Characteristic Hexasaccharide Sequences in Octasaccharides Derived from Shark Cartilage Chondroitin Sulfate D with a Neurite Outgrowth Promoting Activity*

Satomi NadanakaDagger , Albrecht Clement§, Kimiko MasayamaDagger , Andreas Faissner§, and Kazuyuki SugaharaDagger par

From the Dagger  Department of Biochemistry, Kobe Pharmaceutical University, Higashinada-ku, Kobe 658, Japan and the § Department of Neurobiology, University of Heidelberg, D-69120 Heidelberg, Germany

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

A mouse brain chondroitin sulfate (CS) proteoglycan, DSD-1-PG, bears the DSD-1 epitope and has neurite outgrowth promoting properties. Shark cartilage CS-C inhibits the interactions between the DSD-1-specific monoclonal antibody 473HD and the CS chains of the DSD-1-PG, which is expressed on the mouse glial cells (Faissner, A., Clement, A., Lochter, A., Streit, A., Mandl, C., and Schachner, M. (1994) J. Cell Biol. 126, 783-799). On the other hand, several hexasaccharides isolated from commercial shark cartilage CS-D, which contains a higher proportion of characteristic D units (GlcUA(2-sulfate)beta 1-3GalNAc(6-sulfate)) as compared with CS-C, has the A-D tetrasaccharide sequence composed of an A disaccharide unit (GlcUAbeta 1-3GalNAc(4-sulfate)) and a D disaccharide unit (Nadanaka, S. and Sugahara, K. (1997) Glycobiology 7, 253-263). In this study, the biological activities and the structure of shark cartilage CS-D were investigated. CS-D inhibited the interactions between monoclonal antibody 473HD and DSD-1-PG and also promoted neurite outgrowth of embryonic day 18 hippocampal neurons. Eight octasaccharide fractions were isolated from CS-D after partial digestion with bacterial chondroitinase ABC by means of gel filtration chromatography and anion-exchange high performance liquid chromotography to investigate the frequency and the arrangement of the A-D tetrasaccharide unit in the polymer sequence. Structural analysis performed by a combination of enzymatic digestions with 500-MHz 1H NMR spectroscopy demonstrated that the isolated octasaccharides shared the common core structure Delta HexAalpha 1-3GalNAcbeta 1-4(GlcUAbeta 1-3GalNAc)3 with four, five, and six sulfate esters at various hydroxyl groups in different combinations. In the structure, Delta HexA and GlcUA represent 4-deoxy-alpha -L-threo-hex-4-enepyranosyluronic acid and glucuronic acid, respectively. No D-D tetrasaccharide sequence was found, and discrete D disaccharide units were demonstrated exclusively as A-D tetrasaccharide units in either an A-D-A or an A-D-C hexasaccharide sequence in the five octasaccharides that represented about 5.0% (w/w) of the starting polysaccharides (C denotes the disaccharide GlcUAbeta 1-3GalNAc(6-sulfate)). It remains to be determined whether such characteristic hexasaccharide sequences present in shark cartilage CS-D serve as functional domain structures recognized by some protein ligands.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Chondroitin sulfate (CS)1 chains occur in animal tissues usually as proteoglycans (PGs), in which these polysaccharide chains are covalently attached to a core protein. CS-PGs are present on cell surfaces and in the extracellular matrix of most tissues and are involved in many biological activities. However, the structure of CS chains is only poorly understood. The backbone structure of CS chains consists of repeating disaccharide units, each containing a glucuronic acid (GlcUA) residue and an N-acetylgalactosamine (GalNAc) residue. Diversity in ester-sulfation of both saccharide residues results in structural variability of CS chains, which is assumed to be the basis for its functional diversity. Immunological studies using monoclonal antibodies (mAbs) have revealed that the sulfation profile of CS chains changes with concomitant specific spatio-temporal patterns in various tissues, suggesting that CS isoforms differing in sulfation position and degree perform distinct functions in development (1). Disaccharide units with various sulfation profiles are non-randomly distributed in the CS chains as detected by mAbs (2).

It had been shown previously that the CS-PG named DSD-1-PG from developing mouse brain is located on the surface of immature glial cells in the central nervous system and promotes neurite outgrowth (3). The neurite outgrowth stimulating capacity of DSD-1-PG was strongly reduced by mAb 473HD, which recognizes a glycosaminoglycan epitope present in DSD-1-PG. Surprisingly, shark cartilage CS-C inhibited the interactions between the mAb 473HD and the DSD-1 epitope (3). In agreement with this observation, neurite outgrowth promotion was also abolished by treatment with chondroitinase ABC, which removes the CS chains of the PG (3). It can be concluded from these data that the particular CS structure recognized by mAb 473HD is of physiological relevance in the context of neurite outgrowth. In addition, the rat brain 6B4 CS-PG/phosphacan, which corresponds to the extracellular region of a receptor-like protein-tyrosine phosphatase, PTPzeta /RPTPbeta , was demonstrated to interact with pleiotrophin through the CS chains and the core protein, and to regulate pleiotrophin-induced neurite outgrowth (4). The binding was specifically inhibited by shark cartilage CS-C. Structurally, shark cartilage CS chains possess a unique D disaccharide unit GlcUA(2S)beta 1-3GalNAc(6S) in addition to the common A and C disaccharide units (GlcUAbeta 1-3GalNAc(4S) and GlcUAbeta 1-3GalNAc(6S)), where 2S, 4S, and 6S denote 2-O-, 4-O- and 6-O-sulfate, respectively (5). D units have been found not only in shark cartilage CS but also in the CS of the mouse mast cells derived from immune lymph nodes. The CS-D composed of A and D disaccharide units occurs transiently, functioning as an important phenotypic marker that distinguishes different mast cell subsets (6). CS in the basement membrane of embryonic mouse tooth germ also possesses D disaccharide units and appears transiently. A, C, and D units are produced in this order in a time-dependent manner (1), as demonstrated using mAbs including MO-225, which specifically recognizes the epitope containing a D disaccharide unit (7). Although there is accumulating evidence for the biological importance of CS-D, structural information regarding the sequential arrangement of its disaccharide units and its biosynthetic mechanisms remain unclear.

To investigate the structural features and the biosynthetic mechanism of shark cartilage CS, we have been analyzing the structures of oligosaccharides prepared by bacterial chondroitinase or testicular hyaluronidase digestion of a shark cartilage CS-D isoform, which contains a larger proportion of D disaccharide units than CS-C (21.2 versus 9.62%). Commercial CS-D and CS-C isoforms are purified from cranial cartilages of a shark strain Rhizoprionodon acutus (Rüppell) and a mixture of cartilages from various body parts of multiple shark strains, mainly Prionace glauca (Linnaeus), respectively, according to the manufacturer. Previous findings revealed a unique A-D tetrasaccharide sequence, GlcUAbeta 1-3GalNAc(4S)beta 1-4GlcUA(2S)beta 1-3GalNAc(6S), in one tetrasaccharide and in a few hexasaccharides (8-10). The A-D tetrasaccharide sequence is recognized by the mAb MO-225 (7), and its epitope is transiently expressed and disappears during developmental maturation of mouse odontoblasts, suggesting the epitope to be biologically functional (1). The aim of this study was to investigate the biological activity of CS-D especially in the context of neurite outgrowth, to clarify the distribution of D disaccharide units and A-D sequences along CS-D chains, and to characterize the sulfation profiles surrounding them by analyzing structures of larger oligosaccharides (i.e. octasaccharides). Preliminary data have been reported in abstract form (11).

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- The following materials and enzymes were purchased from Seikagaku Corp., Tokyo, Japan: a sodium salt preparation (super special grade) of shark cartilage CS-D, six unsaturated CS-disaccharides, chondroitinase AC-II (EC 4.2.2.5), conventional chondroitinase ABC, and highly purified chondroitinase ABC (commercialized as a protease-free preparation) (EC 4.2.2.4), and chondro-4-O-sulfatase and chondro-6-O-sulfatase abbreviated as CS-4-sulfatase (EC 3.1.6.9) and CS-6-sulfatase (EC 3.1.6.10), respectively. Delta 4,5Hexuronate-2-O-sulfatase (abbreviated as Delta hexuronate-2-sulfatase) purified from Flavobacterium heparinum (12) was provided by Dr. K. Yoshida, Seikagaku Corp. The CS-D octasaccharide fraction was obtained as reported (8) after digestion with highly purified chondroitinase ABC followed by gel filtration chromatography on a column of Bio-Gel P-10. The following authentic sulfated tetrasaccharides were isolated previously from shark cartilage CS-D (13): Delta HexAalpha 1-3GalNAc(4S)beta 1-4GlcUAbeta 1-3GalNAc(4S), Delta HexAalpha 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc(4S), Delta HexA alpha 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc(6S), Delta HexA(2S)alpha 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc(4S), and Delta HexA(2S)alpha 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc(6S). Delta HexAalpha 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc was prepared by the digestion of Delta HexAalpha 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc(6S) with CS-6-sulfatase.

Cell Culture and Bioassays-- Hippocampal neuron cultures were established from embryonic day 18 rat brain according to the standard procedures (14) with some modifications: hippocampi were dissected in a 37 °C Ca-Mg-free Hank's balanced salt solution supplemented with 0.6% (w/v) glucose and 7 mM HEPES-NaOH, pH 7.4, treated with 0.25% (w/v) trypsin at 37 °C, washed three times with a Ca-Mg-free Hank's balanced salt solution, and dissociated by repeated passage through two fire-polished Pasteur pipettes of different sizes. Cells were plated at a density of 8000 cells/cm2 on glass coverslips precoated with 15 µg/ml poly-DL-ornithine (catalog no. P-0671, Sigma, Deisenhofen, Germany) in 0.1 M borate buffer, pH 8.1, and subsequently with 5 µg/ml uronic acid equivalents of different CS isoforms in phosphate-buffered saline (PBS). The cultures were kept in a humidified atmosphere with 5% CO2 in minimum Eagle's medium containing the N2 supplements of Bottenstein and Sato (15), 0.1% (w/v) ovalbumin, and 0.1 mM pyruvate. After 24 h in culture the cells were fixed with 4% (w/v) paraformaldehyde for 20 min at room temperature, washed three times with PBS, permeabilized with 0.1% (w/v) Triton X-100 in PBS for 15 min, and stained with mAb to alpha -tubulin (clone DM 1A; Sigma) and peroxidase-derivatized secondary antibodies to mouse IgG (Dianova), as described (16). For quantitative analysis, 100 cells were analyzed per coverslip; the neurons growing one fiber exceeding a cell diameter in length were counted as process-bearing, and the corresponding fraction was given as percentage. At least six independent experiments per parameter and condition were carried out.

Enzyme-linked Immunosorbent Assay (ELISA)-- Competition ELISAs were performed as described (3). In brief, DSD-1-PG preparations were absorbed overnight at 4 °C to polyvinylpyrrolidone 96-well plates (Falcon Plastics, Cockeysville, PA) at 0.5 µg/ml uronic acid equivalents, diluted in 0.1 M NaHCO3, pH 8.0 to 100 µl/well. The wells were blocked with 1% (w/v) bovine serum albumin in PBS plus 0.05% (v/v) Tween 20 (PBS-Tween) at least for 1 h at room temperature. The purified mAb 473HD (0.5-1 µg/ml) was preincubated with glycosaminoglycans (GAGs) (10 µg/ml) in the blocking buffer for 2 h at room temperature and used for ELISA on DSD-1-PG in the presence of the competitors for 1 h at room temperature. After three washes with PBS-Tween, a secondary goat anti-rat IgM antibody derivatized with peroxidase (Dianova, Hamburg, Germany) was incubated in the blocking buffer (1:5000) for 1 h at room temperature. Thereafter, the wells were washed three times with PBS-Tween and the ELISA was developed with 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (17). The colored reaction product was quantified in an ELISA reader at 405 nm (Titertec Multiscan, Flow Laboratories, Inc.).

Enzymatic Analysis-- Enzymatic digestion of the isolated oligosaccharides with chondroitinase AC-II or highly purified chondroitinase ABC was carried out using 0.5 nmol of each oligosaccharide substrate and 10 mIU of the enzyme in a total volume of 30 µl of the appropriate buffer at 37 °C for 30 min as described (13). After incubation, the reaction mixtures were boiled at 100 °C for 1 min, cooled to room temperature, mixed with 370 µl of 16 mM NaH2PO4, and analyzed by HPLC. For successive enzymatic digestion of fraction VIb, the fraction (1 nmol as Delta HexA) was first incubated with 10 mIU of highly purified chondroitinase ABC in a total volume of 20 µl of 50 mM Tris-HCl buffer, pH 8.0, containing 60 mM sodium acetate and 100 µg/ml bovine serum albumin at 37 °C for 60 min and boiled at 100 °C for 1 min to terminate the reaction. Half of the sample was analyzed by HPLC, whereas the other half was mixed with 10 µl each of water and a Delta hexuronate-2-sulfatase (2.5 mIU) solution in 20 mM sodium acetate buffer, pH 6.5, containing 0.15% bovine serum albumin and incubated at 37 °C for 120 min. The reaction was terminated and analyzed by HPLC as above. For time-course experiments of chondroitinase AC-II digestion of the oligosaccharides, each fraction containing 0.5 nmol (as Delta HexA) of an oligosaccharide was incubated with 1 mIU of chondroitinase AC-II in a total volume of 30 µl of the appropriate buffer at 37 °C for 1.5, 3, 15, 30, or 150 min. The reaction was terminated and analyzed by HPLC as above.

HPLC and Capillary Electrophoresis (CE)-- Fractionation and analysis of unsaturated oligosaccharides were carried out by HPLC on an amine-bound silica PA03 column using a linear gradient of NaH2PO4 at a flow rate of 1 ml/min at room temperature as described for the separation of CS-di- and -tetrasaccharides (13, 18, 19). Eluates were monitored by absorption at 232 nm. The separated fractions were concentrated and desalted through a column of Sephadex G-25. CE was carried out to examine the purity of each isolated oligosaccharide fraction using a fused silica capillary and 25 mM sodium phosphate buffer, pH 3.0, in a Waters capillary ion analyzer with a negative polarity power supply (20). The electrophoresed fractions were detected by absorption at 185 nm.

Other Analytical Methods-- Oligosaccharides produced by enzymatic digestions were quantified based on the absorbance (E232 = 5500 M-1 cm-1) caused by the Delta 4,5 sites of the uronic acid at the non-reducing ends (21).

500-MHz 1H NMR Spectroscopy-- Octasaccharide fractions were repeatedly exchanged in D2O with intermediate lyophilization. Their 1H NMR spectra were measured on a Varian VXR-500 at a probe temperature of 15, 26, or 60 °C (22-24). Chemical shifts are given relative to sodium 4,4-dimethyl-4-silapentane-1-sulfonate but were actually measured indirectly relative to acetone (delta  2.225) in D2O (25).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Interactions with the mAb 473HD and the Neurite Outgrowth Promoting Activity of Shark Cartilage CS-D-- Previous studies suggested that the mAb 473HD reacted with the DSD-1 epitope, which was contained in CS-polymers covalently linked to the core of DSD-1-PG. The epitope was proved to be necessary for the neurite outgrowth promoting properties of DSD-1-PG on embryonic day 18 hippocampal neurons, as shown by digestion of the culture substrate with chondroitinase ABC (3). The stimulatory effect of DSD-1-PG on neurite elongation was strongly reduced by mAb 473HD (3), supporting the functional importance of the epitope. In this study, to circumscribe the molecular nature of the epitope in more detail, various GAGs were tested for their ability to interfere with mAb 473HD binding to its antigen. Of these, CS-C and CS-D but not the other GAGs competed the binding of mAb 473HD to DSD-1-PG. According to these competition assays, CS-C and -D contained the DSD-1 structure, in contrast to the other GAGs and dextran sulfate (Fig. 1). In view of the asserted functional properties of the DSD-1 epitope, these GAGs were probed for neurite outgrowth promoting properties. In this functional assay, CS-D and CS-E most efficiently enhanced the fraction of neurite-bearing cells, as compared with other CS isoforms (Fig. 2). Only the entire DSD-1-PG was able to promote neurite outgrowth more strongly than CS-D and CS-E. CS-C, which also contains the DSD-1 epitope according to the competition ELISA, was not efficient in this assay. This probably reflects a lower concentration of the DSD-1 structure in CS-C preparations, as shown by competition ELISAs with serially diluted GAGs.2 Therefore, CS-D was chosen for further structural analysis. The observed neurite outgrowth promoting activity of CS-E will be discussed below (see "Discussion").


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Fig. 1.   Competition of mAb 473HD binding by soluble GAGs. mAb 473HD was preincubated with individual various GAGs and used for ELISA on DSD-1-PG in the presence of the GAGs. The percent of control was calculated using the equation: percent of control = OD405 nm test/OD405 nm control. The experiments were performed at least in triplicate. The results of one representative experiment are shown. Dex.S, dextran sulfate; HA, hyaluronic acid; KS, keratan sulfate; HS, heparan sulfate; Hep, heparin.


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Fig. 2.   Promotion of neurite outgrowth of hippocampal neurons by different chondroitin sulfate isoforms. Embryonic day 18 hippocampal neurons were seeded and cultured on glass coverslips, which were precoated with different CS isoforms and DSD-1-PG as described under "Experimental Procedures." The graph shows the percent increase of the fraction of neurite-bearing cells on the different substrates compared with the polyornithine control. The number of experiments is given in parentheses. The experiments were statistically evaluated with Student's t test. ns, not significant; x, 0.01 < p < 0.05; xx, 0.001 < p < 0.01; xxx, 0.001 > p. Abbreviations in parentheses (ns and xx) indicate statistics between the data obtained with the indicated sample and the polyornithine control.

Isolation of the Octasaccharide Fraction-- To isolate octasaccharide fragments from shark cartilage CS-D, chondroitinase ABC rather than testicular hyaluronidase was used because the former does not have transglycosylation activity unlike the latter, which may generate unnatural compounds by such an activity. Two different commercial preparations of chondroitinase ABC are available: a conventional one and a highly purified one. Conventional chondroitinase ABC contains a minor extra protein (Mr 100,000) in addition to the major one corresponding to the highly purified enzyme (Mr 98,000) as examined by SDS-PAGE (see "Discussion" of Ref. 13; see also Ref. 26). Enzymatically, a highly purified preparation does not digest tetrasaccharides (13). It shows an exolytic action on CS-hexasaccharides removing only the disaccharide units on the non-reducing termini and leaving the tetrasaccharides on the reducing side undigested (8), contrary to the previously proposed concept of its non-random endolytic activity (27). However, conventional chondroitinase ABC digests tetrasaccharides efficiently. Hence, the highly purified preparation was used under limited incubation conditions to obtain relatively large fragments (i.e. octasaccharides) from shark cartilage CS-D.

A commercial preparation of shark cartilage CS-D was partially digested with highly purified chondroitinase ABC, and the digest was size-fractionated by gel filtration into di-, tetra-, hexa-, octa-, deca-, dodeca-, and tetradecasaccharides using a column of Bio-Gel P-10 as reported (8). The octasaccharide fraction was subfractionated into fractions I-VIII by HPLC on an amine-bound silica column as indicated in Fig. 3. Fraction II contained at least two compounds: the CS-4-sulfatase-sensitive major (77%) and CS-4-sulfatase-resistant minor compound (23%). Under mild incubation conditions, CS-4-sulfatase acts preferentially on compounds bearing a GalNAc(4S) residue at the reducing end (13, 28). Therefore, fraction II was digested with CS-4-sulfatase, and the digest was subjected to HPLC fractionation to separate the digested and undigested compounds. The desulfated compound resulting from the major component eluted approximately 10 min earlier than the corresponding parent compound on HPLC (not shown). It was isolated separately from the undigested minor compound and designated as fraction II-S. Fractions V and VI were further purified by rechromatography into subfractions Va and Vb in a molar ratio of 44:56, and into VIa and VIb in a molar ratio of 38:62, respectively. Since fraction Va contained at least two components, the major component Va-I that represented 92% of fraction Va was obtained after CS-4-sulfatase digestion followed by HPLC. Among the isolated fractions, the major fractions I, II-S, IV, Va-I, Vb, VIa, VIb, and VIII were seemingly pure, giving a symmetrical single peak on HPLC and CE (data not shown), and subjected to structural analysis as described below.


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Fig. 3.   HPLC fractionation of the octasaccharide fraction. The octasaccharide fraction obtained from gel filtration on Bio-Gel P-10 (8) was separated into fractions I-VIII on an amine-bound silica column using a linear NaH2PO4 gradient from 0.31 to 0.71 M over a 75-min period. For the experimental details, see "Experimental Procedures."

Enzymatic Characterization of the Isolated Octasaccharide Fractions I, II, IV, VIa, VIb, and VIII Using Chondroitinase AC-II and Sulfatases-- Oligosaccharides in the isolated fractions were first characterized by enzymatic digestions in conjunction with HPLC before 1H NMR analysis. The disaccharide composition of each isolated octasaccharide fraction was determined by chondroitinase AC-II digestion in conjunction with HPLC, and the findings are summarized in Table I. All octasaccharides in the isolated fractions were digested completely into disaccharides by chondroitinase AC-II, indicating that all the internal uronic acid residues in each component are GlcUA and not L-iduronic acid (IdceA).

                              
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Table I
Disaccharide composition of the isolated octasaccharide fractions
Each octasaccharide fraction was digested with chondroitinase AC-II, and the products were identified and quantified by HPLC as described under "Experimental Procedures." Similar results were obtained using a conventional commercial chondroitinase ABC instead of chondroitinase AC-II.

The results obtained from enzymatic studies with fraction VIb are described below as representatives. Digestion of fraction VIb with chondroitinase AC-II gave three major products, Delta Di-4S, Delta Di-6S, and Delta Di-diSD, in a molar ratio of 2:1:1 as quantified by HPLC (Table I and Fig. 4D). The peaks around 10 min did not contain Delta Di-0S and were derived from the enzyme preparation. The sequential arrangement of these disaccharide units was first characterized using three types of sulfatases: Delta hexuronate-2-sulfatase, CS-4-sulfatase, and CS-6-sulfatase. The enzyme Delta hexuronate-2-sulfatase removes a sulfate group only from the C-2 position of a Delta HexA residue located at the non-reducing terminus (12), whereas the enzyme CS-6-sulfatase removes only a sulfate group on C-6 of the GalNAc residue at the reducing terminus of oligosaccharides (28). Although CS-4-sulfatase can remove sulfates from internal GalNAc residues under harsh incubation conditions as well as from the GalNAc residue at the reducing terminus, it acts predominantly on the latter GalNAc under limited incubation conditions (28). The major component in fraction VIb was completely digested by CS-4-sulfatase but resistant to CS-6-sulfatase (Table II), indicating that one of the 4-sulfated GalNAc residues is located at the reducing terminus. The major component representing 95% of this fraction was resistant to Delta hexuronate-2-sulfatase (Table II), indicating that it has a non-sulfated Delta HexA residue at the non-reducing end. The sequential arrangement of the disaccharide units was finally determined by time-course digestion experiments using chondroitinase AC-II, which has an exolytic action (27). At the indicated time intervals, the disaccharide units released sequentially from the non-reducing terminus of the octasaccharide were determined by HPLC. It should be noted that relatively mild incubation conditions were used to monitor the early reaction products from partial digestions. The predominant early digestion product was Delta Di-6S followed by Delta Di-4S (Fig. 4, A and B), suggesting that the major compound contained the sequence Delta HexAalpha 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc(4S) on the non-reducing side. Hence, the major compound in fraction VIb contained the following structure with a C-A-D-A sequence, which was also supported by the data from chondroitinase ABC digestion experiments (see below).


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Fig. 4.   Time-course experiments of chondroitinase AC-II digestion of the isolated fraction VIb. Fraction VIb (0.5 nmol) was digested with chondroitinase AC-II (1 mIU) for 1.5 (A), 30 (B), or 150 min (C). Each digest was analyzed by HPLC on an amine-bound silica column using a linear NaH2PO4 gradient from 16 to 800 mM over a 90-min period. Fraction VIb (0.5 nmol) was also digested with 10 mIU of the enzyme for 20 min for complete digestion (D). The elution positions of the following authentic hexa- and tetrasaccharides are indicated by open arrows: Hexa-, Delta HexAalpha 1-3GalNAc(4S)beta 1-4GlcUA(2S)beta 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc(4S); Tetra-, Delta HexA(2S)alpha 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc(4S). Peaks eluted before 8 min are attributable to the buffer salts and those around 10 min are derived from the enzyme preparation. The peak eluted at 25 min in B-D corresponded to Delta Di-diSD. The elution positions of authentic unsaturated disaccharides are indicated in the top panel by arrows. a, Delta Di-0S; b, Delta Di-6S; c, Delta Di-4S; d, Delta Di-diSD; e, Delta Di-diSE; f, Delta Di-triS.

                              
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Table II
Action of sulfatases on the isolated octasaccharides
Each octasaccharide was digested with Delta hexuronate-2-, CS-4-, or CS-6-sulfatase, and the digest was analyzed by HPLC.

Fraction VIb-- Fraction VIb structure is Delta HexAalpha 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc(4S)beta 1-4GlcUA(2S)beta 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc(4S).

Likewise, fractions I, II-S, IVa, VIa, and VIII were successfully analyzed by enzymatic digestions in conjunction with HPLC. The results obtained from sulfatase digestion experiments are summarized in Table II. The enzymes CS-4- and CS-6-sulfatase removed a sulfate group from 70% and 12% of the compounds in fraction IV, respectively, as judged by HPLC (data not shown), indicating that the compounds in the major and the minor subfractions designated as IVa and IVb have a 4-sulfated and a 6-sulfated GalNAc residue at the reducing terminus, respectively. To deduce the structures of the major components in fractions IV, VIa, and VIII, time-course digestion experiments using chondroitinase AC-II were also carried out, and the results are shown in Fig. 5. Notably, in these experiments, the D-A linkage seems to have been cleaved by chondroitinase AC-II relatively slowly when compared with the D-C linkage as shown in Figs. 4 and 5. Fraction IV containing a D-C sequence on the non-reducing side was completely digested into disaccharides under the relatively mild incubation conditions used, whereas fractions VIa, VIb, and VIII containing a D-A sequence was digested relatively slowly showing a certain degree of resistance of the linkage against the enzyme. Based on the results from enzymatic studies (Tables I and II, Fig. 5), the following structures were deduced. The structure of the major compound in fraction II was deduced from that in fraction II-S. The Delta Di-6S unit detected upon chondroitinase AC-II digestion of fractions VIa and VIII and the Delta Di-4S unit generated from fraction I (Table I) were considered to be derived from minor components.


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Fig. 5.   Time-course experiments of chondroitinase AC-II digestion of fractions IV, VIa, and VIII. Individual fractions IV, VIa, and VIII (0.5 nmol each) were incubated with chondroitinase AC-II (1 mIU) for the indicated time intervals, and each digest was analyzed by HPLC on an amine-bound silica column. Each digestion was incomplete since it was carried out under limited conditions. A, fraction IV; B, fraction VIa; C, fraction VIII. Percent recoveries were calculated based on the peak areas in the HPLC chromatograms and are expressed as percentages relative to the amount of each parent octasaccharide fraction used for digestion. open circle , Delta Di-4S; bullet , Delta Di-6S; black-triangle, Delta Di-diSD; triangle , tetrasaccharide; black-square, hexasaccharide; square , octasaccharide.

Fractions I, II, IVa, VIa, and VIII-- Fraction I structure is Delta HexAalpha 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc(6S)beta 1-4GlcAbeta 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc(6S). Fraction II structure is Delta HexAalpha 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc(4S). Fraction IVa structure is Delta HexA(2S)alpha 1-3GalNAc(6S)beta 1- 4GlcUAbeta 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc(6S)beta 1-4GlcAbeta 1-3GalNAc(4S). Fraction VIa structure is Delta HexAalpha 1- 3GalNAc(4S)beta 1-4GlcUAbeta 1-3GalNAc(4S)beta 1-4GlcUA(2S)beta 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc(4S). Fraction VIII structure is Delta HexA(2S)alpha 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc(4S)beta 1-4GlcUA(2S)beta 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc(4S).

Characterization of Highly Purified Chondroitinase ABC Using the Structurally Defined Octasaccharides-- For the structural analysis of fractions Va and Vb, highly purified chondroitinase ABC was an indispensable tool along with chondroitinase AC-II and the sulfatases. This enzyme acts on unsaturated hexasaccharides in an exolytic fashion, removing an unsaturated disaccharide unit from the non-reducing terminus of hexasaccharides irrespective of the sulfation profiles (8). To further investigate the mechanism of action of this enzyme, the structurally defined octasaccharides described above were digested exhaustively by the enzyme and the products were analyzed by HPLC.

The results obtained with fraction VIb are described below as representatives. Digestion of fraction VIb with highly purified chondroitinase ABC yielded two disaccharide units (Delta Di-6S and Delta Di-4S) and two unsaturated tetrasaccharides with recoveries of 86%, 98%, 90%, and 18%, respectively (Table III, Fig. 6A). The major and the minor tetrasaccharides were detected at the elution positions of the authentic tetrasaccharides Delta HexA(2S)alpha 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc(4S) and Delta HexAalpha 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc(4S), respectively. The major tetrasaccharide was shifted to the position of Delta HexAalpha 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc(4S) upon subsequent Delta hexuronate-2-sulfatase digestion (Fig. 6B), confirming the structure of the above major tetrasaccharide derived from the tetrasaccharide portion on the reducing side of the octasaccharide. In contrast, the minor C-A tetrasaccharide was assumed to be derived from the non-reducing side of the major octasaccharide because a D-A, but no C-A, tetrasaccharide was produced in a time-course digestion experiment using chondroitinase AC-II that has an exolytic activity to release disaccharide units, but not tetrasaccharide units (Fig. 5, B and C). Thus, highly purified chondroitinase ABC must have split the major octasaccharide in the case of fraction VIb into two tetrasaccharides by 18%.

                              
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Table III
Analysis of the chondroitinase ABC digests of the isolated octasaccharide fractions
Each octasaccharide fraction was digested with highly purified chondroitinase ABC, and the reaction products were identified and quantified by HPLC as described under "Experimental Procedures."


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Fig. 6.   Enzymatic analysis of the isolated fraction VIb. The isolated fraction VIb was digested with highly purified chondroitinase ABC (A) or successively with highly purified chondroitinase ABC and then Delta hexuronate-2-sulfatase (B). Each digest was analyzed by HPLC on an amine-bound silica column using a linear NaH2PO4 gradient from 16 to 800 mM over a 90-min period. The elution positions of the authentic unsaturated disaccharides are indicated at the top of A. The peaks marked by asterisks were often observed upon high sensitivity analysis and were due to an unknown substance that eluted from the column resin. For the peaks eluted before 15 min, see the legend to Fig. 4. The elution positions of the following authentic tetrasaccharides are indicated in A and B: 1, Delta HexA(2S)alpha 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc(4S); 2, Delta HexAalpha 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc(4S). The elution positions of authentic unsaturated disaccharides are indicated in the top panel by arrows. a, Delta Di-0S; b, Delta Di-6S; c, Delta Di-4S; d, Delta Di-diSD; e, Delta Di-diSE; f, Delta Di-triS.

Likewise, the specificity of highly purified chondroitinase ABC was investigated by digesting fractions I-S, II-S, IV, and VIa, and the results are summarized in Table III. The data altogether indicated that the enzyme digested preferentially the tetrasaccharide portion on the non-reducing side of the octasaccharides into disaccharides in an exolytic fashion, although it split octasaccharides into two tetrasaccharides to a small extent in some cases. The exolytic action seems to be dominant over the endolytic action, if not exclusive. Although the results obtained from digestion of fraction IV demonstrated a minor tetrasaccharide fragment having the same sequence as that of the tetrasaccharide on the non-reducing side, the results may indicate that fraction IV contained at least two octasaccharide components and that the major and one of the minor tetrasaccharides were derived from the above tetrasaccharide sequences on the reducing side of each octasaccharide, respectively. The production of the major tetrasaccharide sequence was in agreement with the notion that the enzyme exolytically digested the tetrasaccharide portion on the non-reducing side of the major octasaccharide component IVa in fraction IV. The production of the minor tetrasaccharide sequence was consistent with the findings from the CS-6-sulfatase digestion experiment where the minor component in fraction IV was susceptible to the enzyme. However, there is the possibility that the enzyme split the major octasaccharide into two tetrasaccharides to a small extent (6%). Structural analyses of fractions Va and Vb were then performed, taking advantage of this mechanism of action of highly purified chondroitinase ABC.

Enzymatic Characterization of Fractions Va and Vb Using Chondroitinase AC-II, Sulfatase, and Highly Purified Chondroitinase ABC-- Fraction Vb gave rise to three major disaccharides, Delta Di-6S, Delta Di-4S, and Delta Di-diSD, in a molar ratio of 2:1:1 upon chondroitinase AC-II digestion as quantified by HPLC (Table I). The sequential arrangement of these disaccharide units was determined by digestions using chondroitinase AC-II and highly purified chondroitinase ABC. The predominant early product generated upon digestion with the former enzyme was Delta Di-6S, followed by Delta Di-4S (data not shown), suggesting that the tetrasaccharide structure on the non-reducing side of the major compound was Delta HexAalpha 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc(4S). Digestion of fraction Vb with the latter enzyme yielded three disaccharide units (Delta Di-6S, Delta Di-4S, and Delta Di-diSD) and two unsaturated tetrasaccharides in molar proportions of 74:80:22:81:24, taking the parent octasaccharide as 100 (Table III). The major and the minor tetrasaccharides were detected at the elution positions of the authentic tetrasaccharides Delta HexA(2S)alpha 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc(6S) and Delta HexAalpha 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc(4S), respectively. Fraction Vb was sensitive to CS-6-sulfatase (Table II), indicating that the major compound in fraction Vb contained a GalNAc(6S) residue at the reducing terminus. Product analysis in the time-course digestion experiments for fraction Vb using chondroitinase AC-II demonstrated a single tetrasaccharide (D-C) species (data not shown), which was most likely derived from the reducing side. Thus, the major compound in fraction Vb was judged to contain the octasaccharide structure with a C-A-D-C sequence shown below. The minor tetrasaccharide structure Delta HexAalpha 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc(4S) produced by highly purified chondroitinase ABC was most likely derived from the non-reducing side of the major octasaccharide due to the cleavage of the central hexosaminidic linkage by the enzyme.

Fraction Vb-- Fraction Vb structure is Delta HexAalpha 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc(4S)beta 1-4GlcUA(2S)beta 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc(6S).

Likewise, fraction Va-I was structurally analyzed. Digestion of fraction Va-I with highly purified chondroitinase ABC yielded three disaccharide units (Delta Di-6S, Delta Di-4S, and Delta Di-diSD) and two unsaturated tetrasaccharides in molar proportions of 22:192:16:93:26, taking the parent octasaccharide as 100 (Table III). The major and the minor tetrasaccharides were detected at the elution positions of the authentic tetrasaccharides Delta HexA(2S)alpha 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc(6S) and Delta HexAalpha 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc(4S), respectively (data not shown). The major octasaccharide component in fraction Va-I contained the structure with an A-A-D-C sequence shown below. The minor tetrasaccharide was probably derived from a minor octasaccharide whether it is from the reducing or non-reducing side.

Fraction Va-I-- Fraction Va-I structure is Delta HexAalpha 1-3GalNAc(4S)beta 1-4GlcUAbeta 1-3GalNAc(4S)beta 1-4GlcUA(2S)beta 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc(6S).

500-MHz 1H NMR Spectroscopy-- Fractions Vb, VIa, VIb, and VIII were obtained in amounts large enough to allow solid structural determination by 500-MHz 1H NMR spectroscopy in addition to the enzymatic characterization described above. The one-dimensional spectrum of fraction Vb recorded at 26 °C is shown in Fig. 7. Signals at 4.4-5.5 ppm were identified as H-1 resonances of the constituent saccharide residues by comparison with the NMR spectra of unsaturated CS-tetrasaccharides (13) and CS-hexasaccharides (8). Since the H-1 resonances of beta GalNAc residues and 2-O-sulfated GlcUA residues as well as the H-4 resonances of the 4-O-sulfated GalNAc residues of the octasaccharides in all the fractions were obscured by the HOD signal when spectra were recorded at 26 °C, they were also recorded at 15 °C for fraction VIb and at 60 °C for fractions Vb, VIa, and VIII. The spectrum of fraction Vb recorded at 60 °C is shown in the inset of Fig. 7. Proton signals in the one-dimensional spectra were assigned using two-dimensional HOHAHA and COSY spectra as described (8, 13).


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Fig. 7.   Structural reporter group regions of the 500-MHz 1H NMR spectrum of fraction Vb recorded in 2H2O at 26 °C. The inset shows the spectrum recorded at 60 °C. The letters and numbers refer to the corresponding residues in the structures. U, GlcUA; Delta U, Delta HexA; G, GalNAc.

The two-dimensional HOHAHA and COSY spectra of fraction Vb are shown in Fig. 8. The assignment pathway for alpha GalNAc-1 from H-1 to H-4 signals is shown in the COSY spectrum measured at 26 °C (Fig. 8A). Beginning with the alpha GalNAc-1 H-1 proton signal at delta 5.213, a cross-peak showing the connectivity between the H-1 and the H-2 (delta  4.280) protons was found. The connectivities of the H-2 to the H-3 (delta  4.030) and then to the H-4 resonance (delta  4.19) were sequentially established. Although the connectivity of the H-4 to the H-5 resonances was not observed, the H-5 signal (delta 4.340) was assigned in the one-dimensional spectrum based on comparison with the corresponding signals belonging to the tetra- and hexasaccharides of the same series, such as Delta HexAbeta 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc(4S) and Delta HexAbeta 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc(4S) (8, 13). The cross-peaks of the H-5 with the H-6 and H-6' atoms were observed at delta  4.19 and 4.14, respectively, as indicated in Fig. 8A. In a similar fashion, other proton signals were assigned starting with the H-1 resonance of each of the other sugar residues, except for Delta HexA-8 and GalNAc-5. The Delta HexA-8 H-1 signal (delta  5.181) was identified by the sequential assignment of the cross-peaks starting with the characteristic H-4 signal (delta  5.874) in the COSY spectrum. The GalNAc-5 H-1 signal was assigned beginning with the H-4 resonance characteristic for a 4-O-sulfated GalNAc residue (see below). The anomeric resonances of the GalNAc and GlcUA residues were easily discriminated by the difference in chemical shifts. Anomeric resonances of the GalNAc-3, -5, and -7 residues or the GlcUA-2, -4, and -6 residues were distinguished from one another, respectively, as described below.


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Fig. 8.   Two-dimensional HOHAHA and COSY spectra of fraction Vb. A, COSY spectrum recorded at 26 °C; B, COSY spectrum recorded at 60 °C; C, two-dimensional HOHAHA spectrum recorded at 60 °C. The letters and numbers refer to the corresponding residues in the structures. For abbreviations, see the legend to Fig. 7. In A, the assignment pathways of proton signals for alpha G-1, U-2, U-6, and Delta U-8 are indicated, where cross-peaks belonging to alpha G-1 are boxed and those belonging to Delta U-8 are connected by bold lines. Cross-peaks between H-1 and H-2 for beta G-1, G-3, G-5, and G-7 are also indicated. In B, the assignment pathways of protons for U-4 and G-5 are indicated, where cross-peaks belonging to the former are circled. In C, protons belonging to G-3, G-5, and G-7 are indicated on the H-3, H-2, and H-2 tracks, respectively, although the G-3 H-3 and G-7 H-2 tracks overlapped each other.

The anomeric resonances of GalNAc-3, 5 and 7 were clustered at delta  4.55-4.58 in the one-dimensional spectrum (Fig. 7). Starting with the H-4 resonance (delta  4.74) of the GalNAc-5 residue, other proton resonances of this residue including the H-1 (delta  4.60) were assigned in the COSY spectrum recorded at 60 °C (Fig. 8B). The interrelationship among the GalNAc-5 H-1, H-2, H-3, and H-4 was confirmed in the HOHAHA spectrum recorded at 60 °C (Fig. 8C). In our previous study, the H-1 signal of GalNAc-5 of the four unsaturated CS-hexasaccharides was localized in a lower magnetic field than that of GalNAc-3 in the same sequence, reflecting the substitution of the GalNAc-5 residue by the Delta HexA residue at the non-reducing terminus (8). Therefore, the strong cross-peak that was observed at delta  4.58 in the COSY spectrum and assigned above to GalNAc-5 H-1 was judged to be overlapped with that of GalNAc-7 of the octasaccharide in fraction Vb (Fig. 8A). The remaining H-1 resonance at delta  4.55 in the COSY spectrum (Fig. 8A) was assigned to GalNAc-3 and was found to have a similar chemical shift to those (delta  4.55) of the 6-O-sulfated GalNAc-3 residues in the hexasaccharides Delta HexAbeta 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc(4S) and Delta HexA(2S)beta 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc(4S) (8). Since no cross-peak was detected between the GalNAc-3 H-4 and H-5 or between the GalNAc-7 H-4 and H-5, the H-6 signal was found at delta  4.22 in the one-dimensional spectrum by comparison with the corresponding signals belonging to the tetrasaccharide Delta HexAbeta 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc(4S) and the hexasaccharide Delta HexAbeta 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc(4S) (8, 13). The GalNAc-3 H-1 and H-6 were observed on the H-3 track, and the GalNAc-7 H-1 and H-6 were found on the H-2 track in the HOHAHA spectrum, although the two tracks overlapped (Fig. 8C), supporting the connectivities between the H-1 and the H-6 protons of each GalNAc residue.

GlcUA-2 was readily identified by the anomerization effects caused by GalNAc-1, which resulted in doubling of the anomeric proton signal of this GlcUA residue. The anomeric resonances of GlcUA-2 were observed at delta  4.56 and 4.510 (Fig. 7). The GlcUA-2 H-1 resonance of the beta -anomer was assigned to the latter based on the closer resemblance to that of the GlcUA-4 H-1 (delta  4.458) that is beta -linked, whereas the remaining proton resonance at delta 4.56 was assigned to that of GlcUA-2 H-1 of the alpha -anomer. The H-1 resonance at delta  4.458 was assigned to GlcUA-6 based on the similarity in chemical shift to that of the corresponding GlcUA-4 H-1 (delta  4.464) of the hexasaccharide Delta HexAbeta 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc(4S)beta 1-4GlcUAbeta 1-3GalNAc(4S) that shares the same tetrasaccharide sequence on the non-reducing side (8). The anomeric signal observed at delta  4.76 in the one-dimensional spectrum measured at 60 °C (Fig. 7, inset) was assigned to the 2-O-sulfated GlcUA-4 residue, based on the two-dimensional spectra and the similar chemical shift to that of the H-1 signals (delta  4.718 and 4.734) of the 2-O-sulfated GlcUA-4 residues belonging to the authentic tetrasaccharides GlcUA(2S)beta 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc(6S) and GlcUA(2S)beta 1-3GalNAc(6S)beta 1-4GlcUAbeta 1-3GalNAc(4S), respectively (9). The assignment pathways of the proton signals belonging to GlcUA-2, -6 and Delta HexA-8 are indicated in Fig. 8A, whereas that for GlcUA-4 is shown in Fig. 8B.

The resonances observed around delta  2.0 are characteristic of the N-acetyl methyl protons of GalNAc. Three NAc signals belonging to the four GalNAc residues were found for each octasaccharide component. Generally, the NAc signal of a penultimate GalNAc residue substituted by Delta HexA or GlcUA is located in a lower magnetic field than that of the other GalNAc residues in the same sequence. In contrast, that of the reducing GalNAc is observed in a higher magnetic field than those of the other GalNAc residues (8-10, 13). Thus, the NAc signals belonging to the GalNAc-7 and GalNAc-1 residues in each octasaccharide were readily assigned, and the remaining signal with stronger intensity observed between the NAc signals of GalNAc-1 and -7 was assigned to both GalNAc-3 and -5 residues.

Modification by O-sulfation causes downfield shifts of protons bound to the O-sulfated carbon atoms by approximately 0.4-0.7 ppm (29). A downfield shift of the H-6, 6' (Delta  delta  0.4 ppm) of GalNAc-1, -3, and -7, the H-4 (Delta  delta  0.5 ppm) of GalNAc-5, and the H-2 (Delta  delta  0.7 ppm) of GlcUA-4 of the octasaccharide in fraction Vb as compared with those of the non-sulfated residues of authentic disaccharides (30) indicated 6-O-sulfation of GalNAc-1, 3 and 7, 4-O-sulfation of GalNAc-5 and 2-O-sulfation of GlcUA-4, respectively. Likewise, the sulfation profiles of the major compounds in the other octasaccharide fractions were firmly established (Table IV).

                              
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Table IV
1H chemical shifts of structural reporter groups of the constituent monosaccharides of the isolated octasaccharides
Chemical shifts are given in ppm downfield from internal sodium 4,4-dimethyl-4-silapentane-1-sulfonate but were actually measured indirectly to acetone in 2H2O (delta  2.225) at 26 °C. ND, not determined.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Previous studies indicated that mAb 473HD strongly reduced the stimulatory effect of DSD-1-PG on neurite outgrowth and that shark cartilage CS-C, which contains D disaccharide units, specifically inhibited the interactions between mAb 473HD and the CS chains of the DSD-1-PG isolated from mouse brains (3). In this study, biological activities and the structure of shark cartilage CS-D, which contains a higher proportion of D units as compared with CS-C, were investigated. Consistent with the interpretation that the DSD-1-epitope is required for the neurite outgrowth promoting properties of DSD-1-PG, CS-D but not CS-C adsorbed to the culture substrate increased the fraction of neurite-bearing hippocampal neurons. For this reason, CS-D was selected for further structural studies. The significant neurite outgrowth promoting activity observed with squid cartilage CS-E was unexpected and seemingly contradictory to the observation that it did not exhibit significant inhibition of the mAb binding to DSD-1-PG (Fig. 1). However, it is noteworthy that a tetrasaccharide isolated from shark fin cartilage contained a unique sequence Delta HexA-GalNAc(4S,6S)-GlcUA(2S)-GalNAc(6S) with both an E and a D disaccharide unit, and was recognized by mAb MO-225 (7). The intriguing but puzzling observations concerning the CS-E activity remain to be pursued.

Commercial shark cartilage CS-D is composed of four kinds of disaccharide units (A, GlcUAbeta 1-3GalNAc(4S); C, GlcUAbeta 1-3GalNAc(6S); D, GlcUA(2S)beta 1-3GalNAc(6S); and E, GlcUAbeta 1-3GalNAc(4S,6S)) in a molar ratio of 26.9:44.2:21.2:2.0 as described in the specification provided by the manufacturer. A D unit has been demonstrated on the immediate reducing side of an A unit, forming an A-D tetrasaccharide sequence characteristic of shark cartilage CS-D. This A-D sequence has been found in three minor unsaturated hexasaccharides, corresponding to A-D-C, A-A-D, A-D-A sequences, prepared by partial chondroitinase ABC digestion as well as in one tetra- (A-D) and two hexasaccharides (D-A-D and C-A-D) prepared by exhaustive testicular hyaluronidase digestion as summarized in Table V. The propriety of the use of testicular hyaluronidase with a transglycosylation activity for preparing oligosaccharides has been discussed (10). In this study, eight octasaccharides were isolated from the same source after partial digestion with chondroitinase ABC, which has no transglycosylation activity. These octasaccharides, composed of A, C, or D units but devoid of an E unit, included two tetrasulfated (fractions I and II), five pentasulfated (fractions IV, Va, Vb, VIa, and VIb), and one hexasulfated (fraction VIII) structure. The A-D sequence was found in five major fractions in the following sequences as shown in Table V; A-A-D-C (Va), C-A-D-C (Vb), A-A-D-A (VIa), C-A-D-A (VIb), and D-A-D-A (VIII). Thus, the A-D sequence was not unusual as previously presumed (10), but appeared rather frequently. Although the octasaccharide fraction analyzed in this study accounted for only approximately 7.5% (w/w) of the starting CS-D, the revealed sequences indicated that the disaccharide unit located on the immediate non-reducing side of an A-D sequence was an A, C or D unit, whereas that on the reducing side was not a D unit but an A or C unit, forming an A-D-A or an A-D-C hexasaccharide sequence (i.e. GlcUA-GalNAc(4S)-GlcUA(2S)-GalNAc(6S)-GlcUA-GalNAc(4S or 6S)). In other words, D units were not found consecutively as a D-D sequence unlike A-A or C-C sequences, as has been suggested based on the structural studies of the tetrasaccharides generated by digestion with specific chondroitinases AC-I, AC-II, and AC-III (31). The characteristic hexasaccharide sequences most likely reflect the biosynthetic mechanisms: specificities of 2-O- and 6-O-sulfotransferases responsible for producing a D disaccharide unit. It remains to be determined whether such hexasaccharide sequences are associated indeed with biological functions such as a neurite outgrowth promoting activity, and whether the D unit is contained in the CS chains of DSD-1-PG. A preliminary disaccharide composition analysis after chondroitinase AC-II digestion of a limited amount of purified DSD-1-PG showed a peak representing approximately 5% of the total disaccharide units at the elution position of the authentic Delta Di-diSD unit on HPLC.3 The D unit has been identified not only in shark cartilage, but also in mouse mast cells derived from immune lymph nodes (6) and in the basement membrane of mouse tooth germ for a brief period (1). Characteristic sequences including D units may serve as functional domain structures recognized by some protein ligands. Thus, the structural arrangement of D units along CS chains, its function, and biosynthetic mechanism are of particular interest.

                              
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Table V
Oligosaccharides isolated from shark cartilage CS-D
The oligosaccharides that have been so far prepared by digestion of commercial shark cartilage CS-D either with chondroitinase ABC or with testicular hyaluronidase and isolated by gel filtration and subsequent ion-exchange HPLC are summarized. Fr., fraction.

Chondroitinase ABC, which is an indispensable analytical tool, has recently become a subject for discussion in terms of its purity and mechanism of action. Until recently, it had been believed to be an endoeliminase that splits beta 1,4-galactosaminidic bonds of CS and dermatan sulfate chains. However, our recent findings raised questions concerning its purity and mechanism of action. The conventional commercial preparation contained a minor protein component in addition to a major one corresponding to the highly purified enzyme as revealed by SDS-PAGE, and the latter enzyme did not digest tetrasaccharides (see the second paragraph of "Results"; see also Ref. 13). In addition, highly purified chondroitinase ABC preferentially acted on the unsaturated hexasaccharides in an exolytic fashion, releasing only the disaccharide units at the non-reducing ends (8). On the other hand, the conventional preparation of chondroitinase ABC has been claimed to have a non-random endolytic action (27). In contrast, the same preparation has also been reported to release di-, tetra-, and hexasaccharides from the non-reducing ends of CS polysaccharides in an exolytic fashion (32). Thus, it had become necessary to define its endolytic or exolytic actions to apply it as an analytical tool. Very recently, after the completion of this work, Hamai et al. (26) reported purifying two chondroitinase ABCs from Proteus vulgaris and crystallizing them. One enzyme, designated exochondroitinase ABC, was demonstrated to be an exoeliminase preferentially acting on CS tetra- and hexasaccharides to yield respective disaccharides, and corresponded to the minor protein bond observed in the conventional enzyme preparation by SDS-PAGE in our previous study (13). The other, designated endochondroitinase ABC, corresponded to the highly purified enzyme and was shown to act on polymer CS and dermatan sulfate chains endolytically, whereas it did not act on tetrasaccharides, which is in good agreement with our findings (13).

In this study, however, exolytic actions of this latter enzyme on octasaccharides were revealed. Structural analysis of highly purified chondroitinase ABC digests of fractions Vb and VIb indicated that the enzyme split the individual octasaccharides into two tetrasaccharide fragments acting on the central galactosaminidic linkages by approximately 20% (Table III), whereas the other octasaccharides were digested exclusively, if not completely, in an exolytic action releasing disaccharide units sequentially from the non-reducing ends. The activity that produced two tetrasaccharides from an octasaccharide is probably attributable to an endolytic activity as claimed by Hamai et al. (26). In conclusion, highly purified chondroitinase ABC (endochondroitinase ABC) that shows primarily an endolytic action on CS/dermatan sulfate polymers (26) has a strong tendency to exert an exolytic action when acting on oligosaccharides such as hexa- and octasaccharides. It cleaves disaccharide units from the non-reducing ends always when acting on hexasaccharides and can cleave tetrasaccharide fragments probably in an endolytic fashion when acting on octasaccharides depending on the sequences although the disaccharide-releasing exolytic activity still dominates the tetrasaccharide-releasing endolytic activity. Thus, the enzyme is useful for determining the tetrasaccharide sequence located on the reducing side of at least hexa-, octa-, and maybe larger oligosaccharides as well. Investigation of the size and sequence dependence of its action will make it more useful. It is interesting to note that chondroitinase ABC from F. heparinum has also been reported to degrade CS and dermatan sulfate chains in an exolytic fashion, but beginning from the reducing end of the molecules (33) in contrast to chondroitinase ABC from P. vulgaris used in this study.

    ACKNOWLEDGEMENTS

We thank Dr. Makiko Sugiura (Kobe Pharmaceutical University) for recording the NMR spectra and Dr. Tonny De Beer (University of Colorado) for the critical review of the manuscript.

    FOOTNOTES

* This work was supported in part by the Science Research Promotion Fund from The Japan Private School Promotion Foundation; by the Sasakawa Scientific Research Grant from the Japan Science Society; by a grant from Hyogo Science and Technology; by Grants-in-aid for Exploratory Research 08877338, Scientific Research (B) 09470509, and Scientific Research on Priority Areas 05274107 from the Ministry of Education, Science, Culture, and Sports of Japan; and by German Research Council (Deutsche Forschungsgemeinschaft) Grant Fa 159/5-3.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a Schilling Professorship for Neuroscience.

par To whom correspondence should be addressed. Tel.: 81-78-441-7570; Fax: 81-78-441-7569; E-mail: k-sugar{at}kobepharma-u.ac.jp.

1 The abbreviations used are: CS, chondroitin sulfate; PG, proteoglycan; GAG, glycosaminoglycan; GalNAc, N-acetylgalactosamine; GlcUA, D-glucuronic acid; HexA, hexuronic acid; Delta HexA or Delta 4,5HexA, 4-deoxy-alpha -threo-hex-4-enepyranosyluronic acid; PBS, phosphate-buffered saline; mAb, monoclonal antibody; ELISA, enzyme-linked immunosorbent assay; CE, capillary electrophoresis; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; COSY, correlation spectroscopy; HOHAHA, homonuclear Hartmann-Hahn; Delta Di-0S, Delta 4,5HexAalpha 1-3GalNAc; Delta Di-4S, Delta 4,5HexAalpha 1-3GalNAc(4-sulfate); Delta Di-6S, Delta 4,5HexAalpha 1-3GalNAc(6-sulfate); Delta Di-diSD, Delta 4,5HexA(2-sulfate)alpha 1-3GalNAc(6-sulfate); Delta Di-diSE, Delta 4,5HexAalpha 1-3GalNAc(4, 6-disulfate); Delta Di-triS, Delta 4,5HexA(2-sulfate)alpha 1-3GalNAc(4, 6-disulfate); Delta hexuronate-2-, CS-4-, or CS-6-sulfatase stands for Delta 4,5hexuronate-2-O-, chondro-4-O-, or -6-O-sulfatase, respectively; Delta U, -G, -U, -2S, -4S, or -6S denotes Delta 4,5HexA, -GalNAc, -GlcUA, -2-O-sulfate, -4-O-sulfate, or -6-O-sulfate, respectively.

2 A. Clement and A. Faissner, unpublished results.

3 A. Clement, S. Nadanaka, K. Sugahara, and A. Faissner, unpublished results.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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