Cartilage and Smooth Muscle Cell Proteoglycans Detected by Affinity Blotting Using Biotinylated Hyaluronan

James Melrose

1. Introduction

Chondrocytes and smooth muscle cells synthesise the large CS-rich proteoglycans aggrecan (1) and versican (2) respectively. Both proteoglycans are capable of interacting with hyaluronan to form molecular aggregates that have important tissue specific functional roles to play. Aggrecan is a major matrix component of cartilage, the aggrecan aggregates are physically entrapped within the collagenous extracellular matrix of this tissue, and it is the collective interplay between this collagenous network and the aggrecan aggregates that equips this tissue with its unique viscoelastic and hydrodynamic properties and the ability to provide an almost frictionless weight-bearing surface to articulating joints (3). Smooth muscle cell versican, in comparison, is a quantitatively minor component of blood vessels but nevertheless it may influence the physicochemical properties of the vessel wall (4). Although the exact functional role of versican within blood vessels has yet to be fully elucidated, it is known to accumulate in intimal lesions during atherosclerosis and is implicated in the entrapment of low-density lipoprotein in the arterial wall during atherogenesis. Such interactions are likely to influence both the viscoelasticity and permeability of the vessel wall.

The interaction of aggrecan and versican with hyaluronan, which forms the basis of the assembly of massive macromolecular proteoglycan arrays within connective tissues, particularly in cartilage, is mediated via an amino-terminal globular domain that extends from the core protein, the so-called G1 domain or hyaluronan-binding region (HABR) (3,6). This interaction is further stabilized via a ternary complex formed with a small glycoprotein link protein that displays an affinity both for hyaluronan and for the G1 domain (7). A further amino-terminal globular domain in aggrecan, termed the G2 domain, has also been identified. This domain is absent in versican, and despite

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

considerable sequence homology with the G1 domain, it does not bind hyaluronan and its exact functional role has yet to be defined. Since both the HABR and the link protein display high affinities for hyaluronan, these hyaluronan-binding proteins (HABPs) have been utilized as probes for the detection and quantitation of hyaluronan in biological fluids by enzyme-linked immunosorbent assays (ELISAs) (8-10), for the localization of hyaluronan in tissue sections by immunohistological techniques (11-17) or for demonstration of hyaluronan species on Western blots (18) (see also Chapter 46).

The recent development of biotinylated hyaluronan (19) and its application as an affinity probe (20-22) now provides a means of monitoring not only electrophoreti-cally resolved intact aggrecan and versican monomer, but also proteolytically derived fragments of these proteoglycans containing functional G1 domains that are likely to be generated in certain disease states. The technique employed, affinity blotting, uses similar methodology to Western blotting but does not utilize specific antibodies. Affinity blotting is so named not only to differentiate it from the related Western technique but also to emphasise the functional nature of the interaction between the G1 domain and a nonsubstituted stretch of the biotinylated hyaluronan affinity probe that forms the basis of the technique. This chapter provides a simplified protocol for the preparation of Gl-containing aggrecan fragments and also demonstrates the utility of the affinity blotting technique for the visualization of Gl-containing proteoglycan species resolved by sodium dodecyl sulfate polyacry-lamide gel electrophoresis (SDS PAGE) or composite agarose polyacrylamide gel electrophoresis (CAPAGE).

2. Materials

2.1. Equipment and Chemicals

1. Electrophoresis system for SDS PAGE (Novex Xcell II mini electrophoresis system).

2. Blotting system for transfer of SDS PAGE gels (Novex Xcell II blot module).

3. Novex model 3540 programmable power supply.

4. Prepoured 4-20% polyacrylamide gradient Tris-glycine gels (Novex).

5. SilverXpress silver staining kit (Novex).

6. Electroelution cell (ISCO little blue tank) (23).

7. Vertical electrophoresis system for CAPAGE (Hoefer SE 600).

8. Blotting system for semidry transfer of CAPAGE gels (Bio-Rad).

9. Platform rocker or orbital shaker.

10. Agarose gel-bond support film (FMC Bioproducts, Rockland, ME, USA).

11. Acrylamide: bis-acrylamide 40% w/v stabilized liquid concentrate (19/1, C = 5%)(Bio-Rad).

12. Agarose, low-electroendosmosis grade (Bio-Rad) .

13. Low-molecular-weight hyaluronan, 170 kDa (Fidia, Abano Terme, Italy) (see Note 1).

14. Avidin conjugated to alkaline phosphatase, 750 U/mg protein (Sigma).

15. EDC (1-ethyl-3-[3-dimethylamino)-propyl]-carbodi-imide) (Sigma).

16. NHS-LC-biotin (sulfosuccinimidyl-6-(biotinamido)-hexanoate) (Pierce).

17. Nitrocellulose (0.22 ^m) (Schliecher and Schuell or Novex).

18. Chromaphor® green dye (Promega).

19. Prestained and protein molecular-weight standards for SDS PAGE (Novex).

2.2. Antibodies and Enzymes

Recombinant proMMP-3 (DuPont Pharmaceuticals) (24). Rabbit anti-VDIPES (DuPont Pharmaceuticals) (see Note 2). Mouse anti-VDIPEN (MAb BC-4) (25) (see Note 2).

Mouse anti-G1 domain of aggrecan (MAb 1-C-6, IgGj iso-type) (see Note 3) (26-27). Mouse anti-KS (MAb 5-D-4, Ig M iso-type, ICN) (see Note 3) (28). Mouse anti-link protein (MAb 8-A-4, IgG2b iso-type) (see Note 3) (26-27). Rabbit anti-bovine versican (29).

Alkaline phosphatase-conjugated goat anti-rabbit IgG (Promega).

Alkaline phosphatase-conjugated goat anti-mouse IgG (Promega).

Alkaline phosphatase-conjugated goat anti-mouse IgM (Kirkegaard and Perry).

2.3. Buffers

1. Buffer A (extraction buffer): 4 M GuHCl buffered with 0.5 M sodium acetate, pH 5.8, 10mM EDTA, PMSF (2 mM), 10mM benzamidine, and 10mM 6-amino hexanoic acid.

2. Buffer B (trypsin digestion buffer): 50 mM Tris-HCl, pH 8.2, 0.15M NaCl, 10mM CaCl2, and 0.02% w/v NaN3.

3. Buffer C (preparative SDS PAGE buffer): 25 mM Tris, 192mM glycine, pH 8.3, containing 0.035% w/v SDS, for use with in-situ band staining with Chromaphor® green (see Note 4).

4. Buffers D1 and D2 (electroelution cell buffers): (24), main tank buffer D1: 25 mM Tris, 192 mM glycine buffer, pH 8.3, 0.035% w/v SDS (90 mL); sample trap buffer D2 (where gel pieces are placed), 2.5 mM Tris, 19.2 mM glycine buffer, pH 8.3, containing 0.0035% w/v SDS.

5. Buffer E (MMP-3 digestion buffer): 50 mM Tris-HCl, pH 7.5, 10 mM CaCl2, 0.2 MNaCl, 0.05% Brij 35, 0.02% NaN3.

6. Buffer F (anion-exchange running buffer): 50 mM Tris-HCl, pH 7.2, 0.15 MNaCl, 7 Murea.

7. Buffer G (SDS PAGE running buffer): 25 mM Tris, 192 mM glycine, 0.1% w/v SDS, pH 8.3.

8. Buffer H (CAPAGE running buffer): 10 mM sodium acetate, pH 6.3, 1 mM sodium sulfate.

9. Buffer I (Western transfer buffer): 12 mM Tris, 98 mM glycine, 20% v/v methanol, pH 8.3.

10. Buffer J (transfer buffer for semidry transfer of CAPAGE gels to nitrocellulose): (BioRad Trans-blot) 25 mM Tris, 192 mM glycine, pH 8.3.

11. Buffer K (affinity-blotting wash buffer): 50 mM Tris, 500 mM NaCl, 0.05% w/v Tween 20, 0.02 % w/v NaN3, pH 7.2.

12. Buffer L (affinity-blotting blocking buffer): buffer K but containing 0.1% w/v Tween 20.

13. Buffer M (Western-blotting wash buffer): 50 mM Tris, 200 mM NaCl, 1% w/v BSA, pH 7.2, 0.02% w/v NaN3.

14. Buffer N (Western blocking buffer): buffer M containing 5% w/v BSA.

3. Methods

3.1. Purification of Cartilage Aggrecan by CsCl Density Gradient Centrifugation

1. Freeze-shattered cartilage powder (5 g) is extracted with 50 mL of buffer A for 48 h at 4°C with constant end-over-end stirring.

2. The extract is recovered by centrifugation (10,000g x 10 min at 4°C) and brought to a starting density of 1.42 g/mL with solid CsCl. Ultracentrifugation is undertaken using a

Fig. 1. (A) CAPAGE (toluidine blue) and (B) biotinylated hyaluronan affinity blots of ultracentrifuge fractions of the 4 M GuHCl ovine and equine articular cartilage extracts (Subheading 3.1). A crude ovine articular cartilage proteoglycan sample (1 ^g hexuronic acid) that contained the Aggrecan-1, -2 (Agg-1, -2) and dermatan sulfate proteoglycan (DS-PG) populations was run in the standard (Std) lanes. Each of the other lanes had a loading equivalent to 1.4 ^L of the original ultracentrifuge fraction except the D1 lanes, which, due to their higher proteoglycan contents, were diluted 1 in 10 and 0.14 ^L of the original ultracentrifuge fraction was run to avoid overloading.

Fig. 1. (A) CAPAGE (toluidine blue) and (B) biotinylated hyaluronan affinity blots of ultracentrifuge fractions of the 4 M GuHCl ovine and equine articular cartilage extracts (Subheading 3.1). A crude ovine articular cartilage proteoglycan sample (1 ^g hexuronic acid) that contained the Aggrecan-1, -2 (Agg-1, -2) and dermatan sulfate proteoglycan (DS-PG) populations was run in the standard (Std) lanes. Each of the other lanes had a loading equivalent to 1.4 ^L of the original ultracentrifuge fraction except the D1 lanes, which, due to their higher proteoglycan contents, were diluted 1 in 10 and 0.14 ^L of the original ultracentrifuge fraction was run to avoid overloading.

TV865B 8 x 17 mL titanium vertical rotor at 205,000g for 20 h at 10°C employing a Sorvall OTD 65 ultracentrifuge.

3. The tubes are manually fractionated into six equal fractions (D1-D6). The density of fractions is determined by weighing a known volume. The hexuronic acid (30) and protein contents (31) of fractions are also monitored by established methods.

4. Individual fractions are exhaustively dialyzed against distilled water and freeze-dried. The D1 fraction of density > 1.55 g/mL serves as purified aggrecan monomer, while the D2-D6 fractions contain G1-containing proteoglycan fragments of intermediate to low buoyant density and link proteins. See Figs. 1 and 2.

3.2. Preparation, Isolation, and Demonstration of G1-Containing Aggrecan Proteoglycan Fragments

1. Freeze-shattered bovine nasal cartilage powder (10 g) is digested with trypsin (0.1 mg/ mL) in buffer B (100 mL)for 24 h at 37°C.

2. The tissue residue is collected and washed three times with 50 mL 1M NaCl.

3. The washed trypsinised tissue residue is then extracted for 48 h with 100 mL buffer A.

4. The clarified extract (100 mL) and HA Sepharose 4B (32) (100 mL gel mixture) are placed inside dialysis tubing and dialyzed overnight at 4°C against 2L of distilled water to reduce the [GuHCl] to ~0.4M (associative conditions).

5. The gel/sample mixture is poured into a column and eluted at room temperature with 0.4 M GuHCl to remove nonbound material. Bound material is fractionated by eluting with a

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