©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of a Novel Glycosaminoglycan Core-like Molecule I
500 MHz H NMR ANALYSIS USING A NANO-NMR PROBE INDICATES THE PRESENCE OF A TERMINAL -GalNAc RESIDUE CAPPING 4-METHYLUMBELLIFERYL-- D-XYLOSIDES (*)

Adriana Manzi (2) (3), Paramahans V. Salimath (1)(§), Robert C. Spiro (3)(¶), Paul A. Keifer (4), Hudson H. Freeze (1)(**)

From the (1) La Jolla Cancer Research Foundation, La Jolla, California 92037, the Glycobiology Program, University of California, San Diego, Cancer Center, and the (2) School of Medicine, La Jolla, California 92093, (3) Telios Pharmaceuticals Inc., San Diego, California 92121, and (4) Varian NMR Instruments, Palo Alto, California 94304-1030

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

-Xylosides compete with endogenous proteoglycan core proteins and act as alternate acceptors for synthesizing protein-free glycosaminoglycan chains. Their assembly on these alternate acceptors utilizes the same glycosyltransferases that make the protein-bound chains. Most studies using alternate acceptors focus on the production of sulfated glycosaminoglycan chains that are thought to be the major products. However, we previously showed that labeling melanoma cells with [6-H]galactose in the presence of 4-methylumbelliferyl (MU) or p-nitrophenyl (pNP) -xylosides led to the synthesis of mostly di- to tetrasaccharide products including incomplete core structures. We have solved the structure of one of the previously unidentified products as, GalNAc (1, 4) GlcA (1, 3) Gal (1, 3) Gal (1, 4) XylMU, based on compositional analysis by high performance liquid chromatography, fast atom bombardment, electrospray mass spectrometry, and one-dimensional and two-dimensional H NMR spectroscopy. The novel aspect of this molecule is the presence of a terminal -GalNAc residue at a position that is normally occupied by -GalNAc in chondroitin/dermatan sulfate or by -GlcNAc in heparin or heparan sulfate chains. An -GalNAc residue at this critical location may prevent further chain extension or influence the type of chain subsequently added to the common tetrasaccharide core.


INTRODUCTION

-Xylosides serve as primers for glycosaminoglycan (GAG)() chain synthesis in animal cells (1, 2, 3, 4, 5, 6) . These acceptors diffuse into cells and into their Golgi apparatus where they compete with endogenous core proteins and make protein-free GAG chains. Previous studies of these acceptors typically focused on the physiological effects of disrupting normal proteoglycan synthesis and on the analysis of the large, highly sulfated GAG chains (1, 2, 4, 5, 6, 7) . Surprisingly, however, several recent studies showed that the major products were not large GAG chains, but rather a series of relatively small, non-sulfated molecules (8, 9, 10) . Some of these were the expected intermediates in the synthesis of the core linkage region of GAG chains, GlcA (1, 3) Gal (1, 3) Gal (1, 4) Xyl-O-3Ser/Thr. Other structures were novel and appeared unrelated to the known route of GAG chain synthesis (8, 9, 10) . Significantly, synthesis of some of these smaller structures also caused selective inhibition of glycolipid synthesis (9) .

Studies from this laboratory showed that 85-90% of the [6-H]Gal-labeled products made on 4-methylumbelliferyl--xyloside (XylMU) by human melanoma cells consisted of Gal (1, 4) XylMU, Gal (1, 3) Gal (1, 4) XylMU, GlcA (1, 3) Gal (1, 3) Gal (1, 4) XylMU, and primarily an unexpected novel compound, Sia (2, 3) Gal (1, 4) XylMU (9) . About 10-15% of the anionic material remained uncharacterized in this study because it was resistant to a variety of enzymatic digestions used to successfully analyze the others. Here we report the purification and structural analysis of one of the components of this material. The results explain its resistance to enzymatic digestions and demonstrate the existence of a previously unidentified core-related structure. In addition, we present evidence that XylMU also primes the synthesis of molecules that lack the typical tandem galactosyl residues found in the core oligosaccharide region of GAG chains.


EXPERIMENTAL PROCEDURES

Materials

Human melanoma cells UACC-903 were obtained from Dr. J. M. Trent, University of Michigan, Ann Arbor, MI. Arthrobacter ureafaciens sialidase was obtained from Oxford Glycosystems, and bovine -glucuronidase was the generous gift of Dr. Phil Stahl, Washington University, St. Louis, MO. C-18 (SPICE) cartridges (400 mg and 2 g) were purchased from Analtech Inc. [6-H]Galactose (15 Ci/mmol) was obtained from American Radiolabeled Chemicals; and QAE-Sephadex, XylMU, and routine chemicals were obtained from Sigma or Fisher.

Isolation of Anionic Material

Six T450 culture flasks each containing 1.8 10melanoma cells (UACC-903) at 80% confluency were incubated for 5 days at 37 °C in 7% COatmosphere. Each flask contained 200 ml of serum-free Dulbecco's modified Eagle's medium supplemented with 1 m M XylMU. The cells were removed by centrifugation and the medium was lyophilized and then reconstituted in 40 ml of water. A mixture of 54,000 cpm of [H]Gal-labeled anionic MU--xylosides previously purified from another batch of these cells was added to the reconstituted medium to monitor purification. The radiolabeled material had been purified by C-18 chromatography and ion-exchange chromatography on QAE-Sephadex as described below and in previous work (9) . The material contained a single negative charge as determined by ion-exchange HPLC on a Varian AX5 column.

The mixture of labeled and unlabeled material was dialyzed extensively against water using 3500 Mcut-off dialysis tubing. The material retained within the tubing was chromatographed on a 5-ml column of QAE-Sephadex and the anionic molecules were eluted with 100 m M NaCl. The eluate was desalted on a 2-g bed C-18 cartridge (Alltech) by washing with water and the MU--xyloside-based products were eluted with 40% MeOH. The sample was concentrated by evaporation to 0.5 ml and digested with 40 milliunits of A. ureafaciens sialidase and 50 milliunits of bovine testicular -glucuronidase together in 0.1 M sodium acetate, pH 6.0, with 4 m M CaClat 37 °C for 16 h. It was again chromatographed on QAE-Sephadex to remove the neutralized material and the remaining anionic material was eluted with 100 m M NaCl. This was desalted on a C-18 cartridge, eluted with 40% MeOH, dried, reconstituted with 40% MeOH, and stored at 4 °C.

Digestion of Anionic Material with -N-Acetylgalactosaminidase and -Glucuronidase

About 15 µg of the anionic material described above was digested with a combination of 0.3 units of -glucuronidase and 25 milliunits of - N-acetylgalactosaminidase in 100 m M sodium citrate-phosphate buffer, pH 4.0 (Supplied by Oxford Glycosystems), in a volume of 50 µl for 20 h at 37 °C. Following digestion the neutral and anionic xylosides were separated on QAE-Sephadex as described above, desalted on a C-18 cartridge, and dried for analysis.

Compositional Analysis

Compositional analyses were carried out at the UCSD Glycobiology Core. The sample was hydrolyzed in 2 M trifluoroacetic acid, 100 °C, for 4 h and the hydrolysate was analyzed by HPAEC-PAD on a Dionex DX500 (Dionex Corp.) on a CarboPac PA-1 column. All monosaccharide components and the MU residue were eluted using a linear gradient of sodium acetate from 0 to 100 m M between 5 and 25 min with constant, 16 m M sodium hydroxide concentration throughout the run. The elution position and detector response were determined with authentic standards. Initial qualitative analysis of the hydrolysate was done on silica gel plates developed in EtAC:Pyr:AcOH:HO, 5:5:1:3, solvent system. Sugars were detected either by silver nitrate spray or by ninhydrin spray for amino sugars.

H NMR Spectroscopy

The samples analyzed were: I, 25 µg of the undigested anionic xyloside; IA, 15 µg of the material neutralized by the combined - N-acetylgalactosaminidase and -glucuronidase digestions; IB, 10 µg of the material which remained anionic after the combined enzyme digestion. Each sample was repeatedly exchanged in DO (99.96%, Aldrich Chemical Co.) with intermediate lyophilization, and finally dissolved in 40 µl of 99.996% DO. Spectra were recorded on a Varian Unity Plus 500 MHz spectrometer (Varian Applications Laboratories, Palo Alto, CA) using a Nano-NMR probe. The Nano-NMR probe spins samples rapidly (1-2 kHz) at the magic angle to remove the magnetic-susceptibility contributions to the H NMR line widths (11) . This unique technology produces high-resolution spectra in sample volumes <40 µl, thus lowering solvent contaminants and background. The probe temperature was 25 or 30 °C (as indicated in the figure legends) and the spin rate was typically about 1850 Hz. Proton one-dimensional NMR spectra were obtained in about 1000 scans using presaturation of the HOD peak. TOCSY two-dimensional spectra (12) were obtained using a 10-kHz MLEV17 spin lock of either 50- or 100-ms duration, 1.6 s (sample I), or 1.0 s (samples IA, and IB) of presaturation, 88 (sample I) or 48 (sample IA and IB) scans per tdata point, and 300 complex data points in t; total measuring time was 30 h for sample I, and 13 h for samples IA and IB. Double quantum-filtered COSY (DQFCOSY) spectra (13) were obtained using either 300 (samples I and IA) or 350 (sample IB) complex tdata points having 48 scans each; measurement times were 18 and 21 h, respectively. ROESY two-dimensional data (14) for sample I were obtained using a 2-KHz pulsed spinlock of 200 ms duration, using 192 scans per 300 complex tdata points (60 h experiment). All two-dimensional spectra were obtained in a phase-sensitive mode using hypercomplex sampling in t, and included presaturation of the HOD resonance. Line broadening (0.2 Hz) was applied to one-dimensional spectra, while two-dimensional spectra were weighted with gaussian functions. Chemical shifts are given relative to 4,4-dimethyl-4-silapentane-1-sulfonate added as internal standard to xyloside I. In the case of xylosides, IA and IB were actually measured relative to the residual HOD peak at 4.80 ppm. When analyzing MeSO- dsolutions chemical shifts were referenced to the MeSO multiplet at 2.49 ppm.

LSI-MS and Electrospray MS

LSIMS analysis was performed with a VG 70-SE (VG Instruments, United Kingdom) magnetic sector mass spectrometer, in both positive and negative ion modes. The instrument was equipped with a cesium ion gun operated at 23 kV and with an emission current of 2-3 µA, in the positive mode and 18 kV, with an emission current of 4-5 µA in the negative mode. Spectra were recorded in the mass range 200-2000 at constant magnetic field. The xyloside (10 µg) dissolved in 5 µl of 40% methanol was applied to the stainless steel target holding 2 µl of glycerol/thioglycerol matrix. The scans were repeated after addition of sodium chloride to confirm the identity of the molecular ion. Electrospray experiments were performed at the Scripps Research Institute Mass Spectrometry Facility using an API III Perkin Elmer Sciex triple-quadrupole mass spectrometer with an upper mass range of m/z 2400. The ion spray interface was used for sample introduction with a potential of the interface sprayer at 3.4 kV. All solvents and reagents were of 99.9% purity and were obtained from Aldrich or Curtin Matheson. Water was purified on a Nanopure filtration system. The sample was dissolved in a methanol/water solvent at 20-50 µ M and injected into the spectrometer at 4.0 µl/min.


RESULTS

Preliminary Analysis of the -Xyloside-based Products

In a previous study we labeled and purified a group of [H]Gal-labeled -xyloside-based products that were secreted into the medium by human melanoma cells (9) . We determined the structures of 90% of these products by sequential enzymatic digestions and HPLC analysis, as reported. Applying the same methods to analyze the size and charge of the remaining 10-15% of anionic species showed that this labeled material contains 3-5 monosaccharides and a single negative charge by anion-exchange HPLC (data not shown). In the previous study, one of the keys to solving the structures of the other components was sequential enzymatic or chemical degradations of the anionic molecules. However, the negative charge on the uncharacterized material was resistant to all treatments and digestions which included: mild acid hydrolysis that cleaves sialic acids, Newcastle Disease Virus sialidase, A. ureafaciens sialidase, bovine -glucuronidase, Escherichia coli alkaline phosphatase, sequential mild acid hydrolysis and alkaline phosphatase digestion, jack bean -galactosidase digestion, or a sequential digestion with human placental -hexosaminidase A followed by -glucuronidase, and treatment with mercuric acetate (or chloride). The last procedure cleaves the glycosidic linkage of terminal uronic acids that contain a 4,5-unsaturated bond (15, 16) . In addition, this material cannot be metabolically labeled when cells are incubated for 12 h with up to 1 mCi/ml SOunder conditions that heavily label chondroitin sulfate chains at the same time (data not shown). Thus, the molecule(s) does not appear to contain sulfate esters. Since these treatments failed to provide any structural information, we turned to the purification of larger amounts of nonradiolabeled material for direct chemical analysis.

Purification of Unknown Xyloside-based Product(s)

Human melanoma cells were grown in the presence of 1 m M XylMU for 5 days as described under ``Experimental Procedures.'' To monitor purification and the completeness of sialidase and -glucuronidase digestions of the nonradiolabeled material, 54,000 cpm of [H]Gal-labeled -xylosides with one negative charge were added to the culture medium at this point. This labeled tracer was purified from a separate batch of cells by C-18 and ion-exchange chromatography on QAE-Sephadex and a small portion was characterized by prior -glucuronidase and sialidase digestions. This provided an estimate of the amount of the tracer material that should be resistant to the digestion in the mixture and, therefore, a measure of the expected yield. The flow chart in Fig. 1shows the purification strategy based on the methods used to obtain the radiolabeled material. A combination of dialysis, C-18, and QAE-Sephadex anion-exchange chromatographies coupled with -glucuronidase and sialidase digestions gave nearly the expected yields based on our previous results (9) and prior analysis of the radiolabeled tracer itself (see ). At this point, we could not determine the purity of the nonlabeled material in the preparation because its isolation was solely based on the use of the radioactive tracer.


Figure 1: Purification of anionic xyloside products. Flow chart of the purification of the XylMU products by C-18 and QAE-Sephadex chromatographies combined with exoglycosidase digestions. The final product was desalted on a C-18 cartridge and used for the analyses described here. Details are given under ``Experimental Procedures'' and in the text.



Compositional Analysis

The monosaccharide composition of the preparation was determined as described under ``Experimental Procedures.'' MU, Xyl, Gal, GlcA, and GalNHwere detected in molar ratios of 1.0:1.1:2.1:0.6:1.6. Stronger hydrolysis conditions (6 N trifluoroacetic acid, 18 h, 100 °C) did not show the presence of any other monosaccharide.

FAB-MS and Electrospray MS Analysis of the Major Component

To further characterize the molecule, a portion of the sample was subjected to FAB-MS analysis in both positive ion and negative ion modes. These analyses indicated that the major component has a native molecular mass of 1011 (data not shown). The sample was then analyzed by electrospray mass spectrometry in both negative and positive ion modes. The results in the negative ion mode (Fig. 2 A) showed a major pseudomolecular ion at m/z 1010 and minor peaks at m/z 1032 ([M - 2H+ Na]) and 1054 ([M - H+ 2Na]) resulting from the substitution with Naions (22 µm each). Detailed analysis of the structure of the minor components (peaks between 1068 and 1196 atomic mass units) is in progress. Fig. 2 B, shows the results in positive ion mode with the expected [M + H]( m/z 1012), [M + Na]( m/z 1034), and [M + 2Na- H]( m/z 1056). Thus, the native molecular mass for the major component by electrospray mass spectrometry is 1011 and agrees with that obtained by FAB-MS. This mass is consistent with the following structure, HexNAcHexAHexPentMU.


Figure 2: Electrospray mass spectrometry. The purified xylosides I, IA, and IB were subjected to electrospray mass spectrometry as described under ``Experimental Procedures.'' A, negative ion mode spectrum of xyloside I; B, positive ion mode spectrum of xyloside I; C, positive ion mode spectrum of xyloside IA; and D, negative ion mode spectrum of xyloside IB. The molecular mass of the major compound in sample I is calculated to be 1011 atomic mass units, which is consistent with a molecule composed of HexNAc-HexA-Hex-Hex-Xyl-MU.



Minor Components of Xyloside I Detected by Electrospray MS Analysis

Several other minor components were detected (less than 10% each): m/z 994, corresponding to HexNAcPentMU; m/z 818, corresponding to HexNAcHexAPentMU; and m/z 747 and 769, corresponding to HexAPentMUpseudomolecular ion and its monosodiated form. Other fragments detected in the negative ion mode (Fig. 2 A) are consistent with the following structures: HexNAcHexAHexPentMU(SOH)( m/z 1092, [M - H]; m/z 1114, [M - 2H+ Na]; m/z 1136, [M - 3H+ 2Na]), HexNAcPentMU(SOH)( m/z 1092 [M - H]; m/z 1114, [M - 2H+ Na]) and HexNAcHexAHexPentMU(CH)( m/z 1068, [M - H+ 2Na]). This indicates that a pentasaccharide with the same primary structure as the major compound can be substituted with either a monosulfate ester, or a methyl ester (or ether) in some cases. Very little fragmentation of the major molecular ion is observed ( m/z 806).

Electrospray MS Analysis of Neutral and Anionic Species after Glycosidase Digestion

The anionic xyloside mixture (sample I) was digested with a combination of -glucuronidase and - N-acetylgalactosaminidase and then separated into neutral (IA) and anionic (IB) fractions by QAE-Sephadex chromatography (see ``Experimental Procedures''). Electrospray analyses of these two fractions are shown in Fig. 2 C (xyloside IA, positive ion mode), and Fig. 2 D (xyloside IB, negative ion mode). Since these samples were analyzed after exchanging the hydroxyl protons with DO for NMR analysis, and dissolved in HO for electrospray analysis, partial deuteration leading to heterogeneity of individual peaks is observed. The two major ions seen in the neutral fraction shown in Fig. 2 C at m/z 657 and 331, are consistent with a primary structure containing HexPentMUand PentMU, respectively, where a proton has been substituted by sodium. The two corresponding pseudomolecular ions are observed at m/z 309 and 640.

Electrospray mass spectrometric analysis of remaining anionic sample IB in the negative ion mode (Fig. 2 D) showed the presence of some remaining major product ( m/z 1011). In addition, other pseudomolecular ions seen are consistent with the following structures: HexNAcHexAHexPentMU( m/z 849, minor), HexAPentMU( m/z 747), HexNAcHexAPentMU( m/z 687, major), HexAPentMU( m/z 615), and PentMU ( m/z 307, major). Fragment ions arising from the cleavage of the N-acetylhexosamine glycosidic linkage are also observed at m/z 806 (1011-HexNAc), m/z 645 (849-HexNAc), and 483 (687-HexNAc). The minor novel MU glycans detected by electrospray mass spectrometry are listed in .

500-MHz H NMR Spectroscopy

Because of the small amount of material available (estimated between 10 and 25 µg for the different samples analyzed), one-dimensional and two-dimensional H NMR analysis were carried out using a Nano-NMR probe containing 40 µl of the sample solution. The standard, 7-hydroxy-4-methylcoumarin -xyloside (XylMU) was analyzed in the same fashion for comparison (Fig. Z1).


Figure Z1: Structure 1



Standard 7-Hydroxy-4-methylcoumarin -Xyloside

H NMR data for 7-hydroxy-4-methylcoumarin is available in the literature (17) . The one-dimensional H NMR of XylMU was recorded in MeSO- d. The signals corresponding to the methyl and aromatic hydrogens were readily assigned as indicated in I. The anomeric signal of the -xyloside appears at 5.045. This represents a downfield shift of 1.132 ppm relative to the anomeric resonance of a terminal non-reducing -xylose residue linked to OH-3 of an underlying mannose residue, recorded in the same solvent (18) . However, not all of the expected signals were found in the one-dimensional spectrum. Only two sharp doublets at 3.364 and 3.770, and two broad signals at 5.37 and 5.10, corresponding to hydroxyl protons, were observed. These signals were resolved by a combination of two-dimensional DQFCOSY and TOCSY experiments (not shown) run at different temperatures (between 22 °C and 65 °C). In this way, the remaining resonances overlapping the residual HOD peak could be assigned. The DQFCOSY experiment showed the expected coupling between the aromatic protons d and e, and between the aromatic proton b and the methyl proton in the coumarin ring. The DQFCOSY spectrum also located the Xyl H-2 signal at 3.24 ppm, which represents a downfield shift of 0.2 ppm relative to the value reported in Ref. 2. The broad doublets at 5.100 and 5.370 were assigned to OH-3 and OH-2, respectively, on the basis of their coupling with H-1, H-2, and H-3. From the analysis of the connectivities of these hydroxyl groups it was also evident that the signal of H-3 is superimposed to that of H-2 ( 3.24). Thus the signal at 3.364 was assigned to Xyl H-4, and that at 3.770 to Xyl H-5. H-5in a -linked xylose is expected to produce a doublet of doublets slightly downfield from the H-2 resonance. The TOCSY spectrum showed a correlation between H-4 and a signal at 3.260 that was assigned to H-5. The TOCSY spectrum also showed a cross-peak between H-4 and a signal overlapped to H-1, this signal most likely corresponds to OH-4. It has to be pointed out that a downfield shift of 0.17-0.35 ppm from the values obtained in DO was reported when using MeSO as the solvent (18) . The assignments of the proton resonances in the xylose ring (with the exception of H-1 and H-2, deprotected by the aromatic ring) correspond very well with the reported assignments for a 4-substituted methyl xyloside which spectra were also obtained in MeSO- dat 30 °C (19) .

One-dimensional and Two-dimensional H NMR Analysis of the Unknown Xyloside I

The one-dimensional H NMR spectrum obtained with approximately 25 µg of xyloside I using a Nano-NMR probe is shown in Fig. 3, and the H NMR data are summarized in I. This spectrum is consistent with the electrospray mass spectrometric analysis (see above), indicating the presence of a mixture. However, considerable amount of information can be obtained from the spectrum. The signals expected for the MU aromatic ring, as well as those characteristic regions of the oligosaccharide ``reporter groups'' are readily recognized. From the integration of the anomeric region it is possible to conclude that at least two components (in ratio 2:1) are present.


Figure 3: One-dimensional H NMR spectrum of the -xyloside I recorded at 500 MHz in DO at 25 °C. A, complete spectrum; B, expansion of the anomeric region; C, expansion of the aromatic region; and D, expansion of the 3.30-4.75 ppm region.



The first striking feature of this spectrum is the presence of a pair of -anomeric resonances at 5.480 ( J= 3.91 Hz) and 5.463 ( J= 3.67 Hz), in a ratio 2:1 (Fig. 3 B). This -anomeric signal is unexpected because there is no precedent for such a residue in the known sequence of the GAG chain core. The same 2:1 ratio is observed for the two pairs of -anomeric signals at 5.243 ( J= 7.57 Hz) and 5.254 ( J= 7.09 Hz), shown enlarged in Fig. 3 B. Two types of aromatic signals d are also observed (Fig. 3 C). The signal at 4.598 was assigned to a -Gal anomeric proton (Fig. 3 D). The remaining anomeric signals at 4.701 and 4.690 could be tentatively assigned to another -Gal residue, and to a -glucuronic acid residue on the basis of the known monosaccharide composition, and the known structure of a GAG chain core region. However, it is not possible to decide which is which. The signal at 2.496 was assigned to the MU methyl protons, and that at 2.080 to an N-acetyl group.

The connectivities of the anomeric protons in the two-dimensional TOCSY NMR spectrum of the same sample are shown in Fig. 4 . It should be pointed out that the signal at approximately 4.4-4.5 ppm observed in the one-dimensional experiment is not found here. Since the spinning rate for this particular experiment was 2240 Hz, this corresponds to a spinning side band located at 2240 Hz (about 4.4 ppm) from the origin (4,4-dimethyl-4-silapentane-1-sulfonate signal). In the two-dimensional TOCSY experiment, since the spinning rate is different, this is producing the tnoise line at 4.9 ppm. The -anomeric signal at 5.480 presents cross-peaks with only three other signals. The typical pattern of a galactose-type ring where the coupling constant between H-4 and H-5 is very small and cannot produce an observable magnetization transfer. Furthermore, comparing the spectra acquired with 50 and 100 ms mixing time (not shown), it is possible to assign the resonance at 4.173 to H-2, the resonance at 3.897 to H-3, and the one at 4.016 to H-4.


Figure 4: Two-dimensional TOCSY of the -xyloside I recorded at 500 MHz in DO at 25 °C. Expansion of the region between 3.2 and 5.6 ppm showing the connectivities in each monosaccharide residue. The numbers and letters in the spectra refer to the corresponding residues in the structure.



Very few examples of -linked N-acetylgalactosamine residues have been reported (20, 21, 22, 23) , and certainly none of them in GAG chains. Thus, other experiments were carried out to confirm this assignment (see below). The pattern of cross-peaks for the anomeric signal at 5.243 is similar to that of the xylose ring on the standard MU -xyloside, but with a considerable downfield shift explained by the different solvent used in both experiments. A detailed analysis of the two-dimensional TOCSY slices obtained in the fdirection, at 50- and 100-ms mixing times (not shown), allowed assigning of the different resonances as indicated in I. The group of cross-peaks correlated to the anomeric signals at 4.6-4.7 is very complex due to the presence of a mixture of compounds. As shown in Fig. 4, there is considerable overlapping of signals between the glucuronic acid and galactose residues. Furthermore, it is clear from the expansion of the two-dimensional TOCSY spectrum that there are two different uronic acid signals characterized by the typical triplets of H-2 between 3.1 and 3.3 ppm. These triplets were also observed in the one-dimensional spectrum (see Fig. 3A). In fact, the integration of the one-dimensional spectrum allows us to determine that the uronic acid with H-2 resonance at higher magnetic field corresponds to the main component in this mixture. From the expansion of the slices obtained in the fdirection at 50 and 100 ms (not shown), it was possible to assign the resonances of Gal-2, Gal-3, and the two GlcA residues of the major and minor components of the mixture, as indicated in I. The resonances observed for the two galactose residues, as well as those for the glucuronic acid residue, correspond better to the data reported in the literature for serine-linked oligosaccharides obtained from proteoglycan linkage regions than with those reported for reduced oligosaccharides (24, 25, 26, 27) . However, there is considerable influence of the MU aglycone.

NMR Analysis of Fractions IA and IB

To confirm the structure of the major component in sample I, approximately 20 µg of this material was digested with a combination of - N-acetylgalactosaminidase and -glucuronidase. This treatment neutralized 66% of the [H]galactose tracer in the preparation and showed that the major component of the tracer was sensitive to this digestion. The neutralized material was separated from the remaining anionic material on QAE-Sephadex and desalted on C-18. Both the neutral component, IA (15 µg), and the remaining anionic component, IB (10 µg), were analyzed by one-dimensional and two-dimensional H NMR after purification.

As indicated by the one-dimensional H NMR, the neutral product is still a mixture with anomeric signals for -xylose at 5.260 and 5.290 in a ratio 2:1. However, the anomeric signal at 5.480 has disappeared, as well as the methyl signal of the N-acetyl group. This result confirms the assignment of the resonance at 5.480 to the anomeric proton of a -GalNAc residue. As expected for this neutral compound, no resonances that could be attributed to a glucuronic acid residue were observed. On the contrary, a clear -galactose anomeric region shows two resonances at 4.660 and 4.715 in a 1:1 ratio (Fig. 5 A). Resonances were assigned using the two-dimensional DQFCOSY and TOCSY experiments shown in Fig. 5, B and C. The corresponding H NMR data are summarized in I. Thus, the H NMR results indicate that one of the components in this mixture has the structure, Gal (1, 2, 3) Gal (1, 2, 3, 4) Xyl (1, 2, 3, 4) MU, while the other one resembles the starting material (MU--xyloside). These results are in agreement with the mass spectrometry study described above.


Figure 5: NMR spectra of the neutral fraction ( IA) of the -xyloside recorded at 500 MHz in DO at 30 °C. A, expansion of the carbohydrate region; B, two-dimensional DQFCOSY spectrum. In the figure, the assignment pathways for the xylose and galactose residues are drawn; C, two-dimensional TOCSY spectrum obtained with 100 ms mixing time. The numbers and letters in the spectra refer to the corresponding residues in the structure.



The spectra obtained for the acidic product showed the presence of a complex mixture of several compounds in similar ratios, and complete assignment could not be achieved. However, several pieces of information could be obtained from these experiments.

1) The sample still contains N-acetylgalactosamine, as indicated by the presence of a N-acetyl methyl signal in the one-dimensional spectrum (not shown), together with a clear TOCSY pattern (not shown), which chemical shifts are listed in I. Furthermore, a second -anomeric resonance of N-acetylgalactosamine is observed in the one-dimensional spectrum in a ratio 0.75:1 with the main component.

2) Only one set of xylose signals was found with a -anomeric resonance (see I). H-5could not be detected (data not shown). The higher intensity of this signal is consistent with the electrospray mas spectrometry data that show the presence of sequences containing two or three xylose repeats.

3) The region of the TOCSY spectrum corresponding to the -galactose and -glucuronic acid cross-peaks looks similar to that of the original mixture. However, in this case all resonances could not be completely assigned because of lack of sensitivity. It is possible to observe two galactose patterns, one of which has a superimposed set of signals that should correspond to the glucuronic acid residue with H-2 resonance at 3.46. A second uronic acid was not found in this product, but a new set of signals was observed in this region with anomeric resonance at 4.55 (not shown). This chemical shift is similar to the reported values for a -GalNAc residue sulfated at position 6 (26) .

Linkage Analysis of Xyloside I by Two-dimensional ROESY

The two-dimensional ROESY experiment allowed us to identify the linkage positions. Interresidue cross-peaks were found between H-1 of GalNAc and H-4 of GlcA, H-1 of GlcA and H-3 of Gal-3, between H-1 of Gal-3 and H-3 of Gal-2, and between H-1 of Gal-2 and H-4 of Xyl. Furthermore, the H-5 signal of the GalNAc residue could be assigned to 3.70 ppm since the closeness in space of the H-4 and H-5 affords a cross-peak in this spectrum (Fig. 6).


Figure 6: Two-dimensional ROESY spectrum of the -xyloside I recorded at 500 MHz in DO at 30 °C, with a mixing time of 200 ms. Interresidue ROEs are indicated by arrows. ROESY correlations (phase opposite the diagonal) are indicated with 10 contour lines; TOCSY artifacts, and the diagonal are indicated with a single contour line.




DISCUSSION

This paper extends our previous work showing that human melanoma and Chinese hamster ovary cells incubated with MU- or p-nitrophenol--xylosides and metabolically labeled with [6-H]Gal make a series of neutral and anionic labeled di- to tetrasaccharides on the added acceptors (9) . These include the expected biosynthetic intermediates of the GAG core glycan. In the previous study, most of the anionic [H]Gal-labeled -xylosides were structurally analyzed by enzymatic and chemical degradations, but 10-15% were resistant to these degradations. Preliminary studies showed that the unknown molecule(s) contained 4-5 sugars and a single negative charge. This paper and its companion (36) focus on the study of these products.

Since sulfate and phosphate esters have been identified in the core regions of some chondroitin sulfate chains (24, 25, 26, 27) , either of these groups might account for the negative charge in the before mentioned molecules. However, we found no evidence for sulfate by metabolic labeling, or for acid-labile phosphodiesters or E. coli alkaline phosphatase-sensitive phosphomonoesters. The other candidate was an acidic sugar residue, the most likely choice being GlcA. However, the resistance of the [H]Gal-labeled molecule to -glucuronidase digestion suggests that the GlcA residue has modified or not the terminal residue. When these products were isolated in enough quantity, and were submitted to monosaccharide compositional analysis, LSIMS, and electrospray MS analyses, the results were consistent with a pentasaccharide core typical of chondroitin sulfate. Further analysis by one-dimensional and two-dimensional NMR showed the expected signals for a typical core structure except that the terminal GalNAc was in an rather than configuration.

This result was unexpected since mature chondroitin/dermatan sulfate chains contain -GalNAc residue at this position (3) . Heparan sulfate/heparin chains also share the same core, but have an -GlcNAc residue at this position. Addition of either -GalNAc or -GlcNAc to the core is considered the first committed step for building up each type of chain. The amino acid sequence of the protein itself (27) or modifications of the core saccharide may control the choice between chondroitin sulfate and heparan sulfate (23, 24, 25, 26, 28) . NMR analyses of the core region of chondroitin sulfate chains following chondroitinase digestion have shown that GalNAc at this location is -linked to the single core GlcA (24, 25, 26, 27, 29) . In vitro biosynthetic studies of the core saccharide synthesis using solubilized membranes also showed that the GalNAc at this location is totally sensitive to digestion with -hexosaminidase (30) . These studies also showed that the -GalNAc residue is added to the core -GlcA by a different enzyme than that responsible for chain elongation in chondroitin sulfate.

Our discovery of an -GalNAc residue at this critical location is intriguing because it provides another, previously unrecognized option for the tetrasaccharide intermediate. It is possible that -GalNAc stops further elongation of the chain. Since glycosyltransferases specifically recognize the anomeric linkage of the sugar acceptor, it is unlikely that the next enzyme, presumably a -glucuronosyltransferase, would recognize -GalNAc and -GalNAc residues equally well. The low amount of -GalNAc terminated xyloside products in these preparations may be due to their rapid elongation to form larger GAG chains, which are not retained on the C-18 cartridges used to prepare these samples.

Although terminal -GalNAc residues are sometimes found in glycolipids and in blood group A oligosaccharides (31, 32) , there are no reports of GalNAcGlcA- disaccharide sequences in any glycoprotein in the CarbBank data base. Furthermore, the melanoma cells that we studied do not make such glycolipids and are not reactive with anti-A antibodies. Thus, it is not likely that other -GalNAc transferases account for the synthesis of the -GalNAc-terminated GAG chain cores.

The H NMR results allowed us to clearly identify the major -GalNAc terminated xyloside. However, definitive assignment of all resonances in the H NMR spectra of xylosides I and IB was not possible because of the complexity of these mixtures. Nevertheless, we gained considerable information from the NMR analysis of the minute amounts of this complex mixture. These results were achieved with only 20 µg of sample I which represents about one-tenth to one-third of the minimum amounts used for such analyses. Complete structural characterization of each product in this mixture of xylosides will require much more work to perform similar analyses. This is under way in our laboratories.

Electrospray mass spectrometric analysis also showed evidence for at least five distinct types of structures made on XylMU. The first three types contain one pentose (xylose) and 0, 1, or 2 hexose (galactose) units along with 1-2 HexNAc and HexA. The fourth type contains 2 pentoses, a HexNAc, and 1-2 HexA. The fifth type contains 3 pentoses and a HexA. This last type represents about 10% of sample I. It appears resistant to -glucuronidase digestion since it remains anionic (1B) after two rounds of -glucuronidase digestions, and the corresponding mass for XylMU is not found in the neutral fraction, 1A.

Several conclusions can be drawn from the electrospray analysis. The molecule HexNAcHexAPentMU ( m/z 818), seen in unfractionated sample I, appears to be susceptible to - N-acetylgalactosaminidase digestion. This conclusion is based in the disappearance of its pseudomolecular ion following digestion, and the presence of the pseudomolecular ion for the same molecule minus a HexNAc residue ( m/z 616) in the anionic xyloside sample IB. On the other hand, this molecule also appears to be resistant to -glucuronidase digestion, since an m/z 440 corresponding to the further loss of HexA is not found in the neutral fraction 1A.

The broad array of minor xyloside products identified by NMR and mass spectrometric analyses raises questions about their significance and relationship to the known route of GAG core biosynthesis. Xylose residues are primarily, but not exclusively, found in the GAG core region of mammalian cells. For instance, bovine coagulation factor IX contains Xyl (1, 3) Xyl (1, 3) Glc linked to serine (33) , and Izumi et al. (10) recently found that human fibroblasts make a novel xyloside, Xyl (1, 4) XylMU, when incubated with MU. A xylosyl transferase in the soluble fraction of rat kidney homogenate glycosylates glycogenin, but this enzyme is not expected to have access to the xylosides passing through the Golgi (34, 35) . It is possible that other xylose-containing sugar chains have been overlooked in the past. On the other hand, the small amounts of these structures may simply be the result of glycosyltransferase promiscuity in the presence of high endogenous concentrations of the artificial acceptors.

Is the presence of the -GalNAc cap such an experimental artifact? Although this is possible, we think this is unlikely, based on the results presented in the companion paper (36) . However, it will be necessary to identify such a structure on a known proteoglycan. These studies are also in progress.

  
Table: Recovery of H-Gal radiolabeled anionic xylosides


  
Table: 1952998688p4in NA, not applicable.

  
Table: H chemical shifts of the constituent monosaccharides of compounds I, IA, and IB and the reference compound MU b-Xyloside

Chemical shifts are given in ppm downfield from 4,4-dimethyl-4-silapentane-1-sulfonate



FOOTNOTES

*
This work was supported by Grants NCI RO1 38701 (LJCRF) with MIS 0951 (to A. M.) and NCI RO1 49243, Telios, and American Cancer Society Grant BE-181 (LJCRF). Ion spray mass spectrometer was funded by the Lucille P. Markey Charitable Trust and a National Institutes of Health Shared Instrumentation Grant 1 S10 RR07273. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Current address: Dept. of Biochemistry and Nutrition, CFTRI, Mysore 570 013, India.

Present address: Orquest, Inc., 365 Ravendale Dr., Mountain View, CA 94043.

**
To whom correspondence should be addressed: La Jolla Cancer Research Foundation, 10901 N. Torrey Pines Rd., La Jolla, CA 92037. Fax: 619-450-2101; Tel.: 619-455-6480.

The abbreviations used are: GAG, glycosaminoglycan; MU, 4-methylumbelliferyl or 7-hydroxy-4-methylcoumarin; XylMU, 4-methylumbelliferyl--xyloside; HPAEC-PAD, high pH anion-exchange chromatography with pulsed amperometric detection; FAB-MS, fast atom bombardment-mass spectrometry; LSIMS, liquid secondary ion mass spectrometry; DQFCOSY, double quantum filtered correlation spectroscopy; TOCSY, totally scalar correlated spectroscopy; MLEV17, Malcom Levitt-17 mixing sequence; ROESY, rotating frame Overhauser enhancement spectroscopy; HPLC, high performance liquid chromatography; pNP, p-nitrophenyl.


ACKNOWLEDGEMENTS

We thank Deepak Sampath for preliminary experiments, Delia Matriano for technical assistance, and Drs. Herman Van Halbeek and Claudio Schteingart for helpful discussions.


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