Maria Jos Hernaiz and Robert J Linhardt

1. Introduction

Glycosaminoglycans (GAGs) are a family of complex linear polysaccharides characterized by a repeating core disaccharide structure typically comprised of an ^-substituted hexosamine and an uronic acid residue. They can be categorized into four main structural groups: hyaluronate, chondroitin sulfate (CS)/dermatan sulfate (DS); heparan sulfate/heparin and keratan sulfate.

The biological roles of chondroitin and dermatan sulfate GAGs are poorly understood and their exact chemical structures have not been determined. Because enzymes are highly specific and act under mild conditions, enzymatic methods are often preferable over chemical methods for determining the structure of GAGs.

Enzymes that degrade GAGs have become increasingly important tools for understanding the biological roles of GAGs and the proteoglycans, including the regulation of various cellular process such as adhesion, differentiation, migration, and proliferation (1). Utilizing these enzymes, design and preparation of GAG-based therapeutic agents might become possible (2). Such drugs could have uses as antithrombotic agents, antiatherosclerotic agents, antiinflammatory agents, inhibitors of complement activation and regulators of cell growth, angiogenesis, and antiviral agents.

CS and DS are the most common type of GAGs in extracellular matrix proteoglycans (1). CS is a hetereopolysaccharide made up largely of repeating disaccharide units, in which one sugar is N-acetyl-d-galactosamine and the other is d-glucuronic. These disaccharides can be sulfated at the 4- or 6-position of the ^-acetylgalactosamine residue. The major classes are CS-A (chondroitin 4-sulfate), DS (CS-B), containing 4-sulfated ^-acetylgalactosamine and iduronic acid, and CS-C (chondroitin 6-sulfate) (see Fig. 1).

Chondroitin Sulfatase
Fig. 1. Glycosidic linkages present in CS/DS and chondroitin lyases that act on these linkages.

Microorganisms are a major source of GAG-degrading enzymes (3-5), particularly in the case of soil bacteria, which may depend on connective tissues in animal carcasses as a nutrient source. Based on their catalytic mechanism, GAG-degrading enzymes are divided into two distinct classes: prokaryotic enzymes, which are lyases that depolymerize GAGs by an elimination mechanism (5), and eukaryotic enzymes, which act by hydrolysis (6) (see Fig. 2). The chondroitin lyases depolymerize the CS and DS, by an elimination mechanism, into oligosaccharides containing a A45-unsaturated uronic acid residue at the nonreducing end (3-5). This residue exhibits an absorbance maximum at 232 nm, permitting the detection of the oligosaccharide products of the chondroitin lyases using ultraviolet (UV) spectroscopy.

Four classes of chondroitin lyases have been biochemically characterized: those that act on chondroitin, chondroitin-4-sulfate and chondroitin-6-sulfate (chondroitinase

Chondroitin Svenska

Fig. 2. Enzymatic mechanisms for chondroitin lyases (eliminative cleavage) and chondroitin hydrolases (hydrolytic cleavage), where B is a basic residue in the enzymes catalytic site, R is a monosaccharide (exolytic) or an oligosaccharide (endolytic), R' is H (exolytic) or an oligosaccharide (endolytic), and R'' is an oligosaccharide or polysaccharide.

Fig. 2. Enzymatic mechanisms for chondroitin lyases (eliminative cleavage) and chondroitin hydrolases (hydrolytic cleavage), where B is a basic residue in the enzymes catalytic site, R is a monosaccharide (exolytic) or an oligosaccharide (endolytic), R' is H (exolytic) or an oligosaccharide (endolytic), and R'' is an oligosaccharide or polysaccharide.

AC or chondroitin AC lyase); dermatan sulfate (chondroitinase B or chondroitin B lyase); chondroitin-6-sulfate and hyaluronate (chondroitinase C or chondroitin C lyase); and an enzyme with broad substrate specificity that acts on both chondroitin and dermatan sulfate (chondroitinase ABC or chondroitin ABC lyase) (see Fig. 1). Most commercial preparations of chondroitin ABC lyase are a mixture of two enzymes with endo (ABC endolyase) and exo (ABC exolyase) activities (7). The activity of these enzymes toward small oligosaccharide substrates differs substantially. Similarly, there are two chondroitin AC lyases, AC-I (endolyase) and AC-II (exolyase) (8-9).


Heraiz and Linhardt

Table 1

Sources of Commonly Used Chondroitin Lyases



Chondroitinase ABC (mixture of endolyase and exolyase)


from Proteus vulgaris


Chondroitinase AC-I from Flavobacterium heparinum



Chondroitinase AC-II from Arthrobacter aurescens



Chondroitinase B from Flavobacterium heparinum



Chondroitinase C from Flavobacterium heparinum


Chondroitin lyases are most commonly obtained from Proteus vulgaris, Arthro-bacter aurescens, Bacteroides thetaiotaomicron, Bacteroides stercoris, and Flavobacterium heparinum. Chondroitin lyases from P. vulgaris, A. aurescens, and F. heparinum have been purified to homogeneity and are commercially available (see Table 1). While little is know about the catalytic machinery of these enzymes, the recent publications of the three-dimensional structure of chondroitinase AC and B should shed light on their mechanism of action (10-12).

Determination of CS/DS oligosaccharide structure is a formidable analytical problem that has limited structure-activity relationship studies, and the development of improved methods is necessary for further progress. Current approaches involve the preparation of CS/DS oligosaccharides using chondroitin lyases followed by separation techniques including gel permeation chromatography (GPC) (13), strong anion exchange (SAX)-high-performance liquid chromatography (HPLC) (14), polyacryla-mide gel electrophoresis (PAGE), (14) and capillary electrophoresis (CE) (13,15), that permit analysis of disaccharide composition. These provide important data on composition and domain structure but generally yield indirect and incomplete sequence information. Mass spectrometry (MS) has also been applied to the analysis of CS/DS oligosaccharides. Fast-atom bombardment (FAB-MS), electrospray ionization (ESI-MS), and matrix-assisted laser desorption/ionization (MALDI-MS) are capable of determining the molecular weight of oligosaccharides (13). Although muclear magnetic resonance (NMR) spectroscopy provides for the accurate determination of the chemical fine structure of small CS/DS oligosaccharides (containing 2-14 saccharide units), it requires a large amount of material (13,16-18).

What follows in this chapter are descriptions of the materials and methods required to use chondroitin lyase enzymes in the degradation of CS/DS-containing sample and how to assay the activity of these enzymes.

2. Materials

Was this article helpful?

0 0
Healthy Chemistry For Optimal Health

Healthy Chemistry For Optimal Health

Thousands Have Used Chemicals To Improve Their Medical Condition. This Book Is one Of The Most Valuable Resources In The World When It Comes To Chemicals. Not All Chemicals Are Harmful For Your Body – Find Out Those That Helps To Maintain Your Health.

Get My Free Ebook

Post a comment