Introduction

The glycosaminoglycan constituents of proteoglycans, heparan sulfate, chondroitin/ dermatan sulfate, or keratan sulfate (1), and the glycosaminoglycan hyaluronan (2) are important constituents of all tissue matrices. They provide tissue hydration for fluid flow and molecular transport, charge repulsions or attractions for intermolecular spacing (3) and polyanionic domains for growth factor or integrin signaling (4,5), cell adhesion, and migration or proliferation (6-8). As a result, studies of extracellular matrix metabolism in genetic, developmental, or tissue engineering experiments also include analyses of glycosaminoglycan components. The most widely used procedures for glycosaminoglycan analyses rely largely on the identification and quantitation of products generated by depolymerization with lyases (9) or hydrolases (10) by gel electrophoresis (11), nuclear magnetic or mass spectrometry (12-15), highperformance liquid chromatography (16-21) or capillary zone electrophoresis (22-24). While sensitive and accurate, such procedures usually require costly or technically specialized equipment, and involve preparative steps that can lead to loss of products. We describe here an alternative procedure for the identification and quantitation of gly-cosaminoglycan lyase products using fluorophore-assisted carbohydrate electrophoresis or FACE (25,26). It is especially applicable for studies when only small amounts of tissue or proteoglycans are available and/or a high sample throughout is needed. While we limit the illustration of this technically simple procedure to chondroitin/dermatan sulfate and hyaluronan in the chapter, it can also serve as an alternative sensitive and specific way to determine the identity and quantity of products of heparan sulfate-spe-cific lyases (9) and keratan sulfate-specific hydrolases (27).

The repeating disaccharide structures of chondroitin/dermatan sulfate and hyaluronan are illustrated in Fig. 1. In chondroitin and dermatan sulfate, the galNAc^i,4glcA repeats

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

HYALURONAN / CO CH2OR x

VHO-^HO NHAc n

CHONDROITIN SULFATE DERMATAN SULFATE

CHONDROITIN SULFATE DERMATAN SULFATE

Fig. 1. Repeating disaccharide structures for hyaluronan, chondroitin sulfate, and dermatan sulfate. The sulfation at C4 and C6 of the hexosamine and at C2 of the hexuronic acid in chondroitin and dermatan sulfate are indicated by R. Dermatan sulfate is characterized by a variable proportion of L-iduronate residues in the chain, while chondroitin sulfate contains only D-glucuronate residues.

Fig. 1. Repeating disaccharide structures for hyaluronan, chondroitin sulfate, and dermatan sulfate. The sulfation at C4 and C6 of the hexosamine and at C2 of the hexuronic acid in chondroitin and dermatan sulfate are indicated by R. Dermatan sulfate is characterized by a variable proportion of L-iduronate residues in the chain, while chondroitin sulfate contains only D-glucuronate residues.

are extensively substituted with sulfate esters at C4 or C6 of the hexosamine residues, and occasionally also on C2 of an adjacent hexuronic acid residue. All three polymer sequences are cleaved at the ^1,4-bonds by chondroitinase ABC (28), generating A-disaccharides from the chain interior, and mono- or disaccharides from the chain terminal (see Fig. 2). Each lyase product contains a free reducing end that can be stoichiometrically coupled to a fluorescent tag, such as 2-aminoacridone, via reductive amination (19,26,29) (see Fig. 3). The fluorotagged products can be resolved into discrete bands by electrophoresis on high percentage polyacrylamide gels (26,30-33). Each product can be detected down to the picomolar range by illuminating gels with ultraviolet light. Such images are electronically recorded and further processed using computerized image analysis software.

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