Proteoglycan Protocols [Methods in Molec io 171] - R. Iozzo (Humana) WW.pdf - książki - Bio Med 22 - bibiariel

Methods in Molecular Biology TM

VOLUME 171

Proteoglycan

Protocols

Proteoglycan

Protocols

Edited by

Renato V. Iozzo, MD

Edited by

Renato V. Iozzo, MD

HUMANA PRESS

Isolation from Cell Cultures and Tissues

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 proper-

ties and by those taking advantages of the presence of glycosaminoglycan moiety.

This chapter focuses on the latter experimental procedures, which are particularly use-

ful 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 pres-

ence 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

Yanagishita

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 radiola-

beled 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

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

and protease inhibitors (1 m M phenylmethylsulfonyl fluoride, 10 m M N-ethylmaleimide,

10 m M disodium ethylenediaminetetraacetic acid (4) .

2. Stock solutions (100 × 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

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

Isolation from Cell Cultures and Tissues

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).

3. Glass wool, #3950 (Corning).

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 M guanidine

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

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.

A preferred solvent is a urea buffer, since it disrupts molecular interactions by interfering

with the formation of hydrogen bonds.

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-

Yanagishita

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 radioac-

tivity detection or colorimetric procedures; a convenient and sensitive colorimetric pro-

cedure 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

Proteoglycans with differing molecular weights purified by Q-Sepharose chromatogra-

phy 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.

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.

The column is eluted at a flow rate of 0.4 mL/min and each 1-min fraction is collected

( see Fig. 3 ).

Eluent fractions are analyzed for proteoglycan content (by radioactivity detection or colo-

rimetric procedures).

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