Iozzo Renato V. Proteoglycan Protocols

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318 Fritz and Esko

Inhibition of GAG assembly on PGs starts at xyloside concentrations as low as

10–30 µM, concentrations not significantly higher than those needed for maxi-

mum 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 β-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 isola-

tion, and (iii) analysis of the extracted PGs/GAGs.

2. Materials

2.1. GAG Priming and Inhibition of PG Assembly

1. 0.2 M p-nitrophenyl, 4-methylumbelliferyl, or 2-napthol-β-D-xyloside in dimethylsulfox-

ide (DMSO).

2. 0.2 M control glycoside (e.g., p-nitrophenyl-α-

D-xyloside or p-nitrophenyl-β-D-arabino-

side) in DMSO.

3. Culture medium appropriate for the cells or tissue being studied.

4. Radioactive precursors (H

, [6-

H]glucosamine or [1-

H]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 200× 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 pro-

tease 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 µg/mL pepstatin A, 0.5 µg/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.

Fig. 1. Structure of 2-naphthol-β-D-xyloside.

Xylosides 319

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.

6. 20 mM Tris-HCl, pH 7.4, 0.2 M NaCl.

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 KH

, pH 6.0, 0.5 M NaCl, 0.2% (w/v) Zwittergent 3-12.

3. Methods

3.1. GAG Priming and Inhibition of PG Assembly

1. Prepare serial dilutions of the stock xyloside in cell culture medium to achieve final con-

centrations 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 µCi/mL [

S]H

, 20 µCi/mL

[6-

H]glucosamine, or 50 µCi/mL [1-

H]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 µg 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 condi-

tions 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 mea-

sured 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/10

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/10

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/10

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 200× stock solutions to achieve final concentrations of 10 mM

NEM, 1 mM PMSF, 1 µg/mL pepstatin A and 0.5 µg/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.

320 Fritz and Esko

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 chroma-

tography 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-elimina-

tion (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).

4. Notes

1. Stock solutions of xylosides in neat DMSO should not be added directly to cells in cul-

ture, 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 com-

pounds can be dissolved directly in culture medium up to ~5 mM (with warming if nec-

essary). 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

Xylosides 321

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

α- and β-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-β-

D-xyloside will bind to basic fibro-

blast 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.

Fig. 2. Inhibition of PG and priming of free GAG chains on a xyloside. (Source: Lugemwa, F. N.

and Esko, J. D. (1991) Estradiol-β-

D-xyloside, an efficient primer of heparan sulfate biosynthesis.

J. Biol. Chem. 266, 6674–6677.)

322 Fritz and Esko

References

1. Esko, J. D. and Montgomery, R. I. (1996) Synthetic glycosides as primers of oligosaccha-

ride biosynthesis and inhibitors of glycoprotein and proteoglycan assembly, in Current

protocols in molecular biology: Preparation and analysis of glycoconjugates (Ausubel,

R., Brent, R., Kingston, B., Moore, D., Seidman, J., Smith, J., et al., eds.), Greene Publish-

ing and Wiley-Interscience, New York, pp. 17.11.1–17.11.6

2. Okayama, M., Kimata, K., and Suzuki, S. (1973) The influence of p-nitrophenyl β-

D-xyloside

on the synthesis of proteochondroitin sulfate by slices of embryonic chick cartilage. J.

Biochem.(Tokyo) 74, 1069–1073.

3. Esko, J. D., Weinke, J. L., Taylor, W. H., Ekborg, G., Rodén, L., Anantharamaiah, G.,

and Gawish, A. (1987) Inhibition of chondroitin and heparan sulfate biosynthesis in

Chinese hamster ovary cell mutants defective in galactosyltransferase I. J. Biol. Chem.

262, 12,189–12,195.

4. Esko, J. D., Stewart, T. E., and Taylor, W. H. (1985) Animal cell mutants defective in

glycosaminoglycan biosynthesis. Proc. Natl. Acad. Sci. USA 82, 3197–3201.

5. Bai, X. M., Wei, G., Sinha, A., and Esko, J. D. (1999) Chinese hamster ovary cell mutants

defective in glycosaminoglycan assembly and glucuronosyltransferase I. J.Biol.Chem. 274,

13,017–13,024.

6. Etchison, J. R., Srikrishna, G., and Freeze, H. H. (1995) A novel method to co-localize

glycosaminoglycan-core oligosaccharide glycosyltransferases in rat liver Golgi. Co-local-

ization of galactosyltransferase I with a sialyltransferase. J. Biol. Chem. 270, 756–764.

7. Etchison, J. R. and Freeze, H. H. (1996) A new approach to mapping co-localization of

multiple glycosyl transferases in functional Golgi preparations. Glycobiology 6, 177–189.

8. Freeze, H. H. and Etchison, J. R. (1996) A new side of xylosides and their close rela-

tives: Co-localization mapping glycosyltransferases in the functional Golgi. Trends

Glycosci.Glyotech. 8, 65–77.

9. Kuan, S. F., Byrd, J. C., Basbaum, C., and Kim, Y. S. (1989) Inhibition of mucin

glycosylation by aryl-N-acetyl-α-galactosaminides in human colon cancer cells. J. Biol.

Chem. 264, 19,271–19,277.

10. Sarkar, A. K., Fritz, T. A., Taylor, W. H., and Esko, J. D. (1995) Disaccharide uptake and

priming in animal cells: Inhibition of sialyl Lewis X by acetylated Galb1-4GlcNAcβ-O-

naphthalenemethanol. Proc. Natl. Acad. Sci. USA 92, 3323–3327.

11. Neville, D. C. A., Field, R. A., and Ferguson, M. A. J. (1995) Hydrophobic glycosides of

N-acetylglucosamine can act as primers for polylactosamine synthesis and can affect gly-

colipid synthesis in vivo. Biochem.J. 307, 791–797.

12. Sarkar, A. K., Rostand, K. S., Jain, R. K., Matta, K. L., and Esko, J. D. (1997) Fucosylation

of disaccharide precursors of sialyl Lewis

inhibit selectin-mediated cell adhesion. J.

Biol. Chem. 272, 25,608–25,616.

13. Lugemwa, F. N. and Esko, J. D. (1991) Estradiol β-

D-xyloside, an efficient primer for

heparan sulfate biosynthesis. J. Biol. Chem. 266, 6674–6677.

14. Fritz, T. A., Lugemwa, F. N., Sarkar, A. K., and Esko, J. D. (1994) Biosynthesis of heparan

sulfate on β-D-xylosides depends on aglycone structure. J. Biol. Chem. 269 , 300–307.

15. Fritz, T. A., Gabb, M. M., Wei, G., and Esko, J. D. (1994) Two N-acetylglucosaminyl-

transferases catalyze the biosynthesis of heparan sulfate. J. Biol. Chem. 269, 28,809–28,814.

16. Fritz, T. A., Agrawal, P. K., Esko, J. D., and Krishna, N. R. (1997) Partial purification and

substrate specificity of heparan sulfate α-N-acetylglucosaminyltransferase . 1. Synthesis,

NMR spectroscopic characterization and in vitro assays of two aryl tetrasaccharides.

Glycobiology 7, 587–595.

Xylosides 323

17. Robinson, H. C., Brett, M. J., Tralaggan, P. J., Lowther, D. A., and Okayama, M. (1975)

The effect of D-xylose, β-D-xylosides, and β-D-galactosides on chondrotin sulphate bio-

synthesis in embryonic chicken cartilage. Biochem. J. 148, 25–34.

18. Kolset, S. O., Ehlorsson, J., Kjellén, L., and Lindahl, U. (1986) Effect of benzyl β-D-xyloside

on the biosynthesis of chondroitin sulphate proteoglycan in cultured human monocytes.

Biochem. J. 238, 209–216.

19. Sobue, M., Habuchi, H., Ito, K., Yonekura, H., Oguri, K., Sakurai, K., Kamohara, S., Ueno,

Y., Noyori, R., and Suzuki, S. (1987) β-

D-xylosides and their analogues as artificial initia-

tors of glycosaminoglycan chain synthesis: Aglycone-related variation in their effective-

ness in vitro and in ovo. Biochem. J. 241, 591–601.

20. Esko, J.D. (1993) Special considerations for proteoglycans and glycosaminoglycans and

their purification, in Current protocols in molecular biology (Ausubel, F., Brent, R.,

Kingston, B., Moore, D., Seidman, J., Smith, J., et al., eds.) Greene Publishing and Wiley-

Interscience, New York, pp. 17.2.1–17.2.9

21. Esko, J. D. and Manzi, A. (1996) Measurement of uronic acids, in Current protocols in

molecular biology (Ausubel, F., Brent, R., Kingston, B., Moore, D., Seidman, J., Smith, J.,

et al., eds.) Greene Publishing and Wiley-Interscience, New York, pp.17.9.8–17.9.11

22. Freeze, H. H., Sampath, D., and Varki, A. (1993) α- and β- xylosides alter glycolipid

synthesis in human melanoma and Chinese hamster ovary cells. J. Biol. Chem. 268,

1618–1627.

23. Izumi, J., Takagaki, K., Nakamura, T., Shibata, S., Kojima, K., Kato, I., and Endo, M.

(1994) A novel oligosaccharide, xylosylβ1-4xylosylβ1-(4-methylumbelliferone), synthe-

sized by cultured human skin fibroblasts in the presence of 4-methylumbelliferyl-β-D-xyloside.

J. Biochem. (Tokyo) 116, 524–529.

24. Nakamura, T., Izumi, J., Takagaki, K., Shibata, S., Kojima, K., Kato, I., and Endo, M.

(1994) A novel oligosaccharide, GlcAβ1-4Xylβ1-(4-methylumbelliferone), synthesized by

human cultured skin fibroblasts. Biochem. J. 304, 731–736.

25. Manzi, A., Salimath, P. V., Spiro, R. C., Keifer, P. A., and Freeze, H. H. (1995)

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 α-GalNAc residue

capping 4-methylumbelliferyl-β-D-xylosides. J. Biol. Chem. 270, 9154–9163.

26. Salimath, P. V., Spiro, R. C., and Freeze, H. H. (1995) Identification of a novel glycosami-

noglycan core-like molecule II. α-GalNAc-capped xylosides can be made by many cell

types. J. Biol. Chem. 270, 9164–9168.

27. Miura, Y. and Freeze, H. H. (1998) α-N-Acetylgalactosamine-capping of chondroitin sul-

fate core region oligosaccharides primed on xylosides. Glycobiology 8, 813–819.

28. Schwartz, N. B., Galligani, L., Ho, P.-L., and Dorfman, A. (1974) Stimulation of synthesis

of free chondroitin sulfate chains by β-D-xylosides in cultured cells. Proc. Natl. Acad. Sci.

USA 71, 4047–4051.

29. Sudhakaran, P. R., Sinn, W., and Von Figura, K. (1981) Initiation of altered heparan sul-

phate on β-D-xyloside in rat hepatocytes. Hoppe-Seyler’s Z. Physiol. Chem. 362, 39–46.

30. Miao, H.-Q., Fritz, T. A., Esko, J. D., Zimmermann, J., Yayon, A., and Vlodavsky, I.

(1995) Heparan sulfate primed on β-D-xylosides restores binding of basic fibroblast

growth factor. J. Cell. Biochem. 57, 173–184.

31. Plotnikov, A. N., Schlessinger, J., Hubbard, S. R., and Mohammadi, M. (1999) Structural

basis for FGF receptor dimerization and activation. Cell 98, 641–650.

Inhibition of Heparan Sulfate Synthesis 325

325

From:

Methods in Molecular Biology, Vol. 171: Proteoglycan Protocols

Edited by: R. V. Iozzo © Humana Press Inc., Totowa, NJ

Inhibition of Heparan Sulfate Synthesis by Chlorate

H. Edward Conrad

1. Introduction

With the exception of hyaluronic acid, all glycosaminoglycans are O-sulfated.

Heparins and heparan sulfates are, in addition, N-sulfated on many of their glu-

cosamine residues. Other naturally occurring structures are also sulfated. For example,

some proteins contain sulfotyrosine residues (1), and some complex lipids are O-sulfated.

In addition, certain N-linked oligosaccharides are sulfated (2). In all of these cases, the

enzymes that add sulfate residues to these structures utilize 3'-phosphoadenosine-

5'-phosphosulfate (PAPS) as the sulfate donor. If PAPS cannot be synthesized, these

biological sulfation reactions will not proceed. Chlorate is a competitive inhibitor of

PAPS synthesis and, as such, can block all sulfation reactions.

PAPS is synthesized by a two-step process utilizing ATP sulfurylase (reaction 1)

and adenosine-5'-phosphosulfate kinase (reaction 2), activities that are in some cases

catalyzed by a single bifunctional enzyme that may localize to the nucleus in mamma-

lian cells (3,4).

ATP + SO

2–

→ APS + PP

(1)

APS + ATP → PAPS + ADP (2)

Chlorate competes with the SO

2–

in reaction 1. Consequently, as the concentration

of ClO

in the cells is increased, the amount of PAPS formed is reduced. Under these

circumstances, the sulfotransferase reactions, which require PAPS as a substrate, will

proceed progressively more slowly as the concentration of PAPS drops. Furthermore,

the sulfotransferases with the highest K

for PAPS will be selectively inhibited com-

pared to the sulfotransferases with lower K

. Such selectivity has been reported in the

biosynthesis of heparan sulfate (5) wherein, with increasing ClO

–

concentrations,

6-O-sulfation of glucosamine residues is inhibited strongly, 2-O-sulfation of uronic

acids is inhibited moderately, and N-sulfation of glucosamine residues is inhibited

326 Conrad

modestly. In the heparan sulfate case, there is a requirement that N-sulfation proceed

before the various O-sulfation reactions, so that the types of sulfation that occur may

not be controlled totally by the K

. Of course, at high ClO

–

concentrations, all

sulfation reactions are inhibited.

Sulfation of glycosaminoglycans occurs in the trans Golgi; i.e., the sulfation is the

final stage in the processing of glycosaminoglycans in the secretory pathway. Thus,

when cells are labeled with

2–

-labeled glycosaminoglycans appear out-

side of the cell within minutes. Consequently, when cells are treated with ClO

–

changes in the structures of secreted glycosaminoglycans can be observed in a few

minutes, and the full effect of the ClO

–

treatment is usually seen in about 30 min.

Since the binding of many growth factors, enzymes, etc., requires sulfated heparan

sulfate, ClO

–

treatment will result in the loss of protein binding to the cell surface.

Thus, ClO

–

is a useful reagent for determining whether various proteins require sul-

fated glycosaminoglycans for cell binding and activity.

2. Materials

1. NaClO

2. Cells in desired culture medium.

3. Methods

3.1. Defining the Concentration of Chlorate Required to Cause Total

Inhibition of Sulfation (

see

Note 1)

Chlorate has been added to cultured cells at concentrations up to 50 mM for 24 h or

more with no apparent adverse effects on the cells (5–10). The concentration of chlo-

rate required to cause total inhibition of sulfation differs in different cell types. Because

of variations in cell types and culture media compositions, the following general

approach may used to determine the effects of varying ClO

–

concentrations on

glycosaminoglycan sulfation.

1. Incubate cultured cells with increasing concentrations of NaClO

(e.g., 5–50 mM) for 1 h.

2. Then incubate the cells for an additional 1 h with a sufficient quantity of

2–

produce ~10,000 cpm of cell surface

–glycosaminoglycan. The amount of

2–

required will vary with the cell type and the type of culture medium used.

4. Remove the culture medium and wash the cells with fresh medium lacking ClO

–

and

2–

5. Treat the cells with trypsin in phosphate-buffered saline to release the cells from the

culture dish. Centrifuge out the cells and determine the number of

–labeled gly-

cosaminoglycan in the supernatant.

6. Repeat these steps for each concentration of ClO

–

7. Choose the concentration of ClO

–

required to give the desired degree of inhibition of

sulfate incorporation.

3.2. Determination of the Effect of Chlorate

on the Activity of Heparan Sulfate-Binding Proteins

Inhibition of sulfate incorporation into heparan sulfate will prevent the binding of

proteins to cell surface heparan sulfate proteoglycans (HSPG) and will block the

Inhibition of Heparan Sulfate Synthesis 327

normal changes in the activities of the cells that result from the protein binding. To

study these changes, the approach used depends on the heparan sulfate-binding protein

that is being studied, the cells that are used, and the activities of the heparan sulfate-

binding protein on the cells. Thus, there is no standard protocol. However, several

literature examples illustrate the general approach to be used.

1. It is known that cell surface heparan sulfate proteoglycan must interact with basic fibro-

blast growth factor in order for the growth factor to bind to its receptor and exhibit its

mitogenic effects (11–13). In the presence of chlorate, basic fibroblast growth factor can-

not bind or exert these effects on cells.

2. Chlorate treatment of rat hepatoma cells prevents the stimulation of β-very-low-density

lipoprotein binding to rat hepatoma cells transfected with hepatic lipase cDNA (14).

3. In cells that produce lipoprotein lipase, newly synthesized lipoprotein lipase normally

adheres to the surface of cells that produce it, but in chlorate-treated cells, there is a direct

secretion of the enzyme into the culture medium (15,16).

4. Notes

1. Heparan sulfate structures that are synthesized by cells in the presence of chlorate are

quite different from any structures that are normally synthesized. When the chlorate con-

centration is high enough, cells produce a completely unsulfated heparan proteoglycan.

The polysaccharide chains that accumulate in the proteoglycan are [GlcA→GlcNAc]n

chains in which a significant proportion of the GlcNAc residues are de-N-acetylated and

ready to receive a sulfate group. Such products have been identified in chlorate-treated

cells in culture. These products, which have anionic carboxyl groups on the GlcA resi-

dues, cationic amino groups on many of the GlcN residues, and very few IdoA residues,

apparently appear on the cell surface but lack the ability to bind proteins. Thus, although

chlorate “disarms” the cell so that it cannot respond to the growth factors or other heparan

sulfate-binding proteins, it also results in the synthesis of an unnatural heparan sulfate

structure that appears on the cell surface. Since heparan sulfate binds to many structures

on and near the surface of cells (collagen, fibronectin, laminin, thrombospondin, etc.), it

is a major contributor to the organization of the extracellular matrix. Consequently, in

the presence of ClO

, the structure of the cell surface and the extracellular matrix may

be altered significantly. Furthermore, all secondary cellular responses that depend on the

primary responses to altered heparan sulfate structure will be altered, even though one

observes only those changes that are measured.

References

1. Mintz, K. P., Fisher, L. W., Grzesik, W. J., Hascall, V. C., and Midura, R. J. (1994) Chlorate-

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