Iozzo Renato V. Proteoglycan Protocols

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288 Lara et al.

45. Sodium azide is an inhibitor of peroxidase activity and should not be included in buffers

used to make the peroxidase substrate or the ABC reagent.

46. Tissue may be harvested after perfusion fixation with 3% paraformaldehyde if desired

and then continue fixation in 3%/0.25%.

47. Never allow the section surface to dry between droplet changes.

48. Include appropriate buffer/no-primary, irrelevant antibody, and positive and negative tissue

controls.

49. Extreme care should be used in handling coated grids, as an intact support film limits

difficulties later with contamination and section deformation. The use of clean, well-aligned

anticapillary forceps to pick up the grids on the rimmed edge will minimize mechanical

damage to the sections and support film.

50. The carbon coat will not only strengthen the coat, but will render this “reverse” side somewhat

hydrophobic, facilitating the subsequent restriction of reagents to the section side of the grid.

51. It is sometimes helpful for later interpretation to collect sections consecutively.

52. Times can vary with block hardness and section thickness, and can be predetermined with

test grids examined in the electron microscope for a loss of defined section edge.

53. Ideally, the carbon side of the grid remains dry during the entire immunostaining process. If

the grid sinks at some point, it is best to proceed through each droplet immersed.

54. Antibody dilution is best determined by bracketing the dilution that gave the best light level

localization.

References

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19. Underhill, C., Nguyen, H., Shizari, M., and Culty, M., (1993) CD44 positive macrophages

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hyaluronan during extracellular remodeling in human restenotic arteries and balloon-injured

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distribution in lesions of atherosclerosis depends on lesion severity, structural characteristics

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versican-rich pericellular matrix is required for proliferation and migration of vascular smooth

muscle cells. Arterioscler. Thromb. Vasc. Biol. 19, 1004–1013.

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electron microscopy and mechanism of action. Anat. Rec. 171, 347–368.

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cellular matrix of human colon carcinoma: an integrated biochemical and stereological

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27. Snow, A. D., Bolender, R. P., Wight, T. N., and Clowes, A. W. (1989) Heparin modulates

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tion of tools and experiments for organ, tissue, cell and molecular biology. Am. J. Physiol.

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arterial smooth muscle cell cultures using cationic dyes. J. Histochem. Cytochem. 32,

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(1988) Arterial chondroitin sulfate proteoglycan: localization with a monoclonal antibody.

J. Histochem. Cytochem. 36, 1211–1221.

32. Gutierrez, P., O’Brien, K. D., Ferguson, M., Nikkari, S., Alpers, C. E., and Wight, T. N.

(1997) Differences in the distribution of versican, decorin, and biglycan in atherosclerotic

human coronary arteries. Cardiovasc. Pathol. 6, 271–278.

33. O’Brien, K. D., Alpers, C. E., Chiu, W., Hudkins, K., Wight, T. N. ,and Chait, A. (1998) A

comparison of apolipoprotein and proteoglycan deposits in human coronary atherosclerotic

plaques: colocalization of apolipoprotein E and biglycan. Circulation 98, 519–527.

34. Mar, H., Tsukada, T., Gown, A. M., Wight, T. N., and Baskin, D. G. (1987) Correlative

light and electron microscopic immunocytochemistry on the same section with colloidal

gold. J. Histochem. Cytochem. 35, 419–425.

35. LR White product instructions.

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Expression and Characterization of Engineered PGs 291

291

From:

Methods in Molecular Biology, Vol. 171: Proteoglycan Protocols

Edited by: R. V. Iozzo © Humana Press Inc., Totowa, NJ

Expression and Characterization

of Engineered Proteoglycans

Wei Luo, Chunxia Guo, Jing Zheng, and Marvin L. Tanzer

1. Introduction

Target proteoglycan gene(s) can be rearranged to fulfill special designs for

expression, purification, or characterization purposes. These are achieved through sev-

eral procedures, including polymerase chain reaction (PCR) amplification of a target

gene, cloning of PCR products into cloning vectors, subcloning of a correct gene frag-

ment into an expression vector, purification of target gene DNA via a large prepara-

tion, transfection into host cells for expression, purification of candidate proteins, and

identification of the proteins. Characterization can be done of core proteins or of the

glycosaminoglycan chains attached to the core proteins. A summary of the expression

of an engineered protien is shown in Fig. 1.

1.1. Construct Assembly of Target Proteoglycans

Gene structure arrangements are accomplished through PCR with specifically

designed oligonucleotides and target template(s) (1). Appropriate PCR products are

engineered into a suitable eukaryotic expression vector using appropriate polylinkers.

The vector containing target gene(s) can express (transiently) proteoglycans through

transfection into candidate eukaryotic cell lines (see Subheading 1.2.).

1.2. Expression of Target Proteoglycans

Large amounts of DNA with molecular-level purity are needed for transfection and

are prepared by a maxiprep kit (Qiagen). The maxiprep-prepared DNA is then used to

transfect eukaryotic cell lines to express/obtain the corresponding proteins. Cell lines

are chosen based on two major criteria: (1) the cell lines themselves do not express the

same target endogenous genes; (2) the cell lines have the capability (e.g., machinery to

add glycosaminoglycan chains) to express proteoglycans.

292 Luo et al.

1.3. Purification of Target Proteoglycans

Expressed products are purified using a nickel column with stringent washing pro-

cedures to eliminate background proteins. A histidine-6 (His6) tag is engineered in the

construct assembly (see Subheading 1.1.) to allow such a purification procedure.

1.4. Identification of Target Proteoglycans

The purified proteins can be run on sodium dodecyl sulfate-polyacrylamide gel elec-

trophoresis (SDS-PAGE) directly to separate the products from background proteins or

processed further for other experiments. Purified products can be characterized by vari-

ous procedures, e.g., quantitation by radioactivity, classification of polysacchrides by

enzymatic digests, and identification of core proteins by immunocapture.

1.5. Characterization of Expressed Proteoglycans

Proteoglycans have unique features that can be individually characterized. Chon-

droitin sulfate, keratan sulfate, dematan sulfate, and heparan sulfate can be digested

with appropriate enzymes to confirm their presence. The example protocol is for chon-

droitin sulfate digestion (see Subheading 3.5.).

1.6. Detection of Proteoglycan Interaction

with Heat-Shock Proteins Part I

Expressed proteoglycans may interact with certain types of heat shock proteins

(Hsp) (2). For example, aggrecan’s G3 domain interacts with Hsp25. The example pro-

tocol is for immunocapture of Hsp25 using anti-Hsp25 antibody (see Subheading 3.6.).

Fig 1. Summary diagram of expression of an engineered proteoglycan.

Expression and Characterization of Engineered PGs 293

1.7. Detection of Proteoglycan Interaction

with Heat-Shock Proteins Part II

Interaction between heat-shock proteins and proteoglycans can also be detected by in situ

cross-linking. This method is suitable only when the interacting candidate Hsp and

proteoglycan are adjacent to each other and they have suitable amino group(s) in proximity

that can be cross-linked. The procedure here uses 3,3'-dithiobis sulfosuccinimidylpropionate

(DTSSP) as a cross-linking reagent that requires lysines or an N-terminal amino group for

reaction. Other reagents may be used for other chemical groups.

2. Materials (

see

Note 1)

2.1. Construct Assembly of Target Proteoglycans

1. Target proteoglycan gene.

2. Specifically designed oligonucleotides with certain features, e.g., restriction sites, His6

tag, etc., as desired.

3. PCR-Script SK(+) cloning vector kit (Stratagene).

4. Escherichia coli (E. coli ) XL-blue MRF' Kan supercompetent cells.

5. X-gal (5-bromo-4-chloro-3-indolyl β-D-galactopyranoside), IPTG (isopropyl β-D-thio-

galactopyranoside), and N,N-dimethyl formamide (Sigma).

6. Restriction enzymes and matching buffers corresponding to the engineered restriction sites.

7. DNA purification system Minipreps (Wizard Plus from Promega).

8. LB broth base (Life Technology).

9. Eukaryotic expression vector (e.g., pcDNA3 from Invitrogen).

10. T4 DNA ligase (Invitrogen).

11. E. coli TOP10F' competent cells (Invitrogen).

12. Ampicillin-containing agar plates (only if ampicillin is the selecting reagent).

13. QiaFilter DNA purification systems (for Maxipreps from Qiagen).

14. Sequencing oligonucleotides (pairing to expression vector or cloning vector or templates).

15. Agarose (Life Technology).

16. NanoSep 0.2-µm DNA filters (Fisher Scientific/Pall Gelman).

17. 125-mL glass flask.

18. 15-mL Falcon tube.

19. DNA sequence verification software (optional).

2.2. Expression of Target Proteoglycans

1. Eukaryotic cell line(s) (e.g., Chinese Hamster Ovary K1 cells, COS cells).

2. Lipofectamine + PLUS reagent (Life Technology).

3. Regular minimal essential culture medium (MEM), regular OPTI-MEM (Life Technology),

and methionine-free and cysteine-free OPTI-MEM (Life Technology, special order).

4. Fetal bovine serum (certified grade).

5. Antibiotic–antimycotic solution (containing100 U/mL penicillin, 100 µg/mL streptomy-

cin, 250 ng/mL amphotericin, Life Technology).

6. Radioactive isotopes

S-Pro-Mix (methionine and cysteine, Amersham, 7.15 mCi, for

4–5 plates of cells) for methionine + cysteine labeling (labeling core proteins) or

S-Na

(sodium sulfate, Dupont NEN, 5 mCi, for 4–5 plates of cells) for sulfate

labeling (labeling sulfated glycosaminoglycan chains).

7. Decolorizing NORIT-A carbon (ACROS Organics).

8. Sterilized 9-in. glass pipets (Fisher Scientific).

294 Luo et al.

9. 10-mL individually wrapped plastic pipets (sterilized).

10. Cell culture dishes (100 mm).

11. Eppendorf tubes (regular and boiling tubes).

12. Table-top centrifuge (up to 14,000g speed).

13. Nonidet P-40 detergent.

14. Phenylmethylsulfonyl fluoride (PMSF, see Subheading 3.2.3.), leupeptin, antipain,

benzamidine, aprotinin, chymostatin, pepstatin, dimethyl sulfoxide (DMSO) (Sigma).

15. Hanks’ Balanced Salt Medium (Life Technology).

2.3. Purification of Target Proteoglycans

1. Ni NTA agarose (Ni resin, Qiagen).

2. Equilibration buffer: 0.1 M NaCl, 44 mM NaHCO3, 1 mM imidazole, 0.5% Triton X-100 in H

3. Met-free and Cys-free OPTI-MEM medium.

4. 10% Triton X-100 solution.

5. pH indicator paper.

6. Stringent wash buffer A:10 mM HEPES, 2 M NaCl, 10% glycerol, 0.1 mM PMSF, 2 mM

imidazole, 0.5% Triton X-100, 6 M urea, 250 mM dithiothreitol in H

O, pH 8.0.

7. Stringent wash buffer B: 10 mM HEPES, 750 mM NaCl, 10% glycerol, 0.1 mM PMSF,

10 mM imidazole, 0.5% Triton X-100, 1 mg/mL bovine serum albumin in H

O, pH 8.0.

8. Stringent wash buffer C: 10 mM HEPES, 750 mM NaCl, 10% glycerol, 0.1 mM PMSF,

10 mM imidazole in H

O, pH 8.0.

9. Elution buffer: 10 mM HEPES, 750 mM NaCl,10% glycerol, 100 mM EDTA, 0.1 mM

PMSF, 250 mM imidazole in H

O, pH 8.0.

10. Roto Torque heavy–duty rotator (Cole Parmer Instruments).

2.4. Identification of Target Proteoglycans

1. 5–15% linear gradient SDS-containing acrylamide gel (can be self-prepared or purchased

from manufacturer).

2. Dithiothreitol (DTT), final concentration 38 mg/mL in loading buffer.

3. Gyrotory shaker (New Brunswick Scientific).

4. Gel fixing solution: 25% Methanol, 10% acetic acid in H

5. Square Petri dish (100 × 100 × 15 mm, Nalge Nunc #4021).

6. Entensify Universal Autoradiography Enhancer Part A and Part B (Dupont).

7. Radiogram cassette and X-ray film (Kodak, Bio-Max films are best).

2.5. Characterization of Expressed Proteoglycans

1. Chondroitinase ABC (Seikagagu) (dissolved in buffer conditions recommended by the

manufacturer).

2. 20× Chondroitinase ABC digest buffer: 2 M Tris-HCl, 600 mM sodium acetate, pH 8.0

(final conditions = 100 mM Tris-HCl, 30 mM sodium acetate, pH 8.0).

3. 10% Triton X-100 solution.

4. 100 mM N-ethylmaleimide (NEM) solution.

2.6. Detection of Proteoglycan Interaction

with Heat-Shock Proteins Part I

1. Hsp25 antibody (Stressgen).

2. Protein A beads (Pierce).

3. Washing buffer D: 50 mM Tris-HCl, 1 M NaCl, 1% Triton X-100, pH 7.5.

Expression and Characterization of Engineered PGs 295

2.7. Detection of Proteoglycan Interaction

with Heat-Shock Proteins Part II

1. DTSSP (Pierce).

2. Washing buffer E: 130 mM NaCl, 20 mM Bicine, pH 8.0, ice cold.

3. Digitonin lysis buffer: 50 mM Bicine, pH 8.0, 150 mM NaCl, 5 mM KCl, 5 mM MgCl

0.2% digitonin, 1.5 mM DTSSP, ice cold.

4. 10 mM glycine solution, ice cold.

3. Methods

3.1. Construct Assembly of Target Proteoglycans

1. The target sequence was amplified by PCR using an upstream oligonucleotide (oligo 1)

and a downstream oligonucleotide (oligo 2). Oligo 1 should contain a specific restriction

site, which corresponds to one of the sites in the polylinker region in the eukaryotic

expression vector. Oligo 2 should contain a specific restriction site, a His6 sequence

(engineered for purification purposes), and a stop codon. The restriction site in oligo 2

can be the same as in oligo 1 or can be different, but it must correspond to a site in the

polylinker region in the eukaryotic expression vector in correct orientation. Usually it is

easier to have two different restriction sites so that you don’t have to verify the orienta-

tion of the sequence after its insertion (see step 22).

2. The PCR reaction is usually a Hot-Start program (see Note 2). A portion of the PCR

products (usually 5 µL from a 50-µL PCR reaction) is denatured and electrophoresed in

an ethidium bromide agarose gel to verify the size of the products.

3. Correct PCR products (the right size) are digested with SrfI and ligated to predigested

pCR-Script SK(+) vector (Stratagene), which is then used to transform E. coli XL1-blue

MRF' Kan supercompetent cells (following the manufacturer’s manual).

4. Prepare LB broth following the manufacturer’s instructions.

5. Dissolve X-gal in N,N-dimethyl formamide (20 mg/mL) and IPTG in water (50 mg/mL).

6. Place transformed products on an ampicillin-containing agar plate (containing X-gal and

IPTG) and incubate at 37°C for 16 h. Blue and white colonies should appear on the plate.

7. Select white E. coli colonies, inoculate one colony into a 15-mL Falcon tube containing

3 mL of LB broth supplemented with ampicillin (50 ng/mL final concentration using

50 mg/mL 1000× stock), shake at 250 rpm at 37°C for 16 h (overnight).

8. Purify miniprep DNA using Wizard Plus Minipreps (Promega) following the manu-

facturer’s instructions.

9. Digest purified DNA (5 µL from 50 µL) with appropriate restriction enzymes at 37°C

for 1–2 h.

10. Electrophorese digested DNA in a 0.8–1% agarose gel to detect and select suitable DNA

fragments, affirming the correct size of DNA fragments.

11. Digest the DNA fragments (16 µL) again with the same restriction enzymes as in step 8.

12. Digest the pcDNA3 vector (Invitrogen) with the same restriction enzymes.

13. Electrophorese (10) and (11) in a 0.8–1% agarose gel.

14. Cut out the agarose bands containing the correct DNA fragments.

15. Purify the DNA using NanoSep filters following the manufacturer’s instructions.

16. Ligate purified DNA fragments and pcDNA3 vector using Invitrogen T4 DNA ligase

following the manufacturer’s instructions.

17. Transform E. coli TOP10F' supercompetent cells using (16) following the manufacturer’s

instructions.

296 Luo et al.

18. Repeat steps 7–10 to verify DNA inserts. Store bacteria-containing liquid broth in an

80/20 ratio glycerol at –80°C.

19. Strip bacteria onto an ampicillin-containing agar plate from glycerol stock and incubate

overnight at 37°C for 16 h (overnight).

20. Inoculate one colony into a 125-mL glass flask containing 50 mL of LB broth supple-

mented with ampicillin (50 ng/mL final concentration) and shake at 250 rpm on a bacte-

rial incubation shaker at 37°C for 16 h (overnight).

21. Prepare a maxiprep using QiaFilter DNA purification systems (Qiagen) following the

manufacturer’s instructions.

22. Quantify the DNA concentration and sequence the DNA to verify the correct

sequence. The sequence must be 100% identical to the original design at the amino

acid level.

23. The verified DNA is ready for the next step (transfection).

3.2. Expression of Target Proteoglycans

3.2.1. Cell Culture

1. (All procedures are performed in sterile-hood cell culture conditions.) Prepare MEM

according to the manufacturer’s instructions (supplemented with 5–10% fetal bovine

serum and 100 U/mL penicillin, 100 µg/mL streptomycin, and 250 ng/mL amphotericin,

filtered).

2. Maintain eukaryotic cells (e.g., CHO) at 37

C in humidified air with 5% CO

3. Seed 1.8 million cells per 100-mm dish in MEM medium 2 d before transfection. Cells

should be 70–80% confluent when transfected.

3.2.2. Transfection Procedure

1. All procedures are performed in sterile-hood cell culture conditions. Mix 9 µg DNA (in

less than 20 µL volume), 460 µL OPTI-MEM, and 30 µL PLUS reagent and invert 5× to

mix well (refer to Life Technology protocol).

2. Incubate at room temperature (RT) for 15 min.

3. Mix 30 µL Lipofectamine and 470 µL OPTI-MEM and invert 5× to mix well.

4. Combine (2) + (3), invert 10×, incubate at RT for 15 min (see Note 3).

5. Aspirate MEM from the cell culture (regular culture medium).

6. Wash cells with 10 mL of regular OPTI-MEM, incubate at 37°C for 20 min and aspirate.

7. Wash cells with 5 mL of regular OPTI-MEM and aspirate.

8. Add 4 mL of regular OPTI-MEM.

9. Add (4) onto cells in a plate-tilted position and swirl medium gently until well mixed.

10. Incubate at 37°C for 5 h. Do not agitate cells when handling dishes.

11. Place twenty 100-mm dishes containing 2 tablespoons of decolorizing NORIT-A carbon

each into another incubator (same conditions as Subheading 3.2.1., step 2) designated

for radioactive isotope usage. Carbon is placed to absorb free radioactive materials

released by metabolizing cells in the incubator.

12. Aspirate medium from (10).

13. Add 5 mL of Met- and Cys-free OPTI-MEM.

14. Add 100 µL of

S-Pro-Mix labeling mix to each dish (about 1.40–1.80 mCi/dish).

15. Incubate at 37°C for 16 h in the incubator supplemented with decolorizing NORIT-A

carbon (step 11). The cells and media are ready for collection.

(The above protocol is for CHO cells.)

Expression and Characterization of Engineered PGs 297

3.2.3. Sample Collection

1. Dissolve 0.5 g of Nonidet P-40 in 100 mL of dH

O to prepare 0.5% Nonidet lysis buffer,

which can be stored at 4°C.

2. Protease inhibitor I: 2 mg/mL leupeptin, 0.4 mg/mL antipain, 2 mg/mL benzamidine, and

2 mg/mL aprotinin dissolved in dH

3. Protease inhibitor II: 1 mg/mL chymostatin and 1 mg/mL pepstatin dissolved in DMSO.

4. 250 mM PMSF: 250 mM PMSF dissolved in 100% ethanol.

5. Prepare 10 mL of Nonidet P-40 lysis buffer (from step 1) with 10 µL of protease inhibitor

I, 10 µL of protease inhibitor II, and 20 µL of 250 mM PMSF.

6. Add 20 µL of 250 mM PMSF, 5 µL of protease inhibitor I, and 5 µL of protease inhibitor

II to a 15-mL tube.

7. Transfer medium (from Subheading 3.2.2., step 15) to the tube.

8. Spin tube for 60s at 925g (1000 rpm) in cell culture centrifuge to eliminate dead cells.

9. Transfer medium (see step 8) to a new 15-mL tube, store at –20°C. Do not disturb the

pellet at the bottom of the tube. The medium samples are ready for purification.

10. Simultaneously, wash cells with 5 mL of Hanks’ medium/dish and discard medium as

radioactive Waste.

11. Add 1 mL of Nonidet P-40 lysis buffer (see step 5) to each dish. Swirl buffer to wet the

entire surface.

12. Completely scrape the cells from the dish and transfer to a 1.5-mL Eppendorf tube; tilt the

dish to decant the cell lysate liquid.

13. Incubate on ice for 10 min and then spin at 14,000g (table-top centrifuge) for 5 min.

14. Transfer supernatant to a new tube, and store the supernatant and debris separately at

–20°C. The supernatant is ready for purification.

3.3. Purification of Target Proteoglycans

All procedures are to be done with extreme care because of the high energy of

radioactive isotope in use.

3.3.1. Ni Resin Equilibration

1. Transfer 100 µL of dispersed resin beads (well-mixed beads with buffer) to a 1.5-mL

Eppendorf tube.

2. Spin at 14,000g for 15 s, then remove liquid.

3. Add 500 µL of equilibration buffer to beads.

4. Place the tube on a Rotator and rotate at RT for 30 min (low speed at 6.5).

5. Spin at 14,000g for 15 s and remove liquid. The resin is ready for sample loading.

3.3.2. Ni Resin Loading

1. Medium loading: 900 µL of medium (from Subheading 3.2.3., step 9) and 47.5 µL of

10% Triton X-100 for final 0.5% Triton-X concentration; adjust pH to 8.0 (usually it

should be pH 7.5–8.0 by itself after collection, using pH paper to monitor).

2. Lysate loading: 700 µL of Met-free medium, 200 µL of lysate (from Subheading 3.2.3.,

step 14), and 47.5 µL of 10% Triton X-100; adjust pH to 8.0.

3. Rotate for 1 h at RT.

4. Spin at 14,000g for 15 s, then transfer supernatant to waste.

3.3.3. Stringent Washes

1. Wash 1: Add 1000 µL of stringent wash buffer A (see Note 4) to each pellet and rotate at

RT for 30 min (notice that color changes from clear to brown at RT).