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 DeltaHexA-GlcNH(3)(+),DeltaHexA-GlcNH(3)(+)(6S),DeltaHexA(2S)-GlcNH(3)(+), and DeltaHexA(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 DeltaHexA-GlcNH(3)(+)(6S),DeltaHexA(2S)-GlcNH(3)(+), and DeltaHexA(2S)-GlcNH(3)(+)(6S) disaccharides, whereas heparinase III yielded only the DeltaHexA-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.