John R Couchman and Pairath Tapanadechopone 1 Introduction

Proteoglycans are quite abundant components of many extracellular matrices, while most cell surfaces also bear these macromolecules. Frequently the profiles are complex. For example, several members of the syndecan and glypican families of cell surface heparan sulfate proteoglycans may be present on a single cell type (1,2). Some extracellular matrices, e.g., from brain, may also contain a variety of proteoglycans including several members of the hyalectans or aggregating proteoglycans such as neurocan, brevican, and versican (3). Frequently it is useful to monitor the nature and variety of proteoglycans in a pool from tissues or cell cultures in a simple manner, before moving on to further purification steps, use of core protein-specific antibodies, or pursuit of a potentially novel core protein.

While proteoglycans require some specialized techniques for analysis, advantage can be taken of their glycanation to identify core proteins even when their precise characteristics remain unresolved. Specific enzymes are readily available, first from bacterial sources but more recently of recombinant origin, which selectively degrade glycosami-noglycans. Chondroitinase ABC will degrade virtually all chondroitin and dermatan sulfates, while leaving heparan and keratan sulfate chains intact. Conversely, heparitinase enzymes will degrade nearly all forms of heparan sulfate, but are unable to degrade chondroitin, dermatan, or keratan sulfate (see Fig. 1). Further, the consequences of glycosaminoglycan removal can be monitored by sodium dodecyl sulfate-polyacryla-mide gel electrophoresis (SDS-PAGE). The heterogeneous nature of proteoglycans ensues largely from the variable number, length and charge of glycosaminoglycans in a pool of a single core protein (e.g., aggrecan from cartilage, or perlecan from a basement membrane preparation). Proteoglycans are frequently seen as broad smears, or sometimes, when large, may not even penetrate a 3% resolving gel (see Fig. 2A). Once glycosaminoglycan lyases have removed most of the chains, the core proteins become much more readily resolved by SDS-PAGE as discrete polypeptides (see Fig. 2).

Fig. 1. Detection of chondroitin/dermatan sulfate proteoglycans core proteins from EHS tumor with R36, a polyclonal antibody recognizing chondroitin/dermatan sulfate stubs remaining after chondroitinase ABC treatment. Lanes 1 and 7 are standards whose molecular weight in kilodaltons is indicated. Lane 2, untreated sample; lane 3, sample treated with chondroitinase ABC and heparinase II and III; lane 4, sample treated with chondroitinase ABC only; lane 5 contains heparinase II and III only, while lane 6 contains chondroitinase ABC alone. The common polypeptide seen in lanes 3 and 5 is present in the heparinase II preparation. The data show that a CS/DS proteoglycan with a core protein of Mr ~ 200,000 (in lanes 3 and 4) is accompanied by a second, large core protein that is revealed only after additional heparinase treatment. This, therefore, represents a hybrid form of perlecan bearing both HS and CS/DS chains.

Fig. 1. Detection of chondroitin/dermatan sulfate proteoglycans core proteins from EHS tumor with R36, a polyclonal antibody recognizing chondroitin/dermatan sulfate stubs remaining after chondroitinase ABC treatment. Lanes 1 and 7 are standards whose molecular weight in kilodaltons is indicated. Lane 2, untreated sample; lane 3, sample treated with chondroitinase ABC and heparinase II and III; lane 4, sample treated with chondroitinase ABC only; lane 5 contains heparinase II and III only, while lane 6 contains chondroitinase ABC alone. The common polypeptide seen in lanes 3 and 5 is present in the heparinase II preparation. The data show that a CS/DS proteoglycan with a core protein of Mr ~ 200,000 (in lanes 3 and 4) is accompanied by a second, large core protein that is revealed only after additional heparinase treatment. This, therefore, represents a hybrid form of perlecan bearing both HS and CS/DS chains.

Chondroitinases and heparitinases are eliminases, so that the remaining core proteins have serine (usually) residues bearing not only the stem oligosaccharide (xylose-galactose-galactose-uronic acid) but also a disaccharide or larger oligosaccharide with a terminal unsaturated uronic acid residue. This, it turns out, is quite antigenic, and monoclonal (4,5) as well as polyclonal antibodies have been raised (6) which recognize the carbohydrate "stubs" remaining after chondroitinase or heparitinase treatments. They are also very specific. An antibody recognizing a heparan sulfate "stub" with a terminal unsaturated uronic acid residue will not recognize the equivalent "stub" generated by a chondroitinase enzyme, and vice versa. Therefore, the combined use of enzymes and antibodies can be used, for example in Western blotting, to estimate the sizes of core proteins, and the type of glycosaminoglycan present. This can be particularly useful where a particular core protein, e.g., perlecan, can be substituted with heparan and/or chondroitin sulfate chains (see Fig. 1). It can provide evidence of hybrid proteoglycans that bear more than one glycosaminoglycan type. Further, since the antibodies do not recognize core protein epitopes, a mixed population of heparan and/ or chondroitin and dermatan sulfate proteoglycan can be quickly analyzed for their number, size, and glycanation profiles. Such evidence can be supported by more traditional metabolic labeling methods, combined with chemical or enzymatic degradation techniques followed by gel filtration analysis.

Heparan Sulfate 3g10 Epitope

Fig. 2. Detection of intact murine perlecan (A) and recombinant domain IV-V of mouse perlecan transfected into COS-7 cells (B) with a rat monoclonal antibody specific to perlecan core protein (A) and monoclonal A-heparan sulfate antibody recognizing HS stub after heparinase III (heparitinase) treatments (B). (A) shows that the intact perlecan core protein is not clearly visible until the HS chains have been removed, while (B) shows HS substitution on the recombinant perlecan. In each blot, the proteoglycans are untreated (U), heparitinase-pre-treated (H), heparitinase- and chondroitinase-pretreated (HC), or chondroitinase-pretreated (C). Lanes E contain heparitinase and chondroitinase enzymes only.

r.iLi AUS

Fig. 2. Detection of intact murine perlecan (A) and recombinant domain IV-V of mouse perlecan transfected into COS-7 cells (B) with a rat monoclonal antibody specific to perlecan core protein (A) and monoclonal A-heparan sulfate antibody recognizing HS stub after heparinase III (heparitinase) treatments (B). (A) shows that the intact perlecan core protein is not clearly visible until the HS chains have been removed, while (B) shows HS substitution on the recombinant perlecan. In each blot, the proteoglycans are untreated (U), heparitinase-pre-treated (H), heparitinase- and chondroitinase-pretreated (HC), or chondroitinase-pretreated (C). Lanes E contain heparitinase and chondroitinase enzymes only.

2. Materials

1. Samples for analysis dissolved in suitable buffers (see Note 1).

2. Heparitinase buffer: 0.1 M sodium acetate, 0.1 mM calcium acetate, pH 7.0.

3. Chondroitinase buffer: 50 mM Tris-HCl, 30 mM sodium acetate, 20 mM ethyleme-diamintetraacetic acid (EDTA), 10 mM NEM, 0.2 mM phenylmethyl sulfonyl flouride (PMSF), and 0.02 sodium azide, pH 8.0 (see Note 1).

4. Protease inhibitor for all heparitinase treatments (see Note 2): 10x trypsin inhibitor type III-0 (ovomucoid 100 ^g/mL, Sigma).

5. Glycosaminoglycan lyases (Seikagaku or Sigma):

a. Heparan sulfate: heparinase III (EC 4.2.2.8). This enzyme is also known as heparitinase and heparitinase I.

b. Chondroitin sulfate and dermatan sulfate: chondroitin ABC lyase (EC 4.2.2.4).

c. Chondroitin sulfate: chondroitin AC II lyase (EC 4.2.2.5).

d. Dermatan sulfate: chondroitin B lyase (no EC number).

6. Glycosaminoglycan carriers (optional): chondroitin sulfate type A (chondroitin 4-sulfate) or type C (chondroitin 6-sulfate), heparan sulfate (Sigma).

7. 2x SDS-PAGE sample buffer (Sigma), with or without reducing agent (e.g., 40 mM dithioerythreitol).

8. Prestained protein molecular-weight standards (Sigma or Bio-Rad).

9. SDS-PAGE gels. If a wide size range of core proteins is suspected, 3-15% gradient gels can be useful.

10. Electroblotting and transfer buffers and apparatus.

11. Transfer membrane: 0.45-^m nitrocellulose (Bio-Rad Trans-Blot® transfer medium or Schleicher & Schuell #BA85), PVDF (Millipore Immobilon P), or positively charged nylon (Bio-Rad Zetabind) membranes.

12. Blocking buffer: 5% nonfat dried milk in 0.1% Tween 20 is optional in phospate-buffered saline, PBS (TPBS).

13. Diluting buffer: 1% nonfat dried milk, 0.1% bovine serum albumin, and 0.1% (v/v) Tween 20 in PBS (for monoclonal antibodies) or triphosphate-buffered saline, TBS (for polyclonal antibodies).

14. Primary antibodies recognizing carbohydrate "stubs" (Seikagaku):

a. Monoclonal A-heparan sulfate (for heparan sulfate GAG).

b. Monoclonal anti proteoglycan A-di-0S, -4S, -6S (for chondroitin/ dermatan sulfate GAGs; see Note 3).

These antibodies are also available as biotin conjugates.

c. Equivalent antibodies recognizing protein of interest.

15. Secondary antibodies: horseradish peroxidase- or alkaline phosphatase-anti-Ig conjugate. Alternately, streptavidin-horseradish peroxidase conjugate should be used where the primary antibodies are biotin conjugates.

16. Chromogenic and chemiluminescence visualization system, e.g., ECL™ Western blotting detection reagents (Amersham Pharmacia Biotech) for peroxidase conjugates or alkaline phosphatase-conjugate substrate kit (Bio-Rad).

3. Methods

1. Divide the proteoglycan sample to be analyzed into equal aliquots. The number depends on the enzyme treatments to be performed. For example, if a proteoglycan pool is suspected to contain chondroitin and heparan sulfate proteoglycans, four aliquots should be used. One sample is left untreated, while others receive chondroitinase ABC, heparitinase, or both enzyme treatments. Ideally, each sample should contain 1-10 ^g of proteoglycan. The choice of buffer depends on the enzymes to be used (see Note 1). Further controls contain buffer with enzyme only (no proteoglycan).

2. Treat samples with appropriate enzymes at 37°C. The amount and duration of enzyme treatment depend on the proteoglycan concentration. For 1-10 ^g of proteoglycan, 2-3 h of incubation with 0.5-1 mU chondroitinase ABC or 1-2 mU heparitinase in the presence of protease inhibitor (1x ovomucoid, see Note 2) is suggested. Where concentrations of proteoglycan are higher, adding a second aliquot of enzyme after 2 h, for a further incubation can be beneficial. Where proteoglycan concentrations are very low (below 100-200 ng per sample), adding approximately 0.5 ^g of appropriate free glycosaminoglycan can be added as carrier to aid efficiency and recovery (but see Note 4).

3. If enzyme activity needs to be verified, set up samples of free glycosaminoglycan (approx 0.5 mg/mL) in buffer, to which the enzymes are added, and incubate simultaneously. Enzyme activity is monitored spectrophotometrically at 232 nm.

4. Enzyme treatments are terminated by adding SDS-PAGE sample buffer (with or without reducing agent, Subheading 2.) and heating to 100°C, if required. Samples can be frozen at -20°C or immediately resolved by SDS-PAGE.

5. Samples are applied to SDS-PAGE gels for conventional electrophoresis and transfer to nitrocellulose or other medium (see Note 5). If a range of core protein masses is suspected, or not known, acrylamide gradients are preferable (e.g., 3-15%).

6. Membranes are blocked conventionally, for example in 5% dried milk powder in phosphate-buffered saline for at least 1 h. They are then probed with monoclonal or polyclonal antibodies recognizing carbohydrate "stubs" created by glycosaminoglycan lyases. These are available as purified IgG, and sometimes in biotinylated form, and should be used at 10-25 ^g/mL. Incubation can be at 4°C overnight, or shorter periods at room temperature or 37°C, but for at least 1 h. Constant gentle agitation is advised.

7. Thorough washing is followed by secondary antibody (e.g., affinity purified goat anti-mouse IgG conjugated to horseradish peroxidase) in the same buffer for 1 h at room temperature. Antibody concentrations should accord with manufacturer's instructions. Extensive washes are then followed by visualization as preferred, such as chemiluminescence.

4. Notes

1. A suitable buffer for chondroitinase ABC or AC II is listed under Subheading 2., as is one suitable for heparitinase (also known as heparinase III). However, where samples are to be treated with both enzymes, we have found the heparitinase buffer to be suitable. It should be noted that chondroitinase B is inhibited by phosphate. Heparinase activity is increased by the presence of calcium ions, but it is reported that the activity of heparinase III is not much decreased by its absence.

2. Polysaccharide lyases are primarily of microbial origin. Protease contamination can be present in the enzyme preparation, especially all heparinase enzymes. This can cause misleading results. Thus, protease inhibitor should be added in case of heparitinase treatments. Chondroitinase ABC is available in protease-free form.

3. Separate, specific antibodies are available that, while all recognizing the terminal unsaturated uronic acid residue, as described, have specificity for the presence and position of sulfate on the adjacent galactosamine residue. The three antibodies can be used combined. At the current time they are only available separately. Most commonly, the prevalence of sulfation is 4S > 6S > 0S.

4. We have found that the use of chondroitinase ABC and heparinase III together leads to a less efficient identification of chondroitin sulfate proteoglycan core proteins than the use of the former enzyme alone. The reasons are not clear, but it may be that products of heparan sulfate lyases are slightly inhibitory to chondroitinase enzymes. It is known that heparin will inhibit chondroitinases, and should therefore not be used as a carrier.

5. Intact proteoglycans transfer poorly to nitrocellulose or similar membrane. The more gly-cosaminoglycan present on a core protein, the more difficult it becomes. This is a result of high mass as well as charge. Therefore, while decorin with one chain can be quite efficiently transferred, aggrecan with >100 chains may not. Transfer to cationic membranes can enhance proteoglycan capture.

References

1. Bernfield, M., Gotte, M., Park, P. W., Reizes, O., Fitzgerald, M. L., Lincecum, J., and Zako, M. (1999) Functions of cell surface heparan sulfate proteoglycan. Annu. Rev. Biochem. 68, 729-777.

2. David, G., Bai, X. M., Van der Schueren, B., Cassiman, J-J, and Van der Berghe, H. (1992) Developmental changes in heparan sulfate expression: in situ detection with mAbs. J. Cell Biol. 119, 961-975.

3. Iozzo, R. V. (1998) Matrix proteoglycans: from molecular design to cellular function. Annu. Rev. Biochem. 67, 609-652.

4. Couchman, J. R., Caterson, B., Christner, J. E., and Baker, J. R. (1984) Mapping by monoclonal antibody detection of glycosaminoglycans in connective tissues. Nature 307, 650-652.

5. Tapanadechopone, P., Hassell, J. R., Rigatti, B., and Couchman, J. R. (1999) Localization of glycosaminoglycan substitution sites on domain V of mouse perlecan. Biochem. Biophys. Res. Commun. 265, 680-690.

6. Couchman, J. R., Kapoor, R., Sthanam, M., and Wu, R. R. (1996) Perlecan and basement membrane-chondroitin sulfate proteoglycan (bamacan) are two basement membrane chon-droitin/dermatan sulfate proteoglycans in the Engelbreth-Holm-Swarm tumor matrix. J. Biol. Chem. 271, 9595-9602.

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