New insights on the specificity of heparin and heparan sulfate lyases fromFlavobacterium heparinum revealed by the use of synthetic derivatives of K5 polysaccharide from E-coli and 2-O-desulfated heparin

The capsular polysaccharide from E. Coli, strain K5 composed of ....--> 4)b eta-D-GlcA(1 --> 4)alpha-D-GlcNAc(1 --> 4)beta-D-GlcA (1 -->....., chemical ly modified K5 polysaccharides, bearing sulfates at C-2 and C-6 of the hexo samine moiety and at the C-2 of the glucuronic acid residues as well as 2-O desulfated heparin were used as substrates to study the specificity of hep aritinases I and II and heparinase from Flavobacterium heparinum. The natur al K5 polysaccharide was susceptible only to heparitinase I forming Delta U -GlcNAc. N-deacetylated, N-sulfated K5 became susceptible to both heparitin ases I and II producing Delta U-GlcNS. The K5 polysaccharides containing su lfate at the C-2 and C-6 positions of the hexosamine moiety and C-2 positio n of the glucuronic acid residues were susceptible only to heparitinase II producing Delta U-GlcNS,6S and Delta U,2S-GlcNS,6S respectively. These comb ined results led to the conclusion that the sulfate at C-6 position of the glucosamine is impeditive for the action of heparitinase I and that heparit inase II requires at least a C-2 or a C-6 sulfate in the glucosamine residu es of the substrate for its activity. Iduronic acid-2-O-desulfated heparin was susceptible only to heparitinase II producing Delta U-GlcNS,6S. All the modified K5 polysaccharides as well as the desulfated heparin were not sub strates for heparinase. This led to the conclusion that heparitinase II act s upon linkages containing non-sulfated iduronic acid residues and that hep arinase requires C-2 sulfated iduronic acid residues for its activity.