Structural studies on the oligosaccharides isolated from bovine kidney heparan sulphate and characterization of bacterial heparitinases used as substrates.

We prepared a series of oligosaccharides from commercial bovine kidney heparan sulphate after limited digestion with heparitinase I from Flavobacterium heparinum, and determined the structures of eight tetrasaccharides and a hexasaccharide by enzymatic analysis, fast atom bombardment mass spectrometry and 500 MHz 1H NMR spectroscopy. The tetrasaccharides share the common core structure delta 4,5HexA alpha 1-4GlcN alpha 1-4HexA1-4GlcN (where delta 4,5HexA is 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid and HexA is hexuronic acid), with zero, one or two sulphate groups. Seven of them contain non-sulphated glucuronic or iduronic acid, and the other, 2-O-sulphated iduronic acid at the internal position. Although they contain ordinary structures which should be widely distributed in the relatively low-sulphated region of heparan sulphate, five of the tetrasaccharides were isolated for the first time as discrete structures. The structure of the hexasaccharide was determined as delta 4,5HexA alpha 1-4GlcNAc alpha 1-4GlcA beta 1-3Gal beta 1-3Gal beta-1-4 Xyl and is derived from the carbohydrate-protein linkage region of the heparan sulphate chains. The hexasaccharide seems to have been released by the alkaline treatment used to prepare the heparan sulphate. The Gal residues were non-sulphated as are those in the porcine intestinal heparin chains, but in contrast to the sulphated Gal structures previously demonstrated in the carbohydrate-protein linkage region of chondroitin sulphate chains. These oligosaccharides were used to investigate the substrate specificity of heparitinases I and II from F. heparinum. The results revealed that heparitinase I cleaves hexosaminidic bonds linked to non-sulphated glucuronic or iduronic acid residues. The glucosaminidic linkage of the hexasaccharide was sensitive to heparitinase I, but resistant to heparitinase II, demonstrating the differential specificity of these enzymes towards the carbohydrate-protein linkage region.