Abstract
GlcUAβ1-3GalNAc(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 Δ4,5HexUAα1-3GalNAc(4S,6S) at their non-reducing ends, indicating that the disulfation of GalNAc abrogates HCLase Er activity on the β1-4 linkage between the E unit and the following disaccharide. Δ4,5HexUAα1-3GalNAc(4S,6S)β1-4GlcUAβ1-3GalNAc(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 (Gly739-Gln796) 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.
Highlights
glucuronic acid (GlcUA)1–3GalNAc(4S,6S) (E unit)–rich domains have been shown to play key roles in various biological functions of chondroitin sulfate (CS)
The Simple Modular Architecture Research Tool (SMART), SignalP 4.0, and LipoP 1.0 analyses indicated that HCLase Er contained a type I signal peptide composed of 21 amino acid residues at its N terminus
The effects of pH on the reaction rate of rHCLase Er were investigated at the optimal temperature of 30 °C, and the results showed that the optimal pH was 8.0 when either hyaluronic acid (HA) or CS-C was used as the substrate, whereas the rate of enzymatic activity was much higher in 50 mM Tris-HCl buffer than in 50 mM NaH2PO41⁄7Na2HPO4 buffer (Fig. 3B)
Summary
The putative GAG-degrading gene named hclase er (GenBankTM accession number MF458894) was 2973 bp in length and had a GC content of 50.6%. The Simple Modular Architecture Research Tool (SMART), SignalP 4.0, and LipoP 1.0 analyses indicated that HCLase Er contained a type I signal peptide composed of 21 amino acid residues at its N terminus. Carbohydrate-active enZYme database (http://www.cazy.org/)3 [55], Simple Modular Architecture Research Tool, and BLASTp search showed that the HCLase Er protein contained a GAG_lyase superfamily module (Glu41–Pro695) and a lyase_8 module (Lys369– Pro617), suggesting that the enzyme is a member of polysaccharide lyase 8 family (Fig. 1A). A BLASTp search showed that HCLase Er shared a sequence identity of higher than 30% with a number of putative chondroitinases from bacteria such as the Vibrio, Bacteroides, Indibacter, Flavobacterium, and Pedobac-. Of the proteins identified, HCLase Er shared the highest percentages of sequence identity (34%) with a chondroitinase AC lyase from Flavobacterium heparinum [36] and followed by xanthan lyase (27%) from Bacillus sp. Compared with the two identified GAG lyases, chondroitinase AC (ACU03008.1) and HCLase (AIL54323.1), there are two long extra fragments (Gly739–Gln796 and Val816–Lys990) at the C terminus of HCLase Er protein (Fig. 1B)
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