Iozzo Renato V. Proteoglycan Protocols

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Cartilage and Smooth Muscle Cell Proteoglycans 55

2.2. Antibodies and Enzymes

1. Recombinant proMMP-3 (DuPont Pharmaceuticals) (24).

2. Rabbit anti-VDIPES (DuPont Pharmaceuticals) (see Note 2).

3. Mouse anti-VDIPEN (MAb BC-4) (25) (see Note 2).

4. Mouse anti-G1 domain of aggrecan (MAb 1-C-6, IgG

iso-type) (see Note 3) (26–27).

5. Mouse anti-KS (MAb 5-D-4, Ig M iso-type, ICN) (see Note 3) (28).

6. Mouse anti-link protein (MAb 8-A-4, IgG

iso-type) (see Note 3) (26–27).

7. Rabbit anti-bovine versican (29).

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

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

10. 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 CaCl

and 0.02% w/v NaN

3. Buffer C (preparative SDS PAGE buffer): 25 mM Tris, 192mM glycine, pH 8.3, contain-

ing 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 CaCl

, 0.2 M NaCl,

0.05% Brij 35, 0.02% NaN

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

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): (Bio-

Rad 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 NaN

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 NaN

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 × 10 min at 4°C) and brought to a

starting density of 1.42 g/mL with solid CsCl. Ultracentrifugation is undertaken using a

56 Melrose

TV865B 8 × 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 pro-

tein 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

Fig. 1. (A) CAPAGE (toluidine blue) and (B) biotinylated hyaluronan affinity blots of ultra-

centrifuge fractions of the 4 M GuHCl ovine and equine articular cartilage extracts (Subheading 3.1).

A crude ovine articular cartilage proteoglycan sample (1 µg 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 µL of the original

ultracentrifuge fraction except the D1 lanes, which, due to their higher proteoglycan contents,

were diluted 1 in 10 and 0.14 µL of the original ultracentrifuge fraction was run to avoid over-

Cartilage and Smooth Muscle Cell Proteoglycans 57

Fig. 2. Analytical 4–20% SDS PAGE of the same ultracentrifuge samples as examined in

Fig. 1. Gels in segments A and D were stained with colloidal Coomassie to visualize the distri-

bution of proteins; proteoglycan species were visualized in B and E by staining with toluidine

blue. Replicate gels were also transferred to nitrocellulose (C and F), and G1 domain-contain-

ing proteoglycan species were visualized by affinity blotting. The top of the figure illustrates

the distribution of protein (31), proteoglycan (as hexuronic acid)

(30), and density of the ultra-

centrifuge samples. The equivalent of 4.1 µL of original ultracentrifuge fraction was loaded

per lane. The migration position of Novex broad-range and See Blue prestained standard pro-

teins are also indicated on the left of the figure.

58 Melrose

linear gradient of 0.4–4 M GuHCl in 50 mM Tris-HCl, pH 7, over ~6 bed volumes.

Fractions of 10 mL are collected at a flow rate of 10 mL/h. Aliquots of individual frac-

tions are assayed for protein and proteoglycan (as sulfated glycosaminoglycan) (33). The

gradient is measured manually using a conductivity monitor.

6. Ice-cold ethanol (3 mL) is added to 1 mL aliquots of individual fractions and the samples

shaken at 4°C. The precipitates are collected by centrifugation, washed with 75% etha-

nol, air-dried and redissolved in SDS PAGE application buffer (0.5 mL).

7. Aliquots (10 µL/lane) of the ethanol-precipitated samples are examined by 4–20% SDS

PAGE and blotted to nitrocellulose, then probed with anti-link protein (Mab 8-A-4).

HABPs are identified by affinity blotting. See Fig. 3.

3.3. Isolation of Aggrecan G1-Proteoglycan Species

by Preparative SDS PAGE

1. Freeze-shattered ovine articular cartilage (10 g) is digested with 0.1 mg/mL trypsin in

100mL buffer B for 24 h at 37°C.

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

3. The washed trypsinized tissue residue is extracted for 48 h with 100mL buffer A, the

extract recovered and dialyzed exhaustively against distilled water and freeze-dried.

4. The freeze-dried material is redissolved in 0.4 M GuHCl and the HABPs isolated by HA

affinity chromatography as indicated under Subheading 3.2 but utilizing isocratic elu-

tion with 4 M GuHCl to remove the bound HABPs; these are dialyzed against distilled

water and freeze-dried (see Note 5).

5. Purified HABP samples are redissolved in 125 mM Tris-HCl buffer, pH 6.8, containing

10% v/v glycerol and 1% w/v SDS application buffer, and heated at 95°C for 5 min.

6. HABP samples from HA affinity (5 mg dry weight) are electrophoresed at 125 V for 90 min

in 1.5 mm 4–20% gradient gels using buffer C as running buffer. Chromaphor

green stain

is added to the upper electrode chamber shortly after commencement of the run in order

to visualize protein band separation within the gel during the run (see Note 4).

7. After electrophoresis, the gel is placed in 5% v/v acetic acid for 10 min to improve the

visualization of the protein bands. Appropriate regions of the gel are then excised and cut

into 1 mm

pieces.

8. Proteins are electroeluted from the gel pieces using a “little blue-tank electroelution

device” (Isco) at 2.5 mA/trap for 2 h at 4°C (23). Each of the electroelution buffer tanks

of this device is filled with 90 mL buffer D1. The sample traps are filled with 10 mL

buffer D2.

9. The electroeluted samples are examined by analytical SDS PAGE and Western and affinity

blotting to confirm the identities of the purified HABPs (see Notes 6 and 7). See Fig. 4.

3.4. Aggrecan MMP-3 Digestions: Detection of G1 Domain

Containing Proteoglycan Species by Western and Affinity Blotting

1. rProMMP-3 samples (24) are activated in 1 mM APMA in buffer E for 3 h at 37°C, then

applied to HiTrap desalting columns (Pharmacia) to remove the APMA and diluted to a

concentration of 40 µg/mL by protein assay (31).

2. D1 aggrecan samples (0.8 mg/mL) are dissolved overnight at 4°C in buffer E.

3. Proteoglycan samples ([0.4 mg/mL] final) are digested at 37°C for up to 18 h with 20 µg/mL

final MMP-3.

4. Aliquots (0.2 mL) of the digests are removed after 0, 4, and 18 h and added to 50 µL of 20 mM

EDTA to stop enzymatic activity and stored at –20°C until required.

Cartilage and Smooth Muscle Cell Proteoglycans 59

Fig. 3. (A) Fractionation by hyaluronan (HA) affinity chromatography (32) of hyaluronan-

binding proteins (HABPs) in an extract of trypsinized bovine nasal cartilage, showing the distri-

bution of proteoglycan species (as sulfated GAG)

(33) and protein (31) and visualization of

HABP pools 1 and 2 by affinity blotting (inset). (B) Aliquots of individual fractions were also

examined by 4–20% gradient SDS PAGE, blotted to nitrocellulose, and link protein species

identified using Mab 8-A-4 and HABPs identified by affinity blotting. The migration positions of

Novex See Blue prestained standards are also indicated on the left-hand side of the figure.

60 Melrose

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 examina-

tion 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 pri-

mary 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 nitro-

cellulose, and versican identified by Western and affinity blotting. See Fig. 6.

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 µg dry weight HABP per lane. (see also Notes 4, 5 and 7).

Cartilage and Smooth Muscle Cell Proteoglycans 61

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) Sil-

ver-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 µg PG monomer in the

starting material prior to digestion/electrophoresis.

62 Melrose

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 µg/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 shak-

ing (see Note 3).

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

M (4 × 5 min).

7. Appropriate secondary detection reagents are then added. For Affinity blots, avidin alka-

line phosphatase, 2 µg/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.

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 µg 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 µg hexuronic acid) was run in lanes 4

and 8 as an internal standard.

Cartilage and Smooth Muscle Cell Proteoglycans 63

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 Na

, pH 6.3, 60% w/v sucrose, and 0.01% w/v bro-

mophenol blue (CAPAGE application buffer).

2. CAPAGE slab gels (0.15 × 14 × 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 electrophore-

sis 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 × 5.5 × 1.5 mm) are electroblotted to nitrocellulose mem-

branes using buffer J in a Bio-Rad semidry blotter at 5.5 mA/cm

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 ε-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 µg 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-over-

end 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.

64 Melrose

4. After 2 h at room temperature there is no further rise in pH; the reaction mixture is there-

fore adjusted to pH 7 with 1N NaOH and exhaustively dialyzed against distilled water

(4 × 5l × 18h). Then the hydrazido-hyaluronan is freeze-dried.

5. Hydrazido-hyaluronan (100 mg) is redissolved overnight in 20 mL 0.2 M NaHCO

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 µmol 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 Hybri-

doma 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 Corpora-

tion, 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 sepa-

rate 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, Univer-

sity 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.