Two Distinct Chondroitin Sulfate ABC Lyases

Abstract

Crude enzyme obtained from chondroitin sulfate-induced Proteus vulgaris NCTC 4636 has been fractionated into 1) an endoeliminase capable of depolymerizing chondroitin sulfate and related polysaccharides to produce, as end products, a mixture of ⌬ 4 -unsaturated tetra-and disaccharides and 2) an exoeliminase preferentially acting on chondroitin sulfate tetra-and hexasaccharides to yield the respective disaccharides. Isolation of the two enzymes was achieved by a simple two-step procedure: extracting the enzymes from intact P. vulgaris cells with a buffer solution of nonionic surfactant and then treating the extract by cation-exchange chromatography. Each of the enzymes thus prepared was apparently homogeneous as assessed by SDS-polyacrylamide gel electrophoresis and readily crystallized from polyethylene glycol solutions. Both enzymes acted on various substrates such as chondroitin sulfate, chondroitin sulfate proteoglycan, and dermatan sulfate at high, but significantly different, initial rates. They also attacked hyaluronan but at far lower rates and were inactive to keratan sulfate, heparan sulfate, and heparin. Our results show that the known ability of the conventional enzyme called "chondroitinase ABC" to catalyze the complete depolymerization of chondroitin sulfates to unsaturated disaccharides may actually result from the combination reactions by endoeliminase (chondroitin sulfate ABC endolyase) and exoeliminase (chondroitin sulfate ABC exolyase).Chondroitin sulfate ABC lyase (EC 4.2.2.4) was first purified from extracts of Proteus vulgaris NCTC 4636 adapted to chondroitin 6-sulfate (1). It is believed to be an endoeliminase that splits ␤1,4-galactosaminidic bonds between N-acetylgalactosamine and either D-glucuronic acid or L-iduronic acid and degrades, therefore, a variety of glycosaminoglycans of the chondroitin sulfate and dermatan sulfate type to the respective unsaturated disaccharides. Using this enzyme, both chondroitin sulfates and dermatan sulfates were shown to contain, in addition to predominant 4-or 6-sulfated disaccharide residues, a smaller proportion of nonsulfated or disulfated disaccharide residues, or both (2). A variety of studies have since shown that the proportion of these disaccharide residues varies greatly with species and anatomical sites, during development and aging, and in pathology (3-11 among others). Many of these data suggest that variation in the carbohydrate sequence and sulfation pattern may be used to specify the functional properties of chondroitin sulfate and dermatan sulfate proteoglycans.There is a commercially available chondroitin sulfate ABC lyase ("chondroitinase ABC" from P. vulgaris, the product of Seikagaku Corp.) which has found wide applications including the quantification of chondroitin sulfate and dermatan sulfate (12), the structural analysis of the carbohydrate moiety of proteoglycans (13-16), the preparation of core proteins from proteoglycans (13, 17), the formation of antigenic epitopes to prepare anti-proteoglycan monoclonal antibodies (18), and ...

Structures of Streptococcus pneumoniae Hyaluronate Lyase in Complex with Chondroitin and Chondroitin Sulfate Disaccharides

“…The distances associated with each individual (67,68). At the other end of the spectrum, P. vulgaris chondroitin sulfate ABC lyase can cleave unsulfated and sulfated chondroitins as well as dermatan sulfate (69). S. pneumoniae HL lies between these two extremes.…”

Binding of a Large Chondroitin Sulfate/Dermatan Sulfate Proteoglycan, Versican, to L-selectin, P-selectin, and CD44

“…These results indicate that versican binds L-and P-selectin and CD44 through its CS side chains. Although chondroitinase ABC can cleave both CS chains and HA, the processing rate for HA is slower (40); thus, we used a concentration of chondroitinase ABC (5 milliunits/ml) that selectively cleaves CS but not HA in Fig. 4C (also see Fig.…”

Structural Analysis of Bikunin Glycosaminoglycan

“…49,50 Endolytic chondroitinase AC acts exclusively on the β-1,4-galactosaminidic bonds between GalNAc and GlcA. 51 In contrast, chondroitinase B only cleaves β-1,4-galactosaminid-ic bonds between 4-O-sulfoGalNAc and IdoA residues, acting exclusively on CS-B.…”