©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Structural Studies on the Hexasaccharide Alditols Isolated from the Carbohydrate-Protein Linkage Region of Dermatan Sulfate Proteoglycans of Bovine Aorta
DEMONSTRATION OF IDURONIC ACID-CONTAINING COMPONENTS (*)

(Received for publication, October 27, 1994; and in revised form, December 16, 1994)

Kazuyuki Sugahara (1)(§) Yumi Ohkita (1) Yuniko Shibata (2) Keiichi Yoshida (2) Akemi Ikegami (1)

From the  (1)Department of Biochemistry, Kobe Pharmaceutical University, Higashinada-ku, Kobe 658 and the (2)Tokyo Research Institute of Seikagaku Corporation, Higashiyamato-shi, Tokyo 207, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Five major hexasaccharide alditols were isolated from the carbohydrate-protein linkage region of bovine aorta dermatan sulfate peptidoglycans after reductive beta-elimination and subsequent chondroitinase ABC digestion. These molecules account for at least 55.3% of the total linkage region. Their structures were analyzed by enzymatic digestion in conjunction with high performance liquid chromatography, electrospray ionization mass spectrometry, and 500-MHz one- and two-dimensional ^1H NMR spectroscopy. Three of these compounds have the conventional hexasaccharide core; DeltaHexAalpha1-3GalNAcbeta1-4GlcAbeta1-3Galbeta1-3Galbeta1-4Xyl-ol. One is nonsulfated, and the other two are monosulfated on C6 or C4 of the GalNAc residue. They represent at least 6.3, 5.2, and 28.8% of the total linkage region, respectively. The other two compounds have the following hitherto unreported hexasaccharide core with an internal iduronic acid residue in common; DeltaHexAalpha1-3GalNAcbeta1-4IdoAalpha1-3Galbeta1-3Galbeta1-4Xyl-ol. One is monosulfated on C4 of the GalNAc, and the other is disulfated on C4 of the GalNAc and of the galactose residue substituted by the iduronic acid residue. These two compounds account for 35% of the five isolated hexasaccharide alditols and at least 4.3 and 10.7% of the total linkage region, respectively. The latter two structures form a striking contrast to the currently accepted conception that heparin, heparan sulfate, and chondroitin/dermatan sulfate share the common linkage tetrasaccharide core GlcAbeta1-3Galbeta1-3Galbeta1-4Xyl. The biological significance of the isolated structures is discussed in relation to the biological functions and the biosynthetic mechanisms of dermatan sulfate.


INTRODUCTION

Dermatan sulfate is an extracellular matrix component of fibrous connective tissues and is also present on cell surfaces as a proteoglycan. Several biological functions of proteodermatan sulfate are known such as extracellular matrix formation through interaction with several types of collagen(1, 2, 3) , inhibition of the mitogenic activity of transforming growth factor-beta(4) , and inhibition of the cell attachment activity of fibronectin(5) . These functions are attributable to the core protein. Dermatan sulfate chains also mediate activities such as anticoagulant activity, self-association activity, and antiproliferative activity. The anticoagulant activity is expressed by binding to heparin cofactor II(6) . Self-association via protein-protein interactions is known, but it also occurs through carbohydrate-carbohydrate interactions(7) . Dermatan sulfate chains rich in iduronic acid have been demonstrated to be antiproliferative and also interact with spermine(8) . However, the structure-function relationship of dermatan sulfate chains is not fully understood mainly because of their complicated structure.

Structurally, dermatan sulfate is similar to chondroitin sulfate and was previously called chondroitin sulfate B(9) . It is generally accepted that chondroitin sulfate and dermatan sulfate as well as heparin and heparan sulfate are covalently bound to serine of a protein core through the common tetrasaccharide structure GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1- in the carbohydrate-protein linkage region(10, 11) . Attached to the linkage region is a long carbohydrate sequence, a so-called repeating disaccharide region, which is composed of alternately arranged uronic acid and N-acetylgalactosamine residues. The uronic acid in the repeating disaccharide units of chondroitin sulfate is glucuronic acid, whereas that in dermatan sulfate is either iduronic or glucuronic acid. Thus, dermatan sulfate contains two types of disaccharide units, -4GlcAbeta1-3GalNAcbeta1- and -4IdoAalpha1-3GalNAcbeta1-. (^1)The disaccharide repeats are modified by sulfation. The former can be sulfated at C4 or C6 of the GalNAc unit, whereas the latter contains almost exclusively 4-sulfated GalNAc units and a minor proportion of IdoA unit which may be sulfated at C2. Combining sequential arrangements of iduronic acid residues and sulfate groups results in a wide variety of domain structures, some of which could have biological activities.

It is known that iduronic acid-rich glycosaminoglycans, like dermatan sulfate and heparan sulfate, inhibit fibroblast proliferation and that the antiproliferative activity appears to be related to iduronic acid content(12) . Although the role of IdoA units is important, their distribution along a given dermatan sulfate chain or the relationship between the structure and biological functions is not well understood. A recent structural study of the binding domain to heparin cofactor II is the best known example showing the relationship between the structure and biological functions of dermatan sulfate(13) . The results of this previous study revealed a unique hexasaccharide structure containing a cluster of three IdoA(2-sulfate)-GalNAc(4-sulfate) repeats, which comprises only 5% of the disaccharides present in intact dermatan sulfate.

The biosynthetic mechanisms of such a domain structure of dermatan sulfate remain obscure. Dermatan sulfate is synthesized basically by a mechanism similar to that established for chondroitin sulfate. Chain initiation occurs by xylosylation of the serine residue of a core protein, followed by the addition of 2 galactose residues yielding the Gal-Gal-Xyl trisaccharide sequence which links each polysaccharide chain via a terminal glucuronic acid residue to the core protein. Chain elongation then occurs by alternate addition of GalNAc and GlcA units. Modification reactions take place at the polymer level. Iduronic acid in dermatan sulfate is formed from glucuronic acid by epimerization, most likely after C4 sulfation of the adjacent N-acetylgalactosamine(14) . Epimerization and C4 sulfation of N-acetylgalactosamine are tightly coupled, whereas C2 sulfation of iduronic acid occurs after the epimerization and does not seem to play any role in the formation of iduronic acid.

We have been analyzing the structure of the carbohydrate-protein linkage region of various sulfated glycosaminoglycans to investigate the structure-function relationship and the biosynthetic mechanisms of these glycosaminoglycans(15, 16, 17, 18, 19, 20) . Since the linkage region is first constructed in biosynthesis, possible differences in the structure of the linkage region may influence that of the repeating disaccharide region to be synthesized thereafter. Recently, we found novel 4-sulfated and 6-sulfated galactose units in the linkage region of chondroitin sulfate chains(15, 16, 17, 18, 19) . In this study, we isolated and characterized the linkage region oligosaccharides from bovine aorta dermatan sulfate to examine possible structural variability and differences in this region from other glycosaminoglycans, especially chondroitin sulfate.


EXPERIMENTAL PROCEDURES

Materials

Dermatan sulfate peptidoglycans were prepared from bovine cardiac aorta as reported previously(21) . The following authentic tetra- and hexasaccharide alditols were obtained from whale cartilage chondroitin 4-sulfate as reported(16) : DeltaHexAalpha1-3Galbeta1-3Galbeta1-4Xyl-ol, DeltaHexAalpha1-3Gal(4-sulfate)beta1-3Galbeta1-4Xyl-ol, DeltaHexAalpha1-3GalNAcbeta1-4GlcAbeta1-3Galbeta1-3Galbeta1-4Xyl-ol, DeltaHexAalpha1-3GalNAc(4-sulfate)beta1-4GlcAbeta1-3Galbeta1-3Galbeta1-4Xyl-ol, DeltaHexAalpha1-3GalNAc(4-sulfate)beta1-4GlcAbeta1-3Gal(4-sulfate)beta1-3Galbeta1-4Xyl-ol. Other materials were obtained from the following sources: chondroitinase ABC (EC 4.2.2.4), chondroitinase ACII (EC 3.1.6.9), chondroitinase B (EC 4.2.2), chondro-4-sulfatase (EC 3.1.6.9) from Seikagaku Corp., Tokyo; NaBH(4) from Nacalai tesque., Kyoto; NaB[^3H](4) (15 Ci/mmol) from American Radiolabeled Chemicals Inc., St. Louis.

Preparation of ^3H-labeled Glycosaminoglycans

The dermatan sulfate peptidoglycan fraction (100 mg containing 1.1 µmol of serine) from bovine aorta was treated with 2 ml of 1 M NaBH(4), 0.05 M NaOH at room temperature for 24 h. For preparation of the ^3H-labeled sample, the peptidoglycan fraction (1 mg) was treated with 100 µl of 0.24 mM NaB[^3H](4) (15 Ci/mmol), 0.5 M NaOH at room temperature overnight; 50 µl of 1.2 mM NaBH(4), 0.5 M NaOH was then added, and the mixture was left for 2 h to complete the reaction. Both labeled and nonlabeled samples were acidified with glacial acetic acid and then neutralized with 1 M NaOH. Following repeated evaporation with methanol, the residues were reconstituted in water. The ^3H-labeled sample was subjected to gel filtration on a column (0.8 times 55.5 cm) of Sephadex G-50 with 0.25 M NH(4)HCO(3), 7% 1-propanol. The flow-through fractions containing ^3H-labeled glycans were pooled, concentrated to dryness by evaporation, and reconstituted in water. A total radioactivity of 1.3 times 10^7 cpm was recovered into this preparation. A portion (6.1 times 10^6 cpm) of the sample was added to the nonlabeled preparation as a tracer, and the mixture was subjected to gel filtration on a column (0.8 times 55.5 cm) of Sephadex G-50 with 0.25 M NH(4)HCO(3), 7% 1-propanol. The fractions containing ^3H radioactivity were pooled and concentrated.

Enzymatic Treatments

Chondroitinase ABC digestion was carried out as follows. The reduced polysaccharide containing 100 mg of the nonlabeled and 6.1 times 10^6 cpm of the ^3H-labeled polysaccharides was incubated with 1 IU of the enzyme in a total volume of 2.6 ml of 0.05 M Tris-HCl buffer, pH 8.0, containing 0.05 M sodium acetate and 100 µg/ml bovine serum albumin as an enzyme stabilizer for 21 h at 37 °C; 0.1 IU of the enzyme was added after 18 h. Then, the mixture was treated in boiling water for 1 min. The digest was subjected to gel filtration on a column (1 times 115 cm) of Bio-Gel P-2 with 0.25 M NH(4)HCO(3), 7% 1-propanol. Chondroitinase ACII digestion of the isolated linkage oligosaccharides (0.5 nmol) was conducted using 3 mIU of the enzyme in a total volume of 30 µl of 0.03 M sodium acetate buffer, pH 6, at 37 °C for 10 min. The reaction was terminated by heating at 100 °C for 1 min. Chondroitinase B treatment of the isolated linkage oligosaccharide in fraction 14 (0.5 nmol) was performed using 4 mIU of the enzyme in a total volume of 30 µl of 0.05 M Tris-HCl buffer, pH 8.0, containing 100 µg/ml bovine serum albumin for 11 h at 30 °C. Chondro-4-sulfatase treatment of the isolated linkage oligosaccharides (0.5 nmol) was carried out using 20 mIU of the enzyme in a total volume of 30 µl of 0.04 M Tris-HCl buffer, pH 7.5, containing 0.04 M sodium acetate and 100 µg/ml bovine serum albumin for 1 or 14 h at 37 °C.

HPLC

Fractionation of the linkage oligosaccharide alditols and analysis of the enzyme digests of the isolated oligosaccharides were carried out by HPLC essentially as reported previously for the separation of the chondrodisaccharides(22) . Chromatography was performed on an amine-bound silica PA 03 column (4.6 times 250 mm) using a linear gradient of NaH(2)PO(4) from 16 to 530 mM over a 60-min period at a flow rate of 1.0 ml/min at room temperature. Eluates were monitored by absorbance at 232 nm.

Xylitol Analysis

Xylitol analysis was carried out according to Yamada et al. (^2)using 0.5 nmol of each fraction corresponding to 1,500-2,000 cpm. Briefly, the ^3H-labeled oligosaccharide fraction was mixed with 50 µg each of nonlabeled xylitol, galactitol, and galactosaminitol and then hydrolyzed with 1 N HCl at 100 °C for 2 h. The sample was subjected to HPLC on an amine-bound silica column. Elution was performed under isocratic conditions using 80% acetonitrile at a flow rate of 1 ml/min. Eluates were monitored by absorption at 195 nm. Three UV-absorbing peaks corresponding to the carrier sugar alditols were collected for radioactivity measurement by liquid scintillation counting.

500-MHz ^1H NMR Spectroscopy

Linkage oligosaccharides were repeatedly exchanged in ^2H(2)O with intermediate lyophilization. The 500-MHz ^1H NMR spectra were measured on a Varian VXR-500 at a probe temperature of 26 °C as described previously(23) . The spectra of fractions 10 and 14 were also recorded at 15 °C to prevent disturbance by the HOD response. Chemical shifts are given relative to sodium 4,4-dimethyl-4-silapentane-1-sulfonate but were actually measured indirectly relative to acetone ( 2.225) in ^2H(2)O(24) .

Mass Spectrometry

The mass spectrometer used was a Hitachi liquid chromatograph/mass spectrometer, M-1200H, equipped with a quadrupole mass filter with a nominal m/z limit of 2,000. Mass spectra were obtained using a pneumatically assisted electrospray ionization (ESI) method in aqueous 50% methanol solution in negative ion mode. The voltage applied on the ESI capillary was -2,700 volts, and the voltage between the first and the second apertures was -30 volts. The flow rate of the analyte solution was 0.05 ml/min.


RESULTS

Isolation of the Linkage Oligosaccharides

To investigate the structure of the carbohydrate-protein linkage region of dermatan sulfate proteoglycans, a peptidoglycan fraction was prepared from bovine aorta as reported previously(21) . The preparation was depolymerized completely by chondroitinase B as examined by gel permeation chromatography (data not shown), indicating the absence of chondroitin sulfate. Disaccharide analysis of the chondroitinase ABC digest of the peptidoglycan fraction by HPLC showed the DeltaDi-4S component to be prevailing as expected for dermatan sulfate (Table 1). A significant amount of DeltaDi-diS(B), which may be associated with an anticoagulant activity, was also identified.



The peptidoglycan fraction (100 mg, 1.1 µmol as serine) was reduced with alkaline NaBH(4), and a small portion (1 mg) was treated separately with alkaline NaB[^3H](4) to prepare tracers by radiolabeling the reducing ends of the polysaccharides. The nonlabeled (100 mg) and ^3H-labeled reduced fractions (1 mg, 6.1 times 10^6 cpm) were isolated by gel filtration on Sephadex G-50, mixed, digested exhaustively by chondroitinase ABC, and the digest was fractionated by gel filtration on a Bio-Gel P-2 column. In addition to the major UV-absorbing disaccharide fraction, the preceding radioactive fractions, presumed to contain linkage oligosaccharides, were observed and separated into three subfractions (Fig. 1). Fractions B-1, -2 and -3, which contained 31, 53, and 16% of the total radioactivity, respectively, were separately pooled and concentrated. In this study the major radioactive fraction, B-2, was used for isolation of the linkage oligosaccharides as described below. Most (70%) of the ^3H label in fraction B-1 was associated with presumable internal oligosaccharides, which were generated probably as a result of an incomplete depolymerization by chondroitinase ABC, and were not investigated further. Most (90%) of the ^3H label in fraction B-3 was recovered in a trisaccharide by HPLC, which was characterized as DeltaHexAalpha1-3GalNAc(4-sulfate)beta1-4[^3H]HexA-ol by enzymatic analyses in conjunction with HPLC, ESI mass spectrometry and ^1H NMR spectroscopy. (^3)It might have been produced by alkali-peeling degradation of the linkage region. Alternatively or in addition it could have been derived from the reducing termini of free glycosaminoglycan chains in the starting peptidoglycan preparation, which had been possibly generated by tissue endoglycosidase as discussed previously for similar sulfated trisaccharides isolated from various chondroitin/dermatan sulfate preparations including the one used in this study(21) .


Figure 1: Gel filtration of the chondroitinase ABC digests of the ^3H-labeled glycans. The chondroitinase ABC digestion was carried out as described under ``Experimental Procedures.'' The digest was chromatographed on a column (1.0 times 115 cm) of Bio-Gel P-2 with 0.25 M NH(4)HCO(3), 7% 1-propanol as the eluent. The fraction size was 1 ml, and 1-µl aliquots were used for determination of the radioactivity (bullet). Aliquots were also used to measure the absorbance at 232 nm (circle). Fractions B-1, B-2, and B-3 were pooled as indicated. V is at around fraction 90 (not shown).



Fraction B-2, which contained 1.26 µmol of oligosaccharides (as DeltaHexA), was fractionated by HPLC (Fig. 2). Of the separated fractions, fractions 3, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 17 were labeled with ^3H, indicating that they probably contained oligosaccharide alditols derived from the linkage region (Table 2). In this study, the five major fractions 3, 8, 9, 10, and 14 were further purified by rechromatography, yielding 69, 57, 317, 47, and 131 nmol (as DeltaHexA), respectively, per 100 mg of peptidoglycans. They represented 6.3, 5.2, 28.8, 4.3, and 11.9% of the total serine of the starting peptidoglycan preparation, respectively.


Figure 2: HPLC separation of the oligosaccharide alditol fraction. The oligosaccharide alditol fraction (B-2) corresponding to 312 nmol of DeltaHexA was chromatographed on an amine-bound silica column as described under ``Experimental Procedures.'' Elution was performed using a linear gradient of NaH(2)PO(4) as indicated by the dashed line. The elution positions of the authentic unsaturated chondrodisaccharides are indicated by arrows.





Characterization of the Oligosaccharides

Aliquots of the isolated fractions 3, 8, 9, 10 and 14 were acid hydrolyzed and subjected to xylitol analysis by HPLC, where xylitol was well separated from galactitol and galactosaminitol. The results showed that the main component in each fraction was xylitol, suggesting that fractions 3, 8, 9, 10, and 14 contained oligosaccharide alditols derived from the linkage region (Table 3). These fractions were next subjected to chondroitinase ACII digestion followed by HPLC analysis. Fractions 3, 8, and 9 were each degraded into equimolar amounts of an unsaturated disaccharide and the presumed common nonsulfated unsaturated tetrasaccharide alditol, DeltaHexAalpha1-3Galbeta1-3Galbeta1-4Xyl-ol (Fig. 3). The disaccharide components derived from fractions 3, 8, and 9 were DeltaDi-0S (Fig. 3B), DeltaDi-6S (Fig. 3C), and DeltaDi-4S (Fig. 3D), respectively. These results indicate that fractions 3, 8, and 9 shared the common core hexasaccharide alditol DeltaHexAalpha1-3GalNAcbeta1-4GlcAbeta1-3Galbeta1-3Galbeta1-4Xyl-ol containing an internal GlcA unit and that the GalNAc unit was nonsulfated in the compound in fraction 3 and sulfated at C6 and C4 in the compounds in fractions 8 and 9, respectively. These structures were confirmed by ^1H NMR spectroscopy (see below).




Figure 3: HPLC analysis of chondroitinase ACII digests of the isolated sugar alditols. Panel A, elution positions of the authentic chondrodisaccharides, acetate, and the authentic tetrasaccharide, DeltaHexA-Gal-Gal-Xyl-ol. Panels B-D, chondroitinase ACII digests of fractions 3, 8, and 9, respectively. The arrows in panels B-D indicate the elution positions of fractions 3, 8, and 9 before digestion, respectively. For the conditions of chondroitinase ACII digestion see ``Experimental Procedures.''



Fraction 14 was partially (10%) degraded by chondroitinase ACII into DeltaDi-4S and the presumed unsaturated tetrasaccharide DeltaHexAalpha1-3Gal(4-sulfate)beta1-3Galbeta1-4Xyl-ol (data not shown), indicating that the structure of this minor component was most likely DeltaHexAalpha1-3GalNAc(4-sulfate)beta1-4GlcAbeta1-3Gal(4-sulfate)beta1-3Galbeta1-4Xyl-ol reported previously for chondroitin 4-sulfate from whale cartilage (16) . However, the major compound accounting for 90% of this fraction remained undigested, and fraction 10 was also resistant to chondroitinase ACII (data not shown), indicating that the structures of the compound in fraction 10 and of the major component in fraction 14 were clearly different from the hexasaccharide alditols isolated previously from the linkage region of chondroitin sulfate in terms of their sensitivity toward chondroitinase ACII. The results suggested that the internal uronic acid in fractions 10 and 14 was not glucuronic acid, but more likely iduronic acid.

ESI/MS of underivatized fractions 10 and 14 were analyzed. Fraction 14 gave doubly charged ions [M-2H], [M+Na-3H], and [M+2Na-4H] at m/z 585, 596, and 607 as well as triply charged ions, [M-3H], [M+Na-4H], and [M+2Na-5H] at m/z 390, 397, and 404 (Fig. 4B). The singly charged ion [M-H] was also observed at m/z 1,172. All these molecular ions corresponded to the approximate molecular weight of 1,173 of the compound in this fraction. Only a doubly charged ion [M-2H] was observed at m/z 546 for fraction 10 (Fig. 4A), which corresponded to the estimated molecular weight of 1,094 of the compound in this fraction.


Figure 4: Negative ion mode ESI/MS of fractions 10 and 14. Panel A, fraction 10; panel B, fraction 14.



To determine the positions of the sulfate groups, aliquots of fractions 10 and 14 were subjected to chondro-4-sulfatase digestion followed by HPLC analysis. After a 1-h incubation, fraction 10 eluted at position a was partially degraded, giving rise to a degradation product at position b which accounted for 30% of fraction 10 used (Fig. 5A). After a 14-h incubation, it was completely converted to the compound at position b (Fig. 5B), corresponding to the authentic nonsulfated hexasaccharide alditol DeltaHexAalpha1-3GalNAcbeta1-4GlcAbeta1-3Galbeta1-3Galbeta1-4Xyl-ol. Thus, the compound in fraction 10 contained one sulfate group on C4 of either GalNAc or one of the 2 galactose residues. It is known that chondro-4-sulfatase acts on C4 of both GalNAc and the galactose substituted by GlcA in the linkage region(16) . Chondro-4-sulfatase treatment of fraction 14, which eluted at position c before digestion, resulted in two peaks after a 1-h incubation, one of which (43%) eluted at position d1 and the other (33%) at position d2 (Fig. 5C). When exhaustively treated, fraction 14 was completely converted to the compound at position d2, which seemed to correspond to position b (Fig. 5D). Position d1 was close to the elution position of fraction 10, indicating that the compound at position d1 was also a monosulfated hexasaccharide alditol. Thus, fraction 14 seemed to contain two sulfate groups on C4 of GalNAc and/or 2 galactose residues. It was noted that only one partially desulfated product was observed, suggesting that chondro-4-sulfatase acted sequentially by first removing one then the other sulfate group. It would appear that this enzyme acted preferentially on one of the two sulfate groups. Which sulfate group, however, remains to be determined. The results indicate that fractions 10 and 14 had the common hexasaccharide core structure DeltaHexAalpha1-3 GalNAcbeta1-4HexA1-3Galbeta1-3Galbeta1-4Xyl-ol, with one or two sulfate groups on either C4 of GalNAc or galactose residue(s) underlined.


Figure 5: HPLC analysis of the chondro-4-sulfatase digests of the isolated hexasaccharide alditols. Fractions 10 and 14 were digested with chondro-4-sulfatase for 1 or 14 h and then chromatographed. Panel A, fraction 10 after a 1-h digestion; panel B, fraction 10 after a 14-h digestion; panel C, fraction 14 after a 1-h digestion; panel D, fraction 14 after a 14-h digestion. Black and white arrows indicate the elution positions of the fractions before and after digestion, respectively. The elution positions of the authentic chondrodisaccharides and hexasaccharide alditols derived from the carbohydrate-protein linkage region of whale cartilage chondroitin 4-sulfate (16) are indicated in panel A by numbered arrows as follows: 1, DeltaDi-0S; 2, DeltaDi-6S; 3, DeltaDi-4S; 4, DeltaDi-diS(D); 5, DeltaDi-diS(E); 6, DeltaDi-triS; 7, DeltaHexA-GalNAc-GlcA-Gal-Gal-Xyl-ol; 8, DeltaHexA-GalNAc(4-sulfate)-GlcA-Gal-Gal-Xyl-ol; 9, DeltaHexA-GalNAc(4-sulfate)-GlcA-Gal(4-sulfate)-Gal-Xyl-ol.



To identify the internal hexuronic acid residue, the sensitivity of fractions 10 and 14 toward chondroitinase B was investigated. Fraction 14 was degraded into two products in equimolar amounts as detected by absorbance at 232 nm (Fig. 6A), while ^3H radioactivity was found only in the faster eluting UV-absorbing peak (Fig. 6B), which corresponded to the position of authentic DeltaHexAalpha1-3Gal(4-sulfate)beta1-3Galbeta1-4Xyl-ol. The nonradiolabeled peak coincided with authentic DeltaDi-4S. Thus, the compound in fraction 14 most likely had the disulfated hexasaccharide alditol structure with iduronic acid at the internal position, DeltaHexAalpha1-3GalNAc(4-sulfate)beta1-4IdoAalpha1-3Gal(4-sulfate)beta1-3Galbeta1-4Xyl-ol. In contrast, fraction 10 was resistant to chondroitinase B digestion (data not shown).


Figure 6: HPLC analysis of the chondroitinase B digest of fraction 14. The isolated fraction 14 was digested with chondroitinase B as described under ``Experimental Procedures,'' and the digests were subjected to HPLC on an amino-bound silica column. Eluates were monitored by absorption at 232 nm (panel A), and ^3H radioactivity (panel B). The elution positions of the authentic chondrodisaccharides, tetrasaccharide alditols obtained from whale cartilage chondroitin 4-sulfate(16) , and fraction 14 before digestion are indicated in panels A and B by numbered arrows: 1, DeltaDi-0S; 2, DeltaDi-6S; 3, DeltaDi-4S; 4, DeltaDi-diS(D); 5, DeltaDi-diS(E); 6, DeltaDi-triS; 7, DeltaHexA-Gal(4-sulfate)-Gal-Xyl-ol; 8, fraction 14 before digestion.



500-MHz ^1H NMR Spectroscopy

Fractions 3, 8, 9, 10, and 14 were analyzed by 500-MHz ^1H NMR spectroscopy. Proton chemical shifts were assigned using two-dimensional HOHAHA and COSY analysis. The NMR data are summarized in Table 4. As presumed from the enzymatic analysis described above, the spectral data of fractions 3, 8, and 9 were in good agreement with those reported for Fr. A, B, and C, respectively, which were isolated previously from the linkage region of chondroitin 4-sulfate from whale cartilage(16) . Thus, the structures of the compounds in fractions 3, 8, and 9 were confirmed to be identical to those from whale cartilage chondroitin 4-sulfate as follows.



The one-dimensional ^1H NMR spectrum of fraction 14 measured at 26 °C is shown in Fig. 7. The inset is the spectrum recorded at 15 °C to suppress the disturbance by HOD line. The resonances at 5.962 and 2.115 ppm are characteristic of the H-4 proton of DeltaHexA (15) and the acetoamide group protons of GalNAc, respectively. Close inspection of the spectrum identified the H-4 signal of Xyl-ol at 3.987 ppm. The resonances between 4.6 and 5.3 ppm are characteristic of anomeric protons, and those at 4.620, 4.686, 4.736, and 5.265 ppm were identified as H-1 resonances of Gal-2, GalNAc-5, Gal-3, and DeltaHexA-6, respectively, by comparison with the spectral data (Table 4) of the reference compound Fr. D (DeltaHexAalpha1-3GalNAc(4-sulfate)beta1-4GlcAbeta1-3Gal(4-sulfate)beta1-3Galbeta1-4Xylol) derived from whale cartilage chondroitin 4-sulfate. The signals at 4.620 and 4.736 had a coupling constant J of approx7.5 Hz for the H-1 of betaGal. The signal at 4.686 had a coupling constant J of approx8.5 Hz for the H-1 of betaGalNAc(23) . The signal at 5.147 had a coupling constant J of approx2.5 Hz, indicating that it was the H-1 signal of alphaIdoA-4, since an internal GlcA or IdoA residue of heparin/heparan sulfate oligosaccharides has been reported to give an anomeric proton signal at around 4.6 or 5.0 ppm and the different coupling constants J of 8.0 or 3.0 Hz, respectively(25) . Starting with these anomeric proton signals, H-2, H-3, and H-4 signals of the corresponding sugar residues were localized unambiguously with the aid of the COSY spectrum recorded at 15 °C (not shown) along the cross-section of the two-dimensional HOHAHA spectrum (Fig. 8). It is worth noting that a further connection from H-4 of IdoA to the H-5 resonance at 4.742 (26) was clearly observed in the COSY spectrum at 15 °C, and it is indicated along the top horizontal line in the HOHAHA spectrum. The NMR data are summarized in Table 4and were in reasonable agreement with those of the reference compound Fr. D, except for the data regarding HexA-4. H-1, H-2, H-3, and H-4 proton signals at 4.686, 4.078, 4.160, and 4.628 are characteristic of 4-sulfated betaGalNAc (see the NMR data of fraction 9 in Table 4). Similarities of the chemical shifts of H-1, H-3, and H-4 of Gal-3 to those of the reference compound D and the large downfield shift of H-4 as compared with that of Gal-3 in fraction 3, 8, or 9 support 4-sulfation of this residue. A significant difference in chemical shift (Delta 0.058) of betaGalNAc-5 H-1 was observed between fraction 14 and the reference compound Fr. D, which was attributed to its linkage to the different HexA isomers. Thus, the NMR data are consistent with the following structure proposed above based upon the results of enzyme digestion. The minor component, disulfated hexasaccharide alditol detected by chondroitinase ACII digestion in this fraction, was not observed by ^1H NMR because of the limited amount.


Figure 7: Structural reporter group regions of the 500-MHz ^1H NMR spectrum of fraction 14 recorded in ^2H(2)O at 26 °C. The inset is the spectrum recorded at 15 °C. The numbers and letters in the spectra refer to the corresponding residues in the structures.




Figure 8: Two-dimensional HOHAHA spectrum of fraction 14 recorded at 15 °C.



The NMR data of fraction 10 are presented in Table 4. Anomeric proton resonances of Gal-2, IdoA-4, and DeltaHexA-6 were identified without difficulty in the one-dimensional spectrum at 26 °C by comparison with the spectral data of fraction 14 and the reference compound D, whereas those of Gal-3 and GalNAc-5 were identifiable in the one-dimensional spectrum recorded at 15 °C (data not shown). Although H-1 signals of Gal-3 and GalNAc-5 were close to each other, they were distinguished based on the slightly different values of the coupling constants J as described above for those of fraction 14. Assignments of most of other resonances belonging to Gal-2, IdoA-4, and DeltaHexA-6 were made using the COSY spectrum at 26 °C (data not shown). A cross-peak between H-2 and H-3 of Gal-2 was not identified in the COSY spectrum because of the spectral complexity in the corresponding region, but connections from H-1 to H-2 and from H-4 at 4.205, a structural reporter group of Gal-2, to H-3 resonances were readily observed. The signal at 4.158 with a coupling constant of 3.0 Hz is characteristic of H-4 of nonsulfated Gal-3. The chemical shifts of protons belonging to the component sugar residues except for HexA-4 of this oligosaccharide were similar to those of the corresponding portion of the hexasaccharide alditol in fraction 9, whereas the chemical shifts of HexA-4 of fraction 10 resembled those of alphaIdoA-4 of fraction 14. A connection of H-4 to H-5 of this residue was also visible in the COSY spectrum. The H-1 and H-4 resonances of the betaGalNAc-5 at 4.683 and 4.623 were consistent with the 4-sulfation of this residue. Based on these NMR data, the following monosulfated hexasaccharide alditol structure was proposed for the compound in fraction 10.


DISCUSSION

In this study, we isolated five major hexasaccharide alditols from the carbohydrate-protein linkage region of a peptidoglycan preparation of bovine aorta, which is assumed to contain dermatan sulfate chains derived from small proteoglycans such as decorin and biglycan(27, 28, 29) in addition to those from large proteoglycans(30) . Three of these (fractions 3, 8, and 9) were isolated previously from chondroitin 4-sulfate(15, 16) , chondroitin 6-sulfate(18) , and oversulfated chondroitin sulfate rich in 4,6-disulfated GalNAc(20) . However, the other two (fractions 10 and 14), which contain an iduronic acid residue as the innermost uronic acid, have not been reported previously. Since the discovery of the unique common carbohydrate sequence in the linkage region of various sulfated glycosaminoglycans including heparin, heparan sulfate, chondroitin sulfate, and dermatan sulfate, it has been accepted that the innermost uronic acid residue immediately adjacent to the linkage trisaccharide sequence of these glycosaminoglycan chains is glucuronic acid(10, 11) . It was demonstrated recently that heparan sulfate shares this tetrasaccharide sequence in the carbohydrate-protein linkage region(25) . In the case of pig skin dermatan sulfate, the first uronic acid residue adjacent to a galactose residue has been demonstrated to be almost exclusively glucuronic acid, although glucuronic acid accounts for only 4-10% of the total uronic acid(31) . No evidence has been obtained for the presence of iduronic acid at this position in such preparations. In contrast, the present study demonstrated iduronic acid at this position in bovine aorta dermatan sulfate, indicating that this structural feature characteristic of dermatan sulfate emerges at the 4th saccharide residue from the attachment site to the core protein. Although how general this structure is among dermatan sulfates of various cells and tissues and how variable it is during development, aging, or pathogenesis remain to be investigated, this structure may have important implications in the expression of biological functions and in the biosynthetic mechanisms of dermatan sulfate. Since a mixed population of proteoglycans from undivided bovine cardiac aorta was used in this study, a next step in these analyses will be to identify the proteoglycan species that manifests the iduronic acid-containing linkage oligosaccharides and to localize such a proteoglycan species in specific tissue areas of aorta.

The dermatan sulfate chain with iduronic acid closer to the linkage region might be more flexible and mobile and thus may be able to swing around the core protein due to the specific conformational properties of iduronic acid. Molecular mechanics and NMR studies indicated that iduronic acid may be present in one of the three low energy conformations, ^1C(4), ^2S(0), or ^4C(1), or in all these three forms in rapid dynamic equilibrium. This equilibrium is highly sensitive to sulfation and carbohydrate sequence as well as to intermolecular factors such as cation binding(32) . Iduronic acid has never been demonstrated in the first uronic acid position of the other iduronic acid-containing glycosaminoglycans heparin or heparan sulfate. Heparan sulfate has a long nonsulfated stretch of more than eight repeating disaccharide units, which are assumed to contain only glucuronic acid(33, 34, 35) , and therefore would be rather rigid in the proximal portion to the linkage region but plastic in the distal portion. In heparin, iduronic acid begins appearing nearer the linkage region than in heparan sulfate, but not in the first uronic acid position(17, 36) .

In the biosynthesis of dermatan sulfate chains, C5 epimerization of glucuronic acid to iduronic acid is considered to be critical(37) . Once one iduronic acid is formed, the C5 epimerase seems to continue to make more iduronic acid thereafter, indicating that the formation of the very first iduronic acid is a key step. Although the selection mechanism of such a target glucuronic acid is unknown, the iduronic acid found at the most proximal position to the linkage region might have been one of the first. Transfer of N-acetylgalactosamine to a dermatan sulfate oligosaccharide with iduronic acid at the nonreducing terminus has been observed(38) , which is in contrast to the biosynthesis of heparin where transfer of N-acetylglucosamine residues occurs only to glucuronic acid and not to iduronic acid residues(39) . Thus, the first epimerization reaction of glucuronic acid to iduronic acid may trigger dermatan sulfate synthesis rather than heparan sulfate synthesis. However, it does not seem to be a prerequisite for the selection of dermatan sulfate over chondroitin sulfate since the innermost uronic acid residue in the linkage region of pig skin dermatan sulfate has been reported to be almost exclusively glucuronic acid(31) . The iduronic acid adjacent to a galactose residue also supports the notion that the C5 uronosyl epimerase for dermatan sulfate is different from that for heparin or heparan sulfate. It has been suggested that microsomes from cultured fibroblasts contain two different uronosyl epimerases, one for biosynthesis of heparan sulfate and the other for that of dermatan sulfate, and that they have different cofactor and pH requirements(40, 41) . Our results indicate that the latter enzyme can recognize the glucuronic acid residue attached to the adjacent galactose unit and may trigger the epimerization reactions.

Both major and minor compounds in fraction 14 contain another novel 4-sulfated galactose structure, which was first demonstrated in chondroitin 4-sulfate of rat chondrosarcoma (15) and afterwards in that of whale cartilage(16) . It has been suggested that epimerization of glucuronic acid to iduronic acid is enhanced by concomitant C4 sulfation of the N-acetylgalactosamine residue(14, 42) . It would be of interest to investigate whether 4-sulfation of the galactose residue accelerates epimerization of the adjacent glucuronic acid residue.

The results of the present study indicate that there are at least five different subclasses of dermatan sulfate chains with respect to the structure of the linkage region. It is likely that different chains have different patterns of modification. It remains to be determined whether biologically active domain structures such as the binding domain to heparin cofactor II is found on a specific subclass chain. The presence of iduronic acid in the vicinity of the linkage region raises the possibility that biologically active domain structures that require iduronic acid may well be found near the linkage region along the dermatan sulfate chains and presented to the corresponding ligands. Another interesting structural feature is 6-sulfation of the first GalNAc residue found in fraction 8. It should be noted that GalNAc-6-sulfate, which is a minor component of dermatan sulfate, can be found in the closest vicinity of the linkage region.

This study revealed the unexpected substrate specificity of chondroitinase B using the iduronic acid-containing linkage fragments as substrates. Previous studies indicated that the enzyme produced hexa- or larger oligosaccharides attached to the linkage glycopeptides (29) . However, in this study it was shown to degrade the major disulfated hexasaccharide alditol in fraction 14 into a disaccharide and a linkage tetrasaccharide alditol. In contrast, it was inert to the monosulfated counterpart in fraction 10. The enzyme seems to recognize not only the iduronic acid but also the 4-sulfate group on the neighboring galactose. The detailed structural requirements remain to be determined for this enzyme which is potentially useful for structural studies of dermatan sulfate, especially for identification of the highly specific structure GalNAc(4-sulfate)beta1-4IdoAalpha1-3Gal(4-sulfate) in the linkage region of dermatan sulfate.


FOOTNOTES

*
This work was supported in part by the Science Research Promotion Fund from the Japan Private School Promotion Foundation and by Grants-in-Aid for Scientific Research(06808061) and Scientific Research on Priority Areas(05274101) from the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 81-78-441-7570; Fax: 81-78-441-7571.

(^1)
The abbreviations used are: IdoA, iduronic acid; HexA, hexuronic acid; DeltaHexA or DeltaHexA, 4-deoxy-alpha-L-threo-hex-4-enepyranosyluronic acid; DeltaDi-0S, DeltaHexAalpha(1-3)GalNAc; DeltaDi-6S, DeltaHexAalpha(1-3)GalNAc(6-sulfate); DeltaDi-4S, DeltaHexAalpha(1-3)GalNAc(4-sulfate); DeltaDi-diS(D), DeltaHexA(2-sulfate)alpha(1-3)GalNAc(6-sulfate); DeltaDi-diS(B), DeltaHexA(2-sulfate)alpha(1-3)GalNAc(4-sulfate); DeltaDi-diS(E), DeltaHexAalpha(1-3)GalNAc(4,6-disulfate); DeltaDi-triS, DeltaHexA(2-sulfate)alpha(1-3)GalNAc(4,6-disulfate); HPLC, high performance liquid chromatography; ESI, electrospray ionization; MS, mass spectrometry; COSY, correlation spectroscopy; HOHAHA, homonuclear Hartmann-Hahn.

(^2)
S. Yamada, M. Oyama, and K. Sugahara, unpublished results.

(^3)
A. Ikegami, Y. Shibata, K. Yoshida, and K. Sugahara, unpublished results.


ACKNOWLEDGEMENTS

We thank Dr. Makiko Sugiura (Kobe Pharmaceutical University) for measuring the NMR spectra and Ken-ichi Shizukuishi (Hitachi Keisoku Engineering Co., Ibaraki, Japan) for recording the ESI/MS of the oligosaccharides, respectively.


REFERENCES

  1. Vogel, K. G., Paulsson, M., and Heinegård, D. (1984) Biochem. J. 223, 587-597 [Medline] [Order article via Infotrieve]
  2. Bidanset, D. J., Guidry, C., Rosenberg, L. C., Choi, H. U., Timpl, R., and Höök, M. (1992) J. Biol. Chem. 267, 5250-5256 [Abstract/Free Full Text]
  3. Font, B., Aubert-Foucher, E., Goldschmidt, D., Eichenberger, D., and van der Rest, M. (1993) J. Biol. Chem. 268, 25015-25018 [Abstract/Free Full Text]
  4. Yamaguchi, Y., Mann, D. M., and Ruoslahti, E. (1990) Nature 346, 281-284 [CrossRef][Medline] [Order article via Infotrieve]
  5. Lewandowska, K., Choi, H. U., Rosenberg, L. C., Zardi, L., and Culp, L. A. (1987) J. Cell Biol. 105, 1443-1454 [Abstract]
  6. Tollefsen, D. M., Pestka, C. A., and Monafo, W. J. (1983) J. Biol. Chem. 258, 6713-6716 [Abstract/Free Full Text]
  7. Frannson, L.-Å., Nieduszynski, I. A., Phelps, C. F., and Sheehan, J. K. (1979) Biochim. Biophys. Acta 586, 179-188
  8. Belting, M., and Franson, L.-Å. (1993) Glycoconjugate J. 10, 453-460 [Medline] [Order article via Infotrieve]
  9. Meyer, K., Davidson, E., Linker, A., and Hoffman, P. (1956) Biochim. Biophys. Acta 21, 506-518 [Medline] [Order article via Infotrieve]
  10. Lindahl, U., and Rod é n, L. (1972) in Glycoproteins (Gottschalk, A., ed) pp. 491-517, Elsevier, New York
  11. Rod é n, L. (1980) in The Biochemistry of Glycoproteins and Proteoglycans (Lennarz, W. J., ed) pp. 267-371, Plenum Publishing Co., New York
  12. Westergren-Thorsson, G., Önnervik, P. O., Fransson, L.-Å., and Malmström, A. (1991) J. Cell. Physiol. 147, 523-530 [Medline] [Order article via Infotrieve]
  13. Maimone, M. M., and Tollefsen, D. M. (1990) J. Biol. Chem. 265, 18263-18271 [Abstract/Free Full Text]
  14. Malmström, A., Fransson, L.-Å., Höök, M., and Lindahl, U. (1975) J. Biol. Chem. 250, 3419-3425 [Abstract]
  15. Sugahara, K., Yamashina, I., de Waard, P., Van Halbeek, H., and Vliegenthart, J. F. G. (1988) J. Biol. Chem. 263, 10168-10174 [Abstract/Free Full Text]
  16. Sugahara, K., Masuda, M., Harada, T., Yamashina, I., de Waard, P., and Vliegenthart, J. F. G. (1991) Eur. J. Biochem. 202, 805-811 [Abstract]
  17. Sugahara, K., Yamada, S., Yoshida, K., de Waard, P., and Vliegenthart, J. F. G. (1992) J. Biol. Chem. 267, 1528-1533 [Abstract/Free Full Text]
  18. Sugahara, K., Ohi, Y., Harada, T., de Waard, P., and Vliegenthart, J. F. G. (1992) J. Biol. Chem. 267, 6027-6035 [Abstract/Free Full Text]
  19. de Waard, P., Vliegenthart, J. F. G., Harada, T., and Sugahara, K. (1992) J. Biol. Chem. 267, 6036-6043 [Abstract/Free Full Text]
  20. Sugahara, K., Mizuno, N., Okumura, Y., and Kawasaki, T. (1992) Eur. J. Biochem. 204, 401-406 [Abstract]
  21. Sugahara, K., Takemura, Y., Sugiura, M., Kohno, Y., Yoshida, K., Takeda, K., Khoo, K.-H., Morris, H. R., and Dell, A. (1994) Carbohydr. Res. 255, 165-182 [CrossRef][Medline] [Order article via Infotrieve]
  22. Sugahara, K., Okumura, Y., and Yamashina, I. (1989) Biochem. Biophys. Res. Commun. 162, 189-197 [Medline] [Order article via Infotrieve]
  23. Yamada, S., Yoshida, K., Sugiura, M., and Sugahara, K. (1992) J. Biochem. 112, 404-447
  24. Vliegenthart, J. F. G., Dorland, L., and Van Halbeek, H. (1983) Adv. Carbohydr. Chem. Biochem. 41, 209-374
  25. Sugahara, K., Tohno-oka, R., Yamada, S., Khoo, K.-H., Morris, H. R., and Dell, A. (1994) Glycobiology 4, 535-544 [Abstract]
  26. Mascellani, G., Liverani, L., Bianchini, P., Parma, B., Torri, G., Bisco, A., Guerrini, M., and Casu, B. (1993) Biochem. J. 296, 639-648 [Medline] [Order article via Infotrieve]
  27. Ehrlich, K. C., Radhakrishnamurthy, B., and Berenson, G. S. (1975) Arch. Biochem. Biophys. 171, 361-369 [Medline] [Order article via Infotrieve]
  28. Register, T. C., and Wagner, W. D. (1990) Connect. Tissue Res. 25, 35-48 [Medline] [Order article via Infotrieve]
  29. Yoshida, K., Arai, M., Kohno, Y., Maeyama, K., Miyazono, H., Kikuchi, H., Morikawa, K., Tawada, A., and Suzuki, S. (1993) in Dermatan Sulfate Proteoglycans (Scott, J. E., ed) pp. 55-70, Portland Press, London
  30. Mörgelin, M., Paulsson, M., Malmström, A., and Heinegård, D. (1989) J. Biol. Chem. 264, 12080-12090 [Abstract/Free Full Text]
  31. Frannson, L.-Å. (1968) Biochim. Biophys. Acta 156, 311-316 [Medline] [Order article via Infotrieve]
  32. Casu, B., Ferro, D. R., Ragazzi, M., and Torri, G. (1993) in Dermatan Sulfate Proteoglycans (Scott, J. E., ed) pp. 41-53, Portland Press, London
  33. Gallagher, J. T., and Lyon, M. (1989) in Heparin (Lane, D. A., and Lindahl, U., eds) pp. 135-158, Edward Arnold, London
  34. Lindblom, A., Bengtsson-Olivecrona, G., and Fransson, L.-Å. (1991) Biochem. J. 279, 821-829 [Medline] [Order article via Infotrieve]
  35. Lyon, M., Deakin, J. A., and Gallagher, J. T. (1994) J. Biol. Chem. 269, 11208-11215 [Abstract/Free Full Text]
  36. Lindahl, U. (1989) in Heparin (Lane, D. A., and Lindahl, U., eds) pp. 159-189, Edward Arnold, London
  37. Malmstr ö m, A., C ö ster, L., Fransson, L.-Å., Hagner-McWhirter, Å., and Westergren- Thorsson, G. (1993) in Dermatan Sulfate Proteoglycans (Scott, J. E., ed) pp. 129-137, Portland Press, London
  38. Malmström, A., and Fransson, L.-Å. (1971) FEBS Lett. 16, 105-108 [CrossRef][Medline] [Order article via Infotrieve]
  39. Helting, T., and Lindahl, U. (1972) Acta Chem. Scand. 26, 3515-3523 [Medline] [Order article via Infotrieve]
  40. Malmström, A., and Åberg, L. (1982) Biochem. J. 201, 489-493 [Medline] [Order article via Infotrieve]
  41. Malmström, A. (1984) J. Biol. Chem. 259, 161-165 [Abstract/Free Full Text]
  42. Silbert, J. E., Silbert, C. K., Humphries, D. E., and Sugumaran, G. (1993) in Dermatan Sulfate Proteoglycans (Scott, J. E., ed) pp. 147-158, Portland Press, London

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