A powerful approach for studying the relationship of proteoglycan (PG) structure to function employs inhibitors to block glycosaminoglycan (GAG) biosynthesis. Although true enzyme-based, active site-directed inhibitors of the glycosyltransferases and sulfotransferases have not yet been described, decoys consisting of (P-d-xylose linked to hydrophobic aglycones have been available for some time (1). As shown over 25 years ago (2), xylosides block PG assembly by serving as alternate substrates, thereby diverting GAG assembly from xylosylated proteoglycan core proteins onto the exogenous xyloside primer. This method of derailing PG biosynthesis has been used to explore PG function in cells, tissues, and animals. The priming of oligosaccha-rides on xylosides has also been used to define the nature of mutations in cell lines deficient in PG biosynthesis (3-5), to co-localize glycosyltransferases in Golgi subcompartments (6-8), and as a model for glycoside primers that affect other kinds of glycoconjugates (9-12).
Beta-d-xylosides consist of xylose in beta linkage to an aglycone (see Fig. 1). The aglycones typically consist of a hydrophobic compound which aids in carrying the polar sugar moiety across cell membranes into the Golgi apparatus, where GAG biosynthesis takes place. While all P-D-xylosides prime chondroitin/dermatan sulfate, studies have shown that their ability to prime heparan sulfate depends on the structure of the aglycone (13,14). Xylosides that prime heparan sulfate require an aglycone containing fused rings that assume a more or less planar configuration. Priming of heparan sulfate also depends on concentration, and usually requires a higher dose than is needed for priming chondroitin sulfate. This structural specificity is thought to arise by recognition of the aglycone by the a-GlcNAc transferase that initiates the formation of the repeating disaccharide units of heparan sulfate (15,16).
Fig. 1. Structure of 2-naphthol-p-D-xyloside.
Inhibition of GAG assembly on PGs starts at xyloside concentrations as low as 10-30 ^M, concentrations not significantly higher than those needed for maximum stimulation of GAG priming (13,17-19). Inhibition of heparan sulfate PG formation typically requires higher concentrations of xyloside than needed for chondroitin/dermatan sulfate PG inhibition, consistent with the higher dose requirements for priming heparan sulfate described above. As might be expected, xylosides that prime heparan sulfate inhibit heparan sulfate PG synthesis at lower doses (13).
The following procedures describe the use of P-D-xylosides to inhibit PG assembly in both cultured cells and tissues. The protocol consists of three steps: (i) addition of the compounds to the growth/incubation medium, (ii) PG/GAG extraction and isolation, and (iii) analysis of the extracted PGs/GAGs.
2.1. GAG Priming and Inhibition of PG Assembly
1. 0.2 M_p-nitrophenyl, 4-methylumbelliferyl, or 2-napthol-p-D-xyloside in dimethylsulfox-ide (DMSO).
2. 0.2 M control glycoside (e.g., _p-nitrophenyl-a-D-xyloside or _p-nitrophenyl-p-D-arabino-side) in DMSO.
3. Culture medium appropriate for the cells or tissue being studied.
4. Radioactive precursors (H235SO4, [6-3H]glucosamine or [1-3H]galactose).
2.2. PG/GAG Extraction
1. Guanidine extraction buffer. 4.0 M guanidine-HCl, 0.2% (w/v) Zwittergent 3-12, 50 mM sodium acetate (pH 6.0), 10 mM EDTA, 10 mM N-ethylmaleimide (NEM), 1 mM phenylmethylsulfonyl fluoride (PMSF), 1mg/mL pepstatin A, 0.5 mg/mL leupeptin. Add protease inhibitors from 200x stocks (2 M NEM in ethanol, 0.2 M PMSF in ethanol, 0.2 mg/mL pepstatin A in ethanol, 0.1 mg/mL leupeptin in water). Store stocks of protease inhibitors at -20°C.
2. Triton X-100 extraction buffer: 20 mM Tris-HCl, pH 7.4, 0.5% (w/v) Triton X-100, 0.15 M NaCl, 10 mM EDTA, 10 mM NEM, 1 mM PMSF, 1 ^g/mL pepstatin A, 0.5 ^g/mL leupeptin.
3. Dialysis membranes, 6- to 8-kDa cutoff.
2.3. PG/GAG Isolation
1. Dialysis buffer: Same as the guanidine extraction buffer but with 4.0 M urea substituted for the guanidine-HCl.
2. DEAE-Sephacel (Amersham Pharmacia Biotech): Equilibrate the resin with 0.2 M NaCl, 50 mM sodium acetate, pH 6.0.
3. DEAE wash buffer: Add solid NaCl to dialysis buffer for a final NaCl concentration of 0.2 M.
4. DEAE elution buffer: Add solid NaCl to dialysis buffer for a final NaCl concentration of 1.0 M.
5. 20 mg/mL chondroitin sulfate A or heparin in 20 mM Tris-HCl, pH 7.0.
2.4. Analysis of PGs/GAGs
1. TSK 4000 SW HPLC gel filtration column (30 cm x 7.5 mm inner diameter).
2. Gel filtration buffer: 0.1 M KH2PO4, pH 6.0, 0.5 M NaCl, 0.2% (w/v) Zwittergent 3-12.
1. Prepare serial dilutions of the stock xyloside in cell culture medium to achieve final concentrations of 0, 0.01, 0.03, 0.1, 0.3, and 1 mM. Prepare similar, separate dilutions of the control glycoside. Add DMSO as necessary to maintain a constant vehicle concentration for all dilutions (see Note 1).
2. Add radioactive precursors to the diluted xylosides (10 ^Ci/mL [35S]H2SO4, 20 ^Ci/mL [6-3H]glucosamine, or 50 ^Ci/mL [1-3H]galactose should be sufficient, but this depends on the sulfate and glucose content of the growth medium. Warm the supplemented medium under culture conditions before adding it to cells or tissue. If sufficient levels (~50 ^g of uronic acid) of PGs/GAGs will be produced, the material can be quantitated chemically, and the radioactive precursors may be omitted (see Note 2).
3. Replace the culture medium in the culture dish or flask with the medium containing xyloside and radioactive precursors, and incubate the samples under appropriate conditions for the cells or tissue under study. Priming occurs rapidly and continues throughout the incubation, since the xyloside is not significantly depleted during the incubation. The number of cells and/or labeling time may need to be varied depending on the cell or tissue type and the sensitivity of the assays employed. If the content of PGs/GAGs is to be measured chemically, the incubation may need to be extended since preexisting molecules must turn over in order to see significant inhibition of PG glycosylation.
3.2. PG/GAG Extraction
1. Extraction of PGs/GAGs from cells and culture medium (20). Chill the cells and medium to 4°C. For most cells, adding Triton X-100 extraction buffer (2 mL/107 cells) will suffice to solubilize membrane and cell-associated PGs and GAGs. The extent of solubilization can be assessed by treating the monolayer (or residual cell pellet) with 0.5 mL/107 cells of 0.1 M NaOH at room temperature for 10 min. Neutralize the solution with 10 M acetic acid, clarify the sample by centrifugation, and measure the amount of residual material in the supernatant. If the Triton extraction fails to solubilize all of the material from the plate, a guanidine extraction procedure should be used. Add 1-2 mL/107 cells of guanidine extraction buffer. Alternatively, add solid guanidine-HCl to achieve a final concentration of 4.0 M, Zwittergent 3-12 to 0.2% w/v, EDTA to 10 mM, and sodium acetate to 50 mM. Add protease inhibitors from 200x stock solutions to achieve final concentrations of 10 mM NEM, 1 mM PMSF, 1 ^g/mL pepstatin A and 0.5 ^g/mL leupeptin. Incubate at 4°C for 1 h. If tissue samples are under study, mince or homogenize the tissue in 5-10 vol of chilled guanidine extraction buffer per gram (wet weight) of tissue. Stir overnight at 4°C.
2. Clarify the extracts by centrifugation at 4°C for 20 min at 12,000 g or 10 min at maximum speed in a microcentrifuge.
3.3. PG/GAG Isolation
1. Samples extracted with Triton X-100 solution may be analyzed by anion-exchange chromatography without further processing. If guanidine-HCl buffer was used, the sample must be dialyzed to lower the ionic strength. Dialyze samples at 4°C against dialysis buffer using membranes with a 6- to 8-kDa cutoff. Alternatively, gel filtration chromatography (e.g., PD-10 columns, Amersham Pharmacia Biotech) or ultrafiltration may be used to exchange buffers.
2. Add 1-2 mg of chondroitin sulfate A or heparin as a carrier to the PG/GAG extract if the samples are radioactively labeled (see Note 3). Note: If PGs/GAGs are to be quantitated chemically by uronic acid assay, omit this step.
3. Prepare a 0.5- to 1.0-mL DEAE-Sephacel column in a disposable pipet tip plugged with glass wool. Remove the bottom few millimeters of the tip to enlarge the opening and improve flow rates. Equilibrate the resin with 5 column volumes of DEAE wash buffer.
4. Apply the PG/GAG extract to the column and wash with 10-20 column volumes of DEAE wash buffer. Elute the PGs/GAGs with 5 column volumes of DEAE elution buffer and collect the eluate as a single fraction.
5. Desalt samples using Sephadex G25 columns or PD-10 columns. These columns can be run in 10% ethanol, which allows the samples to be concentrated.
6. Lyophilize the sample and resuspend it in 0.2 mL of 20 mM Tris-HCl, pH 7.4. Store at 4°C.
3.4. Analysis of Isolated PGs/GAGs
1. Apply samples (<0.2 mL) to a TSK G4000 SW gel filtration column equilibrated in gel filtration buffer. Adjust the flow to 0.5 mL/min, collect fractions (1 min), and assay aliquots for radioactivity. PGs will elute before primed GAGs because of their large size, but baseline resolution may not be achieved. Other FPLC or HPLC columns can be used as well to optimize separation. These columns can be internally calibrated by comparing the elution of intact PGs and free chains liberated from the core proteins by beta-elimination (13). If enough PGs/GAGs are present, their distribution can be measured by the carbazole assay for uronic acid (21).
2. The inhibition of GAG assembly on PG core proteins can be monitored by measuring the decrease in high-molecular weight material corresponding to the intact proteoglycans (see Fig. 2). To separate the effects on heparan sulfate or chondroitin/dermatan sulfate PGs, the samples must be treated with low-pH nitrous acid to depolymerize the heparan sulfate chains (Chapter 34) or by treatment with heparinase(s) or chondroitinase(s) (Chapter 35 and 36).
1. Stock solutions of xylosides in neat DMSO should not be added directly to cells in culture, since DMSO at high concentration can lyse cells. DMSO is also known to cause differentiation of certain cells, which may be associated with a change in PG expression. As an alternative approach, a xyloside stock may be prepared in ethanol or the compounds can be dissolved directly in culture medium up to ~5 mM (with warming if necessary). DMSO stocks of xylosides are stable at -20°C and should be protected from light, since the aglycones absorb in the ultraviolet. Cell monolayers should be visually inspected for signs of toxicity.
2. Recent findings have shown that xyloside priming is not as specific as once thought. A variety of unexpected, small oligosaccharides not necessarily related to GAGs are also
primed and the array of oligosaccharides depends on the cell type (22-27). These unusual oligosaccharides can constitute the majority of the primed material, even at relatively low concentrations of primer (26). Thus, it is important to correlate any change of cellular phenotype with the degree of GAG priming and the actual extent of PG inhibition.
3. Alpha-D-xylosides show little or no ability to stimulate GAG synthesis (17,28,29) but serve as substrates for galactosyltransferase I in vivo (22). High concentrations of both a- and p-xylosides can inhibit glycolipid biosynthesis, and this inhibition may be related to the unusual oligosaccharides produced (22). At high xyloside concentrations the large mass of material generated on the exogenous primer may deplete an internal pool of a nucleotide sugar, although this has yet to been determined. Thus, it is important to work at the minimal xyloside dose required for the desired level of PG inhibition.
A variety of xylosides with different aglycones are available commercially (Sigma or Aldrich) or can easily be synthesized by coupling protected and activated xylose to different aglycones (14). Different aglycones can affect the extent of GAG priming and the composition of the chains, as described above. These differences also may depend on cell type. Thus, it may be important to test more than one xyloside to optimize priming and PG inhibition. By comparing the effects of a xyloside that primes only chondroitin sulfate to one that primes both heparan sulfate and chondroitin sulfate, the role of each type of chain can be potentially assessed (14,30).
GAGs primed by xylosides may have the activities associated with PGs. For example, the heparan sulfate chains primed on 2-naphthol-p-D-xyloside will bind to basic fibroblast growth factor (FGF-2) and enhance growth factor association with high-affinity tyrosine kinase receptors (30). Although the chains differ somewhat in structure from those assembled on natural core proteins (14), they possess the binding sequence for the growth factor and apparently the receptor (31). Therefore, even though PG assembly may be inhibited, a particular proteoglycan-dependent activity may not be altered if the primed GAGs are able to substitute for the PGs.
4. If PGs/GAGs are to be quantitated chemically by uronic acid assay, omit this step.
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