Iozzo Renato V. Proteoglycan Protocols

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Hyaluronan and Its Binding Protein 479

479

From:

Methods in Molecular Biology, Vol. 171: Proteoglycan Protocols

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

Hyaluronan and Hyaluronan-Binding Proteins

Probes for Specific Detection

Bryan P. Toole, Qin Yu, and Charles B. Underhill

1. Introduction

Hyaluronan is a uniformly repetitive, linear glycosaminoglycan (GAG) composed

of disaccharides of glucuronic acid and N-acetylglucosamine: [-β(1,4)-GlcUA-β(1,3)-

GlcNAc-]

. The polymer usually consists of 2,000–25,000 disaccharides, giving rise

to molecular weights ranging from 10

to 10

dalton. Hyaluronan has unusual physi-

cal and biochemical properties, and fulfills several distinct physiological functions

that contribute both to structural properties of tissues and to cell behavior during

formation or remodeling of tissues (1–4). First, hyaluronan contributes directly to tis-

sue homeostasis and biomechanics due to its unique charge characteristics and bio-

physical properties. Second, interactions of hyaluronan with link proteins and

proteoglycans are of fundamental importance to the structural integrity of extracellu-

lar and pericellular matrices. Third, interactions with cell-surface hyaluronan recep-

tors mediate significant influences on cell behavior during morphogenesis, tissue

remodeling, inflammation, and diseases such as cancer and atherosclerosis (1–4).

The potential importance of hyaluronan in development, tissue remodeling and dis-

ease has led to the need for specific detection methods. However, until the mid-1980s

the only method available for detection of hyaluronan was indirect, that is, by using

specific hyaluronidases together with nonspecific staining methods. Subsequently,

specific hyaluronan-binding proteins were adapted for use as morphological probes

(5–7). The most commonly employed probe has been labeled hyaluronan-binding

polypeptides prepared from the cartilage proteoglycan complex of aggrecan, link pro-

tein, and hyaluronan (5,8–10). This method has also been applied to electron

microscopy (11). However, the most widespread use of this preparation has been for

localizing hyaluronan in tissue sections by light microscopy, as described herein.

480 Toole et al.

Many functions of hyaluronan are mediated by hyaluronan-binding proteins (2–4).

The identification and molecular characterization of hyaluronan-binding proteins has

been facilitated by the use of biotinylated hyaluronan (bHA) as a probe in transfer

blots. For example this method has been used to demonstrate the hyaluronan-binding

ability and specificity, and for dissecting the binding domains, of several hyaluronan-

binding proteins (12–15). Although this method can also be adapted to morphologi-

cal localization (16), its major use has been biochemical; this latter application is

described herein.

Binding of hyaluronan to the surface of cells is mediated by hyaluronan receptors,

the major receptor being CD44 (2–4). However the presence of CD44 on the cell sur-

face does not necessarily indicate that a given cell will bind hyaluronan, since many

factors contribute to binding—for example, glycosylation, alternative splicing, and

clustering within the cell membrane (17,18). Consequently, convenient and reliable

means of measuring hyaluronan binding to cells have been developed, the most

common of which involves the use of fluorescein-tagged hyaluronan followed by

analysis by flow cytometry; this application is also described herein.

2. Materials

2.1. Preparation of Biotinylated Hyaluronan-Binding Protein (bHABP)

1. Bovine nasal cartilage (Pel-Freez, Rogers, AR).

2. Surform pocket plane (Stanley Tools); cheesecloth (prewashed); Whatman no. 1 filter paper

(Whatman, Clinton, NJ).

3. 4.0 M guanidium HCl, 0.5 M Na acetate, pH 5.8.

4. HEPES buffer: 0.1 M HEPES containing 0.1 M Na acetate, pH 7.3.

5. Trypsin (type III; Sigma, St. Loius, MO).

6. Soybean trypsin inhibitor (Type I-S; Sigma).

7. Sulfo-NHS-LC-Biotin (EZ-Link; Pierce, Rockford, IL).

8. Hyaluronan-Sepharose (19,20): hyaluronan (100 mg; Seikagaku, Falmouth, MA) is par-

tially digested with testicular hyaluronidase (2 mg [type VIII, Sigma], in 30 mL of 0.05 M

Na acetate/0.15 M NaCl, pH 5.0, at room temperature for 3 h), boiled for 10 min, and then

mixed with 10 mL of EAH-Sepharose 4B (Pharmacia Biotech, Uppsala, Sweden) and 250

mg of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide HCl (Sigma). The mixture is

adjusted to pH 5 with 0.1 M HCl and incubated at room temperature for 24 h, after which

2 mL of acetic acid is added and the mixture is reincubated for 6 h. The gel is then washed

with 1 M NaCl, 0.05 M formate buffer (pH 3), and distilled water.

2.2. Localization of Hyaluronan in Tissue Sections

1. 4.0% formaldehyde, dissolved in PBS-A.

2. PBS-A: calcium and magnesium-free phosphate-buffered saline.

3. 3% hydrogen peroxide, dissolved in methanol.

3. 2 µg/mL bHABP, dissolved in 10% calf serum in PBS-A (filtered through 0.45-µm filter

before use).

5. Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA).

6. 5 U/mL Streptomyces (Calbiochem, San Diego, CA), dissolved in 10% calf serum in PBS-A.

7. 200 µg/mL hyaluronan (Seikagaku) in 10% calf serum in PBS-A; stock solution: 5 mg/

mL in distilled water; store at –20°C.

Hyaluronan and Its Binding Protein 481

8. 1 mg/mL hyaluronan oligomers in 10% calf serum in PBS-A. For preparation of

hyaluronan oligomers (20), hyaluronan is first digested with testicular hyaluronidase

(2 mg [type VIII, Sigma], in 30 mL of 0.05 M Na acetate/0.15 M NaCl, pH 5.0, at 37°C

for 3–24 h). The digest is boiled for 10 min, then passed over a column of Sephadex G-50

(Pharmacia; 1.5 × 250 cm) in ammonium acetate buffer, pH 5.0. Fractions are assayed for

total uronic acid (21) and terminal hexosamine (22), and those fractions with uronic acid

to hexosamine ratios of 5–10 are pooled.

2.3. Preparation of Biotinylated Hyaluronan (bHA)

1. 5 mg/mL hyaluronan (Healon, Pharmacia; or purest preparation from Seikagaku) in PBS-A.

2. MES buffer: 0.1 M 2-N-morpholino ethanesulfonic acid (Sigma), pH 5.5.

3. 50 mM ImmunoPure biotin-LC-hydrazide (Pierce), freshly dissolved in dimethyl sulfoxide.

4. EDC buffer: 100 mg/mL 1,ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride

(Pierce) in 0.1 M MES buffer, pH 5.5.

2.4. Detection of Hyaluronan-Binding Proteins in Blots

1. Routine supplies for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

2. Hybond-ECL nitrocellulose membranes (Amersham, Arlington Heights, IL).

3. Transfer buffer: 0.025 mM Tris, 0.19 M glycine, 10% methanol, 0.005% SDS, pH 8.3.

4. TTBS: 0.2% Tween-20, 0.05 M Tris-HCl, 0.15 M NaCl pH 8.0.

5. Blocking buffer: 20% calf serum (or 1% BSA, 1% nonfat milk) in TTBS.

6. TBS: 0.05 M Tris-HCl, 0.15 M NaCl, pH 8.0.

7. 10% hydrogen peroxide in TBS.

8. 10 µg/mL bHA in TTBS.

9. 200 µg/mL hyaluronan (Seikagaku) in TTBS; stock solution: 5 mg/mL in distilled water;

store at –20°C.

10. 1 mg/mL hyaluronan oligomers in TTBS (see Subheading 2.2.).

11. 1/1000 dilution of Streptavidin-horseradish peroxidase (Amersham) in TTBS.

12. ECL Western blot detection reagents and Hyperfilm-ECL (Amersham).

2.5. Preparation of Fluorescein-Labeled Hyaluronan (Fl-HA)

1. 5 mg/mL hyaluronan (Healon, Pharmacia; or purest preparation from Seikagaku) in water.

2. 3.8 mg/mL fluoresceinamine (Aldrich, Milwaukee, WI) in methanol.

3. 46 mM cyclohexyl isocyanide (Aldrich) in methanol.

4. 250 mM acetaldehyde (Aldrich) in water.

3. Methods

3.1. Specific Localization of Hyaluronan in Tissue Sections (8)

3.1.1. Preparation of Biotinylated Hyaluronan-Binding Protein (bHABP)

(see Note 1)

1. Shred bovine nasal cartilage with Surform pocket plane (or grind under liquid nitrogen in

Wiley mill); mix cartilage with 10 volumes of 4.0 M guanidium HCl, 0.5 M Na acetate,

pH 5.8, and shake overnight at 4°C.

2. Pour mixture through several layers of cheesecloth, then centrifuge at 10,000g, 45 min,

4°C; filter supernatant through filter paper on Buchner funnel; dialyze supernatant thor-

oughly against running tap water followed by distilled water; lyophilize for storage.

3. Dissolve 2.0 g of lyophilized cartilage by stirring in 50 mL of 0.1 M HEPES buffer at 4°C

overnight (or add appropriate amount of solid HEPES and Na acetate to solution if

482 Toole et al.

lyophilization is omitted); add 1.0 mg of trypsin and incubate for 2 h at 37°C; add 1.0 mg

of soybean trypsin inhibitor.

4. Adjust pH to 8.0 with NaOH; add 0.1 mg of sulfo-NHS-LC-biotin per milligram protein

and incubate for 1–2 h at room temperature.

5. Dialyze preparation against 4.0 M guanidium HCl, 0.5 M Na acetate, pH 5.8; mix with 100

mL of hyaluronan-Sepharose (see Subheading 2.1.) equilibrated with the same buffer; dia-

lyze mixture against 9 volumes of distilled water at 4°C overnight on a rotary shaker table.

6. Wash hyaluronan-Sepharose thoroughly with 3.0 M NaCl, then elute bHABP with 4.0 M

guanidine HCl, 0.5 M Na acetate, pH 5.8; dialyze preparation against 0.15 M NaCl.

7. Aliquot and store at -20°C in 50/50 glycerol and 0.15 M NaCl. (See Note 2).

3.1.2. Localization of Hyaluronan in Tissue Sections

1. Fix tissue in 4.0% formaldehyde in PBS-A for 2 h at room temperature; wash twice with

PBS-A; dehydrate through 30%, 70%, 95%, and 100% ethanol, then xylene; embed in

paraffin (see Note 3).

2. Cut sections at 8–10 µm; incubate sections with 3% hydrogen peroxide in methanol for 1

h to inactivate endogenous peroxidases; rinse twice with distilled water; equilibrate in

PBS-A for 5 min.

3. Incubate sections for 1 h with 2 µg/mL bHABP dissolved in PBS-A containing 10% calf

serum. Wash sections 5 times for 1 min each in PBS-A.

4. Use the Vectastain Elite ABC kit for detection of the bHABP.

5. As controls for specificity, pretreat sections with 5 U/mL Streptomyces hyaluronidase in

PBS-A at 37

C for 30 min; or pretreat with 200 µg/mL hyaluronan or 1 mg/mL hyaluronan

oligomers (see Note 4) for 30 min at room temperature, in PBS-A containing 10% calf

serum, then incubate with 4 µg/mL bHABP, together with competing agent, as above.

3.2. Specific Detection of Hyaluronan-Binding Proteins

in Transfer Blots (16)

3.2.1. Preparation of Biotinylated Hyaluronan (bHA)

1. Dissolve hyaluronan in PBS-A at 5 mg/mL, and dialyze against 0.1 M MES buffer over-

night at 4

C (see Note 5).

2. Add 50 mM biotin-LC-hydrazide, freshly dissolved in dimethyl sulfoxide (DMSO), to

give a final concentration of 1 mM (see Note 6).

3. Add freshly prepared EDC buffer to give a final concentration of 10 mM EDC and stir

overnight at room temperature.

4. Dialyze against PBS-A at 4

C for 2 days.

5. Remove any precipitate that forms during the reaction by centrifugation. Aliquot and store

at –20°C for no more than 6 months (it is preferable to make the bHA preparations in small

batches to avoid extended storage).

3.2.2. Detection of Hyaluronan-Binding Proteins in Blots

1. Separate proteins (10–20 µg per lane for crude extracts; 0.1–0.5 µg per lane for purified

proteins) by 10% SDS-PAGE under reducing (0.1 M DTT) or nonreducing conditions.

2. Transfer proteins to Hybond-ECL nitrocellulose membranes in transfer buffer at 300 mA

(constant current) for 1.5 h at 4

C. After transfer, wash the membranes with TTBS, then

block with 20% calf serum (or 1% BSA, 1% nonfat milk) in TTBS at 37

C for 1 h, and

wash 3 more times with TTBS. If interference from endogenous peroxidase activity

occurs, treat the membranes with 10% hydrogen peroxide in TBS at room temperature for

15 min and then wash twice with TBS, before blocking with 20% calf serum in TTBS.

Hyaluronan and Its Binding Protein 483

3. Incubate the membranes with 10 µg/mL bHA in TTBS at room temperature for 2 h. To

establish specificity, preincubate parallel membranes (or strips of membrane) with 200 µg/

mL unlabeled hyaluronan or 1 mg/mL hyaluronan oligomers (see Note 4) in TTBS at

room temperature for 1 h. The bHA is then added, together with unlabeled competing

reagent, and the mixture incubated for 2 h more.

4. Wash the membranes 3–4 times with TTBS for 5–10 min each, and treat with Streptavidin-

peroxidase conjugate in TTBS (1/1000 dilution) for 30 min at room temperature. Wash 3

times with TTBS for 5 min each, incubate the membranes with ECL Western blot detection

reagents for 1 min, and expose using Hyperfilm-ECL (see Note 7).

3.3. Measurement of Hyaluronan Binding to Cell Surface Receptors

by Flow Cytometry (23)

3.3.1. Preparation of Fluorescein-Labeled Hyaluronan

1. Dissolve 3.8 mg of fluoresceinamine in 1 mL of methanol; add dropwise to hyaluronan

solution (~50 mg in 10 mL of water) with constant stirring (see Note 5).

2. Add 1.5 mL of 46 mM cyclohexyl isocyanide in methanol and 0.5 mL of 250 mM acetal-

dehyde and stir for 1 h.

3. Add 3 volumes denatured alcohol, centrifuge, and wash precipitate several times with

alcohol; dry under vacuum (see Note 8).

3.3.2. Measurement of Hyaluronan Binding by Flow Cytometry

1. Detach cells from tissue culture plates with EDTA, wash with media and then with PBS-A.

2. Add 5–50 µg/mL fluorescein-labeled hyaluronan; incubate on ice for 2 h.

3. Wash 3 times with PBS-A; resuspend in PBS-A; analyze in FACScan (see ref. 24).

4. Controls for specific binding include pre-incubation with antibody to CD44, unlabeled

hyaluronan or hyaluronan oligomers (see Note 4).

4. Notes

1. The bHABP is prepared from the proteoglycan complex of cartilage, a ternary complex

containing proteoglycan (aggrecan), link protein, and hyaluronan. Aggrecan and link protein

contain specific hyaluronan-binding domains that bind the three components together. In the

method described, the complex is first extracted in 4 M guanidinium HCl, conditions that

dissociate the components of the complex. The complex is then reassociated by dialysis

against water and treated with trypsin. Trypsin treatment is performed in the associated state,

so as to protect the hyaluronan-binding sites in link protein and aggrecan. The resulting

complex, that is, link protein plus the hyaluronan-binding domain of aggrecan bound to

hyaluronan, is biotinylated while the hyaluronan-binding sites are still protected. The complex

is dissociated again in 4 M guanidinium HCl and then biotinylated link protein plus

hyaluronan-binding domain of aggrecan are purified by affinity chromatography on

hyaluronan-Sepharose. Purified bHABP is now available from Seikagaku.

2. SDS-PAGE analysis of the final product shows two bands, one at ~70–80 kDa (the

hyaluronan-binding domain of aggrecan) and one at ~43 kDa (link protein).

3. Other methods of processing and sectioning, (cryostat, plastic, etc.), may be used.

4. A higher concentration of oligomer than polymer is used for competition, since the former

has a lower affinity for most hyaluronan-binding proteins (25). Competition with chon-

droitin sulfate and heparin can also be used for comparison.

5. It is very important that pure hyaluronan be used for preparation of biotinylated and fluo-

rescein-labeled hyaluronan.

484 Toole et al.

6. We calculated the amount of biotin-LC-hydrazide to be used from the approximate molar

concentration of carboxyl groups in the HA preparation, such that a maximum of 1 out of

10–20 carboxyl groups in the HA would be labeled. Thus, 80–90% of the carboxyl groups

would remain unaltered. We have found that this degree of conjugation yields sufficient

labeling for sensitive detection while preserving full reactivity of the bHA.

7. If high background occurs, perform each step in TTBS containing 10% calf serum.

Sometimes biotin or streptavidin also causes nonspecific binding in the procedure. In

such cases the sample should be preabsorbed, before reaction with bHA, by mixing the

protein extract with biotin-agarose beads (Sigma; 3/1, vol/vol) for 20 min at room

temperature, followed by removal of the beads by centrifugation. The supernatant is

then mixed with Streptavidin-agarose beads (Sigma; 3/1, vol/vol) for 20 min, followed

by removal of these beads.

8. Approximately 7% of the carboxyl groups of hyaluronan are labeled with fluorescein under

these conditions.

References

1. Hascall, V. C. and Laurent, T. C. (1999) Hyaluronan: structure and physical properties.

Glycoforum: Science of hyaluronan. http://www.glycoforum.gr.jp

2. Knudson, W. and Knudson, C. B. (1999) The hyaluronan receptor, CD44. Glycoforum:

Science of hyaluronan. http://www.glycoforum.gr.jp

3. Sherman, L., Sleeman, J., Herrlich, P., and Ponta, H. (1994) Hyaluronate receptors: key

players in growth, differentiation, migration and tumor progression. Curr. Opin. Cell Biol.

6, 726–733.

4. Toole, B. P. (2000) Hyaluronan, in Proteoglycans: Structure, Biology, and Molecular

Interactions (Iozzo, R., ed.), Marcel Dekker, New York, NY, pp. 61–92.

5. Knudson, C. B. and Toole, B. P. (1985) Fluorescent morphological probe for hyaluronate.

J. Cell Biol. 100, 1753–1758.

6. Delpech, B., Bertrand, P., Maingonnat, C., Girard, N., and Chauzy, C. (1995) Enzyme-

linked hyaluronectin: a unique reagent for hyaluronan assay and tissue location and for

hyaluronidase activity detection. Anal. Biochem. 229, 35–41.

7. Fenderson, B. A., Stamenkovic, I. and Aruffo, A. (1993) Localization of hyaluronan

in mouse embryos during implantation, gastrulation and organogenesis. Differentia-

tion 54, 85–98.

8. Green, S. J., Tarone, G., and Underhill, C. B. (1988) Distribution of hyaluronate and

hyaluronate receptors in the adult lung. J. Cell Sci. 90, 145–156.

9. Alho, A. M. and Underhill, C. B. (1989) The hyaluronate receptor is preferentially expressed

on proliferating epithelial cells. J. Cell Biol. 108, 1557–1565.

10. Yu, Q. and Toole, B. P. (1997) Common pattern of CD44 isoforms is expressed in morpho-

genetically active epithelia. Dev. Dyn. 208, 1–10.

11. Ripellino, J. A., Klinger, M. M., Margolis, R.U., and Margolis, R.K. (1985) The hyaluronic

acid binding region as a specific probe for the localization of hyaluronic acid in tissue

sections. J. Histochem. Cytochem. 33, 1060–1066.

12. Yang, B., Yang, B. L., Savani, R. C., and Turley, E. A. (1994) Identification of a common

hyaluronan binding motif in the hyaluronan binding proteins RHAMM, CD44 and link

protein. EMBO J. 13, 286–296.

13. Grammatikakis, N., Grammatikakis, A., Yoneda, M., Yu, Q., Banerjee, S. D., and Toole,

B. P. (1995) A novel glycosaminoglycan-binding protein is the vertebrate homologue of

the cell cycle control protein, Cdc37. J. Biol. Chem. 270, 16198–16205.

Hyaluronan and Its Binding Protein 485

14. Yang, B., Yang, B. L., and Goetinck, P. F. (1995) Biotinylated hyaluronic acid as a probe

for identifying hyaluronic acid-binding proteins. Anal. Biochem. 228, 299–306.

15. Melrose, J., Little, C. B., and Ghosh, P. (1998) Detection of aggregatable proteoglycan

populations by affinity blotting using biotinylated hyaluronan. Anal. Biochem. 256,

149–157.

16. Yu, Q. and Toole, B. P. (1995) Biotinylated hyaluronan as a probe for detection of binding

proteins in cells and tissues. BioTechniques 19, 122–129.

17. English, N. M., Lesley, J. F., and Hyman, R. (1998) Site-specific de-N-glycosylation of

CD44 can activate hyaluronan binding, and CD44 activation states show distinct threshold

densities for hyaluronan binding. Cancer Res. 58, 3736–3742.

18. Kincade, P. W., Zheng, Z., Katoh, S., and Hanson, L. (1997) The importance of cellular

environment to function of the CD44 matrix receptor. Curr. Opin. Cell Biol. 9, 635–642.

19. Tengblad, A. (1979) Affinity chromatography on immobilized hyaluronate and its appli-

cation to the isolation of hyaluronate binding proteins from cartilage. Biochim. Biophys.

Acta 578, 281–289.

20. Banerjee, S. D. and Toole, B. P. (1991) Monoclonal antibody to chick embryo hyaluronan-

binding protein: changes in distribution of binding protein during early brain develop-

ment. Dev. Biol. 146, 186–197.

21. Bitter, T. and Muir, H. (1962) A modified uronic acid carbazole reaction. Anal. Biochem.

4, 330–334.

22. Reissig, J. L., Strominger, J. L., and Leloir, L. R. (1955) A modified colorimetric method

for the estimation of N-acetylamino sugars. J. Biol. Chem. 217, 959–966.

23. Zeng, C., Toole, B. P., Kiney, S. D., Kuo, J. W., and Stamenkovic, I. (1998) Inhibition of

tumor growth in vivo by hyaluronan oligomers. Int. J. Cancer 77, 396–401.

24. Lesley, J., Kincade, P. W. and Hyman, R. (1993) Antibody-induced activation of the

hyaluronan receptor function of CD44 requires multivalent binding by antibody. Eur. J.

Immunol. 23, 1902–1909.

25. Underhill, C. B., Chi-Rosso, G., and Toole, B. P. (1983). Effects of detergent solubiliza-

tion on the hyaluronate-binding protein from membranes of simian virus 40-transformed

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Carbohydrate–Protein Interactions 487

487

From:

Methods in Molecular Biology, Vol. 171: Proteoglycan Protocols

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

Novel Confocal-FRAP Analysis

of Carbohydrate–Protein Interactions

Within the Extracellular Matrix

Philip Gribbon, Boon C. Heng, and Timothy E. Hardingham

1. Introduction

Extracellular matrices (ECMs) contain a mixture of fibrillar and nonfibrillar mac-

romolecular components, which interact through a range of covalent and noncovalent

associations to form a composite structure (1–3). It is the ECM that defines the archi-

tecture, the form, and the biomechanical properties of many tissues. In order to per-

form their functional roles, many of the important noncollagenous components of

extracellular matrices are required to be immobilized or focally located within tissues

(4,5). Positioning of macromolecules within tissues occurs as a consequence of spe-

cific protein–protein and protein–carbohydrate interactions. Aggrecan, the large

aggregating proteoglycan, is noncovalently associated with hyaluronan via its N-ter-

minal domain and this association is further stabilised by link protein (6). A large

immobilized hydrated aggregate structure is formed, with up to 50 aggrecan molecules

per hyaluronan chain, which gives articular cartilage the ability to resist the high com-

pressive loads generated during joint articulation. Other examples of specific intermo-

lecular associations include the binding of cell surface integrins to fibronectins and

collagenous proteins (7) and growth factors to heparan sulphate (8). A variety of meth-

ods have been developed to investigate the affinity and specificity of these associa-

tions and their sensitivity to environmental factors such as pH and metal ion

concentration. Many techniques for analyzing binding equilibria or kinetics require

either the ligand or substrate to be linked to a solid support. As a consequence of

derivitization and high local concentrations, binding equilibria can be significantly

perturbed.

Confocal fluorescence recovery after photobleaching (confocal FRAP) offers a

method for investigating the interactions of molecules at high concentrations, the

formation of molecular networks, and the permeability of such networks to “reporter”

488 Gribbon et al.

molecules of different sizes. Additionally, confocal FRAP can be used to quantify

protein–protein binding and protein–carbohydrate binding under true equilibrium

conditions with a minimum of chemical modifications (9–13).

Experimentally, confocal FRAP involves viewing a solution of a fluorescently labeled

molecule with a confocal microscope, locally creating a bleached area in the solution,

followed by observing the subsequent redistribution of fluorescence (see Notes 1 and 2).

The bleaching can be achieved rapidly by brief high-power laser illumination. In simple

solutions of monodisperse macromolecules, the measurement of the recovery of fluores-

cence yields a long time translational lateral self-diffusion coefficient. This describes the

movement of the macromolecule through a matrix of similar macromolecules and can be

used to investigate self-association and molecule entanglement at high concentration (11).

The lateral tracer diffusion coefficient describes the movement of a macromolecule within

a matrix composed of a high concentration of another macromolecule. Self and tracer

diffusion coefficients are a function of the hydrodynamic friction caused by the solvent

and the hindrances, steric or otherwise, offered by other components in the solution. If a

macromolecule associates specifically with one or more components in a matrix, and its

diffusion coefficient is sufficiently changed in the process, then confocal FRAP can be

used to determine the affinity and specificity of the interaction (13).

The application of confocal FRAP to characterize the specific binding of a low-

molecular-weight protein to a high-molecular-weight ligand will be discussed in

detail here. The movement of a fluorescently labeled macromolecule A, diffusing

within a matrix of an unlabeled macromolecule B, will be considered. The transla-

tional diffusion coefficients of A, B, and the complex AB are D

, D

and D

, respec-

tively. The binding equilibrium between A and B is defined as

A + B ⇔ AB (1)

and at equilibrium, the dissociation constant (K

) is given by:

(2)

where [ ] represents concentration. In a confocal FRAP experiment only the fluores-

cence recovery of component A is measured. At equilibrium, A exchanges rapidly

between bound and free states that have characteristic lifetimes of τ

bound

and τ

free

respectively. If the fluorescence recovery of A is observed over time t, where t >>

(τ

bound

+ τ

free

), then the redistribution of A results from diffusion during multiple free

and bound phases. The measured diffusion coefficient (D

) can then be defined as

= [D

× 〈T

free

〉] + {D

[1 – 〈T

free

〉]} (3)

where 〈T

free

〉 is the fraction of time t that macromolecule A was free during time t.

Since t >> (t

bound

+ t

free

), then 〈T

free

〉 ⯝ A

free

, the fraction of free A at equilibrium.

Similarly, if D

>> D

, then D

⯝ D

and Eq. 3 can be re-expressed as

(4)

The expression for the inverse Scatchard plot is then

[A][B]

[AB]

free

– D

Carbohydrate–Protein Interactions 489

(5)

where [B]

is the concentration of binding sites on B, and n is the number of molecules

of A bound per molecule of B. Values for K

are determined by measuring D

as a

function of the concentration of substrate B. Magnitudes of D

and D

are determined

independently.

Confocal FRAP can be used to characterise competitive binding. Consider an equi-

librium between A and B, in the presence of a competitor, C:

A + B + C ⇔ AB + AC + BC (6)

This equilibrium can be analyzed by confocal FRAP if (1) the translational diffusion

coefficient of C, (D

), is very different from that of B, and (2) binding between B and

C is minimal, that is, [BC] = 0.

Confocal FRAP has recently been used to analyze the association between the G1

domain of aggrecan and hyaluronan. This interaction is ideally suited to analysis by

this technique because (1) the interaction is noncovalent, (2) the diffusion coeffi-

cients of G1 and HA differ considerably, and (3) the diffusion coefficient of the G1-HA

complex is similar to that of hyaluronan (HA) (14). The G1 domain of aggrecan was

labeled with fluorescein isothiocyanate (FITC) at 2.5 mol FITC per mole of G1 (see

Subheading 3.1.1.) and its diffusion coefficient in increasing concentrations of

hyaluronan (800 kDa) at pH 7.4 was determined (see Fig. 1). The diffusion coefficient

of the FITC-G1 was reduced with increasing hyaluronan concentration. Tracer diffu-

sion studies on nonbinding FITC-G1, which had been reduced and alkylated, showed

the reduction in FITC-G1 mobility was not due to the steric effect of the hyaluronan

(14). The self-diffusion of fluoresceinamine (FA)-labeled hyaluronan (800 kDa) was

then determined independently by conventional confocal FRAP. An inverse Scatchard

plot was constructed from the FITC-G1-binding isotherm (see Fig. 2). This gave K

4 × 10

-8

M, and n = 1, (see Fig. 2) which agrees well with dissociation constants

determined by solid-phase-type assays (15,16).

The confocal FRAP technique was used to determine the minimum length of

hyaluronan oligosaccharide required for binding to a single G1 domain. The low

molecular weight of the competitor oligosaccharides compared to the FITC-G1 and

the high-molecular-weight hyaluronan makes this an ideal system for analysis by

confocal FRAP. A mixture of FITC-G1 (5.5 µM) and 800-kDa hyaluronan (12.5 mM

of HA

binding sites) was prepared and >99% of the FITC-G1 was found to be bound

to the hyaluronan. Hyaluronan oligosaccharides (21 mM) of length 4–14 monosaccha-

rides were added (17), and the diffusion coefficient of the FITC-G1 was determined

(see Fig. 3). It can be seen that hyaluronan oligosaccharides compete effectively with

full-length hyaluronan and cause an increase in the diffusion coefficient of FITC-G1.

The method shows that a minimum length of 10 hyaluronan monosaccharides is

needed for maximal binding to FITC-G1 (15).

The confocal FRAP technique is a novel and direct method for investigating the

many protein–protein and protein–carbohydrate interactions that contribute greatly

to the organization and function of extracellular matrices. It can be applied over a

– D

n[B]