Purification of Perlecan from Endothelial Cells

John Whitelock

1. Introduction

Perlecan, the major heparan sulfate (HS) proteoglycan of basement membranes and other connective tissues, is a modular molecule with five structural domains. It has been isolated from various sources including kidney and placenta, but most commonly from the mouse Englebroth Holm Swarm (EHS) tumor (1,2). It has also been purified from cultured cell lines such as endothelial cells (3) or fibroblasts (4). When it has been extracted from tissue or from the extracellular matrix of cultured cells, it has been first solubilized using chaotropic agents such as urea or guanidine. The effect of these dissociating agents on the structure and function of isolated proteoglycans is not known, but it is thought that some of the denaturation may be irreversible. Human perlecan is found predominantly as a heparan sulfate proteoglycan, but has been isolated from carcinoma cell lines as an undecorated protein core and from placental tissue decorated with chondroitin sulfate/dermatan sulfate. The molecular weight of the protein core is approximately 470 kDa, while the size of the mouse homolog is around 400 kDa. Domain I is the N-terminal domain, and it contains a cluster of three potential glycosaminoglycan attachment sites. Domain II has homology to the LDL receptor, with strict conservation of the cysteine residue positions. Domain III shares homology with the short arm of the laminin a-chain. Domain IV is the largest domain with a molecular weight in excess of 200 kDa and is made up of numerous immunoglo-bulin-like repeats similar to those found in the neural cell adhesion molecule. Domain V is the C-terminal domain, and is made up of three regions with homology to the globular domains of the laminin a-chain, interspersed with four epidermal growth factor-like repeats.

From: Methods in Molecular Biology, Vol. 171: Proteoglycan Protocols Edited by: R. V. Iozzo © Humana Press Inc., Totowa, NJ

2. Materials

1. Human endothelial cells were isolated from either umbilical veins or arteries as described previously (5). Dr. J. Gamble provided C11-STH, the endothelial cell line at the Hanson Cancer Center in Adelaide, Australia.

2. Endothelial cell growth medium: For 1 L of Medium 199 (Earles salts), open 1 packet of Medium 199 powder (Gibco) and dissolve powder in 900 mL of pyrogen-free H2O (Baxter), add 2.2 g of NaHCO3, 100 U of penicillin, and 100 ^g of streptomycin. Adjust the pH to 7.2, make the total volume up to 1 L, sterilize by filtration using a 0.22-^m filter and store at 4°C. Before using the medium in cell cultures, add 0.3g/L of bovine brain extract (BBE), prepared as described (6) (see Note 1), 1 mg/L of heparin (Sigma cat. no. H3149) (see Note 2), 15% v/v fetal bovine serum (see Note 3).

3. Flasks that were used to grow endothelial cells were first coated with 10 ^g/mL of fibronectin for 2 h at 37°C. A 2% gelatin solution can be used in place of fibronectin.

4. DEAE Sepharose column (Pharmacia).

5. Anti-perlecan immunoaffinity column. Weigh out 2 g of dry CnBr-activated Sepharose 4B matrix (Pharmacia). This makes a column of bed volume 7 mL. Swell and wash the powder in 1 mM HCl and then equilibrate the gel with approx. 50 mL of 0.1 M NaHCO3, 0.5 M NaCl, pH 8.3 (coupling buffer). Dialyze the anti-perlecan monoclonal antibody (A71, A74, or A76) against the coupling buffer (allow approx. 5 mg of antibody per milliliter of swelled Sepharose). Mix the antibody solution with the prepared gel and mix overnight at 4°C on a rocking platform. The ratio of buffer to swelled gel should be at least 2/1 to achieve efficient mixing. This time can be shortened to 4 h at room temperature. Centrifuge at 1000g, aspirate the supernatant, and wash the gel on a sintered glass funnel under vacuum. Block the remaining active binding sites by incubating the gel with 1 M ethanolamine, pH 8.0 (this should be made fresh) for 2 h at room temperature on a rocker. Centrifuge at 1000g, aspirate the supernatant, and wash the gel with coupling buffer. Perform alternate washes of the elution buffer to be used and the coupling buffer. Equilibrate the column in phosphate-buffered saline and store the column at 4°C

6. Dialysis tubing—10,000-Mr cutoff membrane (Spectropor).

7. Sucrose.

8. Superose™ 6 prepackaged HR 10/30 column and Pharmacia FPLCTM system.

9. Coomassie Plus Protein Assay Kit (Pierce).

10. SDS-PAGE gradient gels, reagents, and electrophoretic equipment (Bio-Rad).

11. Composite agarose-acrylamide gels (see Note 4).

12. Anti-perlecan protein core antibodies; A71 (specific for domain I), A74 (specific for domain V) and A76 (not characterized) (7).

13. Anti-heparan sulfate antibody, 10E4 (Seikagaku).

14. Anti-chondroitin sulfate antibody, CS-56 (Sigma).

15. ELISA plates, reagents, and microtiterplate reader

3. Methods

3.1. Human Endothelial Cell Culture

1. Cultures of human arterial and venous cells are maintained by replacing the conditioned medium three times every week.

2. The cells can also be grown in roller culture bottles (900 cm2). These will need to be coated with fibronectin (10 ^g/mL; 10 mL per roller bottle) for 2 h at 37°C before seeding the cells.

3. Roller cultures are fed twice weekly with 120 mL of endothelial cell growth medium.

4. Once the cells condition the medium, it is removed, centrifuged at 1500g for 10 min to remove cell debris and stored at -20°C. (see Notes 5 and 6).

3.2. DEAE Chromatography

1. Equilibrate a 100 mL bed volume DEAE Sepharose column with approximately 4 volumes of 20 mM Tris, 250 mM NaCl, 10 mM EDTA, 1 mM benzamidine, pH 7.5 (DEAE running buffer) at 4°C.

2. Thaw and filter the conditioned medium (approx. 2-L batches) using either glass wool or a sintered funnel over a vacuum.

3. Apply the medium to the column at a flow rate of 1-2 mL/min.

4. Wash the medium through the column with the DEAE running buffer and continue to wash the column with this buffer until a baseline is achieved (measuring the absorbency at a wavelength of 280 nm) (see Note 7).

5. Elute bound molecules (including the perlecan) from the column with 20 mM Tris, 1 M NaCl, 10 mM EDTA, 1 mM benzamidine, pH 7.5 into a collection vessel which is on ice.

6. Pool the perlecan containing fractions (approx. 60-80 mL) and store at 4°C.

7. Remove a small sample (~200 ^L) for testing in the ELISA based screening assay.

8. Regenerate the DEAE column by washing it with 2 volumes of 20 mM Tris, 2 M NaCl, 10 mM EDTA, 1 mM benzamidine, pH 7.5 and reequilibrating it with the DEAE running buffer.

3.3. Anti-Perlecan Immunoaffinity Chromatography

Equilibrate the column with 20 mM phosphate buffer with 1 M NaCl, pH 7.5 (immuno-affinity running buffer).

Apply the pooled perlecan-containing fractions directly to the immunoaffinity column and recirculate over the column for approx. 6 h or overnight at 4°C, at a flow rate of 1 mL/min.

3. Keep the flow through for testing in the ELISA-based screening assay.

4. Wash the column with approx. 50 mL of the immuno-affinity running buffer or until a baseline is achieved.

Elute the bound perlecan from the immunoaffinity column with either 0.1 M Tris, pH 7.5 containing 3 M MgCl2 or PBS containing 6 M urea. (see Note 8).

6. Pool the perlecan peak (approx. 15 mL).

7. Wash the column with the immunoaffinity running buffer. (Another perlecan peak may elute at this stage; the two peaks should be pooled.)

The column is washed with 50 volumes of immunoaffinity running buffer containing 0.08% NaN3 and stored at 4°C.

Dialyze the perlecan containing peak against three changes of PBS at 4°C overnight and then concentrate to approx. 1-2 mL by removing the PBS through the dialysis tubing by laying it on a bed of sucrose.

10. Once the volume is reduced to approx. 1-2 mL, the clips on the dialysis tubing can be readjusted (to prevent reswelling) and redialyzed against PBS overnight at 4°C to remove any sucrose.

11. The immuno-purified perlecan can be assayed for purity and quantity, and stored in small aliquots at -70°C in siliconized tubes (see Note 9).

3.4. Gel Filtration Chromatography

1. Equilibrate a Superose 6 prepackaged HR 10/30 (Pharmacia) column with 0.5 M CH3COONa, 0.05% Tween-20, pH 7.5 at 0.4 mL/min.

Perlecan Endothelial

Fig. 1. Elution profile of immunopurifed human perlecan using a Superose 6 10/30 prepacked column. Samples of 3H-labeled perlecan were run through a Superose 6 10/30 column as described. HUAEC-derived perlecan is shown by the dashed line, whereas HUVEC-derived perlecan is shown by the unbroken line. The V0 of the column was 8 mL (fraction 10), whereas the Vt was 20 mL (fraction 25). Notice the large amount of free 3H-glucosamine in the HUAEC sample.

Fig. 1. Elution profile of immunopurifed human perlecan using a Superose 6 10/30 prepacked column. Samples of 3H-labeled perlecan were run through a Superose 6 10/30 column as described. HUAEC-derived perlecan is shown by the dashed line, whereas HUVEC-derived perlecan is shown by the unbroken line. The V0 of the column was 8 mL (fraction 10), whereas the Vt was 20 mL (fraction 25). Notice the large amount of free 3H-glucosamine in the HUAEC sample.

2. Set the fraction collector to collect fractions every 2 min (0.8-mL fraction volume).

3. Apply sample via the loop while maintaining the flow rate at 0.4 mL/min.

4. Fractions can be monitored for either the presence of proteins by measuring the absor-bency at a wavelength of 280 nm, or for radioactivity by taking a sample and mixing it with scintillant and counting.

5. This method is essentially as described by Melrose and Ghosh (8). It is particularly suitable for separating biosynthetically labeled perlecan from free label (see Fig. 1), as well as separating 3H-labeled or 35SO4-labeled HS chains that are liberated from the protein core of perlecan by alkaline borohydride elimination (see Fig. 2).

6. Perlecan-enriched fractions can be prepared using this methodology. This is particularly suitable if immuno-affinity chromatography is not possible. It is important to remember that these fractions will also contain other large molecules such as the chondroitin sulfate proteoglycan, versican. This can be monitored by the screening of fractions with the anti-chondroitin sulfate monoclonal antibody, CS-56 (see Note 10).

3.5. Preparation of Perlecan HS (Alkaline Borohydride Elimination)

1. Prepare 2 M NaBH4.

3. Mix an equal volume of the above solutions and add this to a volume of labeled perlecan solution in the ratio 2/1 v/v (NaBH4/KOH:perlecan).

4. Cover with parafilm, and incubate overnight at 45°C.

5. Neutralize with an equal volume of 3.6 M CH3COOH (see Note 11).

900 800 700 600 s 500 -O 400 -300 200 100

Fraction

Fig. 2. Elution profile of HS derived from immunopurified perlecan using a Superose 6 10/ 30 prepacked column. Samples of 3H-labeled HS were run through a Superose 6 10/30 column as described. HUAEC-derived HS is shown by the dashed line, whereas HUVEC-derived HS is shown by the unbroken line. The V0 of the column was 8 mL (fraction 10), whereas the Vt was 20 mL (fraction 25). The molecular weight of the two types of HS was estimated to be 40 kDa using previously published standards for chondroitin sulfate (8).

3.6. Ethanol Precipitation for Concentrating HS

1. To the sample to be concentrated, add 4 volumes of 1.3% CH3COOK in 95% ethanol.

3. Isolate the precipitated labeled perlecan by centrifugation in a microfuge at room temperature for 5 min.

4. Aspirate the supernatant, wash twice (1-5 mL) with salt-free 95% ethanol.

5. The perlecan can be either lyophilized or dissolved in buffer.

3.7. Detection and Quantitation of Perlecan

1. The presence of perlecan in purified and semipurified fractions is monitored using an ELISA-based screening assay using one antibody that reacts with the protein core (A76) and another that reacts with the heparan sulfate (10E4). This assay can also be used to screen for the presence of chondroitin sulfate on the protein core or other co-purifying extracellular matrix proteins (see Note 12).

2. We have found that the heparan sulfate attached to the protein core of perlecan interfered with the BCA reaction, resulting in an overestimation of the amount of perlecan present. Therefore, to avoid these errors, use the dye-binding protein assay, Coomassie Plus Protein Assay (Pierce), as it is more reliable on the amount of protein core present.

3.8. Electrophoresis

1. Standard SDS-PAGE methodology can be employed to analyze perlecan in gels and Western blots. It is recommended to run 4-15% gradient gels, as the intact perlecan runs at the

Perlecan Western Perlecan Endothelial

Fig. 3. COA-PAGE immunoblot of perlecan immunopurifed from a HUVEC cell line and primary HUAEC cultures. Lane 1 represents 3 ^g of perlecan immunopurified from a HUVEC cell line (C11-STH). Lane 2 represents 3 ^g of perlecan immunopurified from primary cultures of HUAECs. Both samples were run through CO-A/PAGE gels and blotted to nitrocellulose. The nitrocellulose was cut into strips and probed with the anti-protein core antibodies as indicated.

Fig. 3. COA-PAGE immunoblot of perlecan immunopurifed from a HUVEC cell line and primary HUAEC cultures. Lane 1 represents 3 ^g of perlecan immunopurified from a HUVEC cell line (C11-STH). Lane 2 represents 3 ^g of perlecan immunopurified from primary cultures of HUAECs. Both samples were run through CO-A/PAGE gels and blotted to nitrocellulose. The nitrocellulose was cut into strips and probed with the anti-protein core antibodies as indicated.

bottom of the application wells. The use of 3-8% gradient gels may facilitate the migration of perlecan into the running gel (see Note 13).

2. SDS-PAGE Gels can be stained with either (see Note 14):

a. 0.25% Coomasie blue in 40% methanol, 50% H2O, 10% CH3COOH.

d. Or a combination of the above with a AgNO3 stain.

3. Composite agarose-polyacrylamide gel electrophoresis (COA-PAGE) can also be employed to analyze the purified perlecans (see Fig. 3). These gels have the advantage that the perlecan migrates through the pores of the gel and the distance the molecules travel depends on the ratio of their mass to their charge. This has the advantage that perlecans from different sources may be separated on the basis of charge (see Fig. 3). (see Note 15). They can be blotted to nitrocellulose on a flat bed dry blotter and probed with antibodies (see Fig. 3).

4. Notes

1. Commercially prepared endothelial cell growth supplement or purified growth factors such as recombinant human fibroblast growth factors (FGF) or vascular endothelial cell growth factors (VEGF) may be used in place of the BBE. They are used at a final concentration of 10 ng/mL.

2. Heparin is not required when using either FGF-2 or VEGF. Cells may also be grown heparin-free by using 15% v/v human serum.

3. After 2 weeks of storage at 4°C, the glutamine will have to be re-added at a final concentration of 2 mM (4 mM for smooth muscle cells). The medium should be discarded after 4 wk.

4. These are prepared essentially as described by McDevitt and Muir (9) (also see Chapterl5).

5. Either 3H-glucosamine, 35S-Na2SO4, or 35S-methionine can be added to the cultures (5 ^Ci/ mL) to produce biosynthetically labeled perlecan. The choice of labeling reagent depends on whether you want to label the protein core or the glycosaminoglycan chains.

6. Endothelial cells derived from primary tissue can be maintained at confluence for l wk. The endothelial cell line could be maintained at confluence for longer periods.

7. Biosynthetically labeled perlecan can be prepared using the same methodology as described for the unlabeled molecule. Its purification can be monitored by sampling 100 ^L of each fraction and mixing it with scintillant (Insta-gel; Packard) and counting in a scintillation counter (LKB 1217 Rackbeta). Radiolabeled perlecan can also be concentrated using ethanol precipitation.

8. When using the 3 M MgCl2 to elute the column, make the solution fresh before use and keep the MgCl2 stored in a desiccator.

9. Collect the perlecan fractions in siliconized Eppendorf tubes to minimize loss due to adsorption.

10. The buffers used for the gel filtration step, which may be used instead of the immunoaffinity step and results in the isolation of perlecan-enriched fractions, may contain 6 M urea as a dissociative agent. This is particularly pertinent to the isolation of perlecan from tissue sources.

11. When performing the NaBH4/KOH step (overnight at 45 °C), choose a screw-capped tube or cover the tube with parafilm. This prevents the cap popping due to the pressure build up caused by the release of H2 gas. Also, choose a 15-mL tube (for solutions up to 2 mL) so that during the neutralization step the H2 gas that is liberated is allowed to escape without the loss of the sample.

12. 0.1% casein in PBS is used as both the blocking agent and diluent, due to the fact that it gives superior backgrounds with the anti-HS antibody, 10E4.

13. If you plan to run gels with a separate stacker, do not remove the stacker before staining or blotting to nitrocellulose.

14. The SDS must be washed out of the gels with 3 changes in either H2O or 50% ethanol prior to staining with Azure A, Alcian blue, or silver.

15. These gels can be stained with 0.08% Azure A in H2O by placing them on a sheet of plastic to act as a support.

Acknowledgments

The author would like to acknowledge Dr. Anne Underwood for discussion and critical review of the chapter; Penny Bean, Sue Mitchell, and Debbie Lock for excellent technical assistance; and the Australian Government through the Co-Operative Research Center scheme for funding.

References

1. Hassell, J. R., Robey, P. G., Barrach, H. J., Wilczek, J., Rennard, S. I., and Martin,G. R. (1980). Isolation of a heparan sulfate-containing proteoglycan from basement membrane. Proc. Natl. Acad. Sci. (USA) 77, 4494-4498.

2. Paulsson, M., Yurchenco, P. D., Ruben, G. C., Engel, J., and Timpl, R. (1987). Structure of low density heparan sulfate proteoglycan isolated from a mouse tumor basement membrane. J. Mol. Biol. 197, 297-313.

3. Saku, T. and Furthmayr, H. (1989). Characterization of the major heparan sulfate proteoglycan secreted by bovine aortic endothelial cells in culture. J. Biol. Chem. 264, 3514-3523.

4. Heremans, A., Cassiman, J.-J., Van Den Berghe, H., and David, G. (1988). Heparan sulfate proteoglycan from the extracellular matrix of human lung fibroblasts. J. Biol. Chem. 263,4731-4739.

5. Weis, J. R., Sun, B., and Rodgers, G. M. (1991). Improved method of human umbilical arterial endothelial cell culture. Thromb. Res. 61, 171-173.

6. Maciag, T., Cerundolo, J., Ilsley, S., Kelley, P. R., and Forand, R. (1979). An endothelial cell growth factor from bovine hypothalamus: identification and partial characterization. Proc. Natl. Acad. Sci. (USA) 76(11), 5674-5678.

7. Whitelock, J. M., Murdoch, A. D., Iozzo, R. V., and Underwood, P. A. (1996). The degradation of human endothelial cell-derived perlecan and release of bound basic fibroblast growth factor by stromelysin, collagenase, plasmin, and heparanases. J. Biol. Chem. 271(17), 10,079-10,086.

8. Melrose, J. and Ghosh, P. (1993). Determination of the average molecular size of gly-cosaminoglycans by fast protein liquid chromatography. J. Chromatogr. 637, 91-95.

9. McDevitt, C. A. and Muir, H. (1971). Gel electrophoresis of proteoglycans and gly-cosaminoglycans on large pore composite polyacrylamide-agarose gels. Anal. Biochem. 44, 612-622.

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