Distinct substrate specificities of bacterial heparinases against N-unsubstituted glucosamine residues in heparan sulfate

The rare N- unsubstituted glucosamine ( GlcNH(3)(+)) residues in heparan sulfate have important biological and pathophysiological roles. In this study, four GlcNH(3)(+)-containing disaccharides were obtained from partially de- N- sulfated forms of heparin and the N- sulfated K5 polysaccharide by digestion with combined heparinases I, II, and III. These were identified as Delta HexA- GlcNH(3)(+), Delta HexA- GlcNH(3)(+)( 6S), Delta HexA(2S)- GlcNH(3)(+), and Delta HexA-( 2S)- GlcNH(3)(+) ( 6S). Digestions with individual enzymes revealed that heparinase I did not cleave at GlcNH(3)(+) residues; however, heparinases II and III showed selective and distinct activities. Heparinase II generated Delta HexA- GlcNH(3)(+)( 6S), Delta HexA( 2S)- GlcNH(3)(+), and Delta HexA( 2S)- GlcNH(3)(+) ( 6S) disaccharides, whereas heparinase III yielded only the Delta HexA- GlcNH(3)(+) unit. Thus, the action of heparinase II requires O- sulfation, whereas heparinase III acts only on the corresponding non- sulfated unit. These striking distinctions in substrate specificities of heparinases could be used to isolate oligosaccharides with novel sequences of GlcNH(3)(+) residues. Finally, heparinases were used to identify and quantify GlcNH(3)(+)-containing disaccharides in native bovine kidney and porcine intestinal mucosal heparan sulfates. The relatively high content of O- sulfated GlcNH(3)(+)- disaccharides in kidney HS raises questions about how these sequences are generated.