Iozzo Renato V. Proteoglycan Protocols

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Substrate Gel and Inverse Substrate Gel Technique 395

10. Rinse the gel twice with distilled water.

11. Establish the molecular sizes of hyaluronidase inhibitors by comparison with protein

molecular-weight standards.

4. Notes

1. This protocol is designed for two Bio-Rad minigels (8 × 10 cm.). For other sizes or thick-

nesses, volumes of stacking and separating gels, and operating current, must be adjusted

accordingly.

2. Not all Coomassie blue preparations work equally well. We have found the product of

BDH Chemicals, (Poole, UK) optimal. There are others that do not work at all.

3. Analysis of bovine testicular hyaluronidase on an HA-substrate gel demonstrates two forms

of the enzyme (see Fig.1, lane 1) corresponding to the soluble and membrane-bound forms

of the enzyme (8,9). Analysis of human serum on the HA-substrate gel performed at pH 3.7

reveals a band at 57 kDa, which is Hyal-1 (see Fig. 1, lane 2). On an inverse HA-substrate

gel performed at pH 7.4, two bands, at 120 and 83 kDa, are identified in mouse serum (see

Fig. 1, lane 3). The data can be compared using the pattern obtained from a conventional

HA-free gel (see Fig. 1, lane 4). The 83-kDa band persists, demonstrating that this is an

artifact, corresponding to an endogenous plasma glycoprotein. The 120-kDa band corre-

sponds to one of the hyaluronidase inhibitors in mouse serum, an inhibitor of neutral-active

PH-20 enzyme.

4. Alcian blue is commonly used for staining HA (17–21). Acidification of the Alcian blue

is recommended. This enhances staining and prevents precipitation of dye. For optimal

Fig. 1 Detection of hyaluronidase and hyaluronidase inhibitors using HA-substrate gel

and inverse HA-substrate gel procedures. M), molecular-weight markers. Lanes 1 and 2:

HA-substrate gel for the detection of hyaluronidase activities. Examination of bovine testicular

hyaluronidase, PH-20 (lane 1), and human plasma hyaluronidase, Hyal-1 (lane 2), were per-

formed at pH 7.4 and pH 3.7, respectively. Lanes 3 and 4: Inverse HA-substrate gel for the

examination of hyaluronidase inhibitor in mouse serum. The HA-containing gel, to which mouse

serum had been applied, was digested with 0.5 rTRU/mL of bovine testicular hyaluronidase at

pH 7.4 (lane 3). The position of the hyaluronidase inhibitors corresponds to a band in which the

HA remained undigested (lane 3, A). A false positive corresponds to a band of endogenous

plasma glycoprotein and possible HA-binding protein. This band could be identified by running

a corresponding gel that does not contain HA (lane 4, B). Lane 1, 0.5 rTRU bovine testicular

hyaluronidase; lane 2, 0.5 µL human plasma; lanes 3 and 4; 4 µL human plasma.

396 Mio et al.

results, staining should be performed for 16 h in a solution of 0.5% Alcian blue in 3%

acetic acid. When sequential staining with Alcian blue and Coomassie blue is performed,

staining for 1 h with Alcian blue is sufficient. For the inverse substrate gel procedure, a

single staining step with Alcian blue is recommended.

5. The hyaluronidase digestion step in the inverse HA-substrate gel procedure is obviously

critical but can be difficult, as the gel is very fragile at this stage. Enzyme activity can also

vary with minor changes of pH and temperature. Optimization of the concentration of

hyaluronidase in the gel digestion step may be required with each experiment. We rou-

tinely utilize three different levels of hyaluronidase with each experiment (0.25, 0.50, and

2.00 rTRU/mL) to avoid over- and underdigestion.

6. High levels of a protein can prevent dye penetration into the gels (9,10) and can gener-

ate false positive bands. Albumin introduces such an artifact when plasma and serum

samples are examined. Albumin is also a HA-binding protein (22–24), which may

explain its persistence in these HA-containing gels. Pronase treatment of the gels elimi-

nates such false positives. Proteins present in lesser amounts will appear as blue bands

following Coomassie blue staining. These should disappear if a pronase digestion step

is interposed.

Acknowledgements

This work was supported by Lion Corporation, Kanagawa, Japan, to K. M., and by

National Institutes of Health (USA) Grant 1P50 DE/CA11912, to R. S.

References

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2. Csóka, T. B., Frost, G. I., and Stern, R. (1997) Hyaluronidases in tissue invasion. Invasion

Metastasis 17, 297–311.

3. Frost, G. I., Csóka, T. B., Wong, T., and Stern, R. (1997) Purification, cloning, and expres-

sion of human plasma hyaluronidase. Biochem. Biophys. Res. Commun. 236, 10–15.

4. Csóka T.B., Frost G.I., Wong T., and Stern R. (1997) Purification and microsequencing of

hyaluronidase isozymes from human urine. FEBS Lett. 417, 307–310.

5. Csóka, T. B., Frost, G. I., Heng, H. H. Q., Scherer, S. W., Mohapatra G., and Stern R.

(1998) The hyaluronidase gene HYAL1 maps to chromosome 3p21.2-3p21.3 in human

and 9F1-F2 in mouse, a conserved candidate tumor suppressor locus. Genomics 48, 63–70.

6. Csóka, A. B., Scherer, S. W., and Stern, R. (1999) Expression analysis of six paralogous

human hyaluronidase genes clustered on chromosomes 3p21 and 7q31. Genomics 60,

356–361.

7. Gmachl, M., Sagan, S., Ketter, S., and Kreil, G. (1993) The human sperm protein PH-20 has

hyaluronidase activity. FEBS Lett. 336, 545–548.

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and Overstreet, J. W. (1996) The PH-20 protein in Cynomolgus macaque spermatozoa:

identification of two different forms exhibiting hyaluronidase activity. Develop. Biol. 175,

142–153.

9. Meyer, M. F., Kreil, G., and Aschbauer, H. (1997) The soluble hyaluronidase from bull

testes is a fragment of the membrane-bound PH-20 enzyme. FEBS Lett. 413, 385–388.

10. Haas, E. (1946) On the mechanism of invasion. I. Antinvasin I, An enzyme in plasma,

J. Biol. Chem. 163, 63–88.

11. Dorfman, A., Ott, M. L., and Whitney, R. (1948) The hyaluronidase inhibitor of human

blood. J. Biol. Chem., 223, 621–629.

Substrate Gel and Inverse Substrate Gel Technique 397

12. Moore, D. H. and Harris, T. N. (1949) Occurrence of hyaluronidase inhibitors in fractions

of electrophoretically separated serum. J. Biol. Chem. 179, 377–381.

13. Guntenhoener, M. W., Pogrel, M. A., and Stern, R. (1992) A substrate-gel assay for

hyaluronidase activity. Matrix 12, 388–396.

14. Miura, R. O., Yamagata, S., Miura, Y., Harada, T., and Yamagata, T. (1995) Analysis of

glycosaminoglycan-degrading enzymes by substrate gel electrophoresis (zymography).

Anal Biochem. 225, 333–340.

15. Mio, K., Carette, O., Maibach, H. I., and Stern, R. (2000) A serum inhibitor of hyalu-

ronidase. J. Biol. Chem., July 24.

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bacteriophage T4. Nature 227, 680–685.

17. Wardi, A. H. and Michos, G. A. (1972) Alcian blue staining of glycoproteins in acrylamide

disc electrophoresis. Anal. Biochem. 49, 607–609.

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(1984) Polyacrylamide-gel electrophoresis and Alcian blue staining of sulphated

glycosaminoglycan oligosaccharides. Biochem. J. 221, 707–716.

19. Turner, R. E. and Cowman, M. K. (1985) Cationic dye binding by hyaluronate fragments:

dependence on hyaluronate chain length. Arch. Biochem. Biophys. 237, 253–260.

20. Wall, R. S. and Gyi, T. J. (1988) Alcian blue staining of proteoglycans in polyacrylamide

gels using the “critical electrolyte concentration” approach. Anal. Biochem. 175, 298–299.

21. Ghiggeri, G. M., Candiano, G., Ginevri, F., Mutti, A., Bergamaschi, E., Alinovi, R., and

Righetti, P. G. (1988) Hydrophobic interaction of Alcian blue with soluble and erythrocyte

membrane proteins. J. Chromatogr. 452, 347–357.

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albumin in the ultracentrifuge. Biochem. J. 59, 620–627.

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mixtures of hyaluronic acid and serum albumin. Biochim. Biophys. Acta 75, 436–438.

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Affinity Coelectrophoresis 401

401

From:

Methods in Molecular Biology, Vol. 171: Proteoglycan Protocols

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

Affinity Coelectrophoresis

of Proteoglycan–Protein Complexes

James D. San Antonio and Arthur D. Lander

1. Introduction

Affinity coelectrophoresis (ACE) was developed as a tool to measure the strengths

of interaction between proteoglycans (PGs) or glycosaminoglycans (GAGs) and pro-

teins, and to assess the specificity of the interaction (i.e., to detect and fractionate GAG

or PG sample constituents that differentially bind to protein) (1). In ACE, trace con-

centrations of radiolabeled GAG or PG are subjected to electrophoresis through agar-

ose lanes containing protein at various concentrations. The electrophoretic pattern of

the radiolabeled GAG or PG is then visualized by autoradiography, or using a

phosphorimager, and the apparent dissociation constant (K

) is calculated as the protein

concentration at which the GAG or PG is half-shifted from being fully mobile at very

low protein concentrations (or between protein-containing lanes) to being maximally

retarded at saturating protein concentrations (see Figs. 1–3).

ACE holds many advantages over other means of studying GAG or PG–protein inter-

actions since it: (1) uses only trace quantities of the interacting molecules (typically a

microgram or less of GAGs, and a milligram or less of protein); (2) studies behaviors of

native proteins and of radiolabeled PGs or GAGs that can be essentially unmodified

(e.g., through metabolic radiolabeling) or minimally modified (e.g., by radioiodination);

(3) can measure strengths of binding even for relatively weak interactions characteristic

of GAG or PG-protein interactions (e.g., 100 nM or weaker K

); (4) can detect protein-

binding heterogeneity in a GAG or PG population and can even be used to isolate the

differentially binding subpopulations for further analysis (see Fig. 4); and (5) is a low

cost, simple, and rapid method, and is amenable to quantitative analysis.

The theory of ACE has been described in detail elsewhere (1,2) and will not be

repeated here. Rather, this chapter will serve as a description of ACE methods, and

will also include a protocol for the preparation of radioiodinated heparin samples,

which are often useful in various ACE applications. However, before

attempting ACE, there are several important questions which need to be addressed:

402 San Antonio and Lander

Fig. 1. Analytical ACE schematic. Top panel: ACE gels poured using a casting stand and

Teflon combs and strips as shown in Fig. 5 are used to create nine parallel rectangular wells,

which are filled with protein–agarose mixtures, each at a different protein concentration. Radiola-

beled GAG or PG is loaded into the slot above the protein-containing wells (shown as a dark line),

and after electrophoresis of the GAG or PG through the protein-containing lanes, its migration as

a function of protein concentration is visualized by autoradiography or phosphorimaging (shown

here as a pattern of peaks and valleys). The degree of GAG/PG retardation at the various protein

concentrations is used to calculate the apparent K

of GAG– or PG–protein binding (see text for

details). Artwork by Shawn M. Sweeney.

Fig. 2. (opposite page) ACE analysis can reveal the affinity of interactions between PGs or

GAGs and various proteins. For these experiments, syndecan-1 was electrophoresed through

types I–VI collagens in ACE gels. (A) Images of PG migration patterns were obtained using a

phosphorimager. The electrophoretograms indicate that some collagens bind strongly to

syndecan-1 (e.g., type V), and others bind weakly (e.g., type II). Protein concentrations in nM

are shown beneath gels. (B) Calculation of affinities of syndecan-1 for various human col-

lagens. From each electrophoretogram in panel (A), retardation coefficients (R) for syndecan-1

were determined (see text) and are plotted against protein concentration. Smooth curves rep-

resent nonlinear least-squares fits to the equation R = R

∞

(1 + (K

/[protein])

). Data are

adapted from (5).

Affinity Coelectrophoresis 403

404 San Antonio and Lander

1.1. Do I Have Enough Protein?

There is no way of knowing a priori how much of a protein sample one needs for an

ACE gel, since it depends on a yet to be determined value, i.e., the affinity the protein will

exhibit for GAGs or PGs. However, some general idea about the amounts of protein

required can be derived from the following example. For type I collagen, a protein of

⯝ 300,000 Da that exhibits a heparin-binding K

in the range of 100–200 nM, one

needs 150 µg of protein per ACE gel (using the ACE gel dimensions specified under

Subheading 2.). This amount allows for the creation of nine protein–agarose samples of

250-µL each, at concentrations of 1000, 500, 250, 100, 50, 25, 10, 5, and 1 nM. Since ACE

gels should be repeated at least three times to derive a reasonable estimate of the K

, then

one would need a minimum of 450 µg of type I collagen for three experiments. Other

proteins such as growth factors are much smaller than collagens, and often exhibit much

higher affinities for GAGs and PGs, and thus can require considerably less protein for

three experiments, i.e., on average < 50 µg total, or in some cases even much less—e.g., for

basic fibroblast growth factor, < 1 µg is required.

1.2. Will the Protein Remain Native?

The native state of the protein, its solubility, and propensity to aggregate as a function

of its concentration or solvent are key considerations, and must be determined for each

protein used in ACE. For example, in the case of laminin, which tends to aggregate in

solution, ethylendiamminetetraacetic acid (EDTA) can be supplemented to the protein

samples and the ACE buffers to inhibit aggregation (3). In the case of the collagens,

Fig. 3. ACE analysis can reveal selectivity in PG– or GAG–protein interactions. Example of

ACE analysis of the interactions between a basic peptide and

S-sulfate metabolically labeled

PGs/GAGs secreted by endothelial cells in vitro. ACE gel image was obtained using a

phosphorimager. At least two populations of PG/GAG, seen as two bands of radiolabeled mate-

rial migrating with different mobilities at low protein concentrations (< 50 nM), indicates het-

erogeneity in size and/or charge density within the PG/GAG mixture. Potential heterogeneity

in PG/GAG–peptide interactions is also obvious at a peptide concentration of 250 nM, in which

a fractionation of the PG species through the peptide-containing lane is evident as a broad

smear throughout the lane, and as a sharp band that migrates approximately halfway down the

lane. Thus, components of the PG/GAG sample are binding strongly to the peptide (i.e., are

retained closer to the top of the peptide-containing lane), and others are binding more weakly to

the peptide (i.e., are not significantly retained and migrate further within the peptide-contain-

ing lane). In such cases preparative ACE can be used to recover differentially binding PG/GAG

populations for further characterization. Data are adapted from (11).

Affinity Coelectrophoresis 405

which undergo fibrillogenesis within about 20 min after being brought from an acidic to

a neutral solution, such fibrils are insoluble and are impossible to subject to a serial

dilution, as is required in ACE. Thus, to avoid this problem one must bring collagen

solutions from the acid soluble to the neutralized state, and then mixed into agarose and

pipetted into ACE gels before fibrillogenesis occurs (4,5).

Fig. 4. Preparative ACE schematic. Top panel: A preparative ACE gel is poured using a

casting stand as shown in Fig. 5, except instead of using protein well-forming Teflon combs, a

single Plexiglas block is used to create one large rectangular well to be filled with a single

protein–agarose mixture. Radiolabeled GAG or PG is loaded into the slot above the protein-

containing wells (shown as a dark line to the left in the gel schematic), and electrophoresed through

the protein-containing zone. Middle panel: The agarose gel surrounding the protein-containing

zone is trimmed away, and the remaining protein-agarose block is sectioned into 2-mm-thick

segments. The amount of radiolabeled GAG or PG in each segment is then determined. Bottom

panel: Actual plot of CPM/fraction of heparin octasaccharide mixture electrophoresed through

1000 nM type I collagen, showing the partial resolution of four differentially binding populations.

(From San Antonio and Lander, unpublished data). Artwork by Drew Likens.

406 San Antonio and Lander

1.3. Are the GAGs or PGs of Interest Suitable?

One of the requirements of ACE is that the GAG or PG concentration is much less than

the Kd of GAG– or PG–protein binding (1). Thus, radiolabeled GAGs and PGs are used at

trace concentrations in ACE gels, i.e., generally far less than 1 µg of GAG or PG/gel will

suffice. However, the GAG/PG must be radiolabeled or labeled otherwise, and present in

great enough quantities to be detecteD (generally for radiolabeled samples at least 10,000

cpm/gel). Therefore, the GAGs or PGs must be metabolically radiolabeled with

S-sulfate

C-D-glucosamine in culture and subsequently purified, or purified in their native forms

but derivatized with, for example, Bolton-Hunter reagent (6), fluoresceinamine (1), or

tyramine (3), followed by radioiodination. Here we have presented a method for the

tyramine endlabeling of heparin for use in ACE. Another factor that must be considered in

the analysis of data from ACE gels is the potential multivalency of GAGs or PGs in terms

of their interactions with proteins; this issue is addressed elsewhere (1).

2. Materials

1. ACE casting apparatus (see Fig. 5): casting stage(s), protein well-forming comb(s),

PG/GAG lane-forming comb(s), and tape (autoclave or equivalent). Combs are precision

tooled from Teflon blocks (for protein well-forming combs) or sheets (for PG/GAG

lane-forming combs). The ACE casting apparatus we most commonly use includes a cast-

ing stage made of Plexiglas with a gel platform of 100 long × 75 wide × 6 mm deep;

protein well-forming combs consisting of nine parallel rectangular blocks spaced 3 mm

apart, each 15 long × 4 × 4 mm; and PG/GAG lane-forming combs cut from a 25 × 75 mm

Fig. 5. Oblique view of apparatus for pouring two ACE gels, each with protein-containing

lanes 15 mm in length. Plexiglas casting stand contains a clear piece of gel bond (not visible in this

photograph), on which are placed two Teflon combs that are each used to create 9 agarose–

protein-containing lanes, and two Teflon strips that are each used to create a GAG/PG loading

slot. The stand is bordered on two sides by masking tape, which retains the agarose and Teflon

strips in place. After filling the stand with agarose, upon solidification the combs and strips are

removed, forming two ACE gel templates to be run as described in the text and shown

diagramatically in Fig. 1.

Affinity Coelectrophoresis 407

rectangle of 1-mm-thick Teflon. Rectangles of 4.5 × 10 mm are removed from two

corners to produce a comb with one 66-mm edge, which is stood on its short edge and

held upright in the casting apparatus by pressing the tape used to seal the apparatus against

the overhanging tabs of the comb.

2. At least 1.2 L of running buffer (RB). To make 2 L, add 22.53 g of sodium 3-(N-mor-

pholino)-2hydroxypropanesulfaonate (MOPSO) to 1.8 L of distilled water with

stirring. Add 20.51 g of sodium acetate, anhydrous, or 34.02 g of sodium acetate, trihydrate.

Use 5 M NaOH to bring the pH to 7.0. Bring to 2 L with distilled water. Store at 4°C, will

last no longer than several months. This buffer can also be prepared as a 5× concentrated

stock and diluted before use.

3. GelBond, 85 × 100 mm sheets (FMC, #53734).

4. 1.053% agarose: 1 g of low-melting-point (LMP) (Sea Plaque Agarose; FMC) in 95 mL

of RB.

5. 2.22% agarose: 1 g of LMP agarose (Sea Plaque Agarose; FMC) in 45 mL of RB.

6. 10% CHAPS in distilled water.

7. Leveling device.

8. ACE gel running box (we use the Hoefer Super-Sub apparatus).

9. Low voltage electrophoresis power supply, capable of delivering 75 V.

10. Waterbath set to 37°C.

11. Boiling-water bath or microwave oven.

12. Circulating water chiller (unnecessary if cold-water tap is available at lab bench where the

gel will be run).

13. Small space heater (1500 W).

3. Methods

3.1. Analytical ACE

This protocol is used to estimate the K

of GAG or PG binding to a protein, or to

visualize heterogeneity in binding between a PG or GAG mixture and a protein.

1. Place the casting stand on a level benchtop. On a piece of GelBond, determine which is the

bonding side by placing a drop of distilled water on one of the sides. If the drop beads up,

it is the nonbonding side and is placed down on the casting apparatus; if the bead spreads

out, it is the bonding side and is placed face up. Using scissors, cut the GelBond to fit the

casting apparatus, then use 1–2 drops of distilled water to hold the nonbonding side in place.

Press excess water out from between the GelBond and the casting stage and, using

Kimwipes, make sure that the sides of the stage are dry.

2. Use autoclave tape to seal the edges of the casting stage, making sure to leave about 1 in

(2.5 cm) excess on both ends so it can be folded against itself to make a tab to facilitate its

removal later. Place the sample-forming comb(s) in the casting stand using the tape to

hold it in place, then place the protein-lane forming comb by centering it and leaving a

space of about 2 mm between it and the sample comb. Using the 15-mm-long protein

well-forming combs and the gel casting stand specified here, either one or two ACE gels

can be poured per stand.

3. When agarose is made fresh, to promote its rapid dissolution allow at least 20 min for it

to soak in room temperature RB before the mixture is boiled. Place a glass bottle

containing the 1.053% agarose into the water bath, making sure it cannot tip and that

the cap is very loose, so that upon heating it will not explode. The bottle should be left

in the boiling-water bath or microwaved until the agarose has come to a boil and is