Domain structure of chondroitin sulfate E octasaccharides binding to type V collagen.

We demonstrated previously that chondroitin sulfate E (ChS-E) binds to type V collagen (Munakata, H., Takagaki, K., Majima, M., and Endo, M. (1999) Glycobiology 9, 1023--1027). In this study, we investigated the structure and binding of ChS-E oligosaccharides. Eleven oligosaccharides were isolated from ChS-E by gel filtration chromatography and anion-exchange high performance liquid chromatography after hydrolysis with testicular hyaluronidase. Separately, seven oligosaccharides were custom synthesized using the transglycosylation reaction of testicular hyaluronidase. Structural analysis was performed by enzymatic digestions in conjunction with high performance liquid chromatography and mass spectrometry. This library of 18 oligosaccharides was used as a source of model molecules to clarify the structural requirements for binding to type V collagen. Binding was analyzed by a biosensor based on surface plasmon resonance. The results indicated that to bind to type V collagen the oligosaccharides must have the following carbohydrate structures: 1) octasaccharide or larger in size; 2) a continuous sequence of three GlcAbeta1--3GalNAc(4S,6S) units; 3) a GlcAbeta1--3GalNAc(4S,6S) unit, GlcAbeta1--3GalNAc(4S) unit or GlcAbeta1--3GalNAc(6S) unit at the reducing terminal; 4) a GlcAbeta1--3GalNAc(4S,6S) unit at the nonreducing terminal. It is likely that these characteristic oligosaccharide sequences play key roles in cell adhesion and extracellular matrix assembly.

Proteoglycan exists in tissues of many mammalian species and is widely distributed in the cell surface and extracellular matrix. It is known that its carbohydrate chain, chondroitin sulfate (ChS), 1 has various important biological activities in areas such as cell migration, recognition, and morphogenesis (1)(2)(3)(4)(5). Recently, greater attention has been directed toward the functions of ChS in the brain, optic nerves, and chondrocytes (6 -8). ChS has many types of structural domains that are known to participate in specific physiological functions. However, the relationship between the biological function and structure of ChS domains is not yet fully understood.
Recently, we used a surface plasmon resonance (SPR) biosensor to investigate the interaction of glycosaminoglycans (GAGs) with collagens and glycoproteins from the extracellular matrix (9). It was found that chondroitin sulfate E (ChS-E) has specific affinity for type V collagen, and that a novel characteristic sequence included in ChS-E is probably involved in binding to type V collagen. ChS-E from squid cartilage consists mainly of the disaccharide units GlcA␤1-3GalNAc(4S,6S), GlcA␤1-3GalNAc(4S), GlcA␤1-3GalNAc(6S), and GlcA␤1-3GalNAc, where 4S and 6S represent 4-O-and 6-O-sulfate, respectively (10,11). Thus, since ChS-E has a variety of structures, it is likely that some specific feature of its structure is necessary for binding to type V collagen. However, the structural features within ChS-E that bind to type V collagen remain unclear.
In the present work, we have prepared various oligosaccharides by testicular hyaluronidase digestion of squid cartilage ChS-E. However, not all of the expected oligosaccharides were obtained. Recently, the transglycosylation mechanism of testicular hyaluronidase, which is an endo-␤-N-acetylhexosaminidase, has been investigated with the aim of performing enzymatic synthesis of GAG oligosaccharides (12,13). Using this enzymatic reconstruction system, it was possible to custom synthesize chimeric GAG oligosaccharides with sequences of nonsulfated and monosulfated disaccharide units, GlcA␤1-3GalNAc, GlcA␤1-3GlcNAc, IdoA␣1-3GalNAc, GlcA␤1-3GalNAc4S, and GlcA␤1-3GalNAc6S. Here, we newly synthesized various chimeric oligosaccharides with disulfated disaccharide units, GlcA␤1-3GalNAc(4S,6S), at the internal position of the sugar chain and used them as model molecules to define the structural requirements needed to bind to type V collagen.
Bovine testicular hyaluronidase (Type 1-S) and ␤-glucuronidase (from Escherichia coli) were obtained from Sigma, and bovine testicular hyaluronidase was further purified according to the method of Borders and Raftery (16). ␣-L-Iduronidase was purified from rabbit liver by the method of Rome et al. (17) and it was also free ␤-glucuronidase ␤-Nacetylhexosaminidase activities. Sephadex G-15 was purchased from Amersham Biosciences AB (Uppsala, Sweden). Bio-Gel P-4 (400 mesh) was obtained form Bio-Rad (Richmond, CA). 2-Aminopyridine (PA) was purchased from Wako Pure Chemical Co. (Osaka, Japan) and recrystallized from hexane. A research grade sensor chip CM5 and an amine coupling kit containing N-hydroxysuccinimide, EDC, 1 M ethanolamine hydrochloride adjusted to pH 8.5 with NaOH and HBS buffer containing 10 mM HEPES, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, and 0.005% surfactant P20 were purchased from Pharmacia Biosensor AB (Uppsala, Sweden). All other chemicals were obtained from commercial sources.
Pyridylamination of Oligosaccharides-Fluorescence labeling with PA of the reducing terminal sugar of oligosaccharides was performed as described previously (20), based on the method of Hase et al. (21).
Custom Synthesis of Chimeric Oligosaccharides-Chimeric oligosaccharides were synthesized using the transglycosylation reaction of testicular hyaluronidase as described previously (22,23). A typical transglycosylation reaction was carried out as follows. Briefly, 50 g of one GAG (HA, Ch, Ch4S, Ch6S, or desulfated DS) as the donor, 20 nmol of a PA-oligosaccharide as the acceptor, and 5 units of testicular hyaluronidase dissolved in 50 g of 0.15 M Tris-HCl buffer, pH 7.0, were incubated at 37°C for 1 h. The reaction was terminated by immersion in a boiling water bath at 100°C for 3 min. The resulting product was purified by HPLC, and its structure was confirmed by a method described previously (9,10). Using systematic combinations of the donor and acceptor molecules, we prepared many chimeric type GAG oligosaccharides consisting of different combinations of disaccharide units (22,23).
HPLC-A high-performance liquid chromatograph (Hitachi L-6200, Hitachi) connected to a fluorescence detector (Hitachi F-1050) or a UV detector (Hitachi L-4200) was used. Fractionation of oligosaccharides derived from ChS-E was carried out with a Polyamine-II column (4.6 ϫ 250 mm; YMC Co., Tokyo, Japan) using a linear gradient of NaH 2 PO 4 from 16 to 1000 mM over a 60-min period at a flow rate of 1.0 ml/min at 30°C. The eluate was monitored by absorbance at 210 nm. Analysis of disaccharides was carried out with the same column using the same linear gradient of NaH 2 PO 4 at a flow rate of 1.0 ml/min at 45°C. Eluates containing unsaturated disaccharides were monitored by ab-sorbance at 232 nm (11,24). Eluates containing PA-disaccharides were monitored at excitation and emission wavelengths of 320 and 400 nm, respectively.
Fractionation and analysis of PA-oligosaccharides were carried out with a PALPAK Type S column (4.6 ϫ 250 mm; Takara Shuzo, Kyoto, Japan) under the following conditions. Solution A containing 3% acetic acid, adjusted to pH 7.0 with triethylamine, and acetonitrile at a ratio of 20:80, and solution B containing the same agents at a ratio of 50:50, were prepared. The column was equilibrated with solution A, and the ratio of solution B to solution A was increased linearly to 100% over a 60-min period at a flow rate of 1.0 ml/min at 30°C. The eluates were monitored at excitation and emission wavelengths of 320 and 400 nm, respectively.
Ion-spray Mass Spectrometry-Mass spectra were obtained on an API-100 triple-quadruple mass spectrometer (PE SCIEX, Ontario, Canada) equipped with an atmospheric pressure ionization source, as described previously (18,19). The samples were dissolved in 0.5 mM ammonium acetate-acetonitrile (50:50) and injected at 2 l/min with a micro-HPLC syringe pump (Pump 22; Harvard Apparatus). In negative-ion mode, scanning was done from m/z 0 to 700 during the 1-min scan (six cycles).
Surface Plasmon Resonance Analysis-Binding of oligosaccharides to type V collagen was determined using a BIA core 2000 (Pharmacia Biosensor AB, Uppsala, Sweden). Binding reactions caused a change in the surface plasmon resonance, which was detected optically and measured in resonance units. Immobilization of type V collagen on the sensor surface via primary amine groups was performed as described previously (9). The amounts of type V collagen immobilized onto the sensor surface were controlled at about 3000 resonance units by changing the injection time. All experiments were carried out at a flow rate of 10 l/min at 25°C. Oligosaccharides in the running buffer were injected onto the sensor surface. Association was monitored for 10 s followed by a dissociation phase. To correct for refractive index change and nonspecific binding, the responses obtained from the surface of albumin as a blank control were subtracted from the type V collagen surface data. All responses were expressed relative to this baseline.

Isolation of the Oligosaccharides Derived from ChS-E-A
commercial preparation of squid cartilage ChS-E was digested with testicular hyaluronidase, and the digest was subjected to fractionation by gel filtration using a Bio-Gel P-4 column. The oligosaccharides were monitored for hexuronic acid (Fig. 1). In this study, the fractions I (Fractions I) and II (Fraction II) expected to contain octasaccharides and hexasaccharides, on the basis of previous chromatographic data (14) for Ch6S, were pooled and further purified by HPLC on a Polyamine-II column. They were then subfractionated into five oligosaccharides (numbers 1-5) ( Fig. 2A) and two oligosaccharides (numbers 6 and 7) (Fig. 2B), respectively, and subjected to structural analyses as described below.
Ion-spray Mass Analysis-Ion-spray mass analyses of the oligosaccharides (numbers 1-7) derived from ChS-E in the negative ion mode defined their molecular weights, from which the composition and number of sulfate residues present in each fraction were inferred. Representative ion-spray mass spectra of oligosaccharides numbers 1 and 7 are shown in Fig. 3. In oligosaccharide number 1, multiply charged ions [M-6H] 6Ϫ , [M-5H] 5Ϫ , and [M-4H] 4Ϫ at m/z 334.8, 401.9, and 502.8, respectively, were revealed (Fig. 3A). The molecular weight of oligosaccharide number 1 was computed to be 2015.2 Ϯ 0.6 based on the presence of these ions. The formula for calculating molecular weights from m/z values of multiply charged ions has been reported previously (18). Therefore, oligosaccharide number 1 was presumed to be an octasaccharide with six sulfate residues. In oligosaccharide number 7, multiply charged 4Ϫ , and [M-3 H] 3Ϫ at m/z 310.3, 388.0, and 517.6, respectively, were revealed (Fig. 3B). The molecular weight of oligosaccharide number 7 was computed to be 1556.3 Ϯ 0.9. Therefore, oligosaccharide number 7 was presumed to be a hexasaccharide with five sulfate residues. The assignments of the multiply charged ions afforded by each of the oligosaccharides (numbers 1-7) are summarized in Table I.
Disaccharide analysis of each oligosaccharide was then performed. To investigate the sequential arrangement of the representative disaccharide units in each oligosaccharide, aliquots of the oligosaccharides were used in two separate experiments. The first aliquot of the oligosaccharides was digested with chondroitinase AC-II, and then labeled with the fluorescent reagent PA. PA-labeled unsaturated and saturated disaccharides were analyzed by HPLC and detected by PA fluorescence at excitation and emission wavelengths of 320 and 400 nm, respectively. Chondroitinase AC-II, which is a bacterial eliminase, should degrade the oligosaccharide to a saturated disaccharide unit derived from the nonreducing terminal and a number of unsaturated disaccharide units derived from the internal region and the reducing terminal. The results are summarized as a composition analysis in Table II. The second aliquot was labeled with PA at the reducing terminal of the oligosaccharides, and then digested with chondroitinase AC-II. PA-labeled unsaturated disaccharide units derived from the reducing terminal were analyzed by HPLC. The results are summarized in Table III. This procedure not only gives the disaccharide composition but also gives information about the sequential arrangement of disaccharide units in the oligosaccharides.
The measured average molecular weight of oligosaccharide number 1 was 2015.2, which corresponded to an octasaccharide with six sulfate residues, (HexA) 4 (HexNAc) 4 (OSO 3 H) 6 . HPLC analysis of the chondroitinase AC-II digest of number 1 after derivatization with PA showed PA derivatives of Di-diS E , ⌬Di-6S, and ⌬Di-diS E in a molar ratio of 1.0:2.3:1.0 (Table II). We assume that the saturated disaccharide unit Di-diS E was derived from the nonreducing terminal. HPLC analysis of the chondroitinase AC-II digest of oligosaccharide number 1 labeled with PA before digestion showed ⌬Di-6S (Table III), which was derived from the reducing terminal. The sequential arrangement of the remaining two disaccharide units was determined by digestion with chondroitinase ABC (28). Chondroitinase ABC split the octasaccharide into one saturated disaccharide derived from the nonreducing terminal, one unsaturated disaccharide derived from the second disaccharide unit from the nonreducing terminal, and one unsaturated tetrasaccharide derived from the reducing terminal. The unsaturated disaccharide unit generated upon digestion with chondroitinase ABC was ⌬Di-6S (data not shown), suggesting that the structure of the second disaccharide unit from the nonreducing terminal of number 1 was GlcA ␤1-3GalNAc(6S). Hence, the following structure is proposed for oligosaccharide number 1: GlcA-GalNAc(4S,6S)-GlcA-GalNAc(6S)-GlcA-GalNAc(4S,6S)-GlcA-GalNAc(6S).
The average molecular weight of oligosaccharide number 2 was 2015.2, which corresponded to an octasaccharide with six sulfate residues, (HexA) 4 (HexNAc) 4 (OSO 3 H) 6 . HPLC analysis of the chondroitinase AC-II digest of number 2 after derivatization with PA showed PA derivatives of Di-diS E , ⌬Di-0S, and ⌬Di-diS E in a molar ratio of 1.0:0.9:1.9 (Table II). We assume that the saturated disaccharide unit Di-diS E was derived from the nonreducing terminal. HPLC analysis of the chondroitinase AC-II digest of oligosaccharide number 2 labeled with PA before digestion showed ⌬Di-0S (Table III), which was derived from the reducing terminal. Therefore, the remaining two disaccharide units, ⌬Di-diS E , were located in the internal region.

Binding of Oligosaccharides Containing E (GlcA␤1-
Oligosaccharide solutions were injected onto sensor surfaces bearing immobilized type V collagen and the binding of oligosaccharides to type V collagen was determined from the increased responses on the sensorgrams. Nonspecific binding and the change in refractive index were corrected for by subtracting the response of an albumin surface from that of type V collagen. The results are shown in Fig. 4. No significant binding of oligosaccharides numbers 1 and 2 to a sensor surface coated with type V collagen was detected (Fig. 4, A and B). On the other hand, the increase in resonance units from the initial baseline represents the binding of the injected oligosaccharide  numbers 3-5 to the surface-bound type V collagen (Fig. 4, C-E).
Judging from the change in response (data not shown), the binding of number 5 to type V collagen was higher than that of numbers 3 and 4. Neither the hexasaccharides (numbers 6 and 7) nor the heptasaccharide without a GalNAc(6S) residue at the reducing terminal of number 3 bound to type V collagen (Fig. 4, F, G, and K). Furthermore, the octasaccharides (numbers 8 -10) without E units also did not bind to type V collagen (Fig.  4, H-J).
Judging from these results, it appears that the binding of oligosaccharides to type V collagen requires a sequence of repeating GlcA␤1-3GalNAc(4S,6S) units. As for the structure at the reducing terminal of the oligosaccharide, octasaccharides with GlcA␤1-3GalNAc(4S,6S), GlcA␤1-3GalNAc(4S), or GlcA␤1-3GalNAc(6S) units at the reducing terminal were found to bind to type V collagen, but those with a GlcA␤1-3GalNAc unit did not. The heptasaccharide without a GalNAc(6S) residue at the reducing terminal did not bind to type V collagen.
Custom Synthesis of New Model Oligosaccharides-The above oligosaccharides, which were prepared by hydrolysis with testicular hyaluronidase, have mostly GlcA␤1-3GalNAc(4S,6S) units at the nonreducing terminal. To study the effect of structural differences at the nonreducing terminal on binding to type V collagen, new model oligosaccharides were custom synthesized by using the transglycosylation reaction of testicular hyaluronidase, as described previously (22,23). Ch, Ch4S, Ch6S, or desulfated DS as donors containing various disaccharide units, and a PA-hexasaccharide (PA-oligosaccharide number 6), GlcA-GalNAc(4S,6S)-GlcA-GalNAc(4S,6S)-GlcA-GalNAc(6S)-PA as acceptor, were incubated with hyaluronidase under optimal conditions (0.15 M Tris-HCl buffer, pH 7.0, in the absence of NaCl at 37°C for 60 min). The reaction products were examined by HPLC by monitoring the fluorescence of the PA-oligosaccharides. A typical HPLC chromatogram with Ch used as a donor is shown in Fig. 5. Three peaks (II, III, and IV) of newly synthesized products were observed: PA-octasaccharide, PA-decasaccharide, and PA-dodecasaccharide elongated by GlcA␤1-3GalNAc units derived from Ch added to the acceptor (peak I). Peak II was recovered and used as oligosaccharide number 12. The amounts of the other elongated PA-oligosaccharides as representative reaction products are shown in Table V. Overall, five new oligosaccharide types (PA-octasaccharides numbers 12-16) having different disaccharide units at the nonreducing terminal were custom synthesized. Number 17 was prepared from number 3 by fluorescence labeling with PA at the reducing terminal, and number 18 was obtained by ␤-glucuronidase digestion of number 17. The nonreducing terminal sugar residue of oligosaccharide number 15 was removed with ␣-L-iduronidase but not with ␤-glucuronidase. In the same way, each oligosaccharide was structurally characterized by chemical analysis, enzymatic analysis, ion-spray mass analysis, and HPLC analysis as previously reported (9,19) (data not shown).
Binding of Newly Synthesized Oligosaccharides Containing GlcA␤1-3GalNAc(4S,6S) Unit to Immobilized Type V Collagen-Seven types of oligosaccharide (numbers 12-18) with different carbohydrate structures at the nonreducing terminals were synthesized enzymatically and used as model oligosaccharides in the binding reaction (Table IV). Solutions of these seven types of oligosaccharide were injected onto sensor surfaces bearing immobilized type V collagen, and the binding of the oligosaccharides to the type V collagen was determined from the increased responses on the sensorgrams. The results are summarized in Table IV. Oligosaccharide number 17, with an GlcA␤1-3GalNAc(4S,6S) unit at the nonreducing terminal, was found to bind to type V collagen. A typical concentrationdependent binding is shown in Fig. 6. However, there was no significant binding of numbers 12-16, which have nonsulfated disaccharide units, GlcA␤1-3GalNAc, IdoA␣1-3GalNAc, or GlcA␤1-3GlcNAc, or monosulfated disaccharide units, GlcA-␤1-3GalNAc(4S) or GlcA␤1-3GalNAc(6S), instead of a GlcA-
Among the GAGs, only a few correlations between particular carbohydrate sequences and functions have been shown, such as the pentasaccharides with anticoagulant and antithrombotic activities (29 -31), or the hexasaccharide having affinity for bFGF (32) in heparan sulfate and heparin, the hexasaccharides in the heparin cofactor II-binding domain of DS (33) and the decasaccharides of HA that bind to core proteins of proteoglycan (34). These examples have shown that the essential size of the oligosaccharide in active domains in GAG seems to be from pentasaccharide to decasaccharide.
Recently, squid cartilage ChS-E was demonstrated to possess neurite outgrowth promoting activity in embryonic rat hippocampal neurons (35). It was also demonstrated that cortical neuronal cell adhesion, mediated by heparin-binding neu-roregulatory factor midkine, is inhibited by squid cartilage ChS-E (36). Binding of ChS-E to midkine was also demonstrated using a BIAcore system (37). Oversulfated ChS is not limited to invertebrates or marine vertebrates and has been found in various tissues and cells from higher land-dwelling vertebrates. ChS-E has been found in human rib cartilage (38), bovine brain (39), and chick brain (40). It is known that the GlcA␤1-3GalNAc(4S,6S) unit is present in the GAGs of serglycin proteoglycan of mouse mast cells (36,41) and also at the nonreducing terminal of GAGs of aggrecan proteoglycan of human knee cartilage (42). Moreover, it was demonstrated that the GalNAc(4S,6S) residue content of the nonreducing terminal region changes in relation to age (42). Therefore, it is likely that GlcA␤1-3GalNAc(4S,6S) units also exist in higher animals, and affect the regulation of various biological phenomena such as adhesion, migration, and neurite outgrowth of neuronal cells.
Type V collagen is widely distributed as a minor component of the extracellular matrix in various tissues (43). It is thought to be an important component for connective tissue assembly, as type V collagen generally co-polymerizes with the more abundant type I collagen (44). Therefore, the binding of chondroitin sulfate E to type V collagen may affect the growth of collagen fibrils. Also, type V collagen has been shown to bind to biglycan (45) and NG2 (46). However, only the core protein part of the molecule responsible for binding to type V collagen has been characterized, not the glycosaminoglycan part. The type V collagen mainly consists of ␣1 (V), ␣2 (V), and ␣3 (V) chains. Although, the type V collagen used in this paper consisted of ␣1 (V) and ␣2 (V) chains (data not shown), no data is available at present concerning what part of the type V collagen binds to octasaccharides derived from chondroitin sulfate E.
GAGs have many types of structural domains, and are known to participate in physiological functions such as anticoagulation and antithrombotic activities. It is important to prepare different GAG oligosaccharides as investigational molecules to analyze the relationship between their structure and function. The chemical synthesis of oligosaccharides has been made possible by recent rapid advances in methodology (47,48). However, chemical synthesis is not at present the best way to prepare these molecules, because of the many reaction steps, the time required and the necessity for special technology. In this respect, attention has been directed to the synthesis of oligosaccharides by using glycosidases, which catalyze transglycosylation as a reverse reaction of hydrolysis (49,50).
In this study, the possibility of using the disulfated disaccharide unit GlcA␤1-3GalNAc(4S,6S) as a substrate for custom synthesis of GAG oligosaccharides by testicular hyaluronidase was examined. As a result, it was possible to transfer various disaccharide units to oligosaccharides having GlcA␤1-3GalNAc(4S,6S) units at the nonreducing terminal, thereby synthesizing new ChS-E-related oligosaccharides each having a different disaccharide unit at the nonreducing terminal. Disulfated disaccharide domains are unique and are specifically recognized by other molecules and are involved in various biological processes. The GAG oligosaccharide libraries that  were custom synthesized in this report will facilitate the investigation of the relationship between the biological function and structure of GAG oligosaccharides.