Fig. 4. O-sulfo-S-linked heparosan analog enzyme challenges and inhibition.

a Cleavage sites of the heparosan chain by the human heparanase or the bacterial heparin lyase enzymes, and the position of the sulfur atom substitution in the hemi-A linkage (for the various pendant sulfo-positions in the sulfated products, see Fig. 3). b The chemically O-sulfated S-link analog or natural O-link heparosan (0, starting polymers without enzyme) were tested for cleavage by overnight treatment with either the recombinant human heparanase (H) or a mixture of recombinant Flavobacterium heparin lyases I, II, and III (L) (n = 1; also see Supplementary Fig. 54 for an independent heparanase challenge). PAGE analysis (10% gel) with Stains-All detection showed that the S-link polymer is resistant to heparanase digestion at its thio-glycosidic linkage GlcA-S-GlcNAc sites but cleaved by the lyases at the GlcNAc-O-GlcA sites with natural glycosidic linkages. As expected, both types of enzymes can digest the natural glycosidic linkages of O-sulfo-O-linked HS. c PAGE analysis (20% gel) of the kinetics (20-min point) of heparanase enzyme (Hepase) cleavage of a fluorescent sulfo-O-linked heparosan substrate (starting material marked with arrow) with a titration of a S-linked analog inhibitor at decreasing concentrations from left to right (either chemically, Chem, or enzymatically, Enz, sulfated polymer; Sub/inhib, substrate molecules:inhibitor molecule molar ratio) (one of 7 titrations shown, each with 2 timepoints and 2 gels). Reaction controls: one quarter or half of the enzyme (2 leftmost lanes; the bracket denotes the approximate extent of fragments with ½× enzyme); half of the substrate without enzyme (rightmost lane).