Mw Stds KDfl

Fig. 4. Analysis of G1 domain-containing proteoglycan species isolated by preparative SDS PAGE of a trypsinized ovine articular cartilage sample by 4-20% SDS gradient PAGE and Western (Mab 1-C-6; anti-G1 domain; MAb 5-D-4 anti-KS) and affinity blotting (biotinylated hyaluronan). Lane 1 represents the crude trypsinized cartilage extract, which had been purified by HA affinity chromatography and lanes 2-4 show aggrecan fragments isolated from this by preparative SDS PAGE (see Subheadings 3.2 and 3.3). Lane 2 contained a high-molecular-weight core protein fragment containing the G1 and G2 domains and also some of the KS-rich region of aggrecan. Lane 3 contained a G1-G2 aggrecan core protein fragment with very little KS substitution, while lane 4 contained free G1 domain devoid of KS. Lanes 1-4 were loaded at 10 ^g dry weight HABP per lane. (see also Notes 4, 5 and 7).

5. Aliquots of proteoglycan digests are examined by 4-20% Tris-glycine gradient PAGE and either silver stained or transferred to nitrocellulose membranes prior to examination by Western and affinity blotting to identify G1-containing proteoglycan species. See Fig. 5.

3.5. Detection of Versican in Smooth Muscle Cell Media Samples by CAPAGE and Western and Affinity Blotting

1. Monolayer cultures of smooth muscle cells are established from explant outgrowth primary human aorta explant cultures (22,34).

2. 48 h media changes (100 mL) from fifth-passage subconfluent cultures are collected and stored at -20°C.

3. The media proteoglycans are ethanol precipitated with 3 volumes of ice-cold ethanol with shaking overnight at 4°C. The precipitate is collected by centrifugation, washed with 75% w/v ice-cold ethanol, and air dried.

4. The proteoglycan pellet is redissolved in 20 mL buffer F and applied to a column of 5 mL DEAE Sepharose CL6B preequilibrated in the same starting buffer. Nonbound material is washed from the column with 6 bed volumes of starting buffer. Bound material is then eluted with 4 bed volumes of 4 M GuHCl and the sample is dialyzed exhaustively against distilled water and freeze-dried.

5. The smooth muscle cell media proteoglycans are examined by CAPAGE, blotted to nitrocellulose, and versican identified by Western and affinity blotting. See Fig. 6.

12341 £31 1234

Fig. 4. Analysis of G1 domain-containing proteoglycan species isolated by preparative SDS PAGE of a trypsinized ovine articular cartilage sample by 4-20% SDS gradient PAGE and Western (Mab 1-C-6; anti-G1 domain; MAb 5-D-4 anti-KS) and affinity blotting (biotinylated hyaluronan). Lane 1 represents the crude trypsinized cartilage extract, which had been purified by HA affinity chromatography and lanes 2-4 show aggrecan fragments isolated from this by preparative SDS PAGE (see Subheadings 3.2 and 3.3). Lane 2 contained a high-molecular-weight core protein fragment containing the G1 and G2 domains and also some of the KS-rich region of aggrecan. Lane 3 contained a G1-G2 aggrecan core protein fragment with very little KS substitution, while lane 4 contained free G1 domain devoid of KS. Lanes 1-4 were loaded at 10 ^g dry weight HABP per lane. (see also Notes 4, 5 and 7).

Fig. 5. Assessment of the generation of free G1 domain of ovine articular, bovine nasal, and equine articular cartilage aggrecan monomer by digestion with stromelysin (rMMP-3). (A) Silver-stained gel segment. (B) Western blots identifying the terminal peptide sequences VDIPEN and VDIPES on the G1 fragments generated by cleavage within the interglobular domain by MMP-3 using neo-epitope antibodies BC-4 and anti-VDIPES, respectively. (C) Demonstration of HABPs in the aggrecan digests by affinity blotting. The migration position of Novex broad-range and see blue prestained standard proteins are also indicated on the left of the figure. The aliquots of digestion mixtures electrophoresed each contained 5 ^g PG monomer in the starting material prior to digestion/electrophoresis.

Fig. 5. Assessment of the generation of free G1 domain of ovine articular, bovine nasal, and equine articular cartilage aggrecan monomer by digestion with stromelysin (rMMP-3). (A) Silver-stained gel segment. (B) Western blots identifying the terminal peptide sequences VDIPEN and VDIPES on the G1 fragments generated by cleavage within the interglobular domain by MMP-3 using neo-epitope antibodies BC-4 and anti-VDIPES, respectively. (C) Demonstration of HABPs in the aggrecan digests by affinity blotting. The migration position of Novex broad-range and see blue prestained standard proteins are also indicated on the left of the figure. The aliquots of digestion mixtures electrophoresed each contained 5 ^g PG monomer in the starting material prior to digestion/electrophoresis.

Fig. 6. Detection of smooth muscle cell (SMC) versican in media samples of three human SMC cell line monolayer cultures by immunoblotting using an antibody to bovine versican (29) (lanes 1—4) and by affinity blotting (20,21) (lanes 5-8). The samples (10 ^g hexuronic acid (30) (lanes 1-3 and 5-7) were separated by CAPAGE (21) and transferred to nitrocellulose by semi-dry blotting. Purified ovine cartilage aggrecan (1 ^g hexuronic acid) was run in lanes 4 and 8 as an internal standard.

Fig. 6. Detection of smooth muscle cell (SMC) versican in media samples of three human SMC cell line monolayer cultures by immunoblotting using an antibody to bovine versican (29) (lanes 1—4) and by affinity blotting (20,21) (lanes 5-8). The samples (10 ^g hexuronic acid (30) (lanes 1-3 and 5-7) were separated by CAPAGE (21) and transferred to nitrocellulose by semi-dry blotting. Purified ovine cartilage aggrecan (1 ^g hexuronic acid) was run in lanes 4 and 8 as an internal standard.

3.6. Western and Affinity Blotting (see Note 6)

1. SDS PAGE gels are transferred to nitrocellulose membranes at 200 mA constant current for 2 h using buffer I (35).

2. Affinity blots are blocked for 3 h in buffer L.

3. Western blots are blocked in buffer N for 3 h.

4. Affinity blots are incubated for 2 h with 2 ^g/mL biotinylated hyaluronan diluted in buffer K (see Note 6).

5. MAb 1-C-6 (1/1000 dilution), 8-A-4 (1/1000 dilution), 5-D-4 (1/5000 dilution), or BC-4 (1/1000 dilution) are diluted in buffer M and allowed to bind for 2 h with constant shaking (see Note 3).

6. Affinity blots are then washed in buffer K (4 x 5 min). Western blots are washed in buffer M (4 x 5 min).

7. Appropriate secondary detection reagents are then added. For Affinity blots, avidin alkaline phosphatase, 2 ^g/mL in buffer K; for Western blots, either goat anti-mouse IgG or IgM, or goat anti-rabbit IgG alkaline phosphatase conjugates 1/5000 dilution in buffer M. After a further 1 h, the membranes are washed again as in step 6.

8. A solution of NBT/BCIP in 0.1 M Tris-HCl, pH 9.5, is then added for the development step, which is allowed to proceed for up to 20 min at room temperature.

9. The blots are then washed in several changes of distilled water and dried.

3.7. Composite Agarose Polyacrylamide Gel Electrophoresis (CAPAGE) (21)

1. Proteoglycan samples are dissolved in 8 M urea, 10 mM sodium acetate, pH 6.3, and 1 mM sodium sulfate (CAPAGE dissociation buffer) overnight at 37°C then diluted 1/1 with 10 mM Tris-acetate/1 mM Na2SO4, pH 6.3, 60% w/v sucrose, and 0.01% w/v bro-mophenol blue (CAPAGE application buffer).

2. CAPAGE slab gels (0.15 x 14 x 14 cm) of 0.6% w/v agarose and 1.2% w/v acrylamide are cast in buffer H and left to polymerize for at least 2 h at 4°C. The gel is then preequilibrated overnight in 4 M urea in buffer H.

3. Proteoglycan samples are electrophoresed in a Hoefer SE 600 vertical slab gel electrophoresis system in running buffer (buffer H) at 50 V for ~5 min, then the voltage is increased to 150 V and the sample electrophoresed until the bromophenol blue marker dye has migrated ~2-3 cm (~45 min).

4. The electrophoresis running buffer is maintained at a temperature of 10 ± 2.0°C during the run, using an external cooler.

5. The gels are stained 30 min in toluidine blue (0.02% w/v) in 0.1 M acetic acid, destained in 0.5 M acetic acid 30 min, then distilled water 5 min and dried onto the hydrophilic side of agarose gel-bond support films. Final destaining is then completed on this dried film in a few minutes in distilled water.

3.8. Semidry Transfer of Proteoglycans from CAPAGE Gels to Nitrocellulose

1. CAPAGE gel segments (14 x 5.5 x 1.5 mm) are electroblotted to nitrocellulose membranes using buffer J in a Bio-Rad semidry blotter at 5.5 mA/cm2 for 30 min (21).

2. Immuno and affinity blots are undertaken as outlined under Subheading 3.6.

3.9. Preparation of HA-Sepharose 4B (22)

1. A suspension of 100 mL of hydrated gel E-aminohexyl-Sepharose 4B is gently mixed end-over-end for 12 h at room temp with 100 mL of a 170 kDa HA solution, 1mg/mL, in milli Q distilled water.

2. The pH of the solution is adjusted to 4.75 with 0.1N HCl and 0.3g solid EDC is gradually added, the pH is maintained at 4.75 with small additions of 0.1 N HCl.

3. After 2 h the sample is brought to pH 7 with 0.1 N NaOH and the mixture dialyzed against several changes of distilled water.

4. The HA-Sepharose 4B gel is stored in 0.02% w/v sodium azide at 4°C. One mL of HA Sepharose 4B gel can bind at least 300 ^g of purified bovine nasal cartilage G1 domain under the conditions outlined above in Subheading 3.3.

3.10. Preparation of Biotinylated Hyaluronan

1. The method is a modification of that of Pouyani and Prestwich (1994) (19). Low-molecular weight (170-kDa) hyaluronan (500 mg) is dissolved with overnight end-overend stirring at 4°C in 100 mL distilled water.

2. Adipic dihydrazide (4 g) is added to this solution and the pH is adjusted to 4.75 using 0.1 N HCl.

3. Solid EDC (1-ethyl-3-[3-dimethylamino)-propyl])-carbodi-imide), (0.5 g) is gradually added to this solution and the pH of the reaction mixture maintained at 4.75 by the addition of small aliquots of 0.1 N HCl.

4. After 2 h at room temperature there is no further rise in pH; the reaction mixture is therefore adjusted to pH 7 with 1N NaOH and exhaustively dialyzed against distilled water (4 x 5l x 18h). Then the hydrazido-hyaluronan is freeze-dried.

5. Hydrazido-hyaluronan (100 mg) is redissolved overnight in 20 mL 0.2 M NaHCO3 with end-over-end stirring at 4°C.

6. Sulfosuccinimidyl-6-(biotinamido)-hexanoate (NHS-LC-biotin, 100 mg) is added to the reaction mixture and end-over-end stirring continued for 18 h at room temperature.

7. Five milliliters of a 1M Tris free base solution is then added and the sample dialyzed exhaustively against distilled water to remove free biotin, then freeze-dried.

8. The level of substitution of D-glucuronic acid residues of the biotinylated hyaluronan with biotin typically is 35.7%, which represents 0.92 ^mol biotin/mg biotinylated hyaluronan (20).

4. Notes

1. Tengblad (1979) (32) provides details on how to depolymerize high-molecular-weight HA to an appropriate size range if a small-molecular-weight HA is not available.

2. The Mab anti-VDIPES* recognizes the major MMP cleavage site between the G1 and G2 globular domains of bovine cartilage aggrecan. This antibody is equivalent to Mab BC-4 (anti-VDIPEN*), which recognizes the MMP cleavage site in the interglobular domain in most other mammalian species investigated. *Single-letter code for amino acid nomenclature.

3. The 1-C-6, 8-A-4, and 5-D-4 Mabs are available from the Developmental Studies Hybridoma Bank, Department of Biological Sciences, The University of Iowa, Ames, IA, USA. Mab 5-D-4 is also available commercially from ICN Biomedicals, and Seikegaku Corporation, Tokyo, Japan.

4. A lower concentration of SDS to that conventionally used in SDS PAGE (36) must be used when staining proteins insitu with Chromaphor/green dye, since the levels of SDS normally used in the Laemmli method prevent staining with this dye.

5. HABP pools 1 and 2 (see Fig. 3) may be further fractionated by dissociative gel permeation chromatography (6 M GuHCl) and/or anion-exchange chromatography in 8 M urea to separate link protein and free G1 domain from other G1-containing proteoglycan species.

6. Although reducing conditions are frequently used in Western blotting to facilitate protein unfolding and thus allow appropriate antigen presentations for effective interaction with Mabs (25-28) reducing conditions must be avoided in affinity blotting since binding of HA to the G1 domain requires a native (disulfide bond stabilized) conformation in this region of the aggrecan core protein.

7. Extractigel D affinity columns (Pierce) may be utilized to remove residual SDS from the HABP pools. Free G1 domain generated as under Subheading 3.3 has been used for the histochemical visualization of HA in rat lung tissues (17). The G1-G2 and G1-G2-KS pools prepared by preparative SDS PAGE (see Subheading 3.3) have also been used as substrates for MMPs using similar methodology to that presented in Fig. 5.

Acknowledgments

Professor Bruce Caterson, School of Molecular and Medical Biosciences, University of Cardiff, Wales, UK, is acknowledged for his gift of the neo-epitope antibody BC-4. Dr. Michael Pratta, Du Pont Pharmaceuticals, Wilmington, Delaware, USA kindly provided the anti-VDIPES Mab and recombinant pro MMP-3 (24) used in these studies.

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