©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Novel Method to Co-localize Glycosaminoglycan-Core Oligosaccharide Glycosyltransferases in Rat Liver Golgi
CO-LOCALIZATION OF GALACTOSYLTRANSFERASE I WITH A SIALYLTRANSFERASE (*)

(Received for publication, October 18, 1994; and in revised form, November 15, 1994)

James R. Etchison Geetha Srikrishna Hudson H. Freeze (§)

From the La Jolla Cancer Research Foundation, Glycobiology/Carbohydrate Chemistry Program, La Jolla, California 92037

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

4-Methylumbelliferyl-beta-xyloside (XylbetaMU) primes glycosaminoglycan synthesis by first serving as an acceptor for the addition of 2 galactoses and 1 glucuronic acid residue to make the typical core structure, GlcUAbeta1, 3Galbeta1,3Galbeta1,4XylbetaMU. To investigate the relative localization of these biosynthetic enzymes, intact and properly oriented rat liver Golgi preparations were incubated with XylbetaMU and 1 µM UDP-[^3H]Gal and then chased with 5 µM of unlabeled UDP-Gal, UDP-GlcUA, UDP-GlcNAc, UDP-GalNAc, and CMP-Neu5Ac. Under these conditions, no intervesicular transport occurs and acceptor labeling depends entirely upon transporter-mediated delivery of the labeled sugar nucleotides into the lumen of a vesicle and co-localization of the appropriate glycosyltransferases. The labeled products were isolated from the incubation medium and from within the Golgi and their structures analyzed by C18, anion-exchange, and amine adsorption high performance liquid chromatography in combination with glycosidase digestions. Surprisingly, the major products within the Golgi were two sialylated xylosides (Siaalpha2,3Galbeta1,4XylbetaMU and Siaalpha2,8Siaalpha2,3Galbeta1,4XylbetaMU) rather than the expected group of partially completed GAG core structures. Less than 10% of the products within the Golgi are the expected core structures containing a second Gal residue or, in addition, GlcUA. The amount of the sialylated products is only partially decreased if the chase is omitted or if the chase is done in the absence of added CMP-Sia, suggesting a pool of previously transported CMP-Sia drives synthesis of the major products. Conversely, when detergent permeabilized vesicles are provided with high concentration of the same sugar nucleotides, the ratio of sialylated products is reduced and replaced by an increase in GAG-like products. These results argue that GAG core-specific Gal transferase I and II are not extensively co-localized within the same Golgi compartment. By contrast, glycosaminoglycan core Gal transferase I is substantially co-localized with an alpha-2,3-sialyltransferase and an alpha-2,8-sialyltransferase. Incubating intact Golgi vesicles with exogenous diffusible acceptors offers a novel method to assess the functional co-localization of glycosyltransferases of multiple pathways within the Golgi compartments.


INTRODUCTION

Most of the glycosylation reactions involved in the biosynthesis of glycoconjugates (glycoproteins, proteoglycans, and glycolipids) occur in the Golgi. The sugar nucleotide donors are made in the cytoplasm, transported, and concentrated into the Golgi lumen by specific transporters(1) . Glycosyltransferases located on the lumenal face of the Golgi utilize the transported nucleotide sugars to glycosylate the various glycoconjugate acceptors transiting through different Golgi subcompartments during their biosynthetic maturation. A popular view is that the required glycosyltransferases are sequentially ordered in the Golgi cis, medial, trans compartments roughly corresponding to the known order of glycosylations(2, 3, 4, 5, 6) . This generally accepted view primarily addresses the early steps in N-linked glycosylation(7) , but the later steps in this pathway, O-linked glycosylation, and most of the GAG (^1)synthesis, may occur in the trans-Golgi (8) and trans-Golgi network(9) . An alternative perspective of the Golgi is that the glycosyltransferase organization is more flexible and cell-type dependent(10, 11, 12, 13, 14) . Regardless of the specific distribution of glycosyltransferases, any glycosylation step requires that a functionally active Golgi compartment must have 1) sugar nucleotide transporters to deliver 2) the donor sugar nucleotides to the lumen of the compartment where 3) the acceptors and 4) glycosyltransferases are spatially and temporally co-localized(15) . In the dynamic setting of the living cell, vesicular trafficking (transport) continuously delivers maturing glycoconjugates from one compartment to the next. In an in vitro, non-transporting, static system, a functional compartment defines itself by having all the essential components spatially co-localized.

This concept was recently exploited by Hayes et al.(16, 17, 18) in a series of papers showing that endogenous acceptors in sealed Golgi vesicles could be radiolabeled by a sugar nucleotide transporter-mediated process. A chase with non-labeled sugar nucleotides led to further glycosylation of the labeled endogenous acceptors. Since this occurred in the absence of intervesicular transport, all of the contributing transporters and transferases involved in the labeling and subsequent glycosylation of the products must have resided in a single functional compartment as defined above.

We have extended this approach by using a small freely diffusable glycoside in place of the normal endogenous acceptors. We chose XylbetaMU as a specific acceptor for the GAG core-specific galactosyltransferase I, and then asked whether other glycosyltransferases and sugar nucleotide transporters needed for GAG core synthesis were also functionally co-localized with this transferase. XylbetaMU primes glycosaminoglycan synthesis by first serving as an acceptor for the addition of galactose and glucuronic acid residues to make the core structure, GlcUAbeta1,3Galbeta1,3Galbeta1,4XylbetaMU(19, 20) . The results obtained in the present studies using the rat liver ``freeze-frame'' Golgi incubations suggest that the first GAG core-specific galactosyltransferase is substantially co-localized with one or more sialyltransferases and not with the second galactosyltransferase required for GAG core synthesis. This unexpected co-localization may explain why, in a previous study(20) , sialylated xylosides were the major products made by melanoma and CHO cells incubated with XylbetaMU. Using purified Golgi vesicles and diffusible acceptors offers a novel method to assess the functional co-localization of glycosyltransferases within the Golgi compartments.


EXPERIMENTAL PROCEDURES

Materials

UDP-[6-^3H]galactose (10 Ci/mmol) was purchased from American Radiolabeled Chemicals or prepared by the procedure of Hayes and Varki(21) . Unlabeled UDP-Gal, UDP-GlcNAc, UDP-GlcUA, UDP-GalNAc, CMP-Neu5Ac, and 4-methylumbelliferyl beta-D-xyloside (XylbetaMU) were purchased from Sigma. N-Acetyllactosamine was purchased from Calbiochem. Jack bean beta-galactosidase, Streptococcus pneumoniae beta-galactosidase, bovine testicular beta-galactosidase, Arthrobacter ureafaciens sialidase, Salmonella typhimurium sialidase, and Newcastle disease virus neuraminidase were from Oxford Glycosystems. Bovine testes beta-1,3-glucuronidase was a kind gift from Dr. Phillip Stahl, Washington University, St. Louis, MO. Ilimaquinone was a generous gift from Dr. Vivek Malhotra, University of California, San Diego, La Jolla, CA. Micro-Spin filters were manufactured by Lida Manufacturing Corp., Kenosha, WI. C18 spice cartridges and C18 modified silica gel resin were obtained from Analtech, Newark, DE.

Isolation of Golgi-enriched Subcellular Fraction

Golgi-enriched membranes were prepared from rat liver essentially as described by Leelavathi et al.(22) , Tabas and Kornfeld(23) , and Hayes et al.(16) . The materials, reagents, and purification procedure closely followed those detailed by Hayes et al.(16) . All sucrose solution concentrations were verified by specific gravity measurement. All procedures were carried out at 0-4 °C unless otherwise noted. Four male Harlan Sprague-Dawley rats (3-4 months old; Charles River) were fasted overnight, anesthetized with isoflurane, and decapitated with a guillotine. The blood was drained for about 30 s, and the livers were rapidly excised and placed in ice-cold homogenization buffer (0.5 M sucrose in 50 mM sodium maleate, 5 mM MgCl(2), pH 6.5). The excised livers were then blotted dry, weighed, finely minced with fine scissors and razor blades, and placed in homogenization buffer at 6-7 g/20 ml. The liver was homogenized with a two-speed Bio Homogenizer (M133/1281-0, BioSpec Products Inc., Bartlesville, OK) at high speed for 30 s and centrifuged at 600 times g for 10 min in a Sorvall SA600 rotor. The postnuclear supernatant was carefully decanted from the loose pellet and saved. The pellet was resuspended in one half the original volume of homogenization buffer and re-homogenized and centrifuged as above. The postnuclear supernatants were combined and portions (16 ml) were layered over 8 ml of 1.3 M sucrose in 50 mM sodium maleate, 5 mM MgCl(2), pH 6.5, buffer (MgM) in a centrifuge tube for the Beckman SW28 rotor. The postnuclear supernatant was overlaid with 13 ml of MgM and centrifuged for 2.5 h at 23,000 rpm. The crude smooth membrane fraction (CS) at and just below the 1.3 M sucrose interface was collected and adjusted to a final sucrose concentration of 1.1 M based on specific gravity. Portions (18 ml) of the CS were layered over 8 ml of 1.25 M sucrose in MgM in a centrifuge tube for the Beckman SW28 rotor. The CS was overlaid with 7 ml of 1.0 M sucrose in MgM, and this was overlaid with 4 ml of MgM. The Golgi membrane fraction was floated to the MgM, 1.0 M sucrose interface by centrifugation for 1.5 h at 23,000 rpm in the SW28 rotor. The Golgi membrane enriched fraction was collected from the MgM, 1.0 M interface and adjusted to a final sucrose concentration of 0.37 M and kept on ice.

Purification of Golgi membranes was monitored by assaying for galactosyltransferase enrichment. UDP-Gal:GlcNAc Galbeta1,4-galactosyltransferase was assayed exactly as described previously(16) . Two separate preparations were used in the experiments described in this report, and both had comparable specific activities (2.5 milliunits/mg of protein). Contamination of the Golgi membranes with soluble cytoplasmic enzymes was followed by measuring lactate dehydrogenase activity. Appropriate dilutions of the subcellular fractions were incubated in a 1-ml reaction at 23 °C containing 0.2 M Tris-HCl, pH 7.3, 1 mM sodium pyruvate, and 0.22 mM NADH. Enzyme activity was measured by following the oxidation of NADH spectrophotometrically at 340 nm. One unit equals 1 µmol of NADH oxidized per min at 23 °C.

Incubation of Golgi Fraction with UDP-[^3H]Gal and Xyloside Acceptor

Portions (0.5 mg) of the Golgi-enriched fraction were incubated with 1 µM UDP-[^3H]Gal (2.5 µCi; 10 Ci/mmol) in a final volume of 250 µl containing 5 mM MnCl(2), MgM buffer, approximately 0.32 M sucrose, and varying concentrations of XylbetaMU. Incubations were done at room temperature (22-23 °C) for 15 min and, unless otherwise noted, were chased for an additional 15 min after adding 6.25 µl of a mixture of unlabeled sugar nucleotides containing 220 µM each of UDP-Gal, UDP-GlcUA, CMP-Neu5Ac, UDP-GlcNAc, and UDP-GalNAc in MgM buffer to give a final concentration of 5 µM each. The incubations were stopped by the addition of approximately 10 volumes of ice-cold MgM buffer and immediately centrifuged for 20 min at 70,000 rpm in the Beckman TLA-100.2 rotor in a TL100 ultracentrifuge. The supernatants were collected, frozen at -80 °C, and lyophilized. The pellets were gently surface-washed three times with ice-cold MgM buffer and frozen at -80 °C.

To verify that the incubations with the Golgi-enriched fraction were dependent on the transport of nucleotide sugars, reactions were carried out as above in the presence of 0.2% Triton X-100. To demonstrate the absence of intercompartmental transport, reactions were carried out as above after pretreatment of the Golgi-enriched fraction with 2 mMN-ethylmaleimide (24) or 30 µM ilimaquinone(25) . To control for the possibility that galactose labeled xyloside product could diffuse from one compartment to another, reactions were carried out as above in the presence of 4 units/ml beta-galactosidase (jack bean).

Additional reactions were carried out after preincubating the Golgi fraction with 1 mMN-acetyllactosamine to attempt to deplete the endogenous CMP-Sia pools and thus favor the formation of GAG core products. These reactions were stopped after the primary 15-min incubation and not chased with the cold nucleotide sugars used in the standard incubation conditions described above. The controls for these incubations used the standard incubation conditions without the nucleotide sugar chase. In another reaction, the Golgi fraction was incubated using the standard conditions except that the chase contained only 5 µM UDP-Gal and UDP-GlcUA.

Transport-independent, permeabilized Golgi reactions were carried out with the following modifications to the standard incubation conditions. The Golgi membranes were permeabilized by the addition of 0.2% Triton X-100, the UDP-[^3H]Gal concentration was increased to 50 µM (25 µCi; 2 Ci/mmol), and the concentration of the nucleotide sugars present during the chase was increased to 100 µM.

To verify that the purified Golgi vesicle fraction was properly oriented and sealed, protease protection of radiolabeled endogenous acceptors was assayed using the methods described previously(16) . The capacity of the Golgi vesicles to transport UDP-Gal and transfer radiolabeled Gal to endogenous acceptors was quantified as described by Perez and Hirschberg(26) .

Extraction and Purification of Xyloside Products from Golgi Incubations

The lyophilized supernatants from the Golgi incubations were extracted with two 1-ml portions of 70% ethanol. The supernatant extracts (SEs) were dried in vacuo on a shaker evaporator at 40 °C. The ethanol-insoluble material (SPs) was saved and stored frozen. The XylbetaMU products were purified from the dried supernatant extracts by reverse phase chromatography on C18 spice cartridges (27, 28) which had been pre-washed with 5-10 volumes each of absolute methanol and water, and equilibrated with 1 M NaCl. The SEs were redissolved in 1 ml of 1 M NaCl and passed through the C18 cartridges. The cartridges were washed with two 1-ml portions of 1 M NaCl followed by two 1-ml washes with distilled water. The bound XylbetaMU products were eluted with three 1-ml portions of 40% methanol (SE40s). The remainder of bound products were eluted with two 1-ml portions of absolute methanol.

The frozen pellets from the Golgi incubations were resuspended by sonication in 0.5 ml 70% ethanol. The samples were centrifuged at maximum speed in a microcentrifuge for 5 min. The pellet was again resuspended by sonication in 0.5 ml of 70% ethanol, and the centrifugation was repeated. The supernatants (XTs) were combined and dried in a Savant Speed-Vac. The extracted pellets (XPs) were solubilized by sonication in 250 µl of 2% SDS in 10 mM Tris base for 10-20 s and stored frozen at -20 °C. The XylbetaMU products were purified from the dried supernatants (XTs) by reverse phase chromatography on C18 spin columns which had been pre-washed with four column volumes each of methanol, distilled water, and 1 M NaCl. The XTs were redissolved in 100 µl of distilled water and applied to the top of the C18 resin (0.2-ml bed volume) in microcentrifuge spin filter baskets. The spin columns were microcentrifuged at 3000 rpm for 1-2 min. The columns were washed with three 200-µl portions of 1 M NaCl followed by two 200-µl washes with distilled water. The bound XylbetaMU products were eluted with three 200-µl portions of 40% methanol (XT40s). The remainder of bound products were eluted with two 200-µl portions of absolute methanol.

The presence of detergent in the supernatants of the permeabilized Golgi incubations resulted in the elution of some of the anionic xyloside products in the water washes during the purification on the C18 cartridges. Initially, these were recovered by repurification of the water washes. Subsequently, we found that these anionic xylosides were retained on the C18 cartridges when washing was done with 0.1 M NH(4)COOH, pH 6, instead of distilled water.

Analysis of Xyloside Products

The radiolabeled xyloside products were analyzed by C18 reverse-phase HPLC on a 4.6 mm times 25-cm Microsorb MV-C18 column. The column was eluted isocratically with water for 5 min followed by a 0-50% methanol gradient over 45 min at a flow rate of 1.0 ml/min. Fractions (1 ml) were collected and assayed by liquid scintillation counting. The column was washed for 5-10 min with 100% methanol between runs. Anionic xyloside products with more than one negative charge bound to the C18 cartridges or spin columns during purification but were not retained on the C18 HPLC column. These were analyzed by modifying the above gradient; the water was replaced with 0.1 M NH(4)COOH, pH 6.

Analysis of xyloside products by amine adsorption HPLC was done using a 4.6 mm times 25-cm Microsorb-MV amino-bonded silica column. The gradient was 80-40% acetonitrile in water over 60 min at a flow rate of 1 ml/min.

Ion exchange HPLC analysis was carried out using the above amino column using the following gradient. After injection, the column was washed with water for 2 min; then a gradient of 0-50 mM sodium phosphate pH 4.3 over 25 min; 50-150 mM over 15 min; and, 150-250 mM over 10 min. The flow rate was 1 ml/min, and 1-ml fractions were collected. A small amount of Na(2)[S]SO(4) (50-200 cpm) was used as an internal marker in each run.

Glycosidase Digestions

Sialidase and beta-galactosidase digestions were carried out according to the manufacturer's recommendations in volumes of 10-50 µl using the supplied buffers. All digestions were terminated by heating at 100 °C for 2-5 min, cooled, and microcentrifuged. beta-Glucuronidase digestions were performed in 50 mM sodium acetate, pH 5.5, in 10-50 µl with 1 µl of enzyme (150 Fishman units/ml) overnight. Sialidase and beta-glucuronidase digestions were monitored using QAE-Sephadex in small columns as described previously (28) or in spin columns containing a 0.2-ml bed volume using similar elution conditions. Release of radiolabeled galactose from XylbetaMU products after beta-galactosidase digestion was quantified by separation of the products using C18 spin columns.

Chemical Degradation

Mild acid hydrolysis to cleave sialic acids was carried out using 0.05 M HCl at 80 °C for 30 min. Methanolysis to remove equatorial and axial sulfate esters was carried out as follows(29) : The sample was dried in vacuo over P(2)O(5), treated with 0.5 N methanolic HCl at room temperature for 5 h, and dried in vacuo over KOH pellets. Residual HCl was removed by repeated evaporation from methanol over KOH pellets. Methyl esters were hydrolyzed by treatment with 0.05 N NaOH for 30 min at room temperature followed by neutralization with HCl.


RESULTS

Characterization of the Golgi Fraction

A Golgi-enriched fraction from rat liver was prepared essentially as described by Hayes et al.(16) with minor modifications (see ``Experimental Procedures''). beta1,4-Galactosyltransferase activity was used to monitor Golgi purification, and, as shown in Table 1, its specific activity increased 100-fold with a 25-30% recovery. Since our approach depends on the absence of intervesicular transport to create freeze-frame glycosylation reactions, we also monitored lactate dehydrogenase activity as a measure of cytosolic contamination since cytoplasmic proteins are needed for intervesicular transport(32) . Less than 0.02% of the total lactate dehydrogenase activity co-purified with the Golgi fraction. This low level of cytoplasmic contamination will not support intercompartmental transport. Moreover, we show that inhibitors of intercompartmental transport have no effect on the amount or types of products made (see below).



To further characterize the purification of the ``functional'' Golgi fraction, we monitored labeling of endogenous acceptors. This assay requires not only the galactosyltransferase activity, but also co-localized UDP-Gal transporter and endogenous acceptors. As shown in Table 1, this activity is enriched almost 1000-fold in the Golgi fraction, but not in other fractions from the sucrose gradient. We likewise assayed the various fractions for their ability to galactosylate XylbetaMU and found that only the classical Golgi fraction was active (Table 1).

To demonstrate that the labeling depends on the transport and concentration of UDP-Gal in the lumen, a portion of the Golgi fraction was incubated (see ``Experimental Procedures'') with 0.1% Triton X-100 to permeabilize the Golgi membranes. This reduced incorporation into endogenous acceptors 15-20-fold (data not shown)(16) . We also quantified the ability of the Golgi fraction to transport UDP-Gal as described by Perez and Hirschberg(26) : 187 pmol/mg of protein was transported in our standard incubation conditions (1 µM UDP-Gal, 15 min, pH 6.5 buffer). This compares well with a value of 372 pmol/mg of protein reported by Perez and Hirschberg (26) using different incubation conditions (2 µM UDP-Gal, 10 min, pH 7.5 buffer).

Fig. 1shows that the incorporation into endogenous acceptors occurs inside the Golgi lumen. Portions of one sample were incubated in the presence of Triton X-100 alone, Pronase alone, or Triton X-100 plus Pronase for 1 and 16 h. The results showed that the incorporated [^3H]Gal was susceptible to Pronase digestion only if the Golgi was permeabilized with Triton X-100. The above results show that the Golgi preparations are intact, properly oriented, and that synthesis requires transportermediated delivery of sugar nucleotides into the lumen.


Figure 1: Protection of the UDP-[^3H]Gal labeled endogenous acceptors from protease digestion. The Golgi vesicles from one standard incubation with 1 µM UDP-[^3H]Gal were split into six aliquot portions. Two portions were treated with 0.1% Triton X-100; two with 1 mg/ml Pronase; and two with 0.1% Triton X-100 and 1 mg/ml Pronase. The samples were incubated at 37 °C and a sample from each set analyzed after 1 and 16 h for perchloric acid-precipitable radioactivity(16) . The dark bars represent the 1-h incubations; the light, the 16-h incubations. The 100% of control value is defined as the 1-h incubation in the presence of Triton X-100 only.



Incorporation of [^3H]Gal into Xyloside Products and Their Characterization

The moderately hydrophobic 4-methylumbelliferyl group of XylbetaMU enables the xyloside to ``diffuse'' across membranes and serve as an alternate acceptor (and inhibitor) for GAG chain initiation(20, 36) . This hydrophobicity also facilitates binding to C18 reverse phase media(20, 27) . To characterize the xyloside products synthesized, we devised the fractionation scheme shown in Fig. 2for the quantitative recovery of the xyloside products. The 70% ethanol-insoluble material from the supernatants (SPs) contained less than 0.5% of the incorporated [^3H]galactose label and was not further analyzed. The 70% ethanol-soluble material from the Golgi pellets (XTs) and the insoluble material (XPs) were analyzed by gel filtration on Sephacryl 200 exactly as described by Hayes et al.(16) . The XTs contained only low molecular weight material while the XPs contained only high molecular weight material eluting in or near the column void volume (data not shown). The 40% methanol eluates from the C18 reverse phase purification of the SE and XT fractions (SE40s and XT40s) contained 70-90% xyloside products (e.g. see Fig. 4). Very little material elutes with 100% methanol.


Figure 2: Fractionation scheme and flow diagram for the isolation of [^3H]Gal-labeled xyloside products from the Golgi incubations. The SE40 fraction contains the xyloside products which diffuse from the Golgi lumen after being labeled with [^3H]Gal; the XT40 fraction, those which remain in the Golgi lumen. The insoluble XP products are mainly [^3H]Gal-labeled N-linked oligosaccharides attached to endogenous acceptors.




Figure 4: C18 reverse-phase HPLC fractionation of [^3H]Gallabeled xyloside products from Golgi supernatant and pellet fractions. [^3H]Gal-labeled xyloside products from Golgi supernatant and pellet fractions (SE40 and XT40, respectively, see Fig. 2) were fractionated by preparative C18 HPLC. Panel A shows the products in the supernatant; Panel B, those in the Golgi pellet. The data shown are for the 100 µM XylbetaMU incubation (open symbols); the data for the control incubation without XylbetaMU (solid symbols) are shown for comparison. The arrows mark the elution positions of authentic GalbetaXylbetaMU and GalbetaGalbetaXylbetaMU.



Fig. 3shows the incorporation of [^3H]galactose into xyloside products with different concentrations of XylbetaMU. Maximum incorporation is reached by about 50 µM and half maximal by 18 µM. Approximately half of the labeled xyloside products are found outside the vesicles. Since we have shown that the vesicles are sealed to macromolecules and the GAG-core specific Gal transferase is located within the Golgi, the products found outside must have diffused from the lumen. This is not unexpected; since the substrate, XylbetaMU, is freely diffusable, the product [^3H]GalbetaXylbetaMU may also be able to diffuse across the membranes, albeit more slowly. Based on our estimates and those of others (30) of the volume of the Golgi lumen (0.5-1 µl/mg of Golgi protein), the concentration of the xyloside products inside the lumen is at least 20-40 µM (averaged over the entire lumenal volume of the Golgi vesicles), while that on the outside is only 0.04-0.08 µM. As shown below, the xyloside products found outside the lumen consist almost entirely of GalbetaXylbetaMU, whereas those inside contain one or more additional sugar residues. This probably reflects a higher diffusion rate for the smaller, radiolabeled XylbetaMU product.


Figure 3: Incorporation of [^3H]Gal into xyloside products versus XylbetaMU concentration. Golgi vesicles were incubated with varying concentrations of XylbetaMU and 1 µM UDP-[^3H]Gal using the standard incubation and chase conditions (see ``Experimental Procedures''). The labeled xyloside products were purified from the supernatant and Golgi pellet (see ``Experimental Procedures'' and Fig. 2). Portions of each were assayed for radioactivity and the values expressed as picomoles of [^3H]Gal incorporated per mg of Golgi protein.



The labeled xyloside products from each XylbetaMU concentration and corresponding controls were fractionated by C18 reverse phase HPLC. Fig. 4shows a representative fractionation of products from the incubation supernatant (Panel A) and Golgi pellet (Panel B) samples (SE40 and XT40, respectively) using 100 µM XylbetaMU (open circles). An equal portion of a control incubation without XylbetaMU is superimposed for comparison (filled symbols). The major product (>90%) found in the supernatant coelutes with authentic GalbetaXylbetaMU and/or GalbetaGalbetaXylbetaMU (which are not resolved on this column). Amine adsorption HPLC (Fig. 5, Panel A) shows it is exclusively GalbetaXylbetaMU. By contrast, only a minor portion (5-10%) of the products from the Golgi pellet elutes at the position corresponding to GalbetaXylbetaMU and/or GalbetaGalbetaXylbetaMU (Fig. 4, Panel B). This product is mostly GalbetaXylbetaMU (80%) and some GalbetaGalbetaXylbetaMU (20%) (Fig. 5, Panel B).


Figure 5: Amine adsorption HPLC analysis of the neutral [^3H]Gal-labeled xyloside products from the Golgi supernatant and pellet fractions. The neutral [^3H]Gal-labeled xyloside products eluting with retention times corresponding to authentic GalbetaXylbetaMU and GalbetaGalbetaXylbetaMU (see Fig. 4) were analyzed by amine adsorption HPLC. Panel A shows the analysis of the neutral products in the supernatant; Panel B, the neutral products in the Golgi pellet. The arrows mark the elution positions of authentic GalbetaXylbetaMU and GalbetaGalbetaXylbetaMU.



The major products (50-70%) associated with the Golgi pellet eluted between fractions 15 and 25 (Fig. 4B, peaks II and III). These products are anionic based on their binding to QAE-Sephadex, and 80-90% of the label in peak II was neutralized by digestion with either the broad spectrum A. ureafaciens sialidase or the alpha-2,3-specific NDV sialidase. The neutral product obtained coelutes with GalbetaXylbetaMU on amine adsorption HPLC (Fig. 6B). Taken together, these results show that peak II is mostly (80-90%) Siaalpha2,3GalbetaXylbetaMU. However, approximately 3-7% of peak II was neutralized by beta-glucuronidase digestion and gives the expected GAG core digestion product, GalbetaGalbetaXylbetaMU (60%), and GalbetaXylbetaMU (40%). These results indicate that peak II contains 2-5% GlcUAbetaGalbetaGalbetaXylbetaMU. Similar analyses showed that peak III was also mostly sialylated and only a few percent could be neutralized by beta-glucuronidase digestion.


Figure 6: HPLC analysis of neutral products obtained after sialidase treatment of the major anionic product from the Golgi pellet. A portion of the major anionic product from the Golgi incubation pellet (see Fig. 4B, peak II) was digested with AUS and the neutralized products separated by QAE-Sephadex chromatography. Panel A shows the C18 HPLC analysis of this neutralized product; Panel B, the amine adsorption HPLC analysis of the same product. The arrows in Panel B mark the elution positions of authentic GalbetaXylbetaMU and GalbetaGalbetaXylbetaMU.



Approximately 20-30% of the products in the Golgi pellet (XT40s) were not retained by the C18 HPLC column (Fig. 4B, peak I, fractions 3-6). Most of the products from the control incubated without XylbetaMU also did not bind. QAE-Sephadex analysis showed that peak I was anionic. It was retained on the C18 HPLC column in the presence of 0.1 M NH(4)COOH, pH 6, acting as a charge suppression/ion-pairing agent. Fig. 7A clearly shows that the xyloside products in peak I were efficiently separated from the endogenous acceptor products eluting in fractions 12-22. Three xyloside products, Ia, Ib, and Ic, were analyzed by ion exchange HPLC and found to have a -2 charge (Fig. 7B). There was insufficient material for further analysis of peaks Ib and Ic which are only about 2-3% of the total xyloside products. Peak Ia was analyzed in detail. Digestion with either the broad spectrum A. ureafaciens sialidase or the alpha-2,8- and alpha-2,3-specific NDV neuraminidase converted 60-70% to neutral products. Treatment with 0.05 N HCl at 80 °C for 30 min to selectively hydrolyze sialic acid moieties also neutralized 85-90%. Analysis of these neutral products by amine adsorption HPLC showed that the major desialylated species obtained was GalbetaXylbetaMU (Fig. 7C). These results suggested that peak Ia was structurally related to the Siaalpha2,3GalbetaXylbetaMU product described above but contained an additional sialic acid residue. Considering the similarity of the latter product with ganglioside GM3 (Siaalpha2,3GalbetaGlcbetaCer), we reasoned that it might be similar to ganglioside GD3 (Siaalpha2,8Siaalpha2,3GalbetaGlcbetaCer). To confirm this we digested it with S. typhimurium sialidase (which does not cleave Siaalpha2, 8Siaalpha2-X linkages but will cleave alpha-2,3- and alpha-2,6-linked Sia residues). Peak Ia was resistant to this digestion and retained both negative charges. This means that an alpha-2,8-linked Sia must have been attached to and blocked digestion of the S. typhimurium-sensitive alpha-2,3-linked Sia. Taken together, these results indicate that the structure of peak Ia is most likely Siaalpha2,8Siaalpha2,3GalbetaXylbetaMU. This material represented 10-15% of the sialylated xyloside products.


Figure 7: HPLC analysis of the highly anionic products from the Golgi pellet. The anionic products which were not retained by the C18 HPLC column using the standard water/methanol gradient were further analyzed by C18 HPLC using an ammonium formate/methanol gradient (Panel A, open symbols). An equal portion of the same peak from Golgi vesicles incubated without XylbetaMU and analyzed in parallel is shown for comparison (solid symbols); this shows the label in fractions 12-22 is not due to xyloside products. Peak Ia was further analyzed by anion exchange HPLC (Panel B); the arrow labeled -2 shows the elution position of Na(2)[S]SO(4). The relative elution position for Siaalpha2,3GalbetaXylbetaMU is indicated for comparison. Peak Ia was digested with AUS, the neutralized products were isolated by QAE-Sephadex, and these neutral products were analyzed by amine adsorption HPLC (Panel C). The arrows in Panel C mark the elution positions of authentic GalbetaXylbetaMU and GalbetaGalbetaXylbetaMU.



Table 2shows the products made by the Golgi at various concentrations of XylbetaMU. The concentration of XylbetaMU has little effect on the relative proportion of the products. Most products in the Golgi lumen are sialylated (75-80%). Sialidase digestions (see above) established the linkages shown. Since the broad spectrum AUS and the alpha-2,3-specific NDV both neutralized 85-95% of the SiaalphaGalbetaXylbetaMU, only a small percentage (5-10%) of the Sia could be in alpha-2,6 linkage. The linkage of the Gal in GalbetaXylbetaMU is beta-1,4 since this product is hydrolyzed by the beta-1,4-specific galactosidase from S. pneumoniae, and we have previously shown that XylbetaMU is galactosylated only by GAG core galactosyltransferase I(28) . The second Gal residue in the GalbetaGalbetaXylMU is presumed to be a beta-1,3 linkage by analogy with the GAG core structure. This Gal residue was resistant to hydrolysis by the S. pneumoniae beta-galactosidase and the Galbeta1,3GlcNAc-specific beta-galactosidase from Xanthomonas manihotis, but was hydrolyzed by Aspergillus niger, jack bean, and bovine testicular beta-galactosidases.



Xyloside Products Made during Modified Chase Conditions

The GAG core related products (i.e. GalbetaGalbetaXylbetaMU and GlcUAbetaGalbetaGalbetaXylbetaMU) account for only 10% of the products in the Golgi lumen. To attempt to favor the formation of GAG core products, we incubated Golgi vesicles using the same conditions but modified the chase to contain only UDP-Gal and UDP-GlcUA (Table 3, Limited Chase). There was no increase in the synthesis of products containing the GAG core. The amount of sialylated products did not change, indicating that there is a substantial pool of CMP-Sia within the Golgi lumen even in the absence of adding CMP-Sia during the chase (Table 3). In another incubation we found that, if the chase is omitted, 35% of the products were still sialylated. Preincubation of the Golgi vesicles with 1 mMN-acetyllactosamine to attempt to deplete the endogenous CMP-Sia pools had no effect on the xyloside products made. Apparently, the LacNAc was unable to deplete the CMP-Sia pools in the Golgi lumen and/or serve as an alternate acceptor for the sialyltransferase.



Controls for Intervesicular Diffusion and Transport

Since 50-60% of the [^3H]GalbetaXylbetaMU made in the Golgi lumen diffuses out of the vesicles, it was important to demonstrate that further glycosylation of this product was not due to diffusion from one compartment into another. Therefore, we included 4 units/ml of purified beta-galactosidase in one incubation. As shown in Table 4, this completely degraded all [^3H]GalbetaXylbetaMU in the supernatant while the distribution of products within the Golgi lumen was not decreased. Surprisingly, there appeared to be a 40-50% increase in the amount of sialylated products.



Although the Golgi purification removes >99.9% of the soluble cytoplasmic proteins and presumably other soluble factors required for vesicular transport between Golgi compartments, we incubated the Golgi vesicles in the presence of either 30 µM ilimaquinone (25) or pretreated the Golgi with 2 mMN-ethylmaleimide(24) . Neither inhibitor of vesicular transport had any effect on the xyloside products made (Table 4; N-ethylmaleimide data not shown). Altogether, the above results demonstrate that the synthesis of the xyloside products in these in vitro Golgi incubations does not involve intercompartmental transport or diffusion.

Products Synthesized by Detergent-permeabilized/Transport-independent Golgi Vesicles

The above results show that sialylated xylosides, structurally analogous to gangliosides, are the predominant products made by in vitro ``freeze-frame'' incubations with purified Golgi vesicles. Similar products are made preferentially in tissue culture(20) . Nevertheless, the majority of previous studies focus on utilizing XylbetaMU as an alternate acceptor and inhibitor of GAG biosynthesis(20, 36) . Therefore, it was important to show that the Golgi fraction was capable of synthesizing the GAG core products expected for xylosides. Since selective chase conditions could not promote the synthesis of these products, one explanation is that the necessary enzymes are located in other compartments that are not available to the first labeled product, [^3H]GalbetaXylbetaMU. To test this, we reasoned that permeabilizing the Golgi vesicles with detergent and providing a high concentration of sugar nucleotides should facilitate free diffusion of the products and result in the synthesis of GAG core structures containing GalbetaGalbetaXylbetaMU even if the two galactosyltransferases are in separate Golgi compartments. The results presented below support this. There is a relative decrease in the synthesis of sialylated xyloside products and a corresponding increase in products containing the GalbetaGalbetaXylbetaMU core structure.

Fig. 8A shows the C18 HPLC profile of the products made by the detergent-permeabilized, transport-independent Golgi vesicles. All of the products were in the incubation supernatant; less than 1% remained in the Golgi lumen after centrifugation. The major neutral product was GalbetaXylbetaMU (68%) and very little (<2%) GalbetaGalbetaXylbetaMU. The peak corresponding to Siaalpha2,3GalbetaXylbetaMU and GlcUAbetaGalbetaGalbetaXylbetaMU was much smaller than seen in previous incubations and represented only about 10% of the total products as compared to about 30% in the standard incubation, indicating its synthesis is favored by continued presence in a single vesicle compartment. Approximately 20% of the products did not bind to the C18 HPLC column. The control incubation without XylbetaMU showed that endogenous acceptors accounted for less than 1% of these products. The xyloside products were shown to be anionic by QAE-Sephadex chromatography and, in contrast to the products from the standard incubations, were resistant to sialidase digestion. Fig. 8B shows that these products are heterogeneous by analysis on C18 HPLC using the ammonium formate/methanol gradient.


Figure 8: HPLC fractionation of the [^3H]Gal-labeled xyloside products made by detergent-permeabilized, transport-independent Golgi vesicles. Detergent-permeabilized Golgi vesicles were incubated with 50 µM UDP-[^3H]Gal and 100 µM XylbetaMU then chased with unlabeled sugar nucleotides as described under ``Experimental Procedures.'' The labeled xyloside products were purified and analyzed by HPLC. Panel A shows the fractionation of these products by C18 HPLC using a water/methanol gradient. The anionic xylosides which were not retained by the C18 column (see bar in Panel A) using this gradient system were rechromatographed by C18 HPLC using an ammonium formate/methanol gradient (Panel B).



Analysis of these anionic products by ion-exchange HPLC showed that the majority contained at least two negative charges (Fig. 9A). Treatment with beta-glucuronidase converted about 36% of the labeled products to neutral structures (Fig. 9B). Analysis of these neutral products by amine adsorption HPLC showed that they co-eluted with authentic Galbeta1, 3Galbeta1,4XylbetaMU. This was puzzling since the beta-glucuronidase digestion appeared to have removed two negative charged moieties. One possibility, although we know of no precedent, is that these products contained 2 terminal beta-GlcUA residues. Alternatively, they may have contained one GlcUA and one sulfate ester, and the beta-glucuronidase preparation may have contained a contaminating sulfatase activity. Addition of sulfate was not anticipated, since we intentionally did not add 3`-phosphoadenylylphosphosulfate to the incubations to avoid the difficult task of analyzing sulfated products. To examine the latter possibility, we subjected these products to mild anhydrous methanolysis using conditions known to cleave 4-O-sulfate esters but not the glycosidic linkages(29) . These conditions will also convert carboxyl groups to their methyl esters. Fig. 9C shows the products. Approximately 30% were converted to neutral, 35% were converted to structures with a single negative charge, and 35% still contained at least two negative charges. The neutral products obtained by this treatment were analyzed by amine adsorption HPLC and they co-eluted with authentic Galbeta1,3Galbeta1,4XylbetaMU. A parallel methanolyzed sample was treated with mild base to hydrolyze the carboxylic methyl esters and analyzed as above. Fig. 9D shows that the product having a single negative charge (see Fig. 9C) was absent after hydrolysis of the methyl esters; suggesting that this product may have contained both a GlcUA residue and a sulfate ester resistant to mild methanolysis. In separate series of analyses, treatment with beta-glucuronidase neutralized 35-40% of these products; methanolysis of the glucuronidase-resistant products neutralized an additional 25%. Analysis of these neutralized products by amine adsorption HPLC again showed that these co-eluted with Galbeta1,3Galbeta1,4XylbetaMU. The highly specific glycosidases and sulfatases needed to structurally define these radiolabeled products are not available. Nevertheless, we know that at least 65% of these products contain the GalbetaGalbetaXyl-core structure and most contain GlcUA and, probably, sulfate. On this basis, we refer to these products as ``GAG-like'' xyloside products.


Figure 9: Anion exchange HPLC analysis of [^3H]Gal-labeled xyloside products after beta-glucuronidase digestion and mild acid methanolysis. The anionic xylosides which were not retained by the C18 column (see bar in Fig. 8A) were analyzed by anion exchange HPLC (Panel A). The arrow marks the elution position of Na(2)[S]SO(4) (-2 charge). Panel B shows a similar analysis after digestion with beta-glucuronidase. Panel C shows the analysis after treatment with 0.5 N methanolic HCl for 5 h at 23 °C. Panel D shows the analysis after treatment with methanolic HCl as above followed by treating with 0.05 N NaOH at 23 °C for 30 min to cleave glucuronate methyl esters.



Table 5shows a summary of the products made by the detergent-permeabilized, transport-independent Golgi vesicles in comparison to those made under standard conditions. The most striking difference is the relative decrease in sialylated structures in the permeabilized Golgi and the corresponding increase in GAG-like structures. The structure of the GAG core region is considerably more complex than once thought(19, 37, 38, 39) . The galactose residues may be sulfated in the 4- or 6-position, the xylose residue may contain a phosphate at the 2-position, and the first GalNAc of the chondroitin sulfate core may be sulfated at the 4- or 6-position. In addition, we recently found a novel alpha-GalNAc residue replacing the typical beta-GalNAc in the chondroitin sulfate core made on xylosides by several different cell types. (^2)Essentially nothing is known about the biosynthesis of these modifications, their order of addition, site of synthesis, or their permanence. The results presented above are consistent with [^3H]GalbetaXylbetaMU being converted into a complex mixture of these anionic structures when incubated with a detergent-permeabilized Golgi preparation.




DISCUSSION

The pioneering studies of Hirschberg showed that sugar nucleotide transporters are essential for the synthesis of glycoconjugates within the Golgi(1) . Mutant cell lines deficient in these transporters make incomplete sugar chains(33, 34) . Hayes et al.(16, 17, 18) recently showed that intact and properly oriented Golgi preparations glycosylate endogenous acceptors when supplied with low concentration of labeled sugar nucleotides. When followed by a chase with non-labeled sugar nucleotides, the labeled chains can be further modified by adding more sugar residues to the radiolabeled chains. Since intervesicular transport does not occur under these conditions, all of the transporters, transferases, and acceptors must reside in the same compartment. Most of the products analyzed in those studies were N-linked oligosaccharides whose biosynthetic pathways have been well characterized. The approach showed an unexpected co-localization of some transferase activities with respect to other studies using different methods of localization. However, it was difficult to quantify the amount of this co-localization.

We wanted to adapt this approach to investigate the synthesis of the core regions of GAG chains. Xylosides have been used for more than 20 years to prime the synthesis of GAG chains and compete with endogenous acceptors. The core region, GlcUAbeta1,3Galbeta1,3Galbeta1,4Xylbeta-, is formed and then elongated by the addition of GlcUA and GalNAc or GlcNAc to initiate heparan or chondroitin sulfate chains. We reasoned that the freely diffusable acceptor could enter the Golgi vesicles, but would only be glycosylated in those which contained both the UDP-Gal transporter and the first galactosyltransferase. A subsequent chase with other sugar nucleotides would then result in the extension of the [^3H]Gal-labeled xylosides. The residues added next would depend upon which enzymes were substantially co-localized with the first galactosyltransferase. Considering the known structure of the core region, it would be reasonable to expect a series of partially completed core structures. However, in previous studies with intact human melanoma or Chinese hamster ovary cells(20) , we found that all of these cell lines preferentially made and secreted a sialylated product, Siaalpha2,3GalbetaXylbetaMU, when incubated with XylbetaMU and [^3H]Gal. These studies showed that the addition of Gal was catalyzed only by GAG core specific galactosyltransferase I; a mutant cell line lacking this specific transferase was unable to galactosylate XylbetaMU.

The results of the studies presented here support the hypothesis that the preferential synthesis of a sialylated, ganglioside-like structure is due to the co-localization of the first GAG core specific galactosyltransferase and at least one alpha-2,3-sialyltransferase. The synthesis appears to utilize previously transported CMP-Sia as well as donors supplied and transported during the chase. Even when CMP-Sia is not provided in the chase, the amount of sialylated molecules is not reduced. Most of the molecules labeled in the absence of a chase or chased in the absence of CMP-Sia still have only 1 Gal residue and do not have the second Gal or the GlcUA residues. Since we have shown that no intervesicular transport and no diffusion between vesicles occur, this strongly argues that Gal transferase I and II are not substantially co-localized. Previous studies (35) using subcellular fractionation and enzyme assays support this conclusion since the two activities were overlapping but not coincident. On the other hand there is substantial overlap with one or more sialyltransferases. This is even seen at the lowest concentration of xyloside (10-20 µM), and therefore is not an artifact due to high concentrations of XylbetaMU. We do not know which sialyltransferases carry out this reaction, but addition of sialic acid is restricted to glycoproteins and glycolipids and does not occur on GAG chains. As discussed previously, the major sialylated product resembles ganglioside GM3, though we do not know whether GM3 synthase carries out this sialylation. Approximately 10-15% of the sialylated XylbetaMU products resembles ganglioside GD3 and the alpha-2,8Siaalpha-2,3Sia- moiety occurs only in gangliosides and in those glycoproteins containing polysialic acid structures (e.g. N-CAM). These results show that both glycoprotein and/or glycolipid biosynthetic steps can occur in the same Golgi compartment as the first galactosylation step of the GAG core region. Previous studies by Vertel et al.(31) have shown that xylose can be added to endogenous acceptors in the transitional region between the ER and cis-Golgi. Our results suggest that the next step occurs in a functional compartment that contains both the galactosyltransferase I and sialyltransferase(s), but this method cannot distinguish whether these reactions occur in the cis, medial, trans, or other compartments. Results using other approaches and other cell lines often place sialyltransferases in the later Golgi compartments(2) . Other studies with Brefeldin A and immunolocalization suggest that some sialyltransferases may occur in early compartments of the Golgi(3, 20) . The studies of Sugumaran et al.(35) place GAG core galactosyltransferases in early (cis) Golgi compartments.

The liver and cell lines derived from it synthesize proteoglycans with typical GAG core structures(40, 41, 42) . Although these cells can use xylosides as alternate acceptors for GAG chain synthesis(43, 44) , these studies focus on the synthesis of the elongated GAG chains rather than the smaller, non-sulfated xyloside products. It is not known if they also make shorter, sialic acid terminated xyloside structures. We have previously shown that CHO and melanoma cells do make sialylated xyloside products (20) in addition to normal GAG chains, but it is not known whether any cell type can sialylate a GAG core precursor during proteoglycan biosynthesis. If sialylation does not normally occur in native proteoglycans, the biosynthetic mechanism must be able to circumvent the action of other co-localized glycosyltransferases. In this case, co-localization of glycosyltransferases does not necessarily control structure in the dynamic Golgi environment. Residence time or additional restrictions to free diffusion within a given compartment may determine whether co-localized glycosyltransferases are able to glycosylate the product of another transferase.

The maximum amount of product appears to be made at about 50 µM XylbetaMU. Further increases in the amount of acceptor do not produce a proportional increase in the amount of product. Since it is unlikely that diffusion of the acceptor is limiting, it is more likely that the availability of sugar nucleotide donor and/or the glycosyltransferase becomes limiting. Since this occurs only within the vesicles that have core Gal transferase I, this should locally restrict the amount of available sugar nucleotide within those compartments. In effect, XylbetaMU may act as a sink for the transported sugar nucleotides and thus inhibit the synthesis of any endogenous acceptor that is co-localized in the same compartment and requires available UDP-Gal. Our preliminary analyses of the galactosylation of endogenous acceptors in the presence of XylbetaMU indicate that there is a 25-35% inhibition of incorporation into endogenous glycoprotein products. Using this rationale, we are now using XylbetaMU to co-localize other galactosyltransferases to the same compartment with GAG Gal transferase I. The results presented here also demonstrate that GalbetaXylbetaMU also diffuses across the Golgi vesicle membranes. Using this structure as the acceptor in similar incubations should allow us to determine if the GAG beta-1,3-glucuronyltransferase is co-localized with GAG Gal transferase II.


FOOTNOTES

*
This work was supported in part by a grant from the Mizutani Foundation for Glycoscience and by United States Public Health Service Grant R01-CA38701. 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.

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

(^1)
The abbreviations used are: GAG, glycosaminoglycan; 4MU, 4-methylumbelliferyl; CHO, Chinese hamster ovary cells; HPLC, high performance liquid chromatography; NDV, Newcastle disease virus; AUS, Arthrobacter ureafaciens sialidase; XylbetaMU, 4-methylumbelliferyl-beta-D-xylose; CS, crude cell smooth membrane; SE, supernatant extract; SP, supernatant pellet; XT, extract supernatant; XP, extract pellet.

(^2)
P. V. Salimath and H. H. Freeze, unpublished results.


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