A chondroitin sulfate and hyaluronic acid lyase with poor activity to glucuronyl 4,6-O-disulfated N-acetylgalactosamine (E-type)-containing structures

GlcUA beta 1-3GalNAc(4S,6S) (E unit)-rich domains have been shown to play key roles in various biological functions of chondroitin sulfate (CS). However, an enzyme that can specifically isolate such domains through the selective digestion of other domains in polysaccharides has not yet been reported. Here, we identified a glycosaminoglycan lyase from a marine bacterium Vibrio sp. FC509. This enzyme efficiently degraded hyaluronic acid (HA) and CS variants, but not E unit-rich CS-E, into unsaturated disaccharides; therefore, we designated this enzyme a CS-E-resisted HA/CS lyase (HCLase Er). We isolated a series of resistant oligosaccharides from the final product of a low-sulfated CS-E exhaustively digested by HCLase Er and found that the E units were dramatically accumulate in these resistant oligosaccharides. By determining the structures of several resistant tetrasaccharides, we observed that all of them possessed a Delta(4,5)HexUA alpha 1-3GalNAc(4S,6S) at their non-reducing ends, indicating that the disulfation of GalNAc abrogates HCLase Er activity on the beta 1-4 linkage between the E unit and the following disaccharide. Delta(4,5)HexUA alpha 1-3GalNAc(4S,6S)beta 1-4GlcUA beta 1-3GalNAc (4S,6S) was most strongly resistant to HCLase Er. To our knowledge, this study is the first reporting a glycosaminoglycan lyase specifically inhibited by both 4-O- and 6-O-sulfation of GalNAc. Site-directed and truncation mutagenesis experiments indicated that HCLase Er may use a general acid-base catalysis mechanism and that an extra domain (Gly(739)-Gln(796)) is critical for its activity. This enzyme will be a useful tool for structural analyses and for preparing bioactive oligosaccharides of HA and CS variants, particularly from E unit-rich CS chains.