©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of a Novel Glycosaminoglycan Core-like Molecule II
-GalNAc-CAPPED XYLOSIDES CAN BE MADE BY MANY CELL TYPES (*)

Paramahans V. Salimath (1)(§), Robert C. Spiro(¶) (2), Hudson H. Freeze (1)(**)

From the (1) La Jolla Cancer Research Foundation, La Jolla, California 92037 and (2) Telios Pharmaceuticals Inc., San Diego, California 92121

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The accompanying article (Manzi, A., Salimath, P. V., Spiro, R. C., Keifer, P. A., and Freeze, H. H. (1995) J. Biol. Chem. 270, 9154-9163) reported the complete structure of a novel [Abstract/Full Text] molecule made by human melanoma cells incubated with 1 m M 4-methylumbelliferyl-Xyl (XylMU). The product resembles a common pentasaccharide core region found in chondroitin/dermatan sulfate glycosaminoglycans, except that a terminal -GalNAc residue is found in a location normally occupied by -GalNAc in these chains or -GlcNAc in heparan sulfate chains. In this paper we show that several other human cancer cell lines and Chinese hamster ovary cells also make -GalNAc-capped xylosides. The [6-H]galactose-labeled XylMU product binds to immobilized -GalNAc-specific lectin from Helix pomatia and the binding is competed by GalNAc, but not by Glc. Binding to the lectin is destroyed by digestion with - N-acetylgalactosaminidase, but not -hexosaminidase. The nature of the aglycone influences the amount and relative proportion of this material made, with p-nitrophenyl--xyloside being a better promoter of -GalNAc-terminated product than XylMU. This novel oligosaccharide accounts for 45-65% of xyloside-based products made by both human melanoma and Chinese hamster ovary cells when they are incubated with 30 µ M XylMU, but at 1 m M both the total amount and the proportion decreases to only 5-10%. In both cell lines this product is replaced by a corresponding amount of Sia2,3Gal4XylMU. Preferential synthesis of the -GalNAc-capped material at very low xyloside concentration argues that it is a normal biosynthetic product and not an experimental artifact. This pentasaccharide may be a previously unrecognized intermediate in glycosaminoglycan chain biosynthesis. Since this -GalNAc residue occurs at a position that determines whether chondroitin or heparan chains are added to the acceptor, it may influence the timing, type, and extent of further chain elongation.


INTRODUCTION

In the previous paper (1) we showed that human melanoma cells make a novel pentasaccharide when incubated with XylMU.() This novel structure contains a terminal -GalNAc residue at the nonreducing end of an otherwise typical GAG core tetrasaccharide. In typical GAG core structures, this position is normally occupied by -GalNAc in chondroitin or dermatan sulfate, or -GlcNAc in heparin or heparan sulfate (2) . However, terminal -GalNAc residues have been reported to occur in blood group A-containing oligosaccharides and in some glycolipids (3, 4) . Since several studies have shown that -xylosides can serve as acceptors for the synthesis of small oligosaccharides which are not part of the well established GAG chain core (5, 6, 7) , an important question is whether the -GalNAc-terminated structure is an artifact of the incubation. Here we present evidence that several types of cells can synthesize this molecule when incubated with XylMU. Significantly, the -GalNAc-terminated pentasaccharide is the preferred product when cells are incubated with very low concentration of acceptor. This result argues that it is a natural product rather than an experimental artifact.


EXPERIMENTAL PROCEDURES

Materials C-18 cartridges were from Analtech, Inc. The Helix pomatia agglutinin-agarose beads were from EY Laboratories. Bovine testicular -glucuronidase was kindly provided by Dr. Philip Stahl, Washington University School of Medicine, St. Louis, MO. Arthrobacter ureafaciens sialidase, chicken liver -galactosidase, - N-acetylgalactosaminidase, and jack bean -hexosaminidase were all from Oxford Glycosystems. Human placental -hexosaminidase A was gift from Dr. Don Mahuran, Hospital for Sick Children, Toronto. Human melanoma cells were provided by Dr. J. M. Trent, University of Michigan, Ann Arbor, MI. Human leukemia cell line HL60, and monoblast line U937 were supplied by Dr. Minoru Fukuda, La Jolla Cancer Research Foundation. Chinese hamster ovary cells were provided by Dr. Jeff Esko, University of Alabama, Birmingham, and the neuroblastoma cell line IMR32 was obtained from American Type Culture Collection. Tissue culture medium was purchased from Life Technologies, Inc. and [6-H]galactose (20 Ci/mmol) was purchased from American Radiochemical, St. Louis, MO. Methods

Labeling of Cells with XylMU and XylpNP

Confluent cells were washed twice with PBS and incubated in XylMU or XylpNP at 1 m M in serum-free Dulbecco's modified Eagle's medium containing 0.1 mg/ml glucose. [H]Gal was added (100 µCi to UACC; 10 µCi to IMR) and incubated at 37 °C for 6 h. At the end of the incubation period, medium was centrifuged to remove debris and passed over a 400-mg C-18 SPICE cartridge equilibrated in 0.15 M NaCl. It was washed 5 times with 1.5 ml of 1 M NaCl and then 1 1.5 ml with water to remove the salt. Inclusion of NaCl during the wash improves binding of the xyloside-based products which are then eluted with 5 1.5 ml of 40% MeOH.

QAE-Sephadex Chromatography

2-cm columns of QAE-Sephadex in 2 m M Tris base were prepared in glass wool-plugged Pasteur pipettes and the sample applied in 1.5 ml of water. The column was washed with 4 1.5 ml of water; 4 1.5 ml of 100 m M NaCl in 2 m M Tris base; and, a 2 1.5-ml wash of 1000 m M NaCl.

C-18 Cartridge Chromatography

Samples from the cell labelings were purified on C-18 SPICE as described above and the fractions for analysis were eluted in 40% MeOH. Cartridges were reused many times and regenerated with 10-20 ml of 100% MeOH followed by 20 ml of HO. HPLC Analysis

Anion Exchange Chromatography

Anionic oligosaccharides (about 1000 cpm) were analyzed on an AX-5 column (Varian Instruments) starting with water for 5 min, followed by a linear gradient of 10-50 m M NaHPO, pH 4.3, for 30 min, and a 5-min wash in 100 m M NaHPO. 1 ml/min fractions were collected and counted (6) .

Size Analysis of Neutral Oligosaccharides

Neutral oligosaccharides were analyzed by amine adsorption on a Varian AX-5 anion exchange column using a linear gradient of 80-65% acetonitrile in water at a flow rate at 1 ml/min. 0-5-min fractions were collected and counted (6) .

H. pomatia Lectin Affinity Chromatography

H. pomatia agglutinin linked to agarose beads was obtained from EY Laboratories and packed into a 0.5 7-cm column and equilibrated with PBS, pH 7.2. Samples of 1000 cpm in 0.1 ml of PBS were mixed with internal marker of [S]SO4, applied to the column, and washed immediately with PBS. Approximately 30-40 5-drop fractions were collected. This was followed by elution with PBS containing 50 m M GalNAc which did not elute any additional material. Recovery was always >90%. Protein estimations were by the method of Lowry (8) . Enzyme Digestions For all enzyme digestions the samples were dried and dissolved in 50 µl of buffer, incubated at 37 °C overnight, and analyzed directly on QAE-Sephadex or C-18 cartridges. Samples for HPLC analysis were diluted with an equal volume of water, heated at 100 °C for 2-3 min, and the insoluble material removed by centrifugation in a microcentrifuge for 1 min at top speed. Digestions with individual or combinations of enzymes were as follows: -glucuronidase (0.3 unit) in 100 m M sodium acetate, pH 5.5; sialidase (8 milliunits) in 100 m M sodium acetate, pH 6.0, containing 4 m M calcium acetate; jack bean -hexosaminidase (50 milliunits) in 50 m M sodium formate, pH 5.0; human placental -hexosaminidase A (2 units) in 25 m M sodium formate buffer, pH 4.5; - N-acetylgalactosaminidase (4 milliunits) in 100 m M sodium citrate-phosphate buffer, pH 4.0 (supplied by Oxford Glycosystems); chicken liver -galactosidase in 100 m M sodium citrate-phosphate, pH 4.0, containing 1 mg/ml bovine serum albumin and 0.15 M NaCl (buffer supplied by Oxford Glycosystems for -galactosidase); sialidase and -glucuronidase, 100 m M sodium acetate, pH 6.0, containing 4 m M calcium acetate; - N-acetylgalactosaminidase and -glucuronidase, 100 m M sodium citrate-phosphate buffer, pH 4.0; - N-acetylgalactosaminidase, -glucuronidase, and -galactosidase, 100 m M sodium citrate-phosphate buffer, pH 4.0, containing 1 mg/ml bovine serum albumin. Measurement of [H]Galactos in GAG Chains-[H]Gal-labeled GAG chains made on XylMU after 6 h was measured by precipitation with cetylpyridinium chloride (9) .


RESULTS

Digestion with -GalNAcase

Since the amount of the purified material described in the previous article was small, we again turned to the analysis of the same [6-H]Gal-labeled material that was used as a tracer in the accompanying article (1) . Based on the previous results, this labeled material consisted of approximately 65% -GalNAc-terminated core structure and about 35% of several other partially characterized products. Using the labeled material focuses on galactose-containing xylosides and excludes the others. The results in Table I show that digestion with -glucuronidase alone or in combination with -hexosaminidase did not convert any of the labeled anionic product into a neutral species. This confirms that [6-H]galactose preferentially labels the galactose-containing species. Digestion with -GalNAcase followed by -glucuronidase is the only treatment that neutralized 65-70% of the material. Longer digestion times or increased amounts of enzyme did not change this amount. The neutralized material was recovered and analyzed by amine adsorption HPLC; it co-eluted with a standard of Gal3Gal4XylMU showing that it had the predicted core structure (data not shown). These results show that the majority of the radiolabeled material prepared from these cells had the same structure as the major component analyzed in the previous paper (1) . The structure of the remaining anionic material is under investigation.

Binding to H. pomatia Agglutinin Column

H. pomatia agglutinin is a lectin that binds terminal -GalNAc residues and also recognizes the Tn antigen (GalNAc--Ser/Thr) that initiates O-GalNAc linked oligosaccharides (10) . To determine whether the -GalNAc-terminated molecule would bind to this lectin, an aliquot of the [H]galactose-labeled material was applied to a column of immobilized H. pomatia agglutinin. To find the elution position of unbound molecules, an internal marker of SOwas added to each sample before applying it to the column. Fig. 1 shows that about 40% of [H]galactose-labeled material eluted exactly with SO, while the remainder bound to the column and eluted by continued buffer wash in the absence of any competing saccharide (GalNAc). Recovery was 90-95%. The broad elution profile is typical of molecules that interact weakly with commercial immobilized lectins. To be certain that the samples ran true, they were pooled separately and rerun over the same column again. The bound material completely rebound and the unbound material ran through the column (data not shown). Digestion of the total material with chicken liver -GalNAcase or inclusion of 50 m M GalNAc at the beginning of the run eliminates binding, and all of the label runs in the void volume, exactly co-incident with SO. In contrast, inclusion of 50 m M Glc does not prevent binding of the labeled material to the column (see Fig. 1).


Figure 1:H. pomatia agglutinin affinity chromatography of sialidase and glucuronidase-resistant anionic [H]Gal-labeled XylMU products. Anionic [H]Gal-labeled xylosides which were resistant to sialidase and glucuronidase digestions were passed over a column of immobilized H. pomatia agglutinin which binds oligosaccharides with terminal GalNAc residues as described under ``Experimental Procedures.'' Panel A, sample applied directly to the column and eluted with buffer (), or following digestion with GalNAcase (). Panel B, a similar aliquot is added to a column pre-equilibrated in 50 m M Glc (), or 50 m M GalNAc (). In each case the arrowhead () marks the elution position of an internal standard of SOwhich marks the void volume of the column.



Occurrence of -GalNAc Capped -Xylosides in Other Cells

We previously showed that melanoma, Chinese hamster ovary cells (CHO), and U937 cells make an unusual sialylated xyloside, Sia3Gal4XylMU (6) . These cells and several other cell types also made anionic xyloside products that were resistant to sialidase and -glucuronidase digestions. Considering the results shown in the companion study (1) , we re-examined the resistant products secreted in the presence of 1 m M XylMU. In each case, an aliquot was digested with an individual glycosidase or a combination of glycosidases and then analyzed by QAE-Sephadex ion exchange chromatography to determine the amount converted into a neutral product. Their size was then determined by HPLC. Table II shows the results of these digestions and the likely structure based on that of the core saccharide. It is clear that the relative proportions of the products varies with the cell type. Human neuroblastoma (IMR-32) and human leukemia cells (HL-60) are rich sources of -GalNAc-capped material, while CHO and the human monocyte cell line (U937) appear to make relatively little. It was surprising that none of the cell lines made any labeled material that was neutralized by sequential -hexosaminidase and -glucuronidase digestions, even though a structure with a terminal -GalNAc residue is expected to be one of the intermediates in chondroitin sulfate chain synthesis. The reason for this is not known, but the most likely explanation is that the addition of -GalNAc residues leads to rapid formation of highly charged chondroitin sulfate chains that do not bind to C-18 cartridges and are discarded during preparation (data not shown).

Effects of the Aglycone Structure on the Synthesis of -GalNAc-capped Xyloside

Most studies with -xylosides use a variety of aglycone ( e.g. MU and pNP) derivatives interchangeably, since they generally have the same effects on GAG chain biosynthesis (11, 12, 13, 14) . However, recent evidence clearly shows that the structure of the aglycone and its concentration substantially influence the proportion of chondroitin sulfate or heparan sulfate chains made on these acceptors (14, 15) . To determine whether the aglycone effects the relative amount of -GalNAc-capped xylosides, both melanoma and neuroblastoma cells were incubated with 1 m M MU or pNP-xyloside and labeled with [H]Gal. The low molecular weight secreted products were purified on C-18 cartridges and the results are shown in Table III. XylMU is a better acceptor than XylpNP and both cell lines show a characteristic distribution of neutral and anionic products. Table IV shows the results of digesting the xylosides having one negative charge with various exoglycosidases. Both cells make a greater proportion of -GalNAc-capped material using XylpNP as an acceptor compared to XylMU. In keeping with this finding, the proportion of total anionic labeled pNP product from neuroblastoma cells which binds to the H. pomatia agglutinin column is correspondingly increased, and -GalNAcase treatment eliminates this binding (shown in Fig. 2).

Xyloside Concentration Determines the Proportion of -GalNAc-capped Material

In previous studies we found that the proportion of Sia3Gal4XylMU made by melanoma and CHO cells varied with the concentration of xyloside (6) . At low concentration (<0.1 m M), the amount was very small (10% of total) while at 1-2 m M it became the predominant product (60-65%). Free GAG chains usually accounted for 10% of the label found in the low molecular weight xyloside products. Other cell lines, such as the neuroblastoma cell line (IMR-32) made almost no sialylated material using 1 m M xyloside. At the time of this study, the other anionic products could not be analyzed; however, the present results prompted a reinvestigation. As shown in Fig. 3, GAG chains account for 5-10% of the total incorporation. The total amount of -xyloside product is nearly maximal by 0.3 m M in melanoma cells, but continues to increase up to 1 m M in CHO cells. However, in both cell lines, the proportions of the various products change. The -GalNAc-capped molecule is the major product (45-65%) made by both melanoma and CHO cells, but only at low (0.03 m M) xyloside concentration. At higher concentrations, the proportion decreases to 5-10%, and is replaced by the sialylated molecule which can account for 70% of the products. The absolute amount of -GalNAc capped material decreases also. The basis for this change is not clear, but in rat liver Golgi the first galactosyltransferase (GalT I) is co-localized with an 2,3-sialyltransferase but not with the second galactosyltransferase (GalT II) (16) . Perhaps at high ``effective concentration'' of XylMU the co-localized GalT I and sialyltransferase consume the XylMU before it ever reaches the compartment containing the next enzyme(s) in the GAG-core biosynthesis. Whatever the reason may be for the changes in amounts of different products, the predominant synthesis of the GalNAc-capped pentasaccharide at low acceptor concentration strongly suggests that it is probably a naturally occurring structure, rather than an artifact of incubation with xylosides.


DISCUSSION

We have shown that a novel structure can be made on different -xylosides by human melanoma, leukemia, monoblast, neuroblastoma, and Chinese hamster ovary cells. The structure is identical to the typical carbohydrate core linkage glycan except for the presence of a terminal GalNAc residue. Thus, this modification is probably common to many types of cells. In addition, a search of the CarbBank data base showed there are no other known carbohydrate structures that contain a GalNAc-GlcA- terminal disaccharide. In preliminary experiments, we found that microsomes from melanoma cells could catalyze the addition of -linked [H]GalNAc from UDP-[H]GalNAc to GlcAMU, and this material was sensitive to - N-acetylgalactosaminidase digestion.() Further work is needed to define this enzymatic activity.

The -GalNAc-terminated structure has not been found in naturally occurring GAG chains. All studies to date show the presence of either -GalNAc or -GlcNAc at this position. Perhaps the core resembles another naturally occurring molecule and becomes an alternate, but inappropriate acceptor of the -GalNAc residue. Sia3Gal4Xyl-MU (6) and Glc-Glc-Xyl-MU (5) , and Xyl4XylMU and GlcAXylMU (7) and those reported in the companion article (1) seem to be unrelated to the known biosynthetic pathway of GAG core synthesis. However, preferential synthesis of GalNAc-terminated product at low xyloside concentration (0.03-0.1 m M) argues that its occurrence is not an artifact. These concentrations of -xylosides typically give nearly maximum inhibition of proteoglycan synthesis (11, 13, 14, 17, 18, 19, 20) .

The reason for the preferential synthesis of the sialylated molecule is unclear, but we speculate that the co-localization of the first galactosyl (GalT I) and a sialyltransferases accounts for these results. Several studies have suggested that some galactosyl and sialyltransferases are co-localized in the same Golgi compartments (21, 22, 23, 24) , and we recently found that the first GAG-core galactosyltransferase is substantially co-localized with an 2,3-sialyltransferase in rat liver Golgi (16) .

The addition of the -GalNAc residue occurs at a critical location in the assembly of GAG chains. Even though chondroitin sulfate and heparan sulfate have the same core saccharide structure terminating in GlcA, the next sugar to be added is -GalNAc in chondroitin/dermatan sulfate chains or -GlcNAc in heparan sulfate chains. All of the factors that control the type of chains added to the common core are not known; however, it is clear that the amino acid sequence of the core protein (or structure of the aglycone in -xyloside acceptors) influences the decision (25) . Also, the core saccharide region of some chondroitin sulfate molecules are modified by phosphorylation of xylose residues and sulfation of one or both galactose residues or the first -GalNAc residue (26, 27, 28) . Heparan sulfate core structures do not appear to have these modifications (29) , suggesting that they may serve as a signal and influence the type of chain added. In at least one instance, the enzymes that polymerize and modify chondroitin sulfate are segregated from those used in heparan sulfate biosynthesis (30) , so the reactions may not be strictly competitive.

The significance of -GalNAc residue at this crucial location is unknown. It could be a stable intermediate in more complex pathway or a transient intermediate that is later replaced by the typical -GalNAc or -GlcNAc residues in chondroitin sulfate or heparan sulfate, respectively. If -GalNAc halts further elongation of the GAG chains, this could be involved in the synthesis of part-time proteoglycans where only a portion of the core proteins contain GAG chains (31, 32) . This will require identification of such an oligosaccharide on a protein.

  
Table: QAE-Sephadex chromatography of anionic [H]Gal xyloside

Effects of various enzyme digestions on the binding of anionic [H]Gal-labeled xyloside products to QAE-Sephadex were done using aliquots (1000 cpm) of sialidase and -glucuronidase-resistant material was digested with the indicated glycosidase(s) and then analyzed on QAE-Sephadex to determine the percentage neutralized.


  
Table: Composition of anionic -xyloside products secreted by various cells

Each cell line was incubated for 6 h in 1 m M XylMU and 10 µCi/ml of [6-H]galactose and the anionic xyloside products were purified and then digested with either sialidase, -glucuronidase alone, -glucuronidase plus -hexosaminidase, or - N-acetylgalactosaminidase plus -glucuronidase as described under ``Experimental Procedures.'' The digest was fractionated on QAE-Sephadex to measure the percentage neutralized by the digestion. The size of the neutralized product was determined as described under ``Experimental Procedures.''


  
Table: Distribution of C-18 cartridge bound xyloside products from melanoma and neuroblastoma cells

Each of the cell lines were incubated for 6 h with 1 m M XylMU or XylpNP as described under ``Experimental Procedures.'' The secreted xyloside products were fractionated on QAE-Sephadex into neutral molecules, those with 1 negative charge, and those with at least 2 negative charges. The results are expressed as H cpm x 10/100 µg of protein. Numbers in parentheses are the % of cpm in each fraction.


  
Table: Analysis of xyloside products with 1charge from human melanoma and neuroblastoma cells

Each sample described in Table III with one negative charge was digested with a single or combination of enzymatic digestions to determine the terminal sugar residue(s). The amount neutralized by each digestion is expressed as a percentage of total with one charge. The size of the neutralized product was determined by amine adsorption chromatography on an AX-5 column as described under ``Experimental Procedures.''



FOOTNOTES

*
This work was supported by Grants NCI RO1 CA49094 and NCI RO1 CA49243 from the National Cancer Institute and American Cancer Society Grant BE-181. 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.

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

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

The abbreviations used are: XylMU, 4-methylumbelliferyl--xyloside; GAG, glycosaminoglycan; MU, 4-methylumbelliferyl; XylpNP, p-nitrophenyl--xyloside; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; HPLC, high performance liquid chromatography.

P. V. Salimath and H. H. Freeze, unpublished results.


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