of 0.1 M HCl and heated at 100” for 4 hours. The hy- drolysate was lyophilized and dissolved in 0.1 ml of water, and a lo-p1 aliquot was subjected to high voltage electrophoresis in formic acid-acetic acid buffer, as described above. The com- pound corresponding to the spot marked GG in the electrophoresis pattern (Fig. 7) was isolated by preparative electrophoresis at pH 2.
Reduction of Glucuronosylgalactose with Potassium Borohydride -A preparation of glucuronosylgalactose which had been purified by chromatography on Dowex 1 was used for these experiments. A 4-ml sample containing 210 pg of galactose was mixed with 0.5 ml of a 1 y0 solution of potassium borohydride and kept at room tempera,ture for 2 hours. After the addition of 0.5 ml of 4 nl acetic acid, the reaction mixture was analyzed for galactose (anthrone method) and uranic acid.
Cleavage of Glucuronosylgalactose by Liver fl-Glucuronida.qe-A 2-mg sample of glucuronosylgalactose was dissolved in 0.1 ml of 0.05 M acetate buffer, pH 5.0, containing 0.2 mg of P-glucuron- idase. The solution was incubated overnight at 37” and a lo-p1 aliquot was assayed for free glucuronic acid and galactose by paper chromatography in Solvent A and by high voltage elec- trophoresis in pyridine-acetic acid buffer.
Preparation of Glucuronosylgalactose Methylglycoside-The methylglycoside was prepared in the following way. Glu- curonosylgalactose, 50 mg, was dissolved in 5 ml of anhydrous methanol containing 27, HCl and heated in a sealed tube at 70” for 4 hours. Hydrogen chloride was removed by evaporation under reduced pressure, at a temperature not exceeding 30”, with repeated additions of methanol. The dried material was dissolved in a small amount of water and de-esterified at 4” by the addition of several portions of dilute NaOH until the solution remained permanently alkaline (phenolphthalein as indicator). After dilution to 25 ml, the disaccharide methylglycoside was isolated by chromatography on Dowex l-Xl0 (formate form), followed by desalting on Sephadex, as described above. The yield of glycoside was approximately 40%. It had a molar ratio of uranic acid to galactose of 1.07, as determined by the carbazole and cysteine-sulfuric acid reactions. The reducing power was 1.6% of the galactose content, indicating that nearly complete glycosidation had taken place. After paper chromatog- raphy in Solvent A, staining with the silver reagent followed by steaming showed one spot with an RF value 1.7 times that of the untreated glucuronosylgalactose.
Periodate Oxidation of Glucuronosylgalactose Methylglycoside- A 0.5.ml sample of a solution of the methylglycoside, which con- tained approximately 1 mg of uranic acid per ml, was mixed with 0.5 ml of 0.1 M sodium metaperiodate and the mixture was kept at room temperature in the dark. After 2 hours, 1.0 ml of 0.1 r~r barium acetate was added and the precipitate was removed b> filtration through a sintered glass filter. A 1.5.ml sample of the filtrate was passed through a column (1 x 5 cm) of Dowex 50-X8 (H+ form, 200 to 400 mesh) followed by 8.5 ml of water. The solution was adjusted to pH 6.0 with NaOH, evaporated to dryness, and dissolved in 1.0 ml of water.
The product was then reduced by the addition of three 50.~1 portions of 1% KBH, at l-hour intervals. The reaction was stopped after 3 hours by the addition of 0.2 ml of 4 M acetic acid and the solut,ion was diluted to 5.0 ml. Aliquots of this solution were analyzed for uranic acid by the carbazole method.
A 2-ml sample of the reduced material was passed over a column (1 x 5 cm) of Dowex 50-X8 (H+ form, 200 to 400 mesh)
Issue of January lo,1966 L. Rod&. and G. Armand 67
and evaporated to dryness several times after the addition of methanol in order to volatilize the boric acid. The dried material was hydrolyzed with 2 ml of 1 M HCl at 100” for 5 hours. Bfter neutralization with 2 M NaOH, the hydrolysate was analyzed for galactose by the n-galactose dehydrogenase and the cysteine- sulfuric acid methods. A control sample, in which the methyl- glycoside had been added after precipitation of the periodate with barium acetate, was carried through all the steps of the procedure described above.
Lead Tetraacetate Oxidation of Glucuronosylgalactose-Oxidation of glucuronosylgalactose with lead tetraacetate was carried out according to the procedure described by Charlson and Perlin (22). A I-mg sample of glucuronosylgalactose was dissolved in 10 ~1 of water, and after mixing with 0.1 ml of glacial acetic acid, 0.4 ml of 1% lead tetraacetate in glacial acetic acid was added. Samples of 50 ~1 were withdrawn at the intervals indicated in
%-* + 0 am0 B -- 0
c -- 0
D-- -
FIG. 1. High voltage electrophoresis of chondroitin I-sulfate hydrolysate. A, chondroitin 4.sulfate hydrolysate after treat- ment with Dowex 50; B, glucuronic acid; C, G-O-p-r-glucuronosyl- n-galactose; and D, galactose.
9
8
0 0 200 400 600 800 1000
EFFLUENT VOLUME (ML)
FIG. 2. Fractionation of chondroitin 4.sulfate hydrolysate on Sephadex G-25 after treatment with Dowex 50.
TABLE I
Reduction of 6-O-P-D-~lUCUrOnOSyl-D-galaCtOSe and glucuronosylgalactose from chondroitin Q-sulfate
with borohydride For experimental details, see the text.
Fig. 6 and mixed with 50 ~1 of 27, oxalic acid in glacial acetic acid. After 30 min, the samples were diluted to 3 ml with water and the precipitated lead oxalate was removed by centrifugation. The supernatant was analyzed for pentose by the orcinol method (23). A zero time control was obtained by the addition of a solution of glucuronosylgalactose to a mixture of lead tetra- acetate and oxalic acid.
The reaction product was further characterized by the fol- lowing procedure. Glucuronosylgalactose, 1 mg, was oxidized for l+ hours, as described above, and after dilution of the reaction mixture to 2 ml, lead ions were removed by passage over a column (1 x 5 cm) of Dowex 5OW-X8 (Hf form, 200 to 400 mesh). The effluent was repeatedly concentrated to dryness with several additions of methanol, dissolved in a small amount of water, and neutralized to pH 6 to 7 with NaOH. Samples of this solution were digested with P-glucuronidase and the digest was examined for free glucuronic acid by paper electrophoresis in pyridine- acetic acid buffer, pH 6.3. Separation of pentoses was carried out by electrophoresis in 0.04 M borax buffer, pH 9.2, at 1000 volts for 4 hours, on sheets (27 x 57 cm) of Whatman No. 3MM paper stained with aniline phthalate.
RESULTS
Isolation of Glucuronosylgalactose from Chondroitin 4-Sulfate- Glucuronosylgalactose was isolated from chondroitin 4.sulfate by a relatively simple procedure involving three main steps: (a) hydrolysis in 1 M HCI for 4 hours; (b) removal of cationic material on Dowes 50; and (c) fractionation of the Dowex 50 effluent by gel filtration on Sephadex G-25. The purification achieved after the second step is illustrated in Fig. 1, which shows the paper electrophoresis pattern of the Dowex 50 effluent. Three spots were present, one moving rapidly with the mobility of free glucuronic acid, one with an intermediate mobility identical with that of 6-0-P-r-glucuronosyl-u-galactose, and one which had moved only slightly from the origin. The latter material contained galactose and xylose, as shown by paper chromatog- raphy in Solvent B.
Fractionation of the hydrolysate after Dowex 50 treatment by gel filtration gave the results shown in Fig. 2. The material corresponding to the spot of intermediate mobility emerged first (Peak 1, Fig. 2), closely followed by free glucuronic acid (Peak !z), which in turn was partly separated from the neutral monosac- charides (Peak 3). Finally a small carbazole-positive component (Peak 4) emerged, which was shown by paper chromatography to be glucuronolactone.
Analyses of the material from the first peak gave the following
68 Glucuronosylgalactose from Chondroitin +$-Sulfate Vol. 241, No. 1
results: glucuronic acid, 49.8%; galactose, 46.7 $& (anthrone method) ; reducing power, 50.4yo (glucose as standard) ; and [c# +12”. The molar ratio of uranic acid to galactose (0.99) and the reducing power indicated that the substance was a disac- charide. Hexosamine and amino acids were absent, or were
ORIGIN
1
3 A e k B :(
c ):
D :(e
E
1
8
F 0 0 0
0 i
FIG. 3. Paper electrophoresis of p-glucuronidase digests of 6-O- @-D-glucuronosyl-D-galactose and glucuronosylgalactose from chondroitin 4-sulfate. A, galactose; B, glucuronic acid; C, glucuronosylgalactose from chondroitin 4-sulfate; D, P-glucuroni- dase digest of C; E, 6-C-&D-ghtcuronosyl-D-galactose; and F, p-glucuronidase digest of E.
100
90
A 1
0 2 4 6 8 IO 12 14 16 18 20 22 24 HOURS
FIG. 4. Assay of galactose liberated from 6-C-p-D-glucuronosyl- D-galactose and glucuronosylgalactose from chondroitin -I-sulfate during digestion with p-glucuronidase. A, glucuronosylgalactose from chondroitin 4.sulfate; B, 6-C-o-D-glucuronosyl-D-galactose. Glucuronosylgalactose, 2 mg, in 2 ml of 0.05 M acetate buffer, pH 5.0, was digested with 0.2 mg of p-glucuronidase at 37”. Samples of 0.2 ml were withdrawn at various times and assayed by the D-galactose dehydrogenase method as previously described (11). The total amount of bound galactose present before digestion was determined by analysis with the cysteine-sulfuric acid method.
TABLE II
Periodate oxidation of glucuronosylgalactose methylglycoside
Recovery of galactose
D-Galactose Cyst&e method deh;$t;e;ase
Recovery of uranic acid
Control. Oxidized sample.
% % %
120 70 97 110 90 14
present in trace amounts. The disaccharide nature of the ma- terial was supported by the fact t)hat it emerged from the Sepha-
dex column in the position expected for a disaccharide. The yield of glucuronosylgalactose can be estimated to be approxi- mately 40%, based on the assumption that each chondroitin 4-sulfate chain (mol wt 25,000) contains 1 glucuronosylgalactose unit.
Characterization of Glucuronosylgalactose from Chondroitin &!Mfate-Reduction of the disaccharide with potassium boro- hydride resulted in destruction of 96% of the galactose moiety (Table I), whereas the uranic acid content was reduced by only 11%. A control experiment with 6-@@D-glucuronosyl-n- galactose gave similar results. These findings establish the galactose moiety as the reducing end of the disaccharide molecule.
The nature of the linkage was studied by treatment of the disaccharide with liver P-glucuronidase. The reaction products were examined by paper electrophoresis, as shown in Fig. 3, and by paper chromatography. The fi-glucuronidase digestion ahnost completely cleaved the disaccharide into glucuronic acid and galactose, establishing the presence of a ,&glucuronidic linkage. In contrast, 6-0-P-D-glucuronosyl-u-galactose was cleaved by the enzyme to a small extent only. The liberation of galactose during the enzymatic digestion was followed by quantitative analyses with the use of D-galactose dehydrogenase (Fig. 4). After 24 hours, 99% of the galactose had been released from the chondroitin sulfate disaccharide, whereas under identical conditions 6-O-/-D-glucuronosyl-D-galactose yielded only 8 %.
Paper chromatography in Solvent A of 6-0-P-D-glucuronosyl. n-galactose and glucuronosylgalactose from chondroitin 4-sulfate showed RGill values of 0.31 and 0.40, respectively.
In order t,o determine the linkage position present in glu- curonosylgalactose from chondroitin -l-sulfate, periodate oxida- tion of the methylglycoside was performed. The results are shown in Table II. Under the conditions used (0.05 M periodate, 2 hours at room temperature) the glucuronic acid content was reduced by 86%, whereas no significant decrease in galactose content was observed. The difference in analytical values for galactose obtained by the two methods is probably a reflection of the fact that only free galactose is determined by the D-galac- tose dehydrogenase assay, whereas t,otal galactose and possibly additional chromogens are determined by the cysteine method. Since only the 1 -+ a-linked disaccharide methylglycoside con- tians a periodate-resistant galactose moiety (see Fig. 5), it is concluded that the disaccharide from chondroitin 4-sulfate has a 1 ---f 3 linkage.
Confirmation of these results was obtained by lead tetraacetate oxidation of glucuronosylgalactose from chondroitin 4-sulfate according to the procedure of Charlson and Perlin (23). In a 1 -+ 3-linked disaccharide an aldohexose moiety at the reducing end is oxidized to the corresponding aldopentose, whereas the
FIG. 5. Scheme of results expected on periodate oxidation of glucuronsylgalactose methylglycosides with different linkage positions. Points of cleavage are indicated in each structure.
0’ I I I I, I
0 IO 20 30 120 MINUTES
FIG. 6. Oxidation of glucuronosylgalactose from chondroitin 4-sulfate and 6-&3-D-glucuronosyl-D-galactose with lead tetra- acetate. O----O, glucuronosylgalactose from chondroitin 4-sul- fate; O---O, 6-O-P-D-glucuronosyl-D-galactose.
oxidation of disaccharides with other linkages results in different products. The data shown in Fig. 6 indicate a rapid format.ion of pentose from glucuronosylgalactose from chondroitin 4-sulfate. The reaction was almost complete after 5 min, and at 2 hours the amount of pentose corresponded to 90% of the galactose origi- nally present. In contrast, no pentose formation from 6-O-p-u- glucuronosyl-n-galactose could be detected. (The slow decrease in orcinol color which was observed on oxidation of the latter
compound is presumably due to slow oxidation of its uranic acid moiety.)
High voltage electrophoresis (pyridine-acetic acid, pH 6.3) of the reaction products after lead tetraacetate oxidation of glu- curonosylgalactose from chondroitin 4-sulfate demonstrated the presence of one major component, presumably 2-O-p-n-glu- curonosyl-n-lyxose, which moved slightly faster than glucu- ronosylgalactose and stained pink with aniline-hydrogen phthal- ate. A faint, pink-staining spot was seen at the origin. Treatment of the oxidation product with @-glucuronidase resulted in the formation of glucuronic acid and lyxose, as demonstrated by electrophoresis in pyridine-acetic acid and borate buffers. These findings provide further evidence for a 1 + 3 linkage in glucuronosylgalactose from chondroitin 4-sulfate.
Isolation of Glucuronosylgalactose from Chondroitin &S’ulfate Glycopeptides-In order to establish the presence of the glu- curonosylgalactose unit in the protein-carbohydrate linkage region of the complex, this disaccharide was also isolated from chondroitin 4-sulfate glycopeptides. The identification of glu- curonosylgalactose among the hydrolysis products was facilitated by a comparison of the electrophoresis pattern (Fig. 7) with that obtained from a hydrolysate of chondroitin 4-sulfate tetrasac- charide. Fig. 7 shows that a number of hydrolytic fragments, including those marked GG (glucuronosylgalactose), XS (xylosyl- serine), and GXS (galactosylxylosylserine), were found only in the glycopeptide hydrolysate.
The glucuronosylgalactose component was isolated by prepara- tive paper electrophoresis. Like the disaccharide from chon-
0 XS GXS 0
0 00 @@“A0 @O 0
0 0 000
FIG. 7. High voltage electrophoresis of hydrolysates of chondroitin 4-sulfate glycopeptide B (a) and chondroitin 4-sulfate tetra- saooharide (B). C, galactose; GG, glucuronosylgalactose; XS, xylosylserine; and GXS, galactosylxylosylserine.
70 Glucuronosylgalactose from Chondroitin /,-Sulfate Vol. 241, No. 1
droitin 4-sulfate, it contained equimolar amounts of uranic acid and galactose and had the reducing power expected for a disac- charide. Further confirmation of the identity of this substance with the disaccharide from chondroitin 4-sulfate was obtained by reduction with borohydride and treatment. with ,&glucuron- idase.
DISCUSSION
The present work appears to be the first isolation of the disac- charide 3-0-P-o-glucuronosyl-n-galactose. The data obtained indicate that the disaccharide unit is located in the carbohydrate- protein linkage region of the chondroitin 4-sulfate-protein com- plex. A tentative formulation of the structure of the linkage region may now be proposed on the basis of the following facts: (a) the isolation of glucuronosylgalactose; (b) the finding that chondroitin 4-sulfate glycopeptide B (4) showed a molar ratio of glucuronic acid to galactosamine of 2 : 1; (c) the recent isolation from chondroitin 4-sulfate of a trisaccharide, galactosylgalac- tosylxylose;* and (d) the previously reported isolation of xylosyl- serine and galactosylxylosylserine (2,3). The proposed structure is shown below.
protein
-GlcUA-GalNAc-GlcUA-Gal-Gal-Xyl-serine I I
so, protein
Acknowledgments-The authors are indebted to Drs. A. Dorf- man and J. A. Cifonelli for many stimulating discussions and valuable suggestions in the course of this work. The skillful technical assistance of Mrs. Judy Duh, Mrs. Rita Smith, and Miss Mary Lou Spach is gratefully acknowledged.
2 U. Lindahl and L. Rod&, unpublished data
6. 7.
8. 9.
10.
11. 12. 13.
14. 15. 16.
17. 18.
19.
20.
21.
22. (1956). ’ ’
23. BROWN, A. II., Arch. Biochem., 11, 269 (1946).
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