Isolation of Proteoglycans from Cell Cultures and Tissues

Masaki Yanagishita

1. Introduction

Proteoglycans are a class of glycosylated proteins characterized by the presence of glycosaminoglycans as a carbohydrate component, which endow them with unique biological as well as biochemical properties. Therefore, isolation of proteoglycans from various biological sources such as cell cultures and tissues could be achieved by ordinary molecular purification procedures utilizing their general molecular properties and by those taking advantages of the presence of glycosaminoglycan moiety. This chapter focuses on the latter experimental procedures, which are particularly useful for obtaining total proteoglycan and glycosaminoglycan species from various biological sources. These protocols could be followed by purification procedures specific to individual proteoglycan species (i.e., by using antibodies to core proteins, or binding proteins to specific sequences of glycosaminoglycans) to select specific molecules.

Two major classes of well-established purification procedures aiming at the presence of glycosaminoglycans in the molecule have been used extensively. They include (1) density gradient ultracentrifugation, and (2) anion-exchange chroma-tography. The former procedure makes use of the fact that the glycosaminoglycan moiety of proteoglycans has high specific gravity, so, proteoglycans with a large number of glycosaminoglycans have high molecular densities (often as high as those of nucleic acids). Therefore, such a procedure is particularly suited for the purification of proteoglycans with many glycosaminoglycan chains (e.g., aggrecan, the major proteoglycan in cartilage tissues). On the other hand, the procedure is not suitable for isolating proteoglycans with only few glycosaminoglycans and high protein contents (e.g., "small, leucine-rich proteoglycans" and cell surface heparan sulfate proteoglycans). Purification procedures belonging to the latter class take advantage of high negative charges contributed by sulfate groups and carboxyl groups universally present in glycosaminoglycans. Thus, they can be widely used

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

Proteoglycan Isolation
Fig. 1. A flow diagram of proteoglycan isolation from a radiolabeled cell culture.

for isolating any species of proteoglycans and glycosaminoglycans, and are the main subject of following discussion.

This chapter outlines a proteoglycan-isolating protocol from a metabolically radiolabeled cell culture (see Fig. 1) as an example case, which addresses technical problems often encountered when working with small quantities of proteoglycans. Other reviews on similar subjects can be consulted for more detailed discussions (1-3).

2. Materials 2.1. Extraction

Buffer: 4 M guanidine HCl, 0.05 M Na acetate, pH 6.0, containing 2% (w/v) Triton X-100 and protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 mM N-ethylmaleimide, 10 mM disodium ethylenediaminetetraacetic acid (4).

Stock solutions (100 x concentrated) of phenylmethylsulfonyl fluoride and N-ethylmaleimide are prepared in ethanol and added to the guanidine HCl buffer just prior to use (they are unstable in aqueous solutions).

2.2. Solvent Exchange

1. Sephadex G-50, fine (Amersham Pharmacia Biotech). Preswelling of Sephadex can be done in hot water (off the heater), which achieves sterilization, degassing, and shortening of swelling time. Extreme caution should be exercised when adding Sephadex powder to boiling water, to avoid flushing. A convenient concentration of gel (50% slurry) can be made by mixing 5 g of Sephadex G-50 with 100 mL of water. Bacteriostatic agents (e.g., 0.02% Na azide) should be added for long-term storage.

2. 10 mL plastic disposable pipet (Falcon).

4. Buffer: 8 M urea, 0.20 M NaCl, 0.05 M Na acetate, 0.5% Triton X-100, pH 6.0.

2.3. Anion-Exchange Chromatography

1. Q-Sepharose, fast flow (Amersham Pharmacia Biotech). Q-Sepharose has to be preequilibrated with the low salt buffer as described below.

2. Low salt buffer: 8 M urea, 0.20 M NaCl, 0.05 M Na acetate, 0.5% Triton X-100, pH 6.0.

3. High salt buffer: 8 M urea, 1.5 M NaCl, 0.05 M Na acetate, 0.5% Triton X-100, pH 6.0.

4. Gradient former (a simple configuration can be made with two beakers).

5. Peristaltic pump.

2.4. Gel Filtration Chromatography

1. Superose 6, HR 10/30 (Amersham Pharmacia Biotech).

2. Buffer: 4 M guanidine HCl, 0.05 M Na acetate, 0.5% (w/v) Triton X-100, pH 6.0.

3. Methods

General experimental procedures introducing radioactive precursors into proteoglycans using cell cultures and tissue cultures are reviewed elsewhere (1).

3.1. Extraction

1. After removing media from the cell layer, approximately 2 mL of extraction buffer (per 35-mm-diameter cell culture dish) is added to a culture plate (see Note 1).

2. Proteoglycans are extracted within 2-3 h of constant shaking at 4°C.

3. Passing the extract through a 1-mL pipet tip up and down approximately 10 times reduces the viscosity of the solution caused by DNA.

4. Extraction of proteoglycans from tissues: As used originally in the extraction of proteoglycan from cartilage tissue (3), 4 M guanidine generally provides excellent solu-bilization of proteoglycans.

5. When tissues are to be extracted, ordinarily approximately 10 times volume of 4 Mguanidine HCl buffer successfully solubilizes proteoglycans from finely minced tissues in 12 h at 4°C.

6. Additional consideration should be made when extraction of cell-associated proteoglycans is attempted; i.e., inclusion of sufficient amounts of detergent may be necessary (such as 2% Triton X-100) for the solubilization of proteoglycans (5).

7. Secreted proteoglycans in cell culture media are generally already soluble, but, in order to minimize interactions between highly charged proteoglycans and other molecules, direct addition of solid guanidine HCl (0.53 g of solid guanidine HCl per milliliter of media makes 4 M guanidine HCl solution) is frequently used.

3.2. Solvent Exchange

1. In order to prepare the extracted proteoglycans in 4 M guanidine HCl for the anion-exchange chromatography procedure in the next step, guanidine HCl has to be replaced with a solvent compatible with the procedure.

2. A preferred solvent is a urea buffer, since it disrupts molecular interactions by interfering with the formation of hydrogen bonds.

3. A convenient buffer-exchange procedure can be done by gel filtration (such as Sephadex G-50 chromatography) using a small disposable pipet. This process is also very conve nient to remove unincorporated radioactive precursors, when radiolabeled cell cultures were extracted with a 4 M guanidine HCl solvent.

4. Preparation of Sephadex G-50 column: Pour preswollen Sephadex G-50 (50% slurry) into a 10-mL plastic disposable column (which has been cut at the top with a file and plugged with glass wool at the bottom) to make 8 mL bed volume.

5. Remove excess water and equilibrate the column with 8 M urea buffer (a total of 9 mL is sufficient to equilibrate the column).

6. Carefully prepare a flat gel surface with a glass Pasteur pipet and remove excess urea buffer. Apply a 2-mL sample and discard the eluent.

7. After the entire sample is in the column, carefully overlay 3 mL of buffer and collect eluent until the entire buffer is in the column (3-mL fraction collected). This fraction contains proteoglycans and other macromolecules in 8 M urea buffer, while leaving small molecules in the original sample (guanidine HCl, isotope precursors, etc.) behind in the column.

8. At this point, the column can be safely disposed of as a radioactive waste.

3.3. Anion-Exchange Chromatography

3.3.1. Preparation of Q-Sepharose Column (see Note 2) and Sample Application

1. 2 mL of preequilibrated Q-Sepharose (1 mL of Q-Sepharose can bind up to 3-5 mg of proteoglycans) is packed into a small column (10-mL plastic pipet cut by a file and plugged with glass wool at the bottom).

2. Alternatively, 2 mL of Q-Sepharose is mixed with the sample in 8 M urea buffer and gently shaken for 1 h, then packed into the column; this latter method gives uniform binding of proteoglycans to Q-Sepharose, resulting in a better flow property, especially when a large quantity of materials is used.

3. After sample application, the column is washed with 10 mL of the low-salt buffer.

4. Then the column is connected to a gradient former and eluted with a total of approximately 40 mL of buffer with a flow rate of 10-15 mL/h.

5. Every 1- to 2-mL fraction is collected and monitored for NaCl concentration by conductivity measurement (see Fig. 2). Eluent fractions are monitored for proteoglycans (by radioactivity detection or colorimetric procedures; a convenient and sensitive colorimetric procedure using Safranin O is described in ref. 6).

6. Typically, heparan sulfate proteoglycans are eluted in a peak at approximately 0.5 M NaCl and chondroitin sulfate proteoglycans at 0.65 M NaCl.

3.4. Gel Filtration Chromatography

1. Proteoglycans with differing molecular weights purified by Q-Sepharose chromatography are further separated by gel filtration chromatography. Superose 6 (or equivalent gel) is suitable for resolving small proteoglycans ("small, leucine-rich proteoglycans", cell surface heparan sulfate proteoglycans, etc.) from large proteoglycans (e.g., aggrecan, versican, and perlecan) and partially degraded proteoglycans.

2. Superose 6 is equilibrated in 4 M guanidine HCl buffer containing 0.5% Triton X-100 (see note 2), and up to 0.5 mL of sample can be loaded to the column.

3. The column is eluted at a flow rate of 0.4 mL/min and each 1-min fraction is collected (see Fig. 3).

4. Eluent fractions are analyzed for proteoglycan content (by radioactivity detection or colorimetric procedures).

Proteoglycans Labelled

Fig. 2. Q-Sepharose anion-exchange chromatography. A sample from a cell culture that was metabolically radiolabeled with [3H]leucine and [35S]sulfate was analyzed. A large peak containing 3H-labeled proteins was efficiently separated from 35S-labeled proteoglycan peaks eluting later in high-salt fractions.

Fig. 2. Q-Sepharose anion-exchange chromatography. A sample from a cell culture that was metabolically radiolabeled with [3H]leucine and [35S]sulfate was analyzed. A large peak containing 3H-labeled proteins was efficiently separated from 35S-labeled proteoglycan peaks eluting later in high-salt fractions.

Proteoglycan

Fraction Number

Fig. 3. Superose 6 chromatography of 35S-labeled cell surface heparan sulfate proteoglycan.

4. Notes

1. Extraction of cell-associated proteoglycans from cultured cell requires the use of detergent (5). Usefulness of 4 M guanidine HCl in the presence of 2% Triton X-100 has been rigorously demonstrated.

2. One of the major technical problems associated with anion-exchange chromatography of proteoglycans, especially when purifying molecules present in small quantities (e.g., isolation of proteoglycans from cell cultures), is poor recovery of materials. This can be, in most cases, overcome by the use of detergents (either nonionic or zwitte-rionic). Routinely, the use 0.5% (w/v) Triton X-100 (or NP-40) dramatically improves recovery of proteoglycans (even glycosaminoglycans) from ion-exchange columns. Most nonionic detergents (such as Triton X-100, NP-40) possess strong absorbance in the ultraviolet (UV) range, thus making UV tracing for protein detection difficult. If this causes problems in analyses, non-UV-absorbing, nonionic detergents such as Genapol X-100TM (Calbiochem) can be used. Also, when the removal of detergents is required in later experimental steps, the use of those with high critical micellar concentrations (such as CHAPSTM, Calbiochem) in place of Triton X-100 is beneficial.

References

1. Hascall, V. C., Calabro, A., Midura, R. J., and Yanagishita, M. (1994) Isolation and characterization of proteoglycans. Meth. Enzymol. 230, 390-417.

2. Yanagishita, M., Midura, R. J., and Hascall, V. C. (1987) Proteoglycans: Isolation and purification from tissue cultures. Meth. Enzymol. 138, 279-289.

3. Hascall, V. C. and Kimura, J. H. (1982) Proteoglycans: Isolation and characterization. Meth. Enzymol. 82, 769-800.

4. Oike, Y., Kimata, K., Shinomura, T., Nakazawa, K. and Suzuki, S. (1980) Structural analysis of chick-embryo cartilage proteoglycan by selective degradation with chondroitin lyases (chondroitinases) and endo-beta-D-galactosidase (keratanase). Biochem. J. 191, 193-207.

5. Yanagishita, M. and Hascall, V. C. (1984) Proteoglycans synthesized by rat ovarian granulose cells in culture; isolation, fractionation, and characterization of proteoglycans associated with the cell layer. J. Biol. Chem. 259, 10,260-10,269.

6. Lammi, M. and Tammi, M. (1988) Densitometric assay of nanogram quantities of proteoglycans precipitated on nitrocellulose membrane with Safranin O. Anal. Biochem. 168, 352-357.

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