Department of Chemistry, Division of Medicinal and Natural Products Chemistry and Department of Chemical and Biochemical Engineering, University of Iowa, Iowa City, IA 52242, USA, 2Faculty of Pharmaceutical Sciences, Chiba University, Chiba 263, Japan, and 3Natural Products Research Institute, Seoul National University, Seoul 110460, Korea
Received on March 15, 2000; revised on May 30, 2000; accepted on June 6, 2000.
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Abstract |
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Key words: dermatan sulfate/oligosaccharide structure/chondroitinase ABC/chondroitin ABC lyase/electrospray ionization mass spectrometry
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Introduction |
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The present study is aimed at gaining insights into the chemical structure of DS through the preparation and structural characterization of homogenous DS-oligosaccharides. Similar studies from our laboratory on heparin (Pervin et al., 1995), heparan sulfate (Hileman et al., 1997
) and acharan sulfate (Kim et al., 1998
) has improved the understanding of these related families of glycosaminoglycans. DS-oligosaccharide standards might be useful in facilitating the development of new glycosaminoglycan sequencing technologies (Liu et al., 1995
; Merry et al., 1999
; Turnbull et al., 1999
; Venkataraman et al., 1999
). Homogenous, structurally characterized DS-oligosaccharides might also aid in studying the interaction of DS with biologically important proteins (Hardingham and Fosang, 1992
; Templeton and Wang, 1992
; Hileman et al., 1998
). For example, DS is known to interact with high specificity to hepatocyte growth factor (Lyon et al., 1998
) and annexin V (Ida et al., 1999
) as shown by surface plasmon resonance. Homogeneous, structurally characterized DS-oligosaccharides might be useful in better understanding the specificity, and in particular the size of the binding site within DS responsible for these interactions. Finally, DS-oligosaccharides should help in better understanding the specificity of enzymes acting on DS such as the chondroitin lyases and hydrolases (Yamagata et al., 1968
; Ototani and Yosizawa, 1979
). Recently, a chondroitin sulfate-derived disaccharide prepared in our laboratory has been used to obtain the high-resolution x-ray co-crystal structure with chondroitin B lyase (Huang et al., 1999
). The current study describes the application of chondroitin ABC lyase to prepare DS-derived disaccharide, tetrasaccharide, hexasaccharide, octasaccharide, decasaccharide, and dodecasaccharide. Further chemical treatment with mercuric acetate was also used to afford DS oligosaccharides having an odd number of saccharide residues. The structures of these purified oligosaccharides were unequivocally established by using electrospray ionization (ESI) mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy.
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Results and discussion |
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The 500 MHz 1D 1H-NMR spectra of all the oligosaccharides were acquired for their structural elucidation. Oligosaccharides 2, 4, 6, and the reduced analog of trisaccharide 3 had been previously prepared and their NMR structural data reported (Sanderson et al., 1989; Yamada et al., 1998
). The spectra of oligosaccharides 2, 4, and 6 were identical to those already reported for these compounds. The complete removal of the unsaturated uronate (Figure 1) with mercuric acetate to form trisaccharide 3 could be demonstrated through the disappearance of the signals corresponding to this residue, in particular the H-4 signal of the
UAp residue at 5.9 p.p.m. The NMR spectrum of trisaccharide 3 was easily assigned based on the previously published spectrum of the reduced form of this trisaccharide (Sanderson et al., 1989
). The structure of octasaccharide 8 and decasaccharide 10 (Tables III and IV) were assigned based on the chemical shifts previously reported for the smaller oligosaccharide analogs (Sanderson et al., 1989
; Yamada et al., 1998
). Because of the complexity of the 1D 1H-NMR spectra of pentasaccharide 5 and dodecasaccharide 12, two dimensional (2D) spectroscopy was required for the complete assignment of these compounds. The 2D 1H-NMR TOCSY spectra of oligosaccharides 5 and 12 are presented in Figures 5 and 6, respectively. These spectra were assigned based on the combination of COSY, NOESY and TOCSY experiments. The 2D TOCSY spectrum (Figure 5) shows prominent off-diagonal cross-peaks labeled 110 in Figure 5. Starting on the upper right of the diagonal, the peak at 5.199 can be assigned to the H1 proton of the reducing end GalpNAc4S residue. Off-diagonal cross-peaks 13 correspond to other protons in this residue that show scalar connectivity to the H1 proton. The off-diagonal cross-peaks labeled 47 are used to deduce the scalar connectivity in the adjacent IdoAp residue. Off-diagonal cross-peaks 8 and 9 correspond to connectivity within the single internal GalpNAc4S residue. Finally, off-diagonal cross-peak 10 corresponds to the H5/H6 connectivity in the GalpNAc4S residue at the reducing end. It was possible to completely assign the 1H-NMR spectrum for pentasaccharide 5 based on these and other 2D experiments (Table III). The chemical shifts of the signals of GalpNAc at nonreducing end in pentasaccharide 5 (Figure 5) are very similar to those of penultimate GalpNAc residue, next to unsaturated uronate in hexasaccharide 6. These results suggest that the magnetic anisotropic effects of an adjacent unsaturated uronate residue are not strong. Next the dodecasaccharide 12 was examined by 2D NMR spectroscopy. The 2D TOCSY spectrum is shown in Figure 6. Assignments were made in the same way as described above for pentasaccharide 5. The chemical shifts of the internal GalpNAc4S and IdoAp residues of dodecasaccharide 12 are very similar suggesting that in solution dodecasaccharide 12 may exist in an extended linear conformation. The similar chemical shifts observed for the signals of the internal sequence suggest such a stable conformation of each carbohydrate residue within dodecasaccharide 12. The complete assignment of dodecasaccharide 12 is presented in Table VI. By obtaining spectra at elevated temperature (45°C) and eliminating the overlap from the HOD signal, the H-5 proton signal of the IdoAp residue 2 and the H-1 and H-4 proton signals of GalpNAc4S residues were determined without ambiguity.
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Materials and methods |
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CE was performed using a Capillary Electrophoresis system with advanced computer interface, model I, equipped with high voltage power supply capable of constant or gradient voltage control using a fused silica capillary from Dionex Corporation (Sunnyvale, CA). UV spectrometer was equipped with a thermostatted cell or a JASCO model V550 (Tokyo, Japan). A Varian 500 MHz NMR spectrometer controlled by a SUN SPARC station 2 workstation was used for all 1D and 2D NMR experiments.
Enzymatic depolymerization of porcine intestinal mucosal dermatan sulfate
Dermatan sulfate (500 ml, 20 mg/ml) in 50 mM TrisHCl-sodium acetate buffer, pH 8.0 was treated with chondroitin ABC lyase (20 U) at 37°C. At various time intervals, 10 µl aliquots were removed and each aliquot was used to monitor the reaction by diluting it in 1 ml of 0.03 M HCl and measuring the absorbance at 232 nm. When the absorbance at 232 nm indicated the digestion was 50% completed, the digestion mixture was heated at 100°C for 3 min. The resulting oligosaccharide mixture was concentrated, by rotary evaporation with warming at 40°C, to 100 ml for fractionation by low pressure GPC.
Low pressure GPC fractionation of oligosaccharides
A portion (1.25 g in 12.5 ml) of the oligosaccharide mixture was fractionated on a Bio-Gel P6 (superfine) column (4.8 x 100 cm) eluted with 100 mM sodium chloride at a flow rate of 1.5 ml/min, fractions were collected (10 ml/tube), and absorbance was measured at 232 nm. The fraction numbers were plotted versus absorbance, affording a chromatogram that showed a partial separation of disaccharide through dodecasaccharides. This separation was repeated (eight times) giving reproducible profiles that permitted the pooling of like-fractions. Fractions consisting of disaccharides, tetrasaccharides, hexasaccharides, octasaccharides, decasaccharides and dodecasaccharides were obtained and concentrated by rotary evaporation.
Desalting the size fractionated oligosaccharides
Sized oligosaccharide fractions were next desalted by GPC on a Bio-Gel P2 column (4.8 x 70 cm) eluted with water at a flow rate of 1 ml/min, fractions were collected (5 ml/tube). The eluent was collected and the fractions containing oligosaccharides, having absorbance at 232 nm, were combined. Each fraction was concentrated using a rotary evaporator and the samples were freeze-dried. The resulting size fractionated oligosaccharide mixtures were light yellow colored powders and were stored at 20°C.
Purification of size fractionated oligosaccharides using semipreparative SAX-HPLC
Charge separation of sized oligosaccharide fractions was carried out by semipreparative SAX-HPLC using a linear gradient of sodium chloride at pH 3.5. The desalted, sized oligosaccharide fractions were injected on a semipreparative column equilibrated with water at pH 3.5. The sample was eluted from the column using a 150 min gradient from 0 to 2 M sodium chloride (pH 3.5) at a flow rate of 4.0 ml/min, the elution profile was monitored by absorbance at 232 nm at 0.51.5 absorbance units full scale (AUFS), and the sample eluted between 30 and 80 min (at 0.51.3 M salt). After each run, the column was washed with 2.0 M sodium chloride, followed by a water wash. Each fraction was applied multiple times to the same column, resulting in nearly identical elution profiles. The major peaks were pooled, lyophilized, and desalted on a Bio-Gel P2 column. Each peak was next analyzed by analytical SAX-HPLC and CE.
Analysis of oligosaccharides by analytical SAX-HPLC
Purified oligosaccharides were analyzed by analytical SAX-HPLC to confirm their purity. The SAX-HPLC column was washed as described above and equilibrated with 0.2 M sodium chloride at pH 3.5. Each oligosaccharide sample (520 µg) was analyzed using a 120 min linear gradient of 01.2 M sodium chloride at pH 3.5 at a flow rate of 1.0 ml/min. The elution profile was monitored by absorbance at 232 nm at 0.02 AUFS. Sample purity was confirmed by the presence of a single symmetrical peak.
Analysis of oligosaccharides by CE
The purity of each oligosaccharide was next confirmed by the presence of a single major symmetrical peak on analysis using CE. Experiments were performed on an ISCO capillary electrophoresis system equipped with a variable wavelength ultraviolet detector set at 232 nm. System operation and data handling were fully computer controlled. The CE system was operated in the reverse polarity mode by applying the sample at the cathode and run with 20 mM phosphoric acid adjusted to pH 3.5 with saturated dibasic sodium phosphate as described previously (Pervin et al., 1994). Separation and analysis were carried out in a fused-silica capillary tube. This capillary was 50 µm inner diameter, 360 µm outer diameter, and 62 cm long, with a 42 cm effective length and was externally coated except where the tube passed through the detector. The capillary was washed extensively with 0.5 M sodium hydroxide followed by deionized distilled water, and then running buffer. Samples were injected by vacuum injection (vacuum level 2, 12.79 kPa.s). Each experiment was performed at 20 kV constant voltage.
Analysis of oligosaccharides by analytical GPC-HPLC
Samples (100 µl at 100 pM concentration) were injected into a 20 µl loop connected to a TSK-Gel 2000SW column. Analysis was performed by isocratic elution with 0.2 M NaCl at pH 3.5 and oligosaccharides were detected at 232 nm. The column void and total volumes were determined using blue dextran and sodium azide.
ESI-MS analysis
Negative-ion spectra were performed by using a Micromass, Inc. (England) Autospec equipped with an electrospray interface as described previously (Kim et al., 1998). Nitrogen gas was used both as bath and nebulizer gas, at flow rates of 250 l/h and 12 l/h, respectively. The electrospray ion source was held at 80°C and the spray needle was held at 7.7 kV. Tetraethylammonium iodide in acetonitrile was used as the calibrant. The solutions of dermatan-derived oligosaccharides were prepared for negative ESI-MS by dissolving the solid sample in 1:1 water/acetonitrile with 0.05% NH4OH which was also used for the mobile phase. Spectra were obtained by injecting 20 µl of each solution. Multiple injections were performed. The spectra were obtained by 3040 scans with the use of manufacturers OPUS software.
NMR spectral analysis of oligosaccharides
The pure oligosaccharide samples were dissolved in D2O (99.0 atm %) filtered through a 0.45 µm syringe filter and freeze-dried to remove exchangeable protons. After exchanging the sample three times, the sample was dissolved in D2O (99.96% of atom). 1D 1H-NMR experiments were performed on a Varian VXR-500 spectrometer equipped with 5 mm triple resonance tunable probe with standard Varian software at 298°K and 313°K on 700 µl samples at 0.10.5 mM. The HOD signal was suppressed by presaturation during 3 and 1.5 s for 1D and 2D spectra, respectively. To obtain 2D spectra, 512 experiments resulting in 1024 data points for a spectral width of 2000 Hz were measured, and the time domain data were multiplied after zero filing (data matrix size, 1K x 1K) with shifted sine-bell window functions for 2D COSY, NOESY, or TOCSY experiments. An MLEV-17 mixing sequence of 100 ms was used for 2D TOCSY and NOESY experiments by using 150, 250, and 500 ms mixing times.
Cleavage of terminal unsaturated uronic acid residue with mercuric acetate
DS tetrasaccharide and hexasaccharide were each dissolved at a concentration of 1 mg/ml in double distilled deionized water. Mercuric acetate reagent (35 mM) was prepared by dissolving 113.3 mg of Hg(OAc) 2 in 10 ml of distilled water adjusted to pH 5 with a few drops of acetic acid. In a typical experiment, 1 ml of oligosaccharide solution (1 mg/ml) was treated with 1 ml of mercuric acetate reagent, stirred for 10 min at room temperature. The reaction mixture was passed over a pre-washed Dowex 50W-X8 H+ column (1 x 5 cm), and then washed with 5 column volumes of distilled water. The total effluent was adjusted to pH 7 using sodium bicarbonate solution, and freeze-dried. The resulting oligosaccharide was redissolved in minimal quantity of water and then was applied to a Sephadex G-25 column (2.5 x 50 cm). Fractions were analyzed by UV spectrometer for absorbance at 210 nm and 232 nm.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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