Identification of human hyaluronidase-4 as a novel chondroitin sulfate hydrolase that preferentially cleaves the galactosaminidic linkage in the trisulfated tetrasaccharide sequence

Abstract

Human hyaluronidases have been considered to be the enzymes acting at the initial step in the catabolism of chondroitin sulfate (CS) in vivo. However, human hyaluronidase-1 digests CS more slowly than hyaluronan (HA), and its preferred substrate is HA rather than CS. We have identified a chondroitin hydrolase in Caenorhabditis elegans, which effectively degrades chondroitin but depolymerizes HA to a much lesser extent (Kaneiwa T, Yamada S, Mizumoto S, Montaño AM, Mitani S, Sugahara K. 2008. Identification of a novel chondroitin hydrolase in Caenorhabditis elegans. J Biol Chem. 283:14971-14979), suggesting the existence of CS-specific endoglycosidases in mammalian systems. In this study, human hyaluronidase-4 was demonstrated to be a CS-specific endo-beta-N-acetylgalactosaminidase. This is the first demonstration of a CS hydrolase in higher organisms. The specificity of a purified recombinant form of the enzyme was investigated in detail through the characterization of degradation products. The best substrate of the CS hydrolase was the galactosaminidic linkage in the sequence of a trisulfated tetrasaccharide GlcUA(2-O-sulfate)-GalNAc(6-O-sulfate)-GlcUA-GalNAc(4-O- or 6-O-sulfate), where GlcUA and GalNAc represent D-glucuronic acid and N-acetyl-D-galactosamine, respectively. The disaccharide unit on the nonreducing side, GlcUA(2-O-sulfate)-GalNAc(6-O-sulfate) (D unit), is rich in shark fin cartilage CS-D among various CS isoforms. CS hydrolase will be a useful tool for investigating CS-specific functions in tissues and cells. In addition, it may well be applicable to the treatment of acute spinal cord injuries as in the case of, or instead of, the bacterial CS lyase which has been used for recent clinical trials.

Secondary Storage of Dermatan Sulfate in Sanfilippo Disease

“…3B) (28). Similarly, the first step in chondroitin/dermatan sulfate degradation is thought to involve partial endolytic processing by two enzymes with endo-␤-Nacetylhexosaminidase or endohexuronidase activities, respectively (29,30). Endolytic processing might expedite catabolic turnover by creating more ends on which exolytic hydrolases act, but other studies suggest that the overall rate of catabolism of heparan sulfate may not be affected by reduced endoglycosidic cleavage (31).…”

Glycosaminoglycans in Human and Bovine Serum: Detection of Twenty-Four Heparan Sulfate and Chondroitin Sulfate Motifs Including a Novel Sialic Acid-Modified Chondroitin Sulfate Linkage Hexasaccharide

“…74 A CS specific hyaluronidase has been characterized recently. 75 Free GAG chains with GalNAc at reducing end (Indicated by a GalNAc-OH end in Fig. 4b) may have been generated from proteoglycans by such hyaluronidases.…”

Mass preparation of oligosaccharides by the hydrolysis of chondroitin sulfate polysaccharides with a subcritical water microreaction system

“…The 2AB-derivatized oligosaccharides generated by hydrolysis with subcritical water were digested with CSase ABC and/or AC-II [9,25,37]. Digests were analyzed by anion-exchange HPLC as described above, and eluates were monitored by measuring fluorescence.…”

“…However, they are eliminases not hydrolases; therefore, degradation products contain an artificial unsaturated hexuronic acid at the 4 nonreducing end, contributing to strong antigenicity. Although hyaluronidases are hydrolases and depolymerize not only hyaluronan but also CS [9,10], these enzymes are not suitable for the mass production of CS oligosaccharides because of their lowthroughput. Major chemical methods for the preparation of CS oligosaccharides are acid hydrolysis [11][12][13] and methanolysis [14].…”