John D Sandy

1. Introduction

The full- or partial-length c-DNA and deduced core protein sequence is now available for at least 38 distinct proteoglycans [for review, see (1)]. Many of these can be placed into several large family groupings, such as the 10 members of the small leu-cine-rich repeat proteoglycans (decorin, biglycan, fibromodulin, lumican, keratocan, PRELP, epiphycan, mimecan, oculoglycan and osteoadherin), the glypicans (GPC-1, cerebroglycan, OCI-5, K-glypican, GPC-5, and GPC-6), the hyaluronan-binding proteoglycans (aggrecan, brevican, neurocan, and versican), the syndecans (SYN-1, fibroglycan, neuroglycan/N-syndecan, and amphiglycan/ryudocan), and the gly-cosaminoglycan-substituted collagens (types IX, XII, and XVIII). In addition, there is a group of apparently unrelated species, which includes agrin, perlecan, leprecan, bamacan, betaglycan, serglycin, phosphacan, NG2, CD44/epican, and testican.

Since most extracellular matrix proteins, and presumably also proteoglycans, undergo some degree of proteolytic modification during biosynthesis or catabolism, each of the core species described above probably exists in vivo in both the intact form and in one or more fragmented forms. Proteolysis may indeed be necessary for the conversion of the intact proteoglycan to a product, or a series of products, that can serve one or more functions in the cell or tissue where it is located. Proteolysis will almost certainly be involved in the removal of proteoglycans from cells or tissues, whether this be part of the normal turnover process or in pathological states where degradative pathways may be altered or accelerated. Despite the wealth of knowledge on core structures, tissue distribution, and function of proteoglycans, there is still very limited information on the role of proteolysis in the biology of these molecules. On the other hand, there are now a number of examples of what appear to be physiologically important proteolytic processing events for proteoglycans, and for aggrecan (2) and brevican (3) the precise cleavage sites and the family of proteinases responsible (ADAMTS) appears to have been established.

A 17-kDa N-terminal fragment of decorin accumulates in human skin with aging (4,5) and a 20-kDa N-terminal biglycan fragment is generated by bFGF treatment of bovine aortic endothelial cells (6), but the details of cleavage of these proteoglycans are unknown in both cases. The ectodomain of syndecan-1 is shed from cell surfaces into wound fluids by proteolysis in vivo (7,8), and similar cell -associated proteolysis appears to generate fragments of betaglycan (9), NG2 (10), testican (11), and perlecan (12), but the structural details have not been reported either. The most detailed analyses of proteoglycan core protein degradation in vivo have been done with the family of hyaluronan-binding proteoglycans, aggrecan, brevican, neurocan, and versican. At least two much-studied HA-binding proteins, hyaluronectin (13) and glial hyaluronate-binding protein (GHAP) (14) appear to represent the N-terminal globular domain of versican, although the versican isoform involved, the precise cleavage sites and the protease(s) responsible for the cleavage(s) are unclear. Neurocan has been isolated from rat brain in two fragments (15) which are generated by cleavage by an unknown proteinase near the middle of the core protein (apparently at the methionine 638-leucine 639 bond), and immunostaining with monoclonal antibodies that recognize only the N-terminal fragment (1F6) or the C-terminal fragment (1D1) suggests that the two fragments have very different functions (16).

Studies on brevican/BEHAB (17) and particularly aggrecan (18-20) have provided the majority of the molecular details on the pathways and enzymes involved in vivo in the proteolysis of proteoglycans. Brevican is present in the brain as the full-length protein (about 145 kDa) and a proteolytic fragment (about 80 kDa), which represents the C-terminal portion. The sequence at the cleavage responsible for this fragment (glutamate 400- serine 401) shows a striking similarity to the five cleavage sites that have been identified in aggrecan degradation in cartilage. The enzyme(s) responsible (aggrecanases) have now been identified as members of the ADAMTS family of metalloproteinases. The detailed studies on cartilage aggrecan degradation leading to the discovery of aggrecanase activity (18) and their cloning as ADAMTS-4 (2) and ADAMTS-11 (alias ADAMTS-5) (20) have provided some proven methodological approaches to the study of proteoglycan fragmentation, and these methods would appear to have general applicability to other proteoglycans. Indeed, the protocols to be described here have largely been developed (18,21-23) for analysis of small quantities of aggrecan and fragments (5- to 250-mg glycosaminoglycan [GAG]) such as those present in small tissue biopsies, biological fluids, and cell or tissue explant culture medium.

2. Materials

2.1. Proteoglycan and Core Protein Preparation from Fluids, Culture Medium, and Collagen-Rich Tissues

1. Streptomyces hyaluronidase (Str. hyalurolyticus), chondroitinase ABC (protease-free, Proteus vulgaris), Endobetagalactosidase (Escherichia freundii), and keratanase II(Bacillus sp.) (Seikagaku).

2. DE 52 cellulose, preswollen microgranular (Whatman).

3. Polyprep chromatography columns (Bio-Rad).

4. Dialysis of small volumes in Slide-a-Lyzer minidialysis units, 10,000 MWCO (Pierce) and large volumes (greater than 200 ^L) in Specta/Por dialysis tubing, 12,000-14,000 MWCO (Fisher).

2.2. Proteoglycan and Core Protein Preparation from Brain and Spinal Chord

1. 10-mL glass homogenizer with Teflon plunger (Thomas Scientific).

2.3. Preparation of Tissue Proteoglycans with No Glycosaminoglycan Substitution

1. Eppendorf microfuge (model 5413 or 5415C).

2.4. N-Terminal Sequencing of Proteoglycan Core Proteins

1. Protein assayed by bicinchoninic acid assay kit (Pierce).

2. Pharmacia FPLC Superose 12 (HR-10/30) and a fast desalting column (HR-10/10).

3. PVDF membranes (Hybond PVDF from Amersham or Immobilon from Millipore).

2.5. Western Blot Analysis and Core Identification

1. Sample preparation in 0.6-mL Snap-cap microcentrifuge tubes (Continental Lab Products).

2. 2x Tris-glycine sodium dodecyl sulfate (SDS) sample buffer as a premixed buffer (Novex).

3. Gel-loading pipet tips (200 ^L) (Labsource, Chicago, IL).

4. Mini-Protean II gel assembly (Bio-Rad) and E19001-XCELL II Mini Cell (Novex).

5. Trans-Blot transfer medium (roll of pure nitrocellulose membrane) (0.45 Bio-Rad).

6. Dry nonfat milk, blotting-grade blocker (Bio-Rad), and polyvinyl chloride laboratory wrap (Fisher).

7. Primary antibodies to most proteoglycans are described in the literature, and aggrecan antibodies, including those which detect neoepitopes on specific cleavage products, have recently been reviewed (24). Secondary antibodies for rabbit primary antibodies are HRP-conjugated goat anti-rabbit IgG (Chemicon), and for mouse monoclonals are generally HRP-conjugated goat anti-mouse IgG (Sigma), but HRP-conjugated goat anti-mouse IgM (Sigma) for antibody 3-B-3.

8. Chemiluminescent substrates, ECL (Amersham) or SuperSignal West Pico (Pierce), and high-performance chemiluminescence film, Hyperfilm ECL (Amersham).

2.6. Image Capture and Quantitation

1. ScanJet 3c/T, with DeskScan II software for PC or Mac (Hewlett Packard.) NIH Image (obtainable online from [email protected](for Mac) or from Scioncorp.com (for Windows). Adobe Photoshop for presentation.

3. Method

3.1. Proteoglycan and Core Protein Preparation from Fluids and Culture Medium (Note 1)

1. Typically, biological fluids (synovial fluid) or medium from cell or tissue culture, which contains at least 5 ^g of proteoglycan core protein, will be required. A desirable starting amount for aggrecan analyses is 250 ^g of GAG (as chondroitin sulfate [CS] and keratan sulfate [KS]) or about 25^g of core protein.

2. If the fluid is viscous due to a high content of hyaluronan (such as is found in synovial fluid or fibroblast-conditioned medium), it should be predigested as follows: Add 1/5 vol of 0.5 M ammonium acetate, 20 mM ethylenediaminetetraacetic acid (EDTA), 0.5 mM 4-(2-aminoethyl)benzenesulfonyl flouride (AEBSF), pH 6.0, and 2 TRU of streptomyces hyaluronidase and incubate at 60°C for 3 h.

3. The digest is dried to remove the ammonium acetate, and may be deglycosylated directly for Western analysis (Subheading 3.6.) as in steps 5 and 6 to follow. If the sample contains a high concentration of nonproteoglycan proteins (such as for serum-containing medium), the dried sample is taken up in about 2 mL of 7 M urea, 50 mM Tris-acetate, pH 8.0, and applied to a 0.5-mL-bed-volume column of DE 52 cellulose in Poly-Prep chromatography columns. The DE 52 cellulose is prepared by washing 3 times in distilled water and stored as a 50% slurry in water at 4°C. The 0.5-mL-bed-volume column is equilibrated in at least 2 mL of 7 M urea, 50 mM Tris-acetate, pH 8.0, before the sample is loaded. After sample loading and collection of unbound material (flow-through), the column is eluted as follows: (a) 2 mL of 7 M urea, 50 mM Tris-acetate, pH 8.0, which is added to the flow-through (b) 8 mL of 0.1 M NaCl, 7 M urea, 50 mM Tris-acetate, pH 8.0, (c) 4 mL of of 0.2 M NaCl, 7 M urea, 50 mM Tris-acetate, pH 8.0, (d) 1.5 mL of 0.8 M NaCl, 7 M urea, 50 mM Tris-acetate, pH 8.0, and (e) 1.5 mL of 1.5 M NaCl, 7 M urea, 50 mM Tris-acetate, pH 8.0.

4. All fractions (b, c, d, e) are dialyzed exhaustively against distilled water (at least 24 h at 4°C in 4 L of water with multiple changes) and the retentates are dried.

5. If the samples contain chondroitin sulfate/dermatan sulfate (CS/DS), the dried retentates are dissolved in 50 mM sodium acetate, 50 mM Tris, 10 mM EDTA,pH 7.6, and 25 mU (per 100 ^g GAG) chondroitinase ABC (protease-free) is added, followed by incubation at 37°C for 1-2 h.

6. If the samples contain KS alone or in addition to CS/DS, the above digestion condition is adjusted to pH 6.0 with acetic acid and 0.5 mU (per 100 ^g GAG) of endo-betagalactosidase and 0.5 mU (per 100 ^g GAG) of Keratanase II are added and incubated at 37°C for 2-4 h.

3.2. Proteoglycan and Core Protein Preparation from Collagen-Rich Tissues

1. Typically, a tissue sample containing at least 5 ^g of proteoglycan core protein will be required.

2. The tissue (cartilage, tendon, ligament, meniscus, aorta, etc.) is rinsed in cold PBS, chopped finely, and extracted by rocking at 4°C (15 mL of extractant per gram wet weight) for 48 h in 4 M guanidine-HCl, 10 mM MES, 50 mM sodium acetate, 5 mM EDTA, 0.1 mM AEBSF, 5 mM iodoacetic acid, 0.3 M aminohexanoic acid, 15 mM benzamidine,1^g/mL pepstatin, pH 6.8.

3. A clear extract is obtained after centrifugation to pellet the tissue and the extract is dialyzed exhaustively against distilled water (at least 24 h at 4°C in 4 L of water with multiple changes), and the retentate is either dried and deglycosylated for Western analysis (Subheading 3.6.) as in steps 5 and 6 under Subheading 3.1., or, if purification is required, it is adjusted to 7 M urea, 50 mM Tris-acetate, pH 8.0, and processed for analysis as for proteoglycans in steps 3-6 under Subheading 3.1.

3.3. Proteoglycan and Core Protein Preparation from Brain and Spinal Chord

1. The tissue (typically 50-300 mg wet weight of brain or spinal chord) is finely sliced and added to ice-cold 0.3 M sucrose, 4 mM HEPES, 0.15 M NaCl, 5 mM EDTA, 0.1 mM

AEBSF, 5 mM iodoacetic acid, 0.3 M aminohexanoic acid, 15 mM benzamidine,1 ^g/mL pepstatin, pH 6.8 (about 9 mL of extractant per gram wet weight of tissue).

2. After a brief (3 x 1 min), cold homogenization, the sample is clarified by centrifugation at maximum speed in an Eppendorf microfuge for 30 min at 4°C and the supernatant is applied to a 0.5-mL-bed-volume column of DE 52 cellulose in Poly-Prep chromatography columns which is equilibrated in 50 mM Tris-HCl, 0.15 M NaCl, 0.1% Triton X-100, pH 8.0. After sample loading and collection of unbound material (flow-through) the column is eluted as follows: (a) 4 mL of 50 mM Tris-HCl, 0.15 M NaCl, 0.1% Triton X-100, pH 8.0, which is added to the flow-through,(b) 4 mL of 50 mM Tris-HCl, 6 M urea, 0.25 M NaCl, 0.1% Triton X-100, pH 8.0, and (c) 50 mM Tris-HCl, 1.5 M NaCl, 0.5% CHAPS, pH 8.0.

3. The flow-through pool, the 0.25 MNaCl wash and the 1.5 MNaCl wash are each prepared for Western analysis (Subheading 3.6.) as in steps 4-6 under Subheading 3.1.

3.4. Preparation of Tissue Proteoglycans with No Glycosaminoglycan Substitution (Notes 2-4)

1. Some proteoglycans, and/or fragments, such as the "free" G1 domains of the different hyaluronan-binding proteoglycans, are present in tissues without GAG substitution. Solubilization of these proteins for SDS-PAGE and Western analysis may be achieved by direct extraction in detergent-containing buffer.

2. Tissue (cartilage, tendon, ligament, meniscus, spinal chord, aorta, sclera, cornea, brain, etc.) is washed in cold PBS, sliced finely, and suspended (about 6 mL of extractant per gram wet weight tissue) by mild agitation in 1% Triton X-100, 50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, 0.05%(v/v) BRIJ-35, 0.025% NaAzide, 5 mM EDTA, 0.1 mM AEBSF, 5 mM iodoacetic acid, 15 mM benzamidine, 1 ^g/mL pepstatin, pH 6.8. After 30 min at room temperature the tissue is removed by centrifugation (microfuge at maximum speed) at 4°C and a portion of the extract taken directly for SDS-PAGE and Western blot. Since deglycosylation is not used, only proteoglycans without GAG substitution will be detected.

3.5. N-Terminal Sequencing of Proteoglycan Core Proteins (Note 5)

1. Deglycosylated core proteins from step 6 under Subheading 3.1. can be prepared for N-terminal sequencing as follows: A portion of the deglycosylated product (containing at least 15 ^g of proteoglycan core protein) is fractionated on Superose 12, eluted in 0.5 M guanidine-HCl at 0.5 mL/min/fraction. The eluant is monitored at 214 nm and the high molecular-weight-pool (fractions 4-8) and low-molecular-weight pool (fractions 9-13) are concentrated to 0.5 mL and desalted on a fast-desalting column run in water at 1 mL/min and monitored at 214 nm. The desalted protein is dried and taken directly for N-terminal analysis.

2. If multiple N-terminal sequences are obtained, the proteins can be further separated on SDS-PAGE, electroblotted to PVDF membrane, stained with Coomassie blue, and individual stained bands cut out with a scalpel for direct N-terminal analysis.

3.6. Western Blot Analysis and Core Identification (Figs 1-3; Note 6)

1. Dry samples (0.1-5 ^g of protein) are dissolved in 20-40 ^L of gel sample buffer (prepared fresh with 12 mg of dithiothreitol, 200 ^L of 6 M urea, and 200 ^L of 2x Tris-glycine SDS sample buffer, pH 6.8) heated in a heating block at 100°C for 5-10 min, followed by centrifugation to spin down the liquid.

2. Samples (up to 40 ^L) are applied with gel-loading pipet tips to Novex precast gels (4-12% or 4-20% Tris-glycine gels, 1.0 mm x 10 wells) and run in electrode buffer (50 mM Tris base, 384 mM glycine, 0.2% SDS, pH 8.8) at 200 V for about 45 min at 4°C.

Fig. 1. Western analysis of aggrecan species isolated by guanidine extraction from normal human articular cartilages of different ages. Lanes 1-6 in both panels show analysis of human aggrecan from fetal, 2 mo, 1-y, 5-y, 15-y and 68-yr samples.The left panel is probed with a general aggrecan antiserum (anti-G1-2) raised against bovine aggrecan G1 domain.The right panel shows the same samples probed with the neo-epitope antiserum (anti-KEEE) to the ADAMTS-generated C-terminal neoepitope at Glu1714. The structure of the six peptides (1, a-c, 4, and 6) is shown diagrammatically, although the C-terminals of some major human aggrecan fragments (peptides a, b, and c) have yet to be identified. The figure clearly illustrates the well-established age-dependent C-terminal truncation of aggrecan in articular cartilage. In addition peptide 4, which migrates between peptide 1 and peptide a, is of very low relative abundance and is not detected readily with the anti-G1 antiserum. This illustrates the extreme sensitivity of the anti-KEEE (anti-peptide) antiserum relative to the anti-G1 antiserum for aggrecan. It should be noted that, in general, aggrecan peptides migrate with molecular sizes that are about twice the size predicted from the peptide size.

Fig. 1. Western analysis of aggrecan species isolated by guanidine extraction from normal human articular cartilages of different ages. Lanes 1-6 in both panels show analysis of human aggrecan from fetal, 2 mo, 1-y, 5-y, 15-y and 68-yr samples.The left panel is probed with a general aggrecan antiserum (anti-G1-2) raised against bovine aggrecan G1 domain.The right panel shows the same samples probed with the neo-epitope antiserum (anti-KEEE) to the ADAMTS-generated C-terminal neoepitope at Glu1714. The structure of the six peptides (1, a-c, 4, and 6) is shown diagrammatically, although the C-terminals of some major human aggrecan fragments (peptides a, b, and c) have yet to be identified. The figure clearly illustrates the well-established age-dependent C-terminal truncation of aggrecan in articular cartilage. In addition peptide 4, which migrates between peptide 1 and peptide a, is of very low relative abundance and is not detected readily with the anti-G1 antiserum. This illustrates the extreme sensitivity of the anti-KEEE (anti-peptide) antiserum relative to the anti-G1 antiserum for aggrecan. It should be noted that, in general, aggrecan peptides migrate with molecular sizes that are about twice the size predicted from the peptide size.

3. Proteins are transferred onto nitrocellulose membrane in transfer buffer (25 mM Tris base, 192 mM glycine, 20% (v/v) methanol, pH 8.4) at 100 V for 1 h at 4°C.

4. The membrane is blocked by incubation for at least 10 min at room temperature in 200 mM Tris-HCl, 1.37 M NaCl, 0.1% (v/v) Tween-20, 1% (w/v) dry nonfat milk, pH 7.6, and then incubated for between 1 h and 16 h at 4°C in 200 mM Tris-HCl, 1.37 M NaCl , 0.1% (v/v) Tween-20, 5% (w/v) dry nonfat milk, pH 7.6, containing the primary antibody at a dilution that is generally about 1/3000 .

5. The membrane is washed (3 x 2 min) in 200 mM Tris-HCl, 1.37 M NaCl, 0.1% (v/v) Tween-20, pH 7.6, and then incubated for about 1 h at room temperature in 200 mM

Fig. 2. Western analysis of aggrecan species present in immature pig proximal femoral epiphyseal cartilage and separated by DE 52 chromatography. A guanidine extract of pig cartilage was processed by DE 52 chromatography as detailed in steps 3-6 under Subheading 3.1. The samples shown in lanes 1, 2, and 3 were recovered from the 0.1 M NaCl, 0.2 M NaCl, and 0.8 M NaCl eluants, respectively, and the blot was probed with the general aggrecan antiserum, anti-G1-2. The structure of the four peptides (1, a, b, and 6) is shown diagrammatically, although the precise C-terminal sequence for peptides a and b have not been identified. Peptides b and 6 elute from DE 52 with low-salt buffer because they are not substituted with CS.

Fig. 2. Western analysis of aggrecan species present in immature pig proximal femoral epiphyseal cartilage and separated by DE 52 chromatography. A guanidine extract of pig cartilage was processed by DE 52 chromatography as detailed in steps 3-6 under Subheading 3.1. The samples shown in lanes 1, 2, and 3 were recovered from the 0.1 M NaCl, 0.2 M NaCl, and 0.8 M NaCl eluants, respectively, and the blot was probed with the general aggrecan antiserum, anti-G1-2. The structure of the four peptides (1, a, b, and 6) is shown diagrammatically, although the precise C-terminal sequence for peptides a and b have not been identified. Peptides b and 6 elute from DE 52 with low-salt buffer because they are not substituted with CS.

Tris-HCl, 1.37 M NaCl, 0.1% (v/v) Tween-20, 5% (w/v) dry nonfat milk (Bio-Rad), pH 7.6, containing the secondary antibody at about 1/3000.

6. The membrane is washed (3 x 10 min) in 200 mM Tris-HCl, 1.37 M NaCl, 0.1% (v/v) Tween-20, pH 7.6, and developed with Amersham or Pierce chemiluminescence reagents as follows: mix 5 mL each of distilled water, reagent 1, and reagent 2 from the ECL kit. Submerge the membrane in mix for about 1 min, protein side up. Remove the membrane with tweezers and allow excess liquid to drip off. Wrap the membrane in clear laboratory wrap and expose on Hyperfilm ECL.

7. Multiple exposures (for example, 5 s,30 s,1 min, 5 min) should be developed to optimize signal intensity and separation for major and minor species.

3.7. Image Capture and Quantitation (Note 7)

1. The film images are captured on an HP ScanJet 3c/T with DeskScan II software (set to Black and White Photo) and opened for quantitation in NIH Image and/or presentation in Adobe Photoshop.

2. For quantitation, individual bands are selected and integrated pixel density values obtained with preset value of pixel aspect ratio (generally about 750). Standardization with known loadings of core protein or fragment (where available) can be used to establish the linear detection range for the assay (23).

4. Notes

1. Treatment with Streptomyces hyaluronidase should totally eliminate viscosity due to hyaluronan before application of samples to DE 52, and the concentration of GAG in the applied samples should not exceed 100 ^g/mL. The DE52 flow-through will contain pro-

Fig. 3. Western analysis of aggrecan species generated in rat chondrosarcoma cell cultures treated with IL-1b. Cultures of rat chondrosarcoma cells (each containing about 30 ^g of GAG) were treated with IL-1b and at intervals (0, 24, 42, 48, 66, 72, 93, 98, 110, 114, and 138 h) the total culture was terminated by addition of buffer and digestion with chondroitinase ABC (step 5 under Subheading 3.1.) After removal of cells by centrifugation, the supernatants (1-11, respectively) were taken directly for Western analysis with monoclonal Ab 2-B-6 (reactive with chondroitin-4-sulfate stubs left on the core protein after Chase ABC digestion). The figure illustrates the time-dependent change in composition of immunoreactive fragments. The structure of six of the peptides is shown diagrammatically. In each case the N-terminal and C-terminal of the individual species were established either by N-terminal sequencing of bands or immunoreactivity with polyclonal antisera to G1 and G3 and neo-epitope antisera to the terminals at Ala 374 (anti-ARGSV), Glu1459 (anti-KEEE), and Glu1274 (anti-SELE).

Fig. 3. Western analysis of aggrecan species generated in rat chondrosarcoma cell cultures treated with IL-1b. Cultures of rat chondrosarcoma cells (each containing about 30 ^g of GAG) were treated with IL-1b and at intervals (0, 24, 42, 48, 66, 72, 93, 98, 110, 114, and 138 h) the total culture was terminated by addition of buffer and digestion with chondroitinase ABC (step 5 under Subheading 3.1.) After removal of cells by centrifugation, the supernatants (1-11, respectively) were taken directly for Western analysis with monoclonal Ab 2-B-6 (reactive with chondroitin-4-sulfate stubs left on the core protein after Chase ABC digestion). The figure illustrates the time-dependent change in composition of immunoreactive fragments. The structure of six of the peptides is shown diagrammatically. In each case the N-terminal and C-terminal of the individual species were established either by N-terminal sequencing of bands or immunoreactivity with polyclonal antisera to G1 and G3 and neo-epitope antisera to the terminals at Ala 374 (anti-ARGSV), Glu1459 (anti-KEEE), and Glu1274 (anti-SELE).

tein that is not GAG-substituted whereas the washes with 0.1, 0.2, 0.8, and 1.5 M NaCl will contain proteoglycans of different compositions. The bed volume of the DE 52 can be increased for isolation of larger amounts (capacity is about 500 ^g of GAG-substituted proteoglycan per milliliter of DE 52) but the concentration loaded should not exceed 100 ^g/mL. Also, the volume of wash solutions should be increased proportionately for larger amounts.

2. An alternative approach to isolation of proteoglycans from all tissues described above is extraction in guanidine-HCl as under Subheading 3.2., followed by ethanol precipitation as follows: To a portion of the guanidine extract add 3 vol of ice-cold ethanol (sodium acetate saturated) and let stand at -20°C for 16 h. Centrifuge at 4°C in the Eppendorf microfuge at maximum speed, remove, and discard the ethanol. Dry the pellet and sus pend it in chondroitinase buffer at 37°C and proceed as from step 5 under Subheading 3.1. This protocol isolates both protein and proteoglycan and is most successful with tissues, such as cartilage, that do not contain abundant guanidine-extractable nonproteoglycan proteins.

3. High-yield purification of proteoglycans and fragments substituted only with KS, such as fibromodulin and lumican, will require the addition of 0.5% CHAPS as detergent. Fibromodulin can be purified on MonoQ anion exchanger in buffers containing 6 M urea and 0.5% CHAPS (25) and lumican from corneas on Q-Sepharose in buffers containing 8 M urea and 0.5% CHAPS (26).

4. Reference (27) gives more detail on the isolation of specific proteoglycans and fragments from nervous tissues.

5. Clear N-terminal sequencing requires 10-100 pmol of protein, which is about 1-10 ^g for most proteoglycan core species. This is similar in sensitivity to Coomassie staining of proteins and therefore, if the species of interest can be identified by Coomassie staining, it should be possible to obtain an N-terminal sequence.

6. Optimal separation and detection of proteoglycan core proteins by Western analysis requires strict control of the completeness of the deglycosylation steps (steps 5 and 6 under Subheading 3.1.). This can be monitored by measuring the loss of reactivity in the dimethylmethylene blue assay (28). For all CS/DS- and KS-substituted proteoglycans, greater than 85% loss of the DMMB reactivity should be achieved on deglycosylation before Western analysis is attempted.

7. Since the chemiluminescence signal obtained with each species and antibody is highly dependent on epitope presentation on the nitrocellulose and the reactivity of the antibody in use, each species requires independent standardization. Because of the unavailability of standard preparations of many proteoglycan core proteins and fragments, quantitation by Western analysis is not possible in most cases. Other methods, such as radioimmunoassays and N-terminal quantitation by chemical means, are needed for stricter quantitation of these products.

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