Proteoglycans Analyzed by Composite Gel Electrophoresis and Immunoblotting

George R. Dodge and Ralph Heimer

1. Introduction

Proteoglycans are the protein products of diverse genes posttranslationally modified with highly negatively charged side chains, commonly known as glycosami-noglycans. The latter consist of repeating disaccharides capable of forming polymers of varying size, which, depending on the specific disaccharide composition, are known as the chondroitin sulfates, the iduronate-containing dermatan sulfates, keratan sulfate, and heparan sulfate. Some of the more complex proteoglycans, such as aggrecan, are decorated by additional nonglycosaminoglycan oligosaccharides (1). Aggrecan contains additionally a hyaluronan-binding domain that in the extracellular matrix facilitates formation of large noncovalently-bound complexes containing one hyaluronan molecule and numerous aggrecans (1). Analysis of proteoglycans by standard SDS-PAGE is complicated by the presence of the negatively charged side chains, which prevent the linerarization of molecules usually achieved by treatment with a mercaptoethanol and SDS. Consequently, in SDS-PAGE most proteoglycans with multiple glycosaminoglycan side chains, if they enter the gel at all, migrate as broad bands and appear as smears due to size heterogeneity and large electroendosmotic effects particularly when the acrylamide composition of the PAGE gel is held to a minimum.

The studies of McDevitt and Muir, now more than three decades ago, made the surprising observation that the proteoglycans from an extract of cartilage resolved in a composite gel composed of 0.6% agarose and 1.2% acrylamide into two sharp bands (2). The mechanism for the separation of the proteoglycans of cartilage into two bands appeared to be based not only on size but also on charge distribution of two subspecies of proteoglycans (3). Gels such as these were originally designed to provide a larger pore size than 3.5% acrylamide alone, which is the limit concentration for gel formation. The technique of composite gel electrophoresis is best understood when the historical perspective is considered and its usefulness in previous applications discussed. These types of composite gels had been used for the separation of proteins (4)

From: Methods in Molecular Biology, Vol. 171: Proteoglycan Protocols Edited by: R. V. Iozzo © Humana Press Inc., Totowa, NJ

Table 1

Monoclonal and Polyclonal Antibodies Useful in Characterizing Proteoglycans

Antigen Name Polyclonal/mAb Notes Reference

Table 1

Monoclonal and Polyclonal Antibodies Useful in Characterizing Proteoglycans

Antigen Name Polyclonal/mAb Notes Reference

Perlecan

7B5

mAb

Laminin-like region

19

Versican

2B1

mAb

20

Keratan sulfate

AN9P1

mAb

Bound to aggrecan

21

Aggrecan

BE123

mAb

After c-ABC-treatment

22

Aggrecan, N-terminal

BC3

mAb

After aggrecanase exposure

23

Aggrecan, C-terminal

BC4

mAb

After aggrecanase exposure

23

Aggrecan

IC6

mAb

HA-binding domain

24

Chondroitin sulfate

CS56

mAb

Bound chondroitin 4 or 6 sulfate

25

Chondroitin-4 sulfate

2B6

mAb

After c-ABC digestion

26

Chondroitin-6 sulfate

3B3

mab

After c-ABC digestion

27

Unsulfated dissacharide

1B5

mAb

After c-ABC digestion

28

Biglycan

LF15

Poly

N-terminus

29

Decorin

LF30

Poly

N-terminus

29

and RNA (5). Improvement of this technique, originally done in tube gels, led inevitably to the use of slab gels (6) and also to improvement in sample preparation through use of SDS and reduction (6) or SDS only (7). These modifications facilitated the analysis of tissue extracts and biologic fluids without prior fractionation. Originally the detection of proteoglycans was accomplished with the basic thiazine dye, toluidine blue. Increased sensitivity of the detection of proteoglycans was achieved by techniques such as biosynthetic labeling with 35SO4 (6) transblotting to positively charged Nylon membranes followed by staining with Alcian blue (7), and transblotting to unmodified Nylon and staining with the highly positively charged 125I-cytochrome c (8). The detection achieved with Alcian blue staining of proteoglycans on positively charged Nylon-66 is nearly 100 times more sensitive than staining with toluidine blue (7). With the availability of monoclonal and polyclonal antibodies specific for diverse proteoglycans (the core proteins and associated GAGs), standard Western blotting techniques could be utilized on proteoglycans transblotted from composite gels. Table 1 contains a partial list of antibodies useful for the characterization of a wide range of known proteoglycans.

An interesting application of Western blotting involves sequential testing of a single blot with multiple antibodies. Antibodies bound to the proteoglycan antigen transblotted to nitrocellulose can be removed by exposure to sodium thiocyanate without materially affecting the binding of the immobilized material. The blot can then be probed again by another antibody. Densitometry scans of each blot can then be superimposed on one another, and by visual inspection this procedure can aid in resolving closely migrating species and also in determining the purity of a proteoglycan isolate or a standard (9).

Fig. 1. Demonstration of different chondroitin/dermatan sulfate proteoglycans in myocardial extracts. Western blots (A, B) prepared from samples of primate myocardial extracts following composite agarose-acrylamide gel electrophoresis were incubated with a 1:200 dilution of the monoclonal CS-56 (ICN) (which reacts with intact chondroitin-4 and -6 sulfate chains) (A) or a 1:200 dilution of the monoclonal 2-B-6 (which reacts with terminal unsulfated disac-charides on chondroitinase ABC digestion) (25). Lane 1 is the extract of the primate myocardium, lane 2 is a standard of 50 ng of bovine aggrecan from articular cartilage. (B) Western of duplicate samples that were exposed overnight at 37°C to 0.1 unit of chondroitinase ABC (ICN) in 5 mL of 0.2 M Tris buffer, pH 8.0, before being incubated with monoclonal 2-B-6. This demonstrates the presence of two clearly defined proteoglycan bands in the mycardial extract (lane 1), discernable only after chondroitinase digestion.

Results obtained with agarose/acrylamide gel electrophoresis have greatly enhanced our knowledge of proteoglycan biochemistry, synthesis, and degradation. First, avoiding time-consuming preparative steps has allowed the direct analysis of biologic specimens and tissue extracts. In general, such analyses have been useful in the simultaneous detection of proteoglycans with relatively large core proteins conjointly with detection of proteoglycans of smaller size. While large and small proteoglycans are easily separable, identification within each group can best be made by specific antibodies. The chief difficulty with separating proteoglycans with large core proteins is the presence of multiple glycosaminoglycan side chains and that the side chains often show size heterogeneity, which then affect overall mobility and electroendosmosis. An example of the number of glycosaminoglycan side chains affecting the mobility is illustrated when biglycan with its two glycosaminoglycan side chains has to be distinguished from decorin, with its single side chain. The core proteins of these proteoglycans have a rather similar molecular weight, although the presence of one additional (two) dermatan/chondroitin sulfate side chain biglycan results in biglycan having a molecular weight of approximately 100,000 daltons and decorin, with one such chain, only 70,000 daltons. One would assume, therefore, that this pair would be readily separable. Figure 1 is an example of the power of using composite gels and Western blotting and demonstrates this by using two different antibodies, CS-56 (reactive with intact chondroitin 4- and 6-sulfate chains) and 2-B-6 (reactive with terminal dissacharides remaining on the core protein of proteoglycans after digestion with chondroitinase ABC (26). One can see in Fig. 1A that, when using monoclonal CS-56,

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