THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 243, No. 7, Issue of April 10, pp. 1543-1550, 1968

Printed in C.S.A

Formation of Three Types of Disulfated Disaccharides

from Chondroitin Sulfates by Chondroitinase

Digestion*

(Received for plthlication, November 6, 1967)

SAKARU SUZUKI, HIDEHIKO SAITO, TATSUYA YAMAGATA, KIMIKO ANNO, NOBUKO SEKO, YUMIKO I<aw~r,f

AND TAMOTSU FURUHASHI~

From the Department of Chemistry, Faculty of Science, Nagoya Unive?^sity, Chikusa, Nagoya, and the Department of Chemistry, Faculty of Science, Ochanomixu University, Bunkyo, Tokyo, Japan

SUMMARY

By means of enzymatic digestion with chondroitinase-ABC, a novel unsaturated disaccharide bearing two sulfate residues has been derived from chondroitin sulfate of squid cartilage. Various data, particularly the formation of 2-acetamido-2- deoxy-3-0-(P-n-gluco-4-enepyranosyluronic acid)-4-O-sulfo- D-galactose and its 6-O-sulfate isomer by hydrolysis with chondro-6-sulfatase and chondro-4-sulfatase, respectively, and the formation of 2-acetamido-2-deoxy-4,6-di-O-sulfo-D- galactose by mild acid hydrolysis, indicate that the two sul- fate residues are located at positions 4 and 6 of the hexos- amine moiety.

A distinct unsaturated disaccharide also bearing two sul- fate residues has been obtained from chondroitin sulfate B preparations of bovine lung and pig skin. One of the sulfate residues has been shown to be substituted at position 4 of the hexosamine moiety. The resistance of the second resi- due to both chondro-4-sulfatase and chondro-6-sulfatase and of the uranic acid moiety to Flavobacterium heparinum

glucuronidase suggests that the sulfate residue is substi- tuted at position 2 or 3 of the uranic acid moiety.

These disulfated disaccharides are, therefore, isomers of 2-acetamido-2-deoxy-3-0-(2- or 3-O-sulfa+-D-gluco-4-ene- pyranosyluronic acid)-6-0-sulfa-D-galactose, the compound previously found in the digest of chondroitin sulfate from shark cartilage. The three isomeric disulfated disaccharides were separated from nonsulfated and monosulfated homo- logues and from one another by paper chromatography in I-butyric acid-O.5 N ammonia (5:3). The separation of disaccharides from chondroitinase digests by paper chroma- tography permits comparison of chondroitinase digests from

* This study was supported in part by research grants from the Wnist,ry of Education, Japan.

1 This work was taken in part from a thesis submitted by Yu- miko Kawai in partial fulfillment of the requirements for the de- gree of Doctor of Philosophy, Faculty of Science, Nagoya Uni- versity.

0 Present address, Seikagaku Kogyo Company, Ltd., Kurihama 7-3. Yokosuka, Japan.

different chondroitin sulfate preparations and detection of slight variations in the types of sulfate linkage.

The term “chondroitin sulfates 21, I<, and C” was introduced in 1958 by Hoffman, Linker, and Alever (I) to describe three isomeric polysaccharides containing acetylgalactosamine, uranic acid, and sulfate in equimolar proportions. However, with structural work on chondroitin sulfates from various sources, it became increasingly apparent that polysaccharides of this class may have more heterogeneous structures than was orig- inally assumed.

The finding that most of these substances are covalently bound to protein in the native state (for a review see Reference 2) has suggested the possibility that chondroitin sulfates may occur in nature with various modes of linkage of the pal>-sac- charide chains to the protein barkbones or with various com- positions and Fcquences of amino a(& of the protein hack- bones.

Another type of heterogeneity was revealed early in 1940 by the studies of Soda, Egami, and Horigome (3), which implied the presence of extra sulfate groups in chondroitin sulfa,te pre- pared from shark cartilage. Since this original study, Suzuki (4) has shown that most of thr ester sulfate is at position 6 of the acetylgalactosamine group, but part of the ester sulfate residue is at position 2 or 3 of the glucuronic acid group. For this polysaccharide, the term “chondroitin sulfate D” was suggested. An apparently similar range of Type 1) polysaccharides with variable excess sulfate has been reported by Furuhaxhi (5, 6), Mathews (7), and ilnderson and Meyer (8) to occur in rlasmo- branch cartilages.

The presence of oversulfated chondroitin sulfate is not confined to elasmobranch cartilage. Thus, Suzuki (4) demonstrated that even in a purified preparation of ChS-B1 (with S:N ratio

1 The abbreviations used are: ChS-B, ChS-D, ChS-E, ChS-A, and ChS-C, chondroitin sulfates B, I), E, A, and C; AD-is.

1543

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1544 Disulfated Disacchades from Chondroitin Sulfates Vol. 243, ivo. 7

of 0.94) from bovine lung, a disaccharide fract,ion of digests of chondroitinase was obtained which bears two sulfate residues. Aklthough it was a question at that time whether this disac- charide was derived from the CBS-B chain or from impurities which contained a very high proportion of disulfated disac- charides, available data clearly indicated that it was a compound distinct from the disulfated disaccharide from ChS-D. Chon- droitin sulfate “B” fractions \vith sulfate t,o hcxosamine ratios ranging from 1.42 to 1.62 have later been obtained by Seno and Meyer (9) from the skin of elasmobranches.

Furthermore, oversulfated chondroitin sulfates differing from the elasmobranch chondroitin sulfates in their optical rotations and infrared spectra were obtained by Mathews, Duh, and Person (10) from cartilages of squid and of the horseshoe crab. The chondroitin sulfate of squid cartilage has been purified extensively by Kawai, Seno, and ;\nno (II), and has been shown to be a new type of chondroitin sulfate, with properties suggesting that it contains disulfated acetylgalactosamine residues (foi reasons which will become evident, this compound will be termed “chondroitirr sulfate E”).

Although there is an abundance of literature relating t,o t,he occurrence of chondroitin sulfates in various tissues, often not,hing more is reported than the classification into the A, I<, and C types based on erroneous simplification. It is obviously necessary to reinvestigate these subjects with respect to the possibilit’y that there might bc some microheterogeneities in this class of polysaccharides.

As shown in the preceding papers (12, 13), all of the acetylga- lact,osaminyl bonds along the il, B, and C type chains are cleaved by chondroitinase-ABC, yielding a populat,ion of the disaccharides which reflects both quality and quantity, with respect t’o t,hc repeating units, of the polysaccharide chain. ,Is will become evident, all the bonds along the D and E type chains are also susceptible to cleavage with chondroitinase-ABC. There is no doubt, therefore, that detailed information on the structure of t’he chondroitin sulfates can be obtained by sub- jecting the compounds in question to chondroitinase-XBC degradation, with subsequent isolation and identification of the disaccharide fragments. The chromatographic and clcctropho- retie separation of the disaccharides on paper (4) in conjunct,ion with the selective desulfation of the disaccharides b-ith chondro- 4- and -6-sulfatases (12, 13) appears to have great potential usefulness in t,he identification of the fragments.

The present paper is concerned with t,he adaptation of these techniques to the chemistry of ChS-B from bovine lung and pig skin, ChS-D from shark cartilage, and ChS-E from squid cartilage. These polysaccharidcs have been quantitatively degraded by the enzyme to yield, in each case, a characteristic disulfatcd disaccharide in addition to either of the known monosulfated disaccharides, ADi-4s and ADi-6S. The di- sulfated disaccharides isolated from chondroitin sulfates B, I), and E have been shown to differ from one another in their chromatographic mobilities as well as in their susceptibilities to degradation with chondro-4-sulfatase, chondro-6-sulfatase, and E’lavobacteriunz heparinum glucuronidase (12). Evidence

2-acetamido-2-deoxy-3-0-(~-D-g~uco-4-enep~~ranosyl~~ro~~i~ acid). 4-0-sl~lfo-u-galactose; ALli-fiS, 2-acetamido-2-deoxy-3.O-(p-n- glnco-4-enepyranosyluronic acid)-6-o-sulfa-D-galactose; Alli-OS, 2-acetamido-2-deoxy-3-0-(p-n-gluco-4-enepyrarlosylllronic acid)- D-galactose. The remainder of the abbreviations are explained in the text.

that the differences arc due to the nonidentity of the position of sulfate in the three disulfated disaccharides is presented in this paper.

CXPI~:RlhIl’,NTAI, PROCEDURE

Xaferials-The following materials nere prepared by pre- viously described methods: sodium ChS-;2 from bovine nasal septa (12) ; calcium ChS-B from bovine lung (14), purified by ethanol fractionat,ion (15) (for analytical figures see Reference 12); sodium ChS-C from shark cartilage (16); sodium ChS-D with a sulfate to hexosamine ratio of 1.45 from shark cart,ilage (6, 17); sodium ChS-E with a sulfate to hesosamine ratio of 1.55 from squid cartilage (II); ADi-OS, ADi-4S, and ADi-GS from chondroitin sulfates (4, 12) ; chondroitinase-dBC, chondro- 4-sulfatase, and chondro-6-sulfatase from Proteus vulgaris (12) ; and glucuronidase from If’. heparinunl (12).

The optical rotatory dispersion of the ChS-A, -B, -C, -D, and -E preparations is shown in Fig. 1. ls pointed out by David- son (I@, ChS--1 and ChS-C exhibited spectra with negative Cotton effect between 200 mp and 230 mp. There were no marked diffcrcnces in wave length at t’he trough of the Cotton effect among chondroitin sulfat,es 11, C, D, and E. In the dispersion curve of ChS-I$ however, no Cotton effect was en- countered at wave lengths as short as 200 ml*. This is not in accord with the results of Davidson (18), which indicate that a trough of the Cotton effect occurs at 220 rnp wit,h ChS-1% The discrepancy between our data and those of Davidson is difficult t,o explain.

The specific rotations, la]:‘, are -30.0” (c, 2.5, in water) for ChS-.$, -52.3” (c, I, in water) for ChS-B, -14.0’ (c, 2.5, in water) for ChS-C, -18.0” (c, 2.5, in water) for ChS-D, and -36.5” (c, I, in water) for ChS-E.

,Icetylgalactosamine B-sulfate was synthesized by the method of Lloyd (19). Scetylgalact,osaminc 4-sulfate was prepared by hydrolysis of UDl’-acetylgalactosamine 4-sulfate in weak acid essentially as described by Strominger (20), and acetylga- lactosamine 4,6-disulfate from UDP-acetylgalactosamine 4,6- disulfate by the method of Harada et al. (21).

Chemicals other than listed above were obtained from Sigma; Wake Chemical Company, Osaka; and other commercial sources.

:Ilethods--The optical rotatory dispersion curves of chon- droitin sulfates were determined with a Jasco optical rotatory dispersion recorder, model OKI)/GV-5, 11.ith t,he use of 0.574 (w/v) aqueous solutions in a IO-mm thick cell (at 700 to 300 mp) or in a l-mm thick cell (at 300 to 200 mp), and the curves of unsaturated disaccharides wit,h the use of 4 Inn5 aqueous solutions in a l-mm thick cell. All measuremel& were made at room temperature, which was maintained between 19” and 21”. Rotations arc expressed as specific rotations or, altcr- natively, as molecsular rotations when the molecular weights are known.

Descending paper chromatography was carried out on Toyo No. 5111 or Xo. 50 paper (60 cm long) in the following solvents: Solvent I, I-butyric acid-O.5 N ammonia (5:3) ; and Solvent II I-butanol-ethanol-water (52: 32 : 16).

Unless otherwise specified, paper clcctrophoresis was carried out on Toyo Xo..51~~~ paper (60 cm long) in 0.05 M ammonium acetate-acetic acid buffer, pH 5.0, at a potential gradient’ of 30 volts per cm for 45 min (22). Compounds containing A4,5- glucuronic acsid were detected by ultraviolet absorption photog-

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I0 -1800

3 i’

I 200 220 240 260 280 300 380 LOO

WAVELENGTH (mp)

FIG. 1. Optical rotatory dispersion curves of sodium cholldro- itin sulfates A, B, C, L), and E in aqueous solution (c, 0.5). The sample of ChS-B was prepared from the calcium salt by the use of ion exchange resin (12). The values presented were calculated for each sample on the basis of a dry weight of soctilun salt, which was measured hy allowing the sample of sodium chorldroitill sulfat,e to dry to a constant weight at) room t,emperature in a vac- uum over P&j.

raphy (23) or by vicning under a Xlilleralight model S-2837.

Reducing sugars were detected by staining with the aniline

hydrogen phthalate reagent (24) and L&O with the silver nitrate

reagent, (23).

Conditions for the digestion of polysaccharides by choll-

droitinase-AK and methods for the assay of the reactions

(based on determination of increase in absorption at 232 mp)

have already been described (12, 13). Conditions for the de-

sulfntion of unsaturated disaccharides by chondrosulfatases

have also been described previously (12, 13).

The procedures for measureme& of hexosamincs (25),

sulfate (26), and glucuronic acid (27) were modified to a mi-

croscale so that the amounts of sample were in the range of 0.01

to 0.1 pmole.

Digestion by Chow&o&use-ABC axd Separation of Disaccharide Products

Chondroit,in sulfates B,2 D, and E were incubated in buffer

with the purified preparation of chondroitinase-UC. M

substrates were used at 2 mM (as uranic acid), and the increase

in absorption at 232 rnp with t,ime was measured. The result,s

are shown in Fig. 2, which includes comparison curves showing

the degradation of ChS-A and ChS-C. The degradation of

ChS-D took place as quickly as that of ChS-A or ChS-C, while

ChS-B and ChS-E were degraded much more slowly. After

prolonged incubation, the level of unsaturated disaccharides

in all the digests, estimated from ultraviolet absorption measure-

men& with the use of the millimolar absorpt,ion coefficients of

unsaturated disaccharides (see Table I, below, and Reference

* A ChS-B sample was also prepared from pig skin by the met,hod of Meyer et al. (15). The close chemical similarity of this prep- aration to the bovine lung preparation has been indicated by analysis of products of the digestion with chondroitinase-ABC (see the t,ext). Optical rot,atory dispersion is also similar to that of the bovine lung preparation.

I I

,lOO% DEPOLYMERIZATIOI

. D . D

D E D E

I I

20 40 60

TIME (MtN

FIG. 2. Bate and extent of degradation of chondroitin sulfates A, B, C, L), and E with chontfroititlase-ABC. Incubation mix- tures cont,ained, in final volumes of 50~1, substrate polysaccharide, 0.1 pmole (as uranic acid); Tris-HCl, PIT 8.0, 2.5 pmoles; sodium acetate, 3 pmoles; bovine serum alblunin, 5 pg; and 0.008 unit of pnrified chondroitinnse-ABC. Blank mixtures contained heat- inactivated enzyme. Incubation was carried out at 37” and was stopped at the indicated times by adding 0.45 ml of 0.05 M KCl- HCl buffer, pH 1.8. The reaction mixture was then centrifuged at 10,800 X 9 for 10 min, and absorption of the supernatant of each sample was mensllred at 232 ,111~. against the corresponding blank mixture. At, the time indicated by the UUYRU, 0.075 rltlit of enzyme was added to the incubation mixtures of CM-B and ChS-E.

12), rose to nearly 100% of the total uronics acid in the polysac-

charides. This indicates that all the repeating units of the

substrate molecules are converted quantitatively to unsaturated

disaccharides.

Aliquots containing 0.5 pmolc (total) of disaccharides were

withdrawn after the maximum values for absorbanre were ob-

tained and \T-cre chromatographcd on paper in Solvent I. X

reproduction of the chromatogram is shown in Fig. 3. ,Umost

all of the disaccharides produced from ChS-S and ChS-C are

ADi-4S and AI&6S, respectively.3 The identification of these

disaccharides has already been described (4, 12).

The chromatogram of the digest of ChS-B showed the presence

of two components in the digest, and those of ChS-D and ChS-E,

three components in each digest (Fig. 3). The faster moving

component of ChS-B and the fastest moving components of

ChS-I> and ChS-E were as mobile as ADi-4S (t,hese componcnt,s

will be referred to as ADi-4SU, ADi-4SD, and ADi-4SE, respec-

tively). In each digest of ChS-D and ChS-E, a component

moving at the same rate as ADi-6S was also present (to be

referred to as ADi-6SD and ADi-6SE, respectively).

The slower moving component from ChS-B and the slowest

moving components from ChS-D and ChS-E were different

from one another in their chromatographic mobilities (for RF values see Table I; these components will be referred to as

ADi-di&, ADi-di&, and ADi-di&, respectively).

3 Although the ChS-A and ChS-C preparations used were of high purity, faint spots corresponding to ADi-6S and ADiX3, respectively, were revealed on the chromatogram (Fig. 3, dashed circles), It is not clear at present whether the compounds were derived from the same polysacchnride chains as those yielding the principal products or derived from impurities.

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I 546 Disuljated Disaccharides jrom Chondroitin Suljates Vol. 243, Ko. 7

After development, of the chromatogram, each of the ultra- violet light-absorbing regions was cut out into strips, eluted by immersion in 0.01 N HCl at 50” for 10 min (13), and examined for its content of ultraviolet-absorbing material. The apparent yields of the components from 0.5 pmole (as uranic acid) of chondroitin sulfates, calculated from glucuronic acid deter- minat,ion (27), were 0.48 pmole of ADi-4Sn and 0.03 pmole of ADi-diSB from ChS-B; 0.15 @mole of ADi-4SD, 0.18 pmole of ADi-GS,,, and 0.18 pmole of ADi-diSD from ChS-D; and 0.15 pmole of ADi-4SE, 0.04 pmole of ADi-6&, and 0.30 pmole of ADi-di& from ChS-E.

It should be noted here that ADi-4Sn, ADi-4SD, ADi-4SE, ADi-diSB, and ADi-di& gave purple colors on paper chro- matograms and paper electrophoretic strips when sprayed with the aniline hydrogen phthalate reagent (24) and heated at, 110”. ADi-6SD, ADi-6SE, and ADi-diSD, in contrast, gave brown colors. Acetylgalactosamine 4-sulfate and acetylgalactosamine 4,6-disulfate (markers) yielded a purple color, whereas acetylga- lactosamine g-sulfate yielded a brown color under t,he same conditions (28).

To isolate the degradation products for further characteriza- tion, 150 mg of the chondroitin sulfate preparation (B, D, or E) were incubated in 15 ml of 0.05 M Tris-HCl, pH 8.0, containing 1.5 mg of bovine serum albumin, 0.75 mmole of sodium acetat,e, and 18 units of chondroitiuase-ABC. The mixture was incu- bated at 37” for 2 hours, and heated in a boiling water bath for 2 min. The precipitate was removed by centrifugation and discarded. The supernatant solution was concent’rat’ed to about 0.1 volume over PZ05 in a vacuum. The solution was chro- matographed as zones (50 cm) on six sheets of Toyo No. 50 filter paper in Solvent I for 45 hours. The products were lo- cated by viewing under ultraviolet light, and were eluted from the chromatograms with wat,er. Chromatography in Solvent I was repeated with each product to remove small amounts of contaminants, after which the product was eluted with water. The samples were then desalted separately by paper chroma- tography in Solvent II, in which all the samples had little mobility. After the chromatograms were dried, the ultraviolet- absorbing zones near the origin were cut out and eluted with water. The samples t,hus obtained were further purified by paper electrophoresis in 0.05 &I ammonium acetate-acetic acid

A B C 0 E STANDARDS

+ j. 4 j. + i

,--. ;‘-‘; - - :.. ,i A Di-6s ‘../

0

1

I. GA

FIG. 3. Tracing of a paper chromatogram in Solvent I of the chondroitinase digests of chondroitin sulfates A, B, C, L), and E. GA, glucuronic acid standard. Ultraviolet absorption print is represented.

Preparation RCA” in Soivent I

Alli-di& 0.29 Al)i-diSn 0.19 AI>i-diSE 0.35

Compound 1~. 0.52

Compolmd IITd 0.52 -

Electro- phoretic

mobility”

cm

18

18 17.5 14 1t

Molar ratio to glucuronic acid

G&C- tos- Sulfate

amine

0.96 2.06

0.95 2.08

0.87 1.94 0.97 1.02

1.00 1.10

reaction

320 G,OOO 20,000 6,700

0 6,000 11,000 6,900 11,000 0,903

11 Mobility of sample (in cerlt~inleters)/mohility of glrlcuronic acid (GA) standard (in centimeters).

b Electrophoresis was carried out under the conditions described in the text.

c Obtained from ADi-di& by digest,ion with chondro-4-s& fatase (see the legend to Fig. 5).

d Obtained from Al>imdiSI, by digestion with chorrdro-(i-sol- fatase (see the legend to Fig. 5).

buffer, pH 5.0 (cf. Fig. 5), and thoroughly desalted by papel chromatography in Solvent II as described above. Afberwards, the eamples were eluted from t,he papers with water and lyoph- ilizcd.

Iden.t~&ation of Degradafion, Products

Evidence was presented previously (4) that ADi-4SH and ADi-6S, were 2-acetamido-2-deoxy-3-O-(P-n-gluco-4-enepy- ranosyluronic acid)-4.O-sulfo-n-galactose and 2-acetamido-2- deoxy-3-O-(P-n-gluco-4.enepyranosyluronic acid)-6-O-sulfo-D- galactose, respectively. This evidence rested on t,he shady of (a) chemical compositions, (b) ultraviolet and infrared spectral properties, (c) bromine uptake, (d) desulfation by the crude P. vulgaris e&acts (containing chondrosulfatases) followed by ident,ification of the product as 2-acetamido-2-deoxy-3-G(p-n- gluco-4-enepyranosyluronic acid)-n-galactose (ADLOS), and (e) degradation by acid or periodate followed by identification of the products as acetylgalactosamine 4-sulfate (from ADi-4Sn) and acet,ylgalactosamine 6-sulfate (from ADi-6&,). Informa- tion presented at that time also led to the conclusion t,hat ADi- diSD was a derivative of ADi-6s bearing a second sulfat’e residue on the glucuronic acid moiety. New evidence which supports the above conclusions and, furthermore, indicates the isomeric disulfated disaccharide nature of ADi-diSD, ADi-diSn, and ADi-di& is presented below.

Optical Rotatory Dispersion-In Fig. 4 are shown the optical rotatory dispersion curves of the disaccharides in question. L1 markedly negative Cotton effect can be seen in all. The wave lengths at which the cross-over points are found are all close to 232 rnk, suggesting that the occurrence of a double bond (A4,5) at the position conjugating to the carboxylic acid group may cause this Cotton effect,. Apparently the Wacetyl substituent, which is thought to be the source of the Cotton effect exhibited by chondroitin sulfates (cf. Reference 29), is far less effective in the disaccharides than t,his chromophore.

ilnalysis-Analyses of ADi-diSu, ADi-diS,, and ADi-di& gave the result,s shown in Table I. Sulfate analyses clearly indicated that, AlLdiSH, ADi-diSr,, and ADi-diSE were di- sulfated. That ADi-4SB, ADi-4SD, ADi-4SE, ADi-6S,, and

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Issue of April 10, 1968 Xuxuki et al. 1547

ADi-6& are monosulfated was also indicated by sulfate analyses which are not shown in this table. All the compounds had the characteristic ultraviolet absorption spectrum of a disaccharide, unsaturated (Y, /3 to the carboxyl group of the uranic acid moiet)y (4, 30, 31). The millimolar absorption coefficients at 232 rnp, based on A4,5-glucuronic acid content (measured by the car- bazole reaction (27), in which test its molar absorption coefficient is similar to that of glucuronic acid (30)), ranged from 6.0 to 6.9.

ADi-diSB, ADi-di&, ADi-4Sn, ADi-4Sn,, and ADi-4& had extremely low absorption coefhciems at 585 mp in the modified MorganElson reaction (25), whereas ADi-diSD, ADi-6Sn, and ADi-6Ss had millimolar absorption coefficients of about 20. The low values for ADi-diSH and ADi-di& suggest that position 4 of the acetylgalactosamine residue is substituted in each of these compounds (4, 28).

Digestion by Sulfatases-The most highly purified prepara- tions of chondro-4-sulfatase and chondro-6-sulfatase were in- cubated with each of the disaccharides.

On exhaustive digestion with chondro-4-sulfatase, ADi-diSn and ADi-di&, as well as ADi-4Sn, ADi-4Sn, and ADi-4SE, were each converted to compounds with lower electrophoretic mobili- ties (referred to as Compoulrds I, II, and III, respectively) (Fig. 5), while ADi-diSn, alX6SD, and ADi-6S’, were unaffected.

With chondro&sulfatasc, on the other hand, ADi-diSn and ADi-di&, as well as ADi-6So and ADi-6&, were converted to

compounds with lower electrophoretic mobilities (referred to as Compounds IV, V, and VI, respectively) (Fig. 5), while ADi-diSn, ADi-4SB, ADi-4Sn, and ADi-4Sn were not affected.

Compounds III and VI moved at the same rate as ADi-OS on paper electrophoresis and on paper chromatography in

4 : ADi-4S

16

12

6 : ADi-6S B : ADi-diSB

D : ADi-diSr,

E : ADi-diSE

8

- I ‘: 4

0

0 X

s

r::iL 220 2LO 260 280 300

WAVELENGTH (mp 1

FIG. 4. Optical rotatory dispersion curves of unsaturated di- saccharides. The sample was dissolved in water at a concentra- tion of 4 mM with respect to the glucuronic acid content. Rota- tions are expressed as molecular rotations, [4]. The data for ADi- and ADi-6S represent the values for the preparations ob- tained from ChS-A and ChS-C, respectively.

z z * z -v 9 v- 0 IO- m VI

5 f 12-

g 14 -w I) WI v w VW WI -

,Q I IV nv-

2 16- 0

18 - wwwv w

I+1 20

FIG. 5. Effect of chondrosulfatases on unsaturated disaccha- rides. Incubation mixtures contained, in final volumes of 60 ~1, the indicated disaccharide, 0.2 pmole; Tris-HCl, pH 8.0, 3 pmoles; sodium acetate, 3 @moles; bovine serum albumin, 5 pg; and enzyme. 1, No enzyme added; 2, 0.08 unit of chondro-4.sulfatase; 3, 0.08 unit of chondro-6-sulfatase; $, 0.08 unit of chondro-4-sulfatase plus 0.08 unit of chondro-6-sulfatase. After incubation for 30 min at 37”, the entire sample was applied on a strip (60 cm long) of Toyo No. 51A paper wetted with 0.05 M sodium citrate-citric acid buffer, pH 5. Electrophoresis was carried out in the same buffer at a potential gradient of 30 volts per cm for 45 min. Ultra- violet absorption prints are represented.

Solvent 1. The compounds isolated from t)he electrophoretic strips all showed a molar ratio of A4,5glucuronic acid to galac- t,osamine of approximately 1: I, but contained no sulfate. On digestion with F. heparinum glucuronidasc, they were converted to acetylgalactosamine and cr-keto acid (see below). There is little doubt, therefore, that all the compounds designated Com- pounds III and VI are ADi-OS. These data then also indicate that the ADi-4s obt,ained from ChS-B, ChS-D, and ChS-E and the ADi-6s from ChS-D and ChS-E are, in fact, derivatives of ADi-OS bearing a sulfate residue at positions 4 and 6, respec- tively, of the acetylgalactosamine moiety.

Compounds I and IV were as mobile as authentic ADi-4s or ADi-6s on paper electrophoresis but were distinct from them in mobility on paper chromatography in Solvent, I. Both com- pounds contained 1 sulfate residue per molecule (Table I), but the sulfate residue was completely resistant to hydrolysis by the sulfatases. They g ave a positive Morgan-Elson color reaction with the same molar absorption coefficient at 585 rnp as ADi-OS, suggesting that neither position 4 nor position 6 of the acetylga- lactosamine moiety is substituted. Since positions 2 and 3 of the acetylgalactosamine moiety are occupied by the acetylamino group and the A4,5-glucuronic acid residue, respectively, and since position 5 of the A4,5-glucuronic acid moiety is in the pyranose ring, the sulfate residue must be located at position 2 or 3 of the A4,5-glucuronic acid moiety. The fact bhat the glucuronidic bond in Compounds I and IV was completely re- sistant to hydrolysis by 8’. heparinum glucuronidase (see below) gave additional support to this assignment. These data then in- dicate that ADi-diSn and ADi-diS, are derivatives of ADi-4S and AT%GS, respectively, bearin, (7 an additional sulfate residue at position 2 or 3 of the A4,5-glucuronic acid moiety.

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1545 Disulfuted Disaccharides from Chondwitin Sulfates Vol. 243, YTo. 7

Compounds II and V had the same RF values as authentic ADi-6S and ADi-4S, resl)ectivclg, in Solvent I. They gave the molar I,atio, A4,5-glucuronic acid to sulfate, approximately 1: I. The sulfate group was easily liberated from Compounds 11 and V by digestion with chondro-6-sulfatase and chondro-4-sulfatase, respectively (Fig. 5). On hydrolysis w&h 0.04 N HCl (see below), Compounds II and V were largely converted to acetylga- lact~o~amine 6-sulfate and acetylgalact,osamine 4-sulfate, rc- spectivcly. All these dat,a indicate that Compound II is ADi- 6S and Compound V, ADi-4S. Apparently, the sulfat,e residues of ADi-diSE are located at positions 4 and 6 of the acetglga- lact,osaminc moiet,y and are removed by chondro&sulfatase

(product, ADi-6s) and chondla-6-sulfat,ase (product, ADi-4s). Digestion by F. heparinum Glucuronidase-It has been shown

in the preceding paper (12) that chromatography on phospho- cellulose and on hydroxylapatite of the crude F. heparinum extracts Feparated a glucuronidase from chondroitinases. The glucuronidase preparation thu, 7 obtained is pure enough for quantitative degradation of AIXOS t,o acetylgalactosaminc and Lu-keto acid, but, as seen from the formation of %-sulfate from 3%ADi-4S (la), it, still contains a chondro&sulfatase-like enzyme

The preparation of Ii’. Izeparinuln glucuronidare was incubated with ADi-4Sn, ADi-4SU, ADi-4&, ALL6SD, ADi-6SE, ADi-di&, ADi-diSD, and ADi-di& at, pH 5.2.

If 0.3 pmole of ADi-OS, ADi-6SU, or ADi-6SE Teas treated for IO min with 0.023 unit of the enzyme, the glucuronic acid moiety was quantitatively removed as measured by the dccreasc in absorbance at 232 mp (Fig. 6). Chromatographic and elcct’ro- phoretic examinat,ion of the digests indicated the appearance of new products with the same RF values as acetylgalactosamine (from ADi-OS) and acetylgalactosamine 6-sulfate (from ADi-GS, and ADi-6&) (Fig. 7). The products reacted lvith the silver nitrate reagent as well as with t,he aniline hydrogen phthalate

reagent,. A product, which is assumed to he cr-keto acid on the basis of its positive react,ion wit,h t,he o-phenglenediamine reagent, (32), was also revealed on the elecbrophoretic strip of each digest. The compound recovered from the strip formed a hydrazone with the 2,4-dinitrophenylhydrazine reagent (33).

ADi- J ADi-LSB,D,E

ADi-6SD,E ADi-diSB,D

ADi-diSE

I * 20 LO 60 80

TIME (MINI

FIG. 6. Effect of F. heparinum glucuronidase on unsaturated disaccharides. Incubation mixtures contained, in final volumes of 0.2 ml, the indicated disaccharide, 0.3 pmole; acetate buffer, pH 5.2, 10 pmoles; sodium fluoride, 10 pmoles; and enzyme, 0.023 unit. Blank mixtures contained heat-inactivated enzyme. The samples were incubated at 37”; 20-~1 aliquots were removed at the indicated times and diluted with 380 ~1 of 0.05 M KCI-HCl buffer. pH 1.8; and the absorption of each simple was measured at 23i ml* against the corresponding blank mixture.

u CHROMATOGRAPHY

05 ’ 01 , my]

ELECTROPHORESIS

FIG. 7. Tracing of paper chromatogram (leff) and paper electro- phoretogram (right) of the glucuronidase digests of unsaturated disaccharides. After treatment of the indicated disaccharides (0.3~mole) for 80 min under the same conditions as those described in the legend of Fin. 6, 100-J alitruots of the reaction mixtures

A

were subjected to p;per chromatography in Solvent I and paper electrophoresis. The chromstogram was slained with the aniline hydrogen phthalate reagent (24), and the electrophoretogram with the o-phenylenediamine reagent (32). The spots correspond- ing to unsaturated disaccharides could be located also by viewing under an ultraviolet lamp, and they are indicated by open symbols. In the electrophoretogram, the spots corresponding lo unsaturated disaccharides, monosaccharides, and a-keto acid showed greyish green, brown, and purple color, respect,ively. Since acetJqg&c- tosarnine G-sulfate had the same mobilitv as cu-keto acid. which gave a strong purple color, the expected l&own spot,s correspond- ing to acetylgalactosamine &sulfate could not b^e seen in the di- gests of AI%?&, AX-I&, and Alli-di&. In the chromntogram, all the spots showed brown or greyish green color also with the o-phenylenediamine reagent, but no spot giving a purple color of a-keto acid was visualized. The standards used are 1, AIli-diSn; 2, Alli-4s; 3, acetylgalactosamine (i-sulfate; 4, acetylgalactosarnine 4-sulfate; and 5, acetylgalactosamine.

Data are also presented in Fig. 6 which indicate that the un- saturat,ed glucuronic acid residue could not be removed from ADi-diSn and ADi-diS, by the glucuronidasc. Since the monosulfated disaccharides (Compounds I and IV) derived from the disulfated disaccharides by enzymatic desulfation (see above)

. . were likewise resistant to the alucuronidase, substitut,ion in these disaccharides at the glucuronic acid moiet,y allpears t,o

prevent hydrolysis. The substitueut is presumably sulfate. The hydrolysis of ADi-4Sn, ADi-4S,,, ADi-4SE, and ADi-diSE

proceeded much more slowly than that of ADi-OS, ADi-&, and ADi-6SE (Fig. 6). Since the enzyme preparation contains the enzyme similar t,o chondro-4-sulfatase and since paper chroma- tography of the digests (Fig. 7) indicates the production of acetylgalactosamine (from Al%4&, AlX4S,, and ADi-4SE) and acetylgalactosamine 6-sulfate (from ADi-di&) instead of the expected glucuronidase product, acetylgalactosamine 4- sulfate, a,nd acetylgalactosamine 4,6-disulfat,e, respectively, it is probable that both disaccharides are susceptible to the glucuroni- dase only after they have been converted to ADi-OS and ADi-6S, respectively, by the associated sulfatase. The difference in

reaction velocity of the reaction with ADi-di& as compared to the react,ion with ADi-4Ss, ADi-4S,, and ADi-4SE may suggest that the sulfate residue at position 4 of the former is removed at the faster rate than the sulfate residue of the latter by this particular sulfatase.

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Issue of April 10, 1968 Suzuki et al. 1549

2c

e5 1g

CHROMATOGRAPHY ELECTROPHORESIS

FIG. 6. Tracing of paper chromatogram (left) and paper elec- trophoretogram (right) showing the acid hydrolysis of unsaturated disaccharides. All the spots were located by staining with the aniline hydrogen phthalate reagent (24). The spots correspond- ing to unsaturated disaccharides could be located also by viewing under an ultraviolet lamp, and they are indicated by open symbols. The standards used are 1, acetylgalactosamine 4,6-disulfate; 8, Compound IV (see Fig. 5); 3, acetylgalactosamine &sulfate; 4, acetylgalactosamine &sulfate; and 5, acetylgalactosamine.

_Icid Hydrolysis--It has been shown previously (4) that when ADi4S and ADi-6S were hydrolyzed with 0.04 N HCl at 100” for 60 min they gave rise to scetylgalactosamine 4-sulfate and acet~ylgalactosamine (i-sulfate, respectively, in a yield of over 8057;. It was of interest, therefore, to see if acetylgalactosamine 4,6-disulfate could be derived from ADi-di& by acid hydrolysis.

d sample of ADi-di& (0.15 pmole), dissolved in 0.04 N HCl (0.2 ml) in a waled tube, was placed in a boiling water bath for 60 min. The hydrolysate was then freed of acid by alternate addition of water and evaporation in a vacuum (three cycles) and chromatographed on paper in Solvent I. Two new spots were regularly observed on the chromatogram after staining with the aniline hydrogen pht,halate reagent or the silver nitrate reagent (Fig. 8). The faster moving spot coincided in mobility with acetylgalactosamine 6.sulfate, and the slo~cr moving spot with acetylgalactosaminc 4,6-disulfate. When the hydrolysat e of ADi-di& was subjected to electrophoresis in paper at, pH 5, two spots again appeared, one of which coincided in mobility with acetylgalactosamine 6-sulfate and the other with acetylgalactosa- mine 4,6-disulfate. Under the same conditions, ADi-4&s, ADi-4&, and ADi-4SE gave two spots, one behaving like acetylgalactosamine and the other like acetylgalactosamine 4-sulfate on paper chromatography as well as on paper electro- phoresis (Fig. 8). ADi-6SD and ADi-6SE, on the ot’her hand, yielded a single spot corresponding to acetylgalactosamine 6-sulfate.

When ADi-di& was treated with acid under the same condi- tions it gave two spot,s, one moving like acetylgalactosamine 4-sulfate and the other like a monosulfated disaccharide, the mobility of which in Solvent I was the same as that of Compound I derived from ADi-di& by chondro-4-sulfatase (see above). Apparently, the glucuronidic linkage of ADi-diSn is more resistant to acid hydrolysis than that of ADi-OS, ADi-4S, ADi-BS, or ADi-di&, suggesting that this compound might cont’ain a

substituted A4,5-unsaturated glucuronic acid moiety. Like- wise, ADi-diSD yielded two sl)ots, corresponding to acetyl- galactosamine B-sulfate and Compound TV.

M the observations described above support the view that ADi-di& is a derivai,ive of ADi-4S bearing a second sulfate residue on the glucuronic arid moiety, AlKdi&, a derivative of ADi-6S bearing a setzond sulfate residue on the glucuronic acid moiety, and ADi-diS,, a derivative of ADi-OS bearing two sulfate residues OJI the awtylgalactosamine residue, presumably at 1)ositions 4 and 6.

DISCUSRION

It is somelT-hat surprising to find in typical ChS-B preparations, the homogeneity and structure of which we believed well established, a structure of such heterogeneity as is seen in the ChS-I> preparation from shark cartilage. i2lthough our data do not permit a decision as to whether the formation of the disulfated disaccharide, ADi-diSI+, is due to contamination with different chondroitin sulfate or the presence of different structure in some of the chondroitin sulfate molecules, our fractionation studies have indicated that the disulfated structure is closely associated with the typicbal monosulfated structure of ChS-U in regard to precipitation by ethanol from aquenous salt’ solutions, elution from cctylp,vridinium chloride-cellulose columns, electrophoretic mobility, and treatment with I’. heparinum chondroitinase-A%C (12). It peems appropriate here also to note that a hybrid structure consisting of D-glucuronic acid, L-iduronic acid, and

sulfated acetylgalactosamine residues has been proposed by Fransson and Rod& (34) for a W-13 fraction obt,ained from pig skin without the use of testicular hyaluronidase. One would expect, therefore, that the extra sulfate groups are located, in part, at least, on the u-glucuronic acid residue. Since the con- figurational difference between n-glucuronic and L-iduronic acids is eliminated in the unsaturated disaccharides, the question of the position of extra sulfate residues has not been anslvered in the present) study.

llnother unusual f’eaturc of thr present investigation is the isolation of a novel type of disulfated disaccharide, ADi-di&, from squid chondroitin sulfate as a major product of chondroit,in- ase digestion. With the assumption that the disulfated di- saccharide, ADi-di&, and the monosulfated disaccharides, ADi-4Sn arid ADi-6&, arc derived from hybrid 1)olysaccharide chaiw with a homogeneous composition, it may be calculated from the analysis of products of chondroitinase digestion that about 3OTL and 8’i; of the acct,ylgalactosamine residues are sulfated at positions 4 and 6, respectively, but the remainder of the acetylgalactoaalnine residues are all disulfated at positions 4 and 6. ‘I’hc occurrerrcc of this unusual type of chondroitin sulfate in invertebrate rartilagc is an interesting example of structural variations of cholldromucoproteins manufactured in a variety of cvolut’ionary conditions. The survey of connective tissues of invertebrates, cyclostomes, rlasmobranches, and osteichthycs has indicated that there are various types of mucopolysaccharide which closely resemble chondroitin sulfates of mammalian tissues in many respects, but which differ in their unusually high sulfate contents (see the recent review by Mathews (35)). In view of this, it, is of interest t,o investigate different chondroitin sulfate preparations from diverse sources with respect to the types of sulfate linkage. The investigation might serve the dual purpose of approaching the molecular evolution of chondromucoproteins and giving information cow

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1550 Disulfated Disaccharides from Chondroitin Sulfates Vol. 243, No. 7

cerning the enzymatic mechanism of sulfation and the associated regulatory system for chondromucoprotein biosynthesis.

It would seem that the disaccharide mapping technique presented in this paper has considerable potentialities for the structural comparison of chondroitin sulfates from different sources. With this method it is possible to compare chondroitin- ase digests from different samples and to detect differences as slight as the variation of a single sulfate residue in each 15 to 20 repeating units. The amount of material used for a single spot of paper chromatography is enough for a qualit,at,ive disaccharide analysis, and the disaccharides from several spots can be pooled for

1.

2. 3.

4. 5. 6. 7. 8. 9.

10.

11.

12.

quantitative analysis.

REFERENCES

HOFFMAN, P., LINKER, A., AXD MEYER, K., Fed. Proc., 17, 1078 (1958).

22. 23. 24. 25.

SHARON, N., Annu. Rev. Biochem., 35, 485 (196G). 26. SODA, T., EGAMI, F., AND HORIGOME, T., Nippon Kagaku 27.

Zasshi, 61, 43 (1940). 28. SUZUKI, S., J. Biol. Chem., 235, 3580 (1960). FURUHASHI, T., J. Biochem. (Tokyo), 50, 546 (1961). FURUHASHI, T., Biochemistry (Tokyo), 33, 746 (1961). MATHEWS, M. B., Biochim. Biophys. Acta, 58, 92 (1962) ANDERSON, B., AND MEYER, K., Fed. Proc., 21, 171 (1962). SENO, N., AND MEYER, K., Biochim. Bioph,ys. A.&a, 78, 258

(1963).

29. BEYCHOK, S., AND KABAT, E. A., Biochemistry, 4, 2565 (1965). 30. LINKER. A., MEYER. K., AND HOFFMAN. P.. J. Bid. Chem..

31.

32. MATHE~VS, M. B., Dun, J., AND PERSON, P., Xature, 193, 378

(1962). 33. KAWAI, Y., SENO, N., AND ANNO, K., J. Biochem. (Tokyo),

60, 317 (1966). 34. YA~IAGATA, T., SAITO, I-I., HABUCHI, O., AND SUZCKI, S., J.

Biol. Chem.,243, 1523 (1968). 35. MATHEWS, M. B., Clin. Orfhop. 48, 267 (1967).

13.

14.

15.

16. Biochim. Biophys. Ada, 21, 506 (1956).

FURUHASHI, T., AND UCHIDA, K., Biochemistry (Tokyo), 33, 537 (1961).

17. FURUHASHI, T., Biochemistry (Tokyo), 34, 46 (1962). 18.. DAVIDSON, E. A., Biochirn. Biophys. Ada, 101, 121 (1965). 19. LLOYD, A. G., Nalure, 183, 109 (1959). 20. S~L’ROMINGER, J. L., J. Biol. Chem., 237, 1388 (1962). 21. HARADA, T., SHIMIZU, S., NAKANISHI, Y., AR’D SUZUKI, S.,

J. Biol. Chem., 242, 2288 (1967). MARKHAM, R., AND SMITH, J. I~., Biochem. J., 52, 552 (1952). SUZUKI, S., J. Biol. Chem., 237, 1393 (1962). PARTRIDGE, S. M., Nature, 164,443 (1949). STROMINGER, J. L., PARK, J. T., AND THOMPSON, It. E., J.

SAITO, H., YAMAGATA, T., >\ND SUZUKI, S., J. Biol. Chem., 243, 1536 (1968).

MARBET, R., AND WINTERSTEIN, A., H&I. Chim. Ada, 34, 2311 (1951).

MEYER, K., DAVIDSON, E., LINKER, A., AND HOFFMAX, P.,

Biol. Chem., 234, 3263 (1959). DODGSON, K. S., Riochem. J., 78, 312 (1961). DISCHE, Z., J. Biol. Chem., 167, 189 (1947). SUZUKI. S.. AND STROMIXGER, J. L., J. Biol. Chem., 235, 2768

(196Oj.

219, 13 (1956). I

NAKADA, H. I., WOLFE, J. B., HOCHSTEIN, L. I., AND ANDRE- OLI, A. J., Bnal. Biochem., 1, 168 (19GO).

LANNING, M. C., AND COHEN, S. S., J. Biol. Chem., 189, 109 (1951).

LINKER, A., HOFFMAN, P., MEYER, K., SAMPSON, P., AND KORN, E. D., J. Biol. Chem., 235, 3061 (1960).

FRANSSON, I,., AND ROD~N, L., J. Biol. Chem., 242, 4161, 4170 (1967).

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Yumiko Kawai and Tamotsu FuruhashiSakaru Suzuki, Hidehiko Saito, Tatsuya Yamagata, Kimiko Anno, Nobuko Seno,

Sulfates by Chondroitinase DigestionFormation of Three Types of Disulfated Disaccharides from Chondroitin

1968, 243:1543-1550.J. Biol. Chem. 

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