A solid-phase substrate of heparanase: its application to assay of human melanoma for heparan sulfate degradative activity.
We found a tumor metastasis-associated heparan sulfate (HS)-degrading endoglycosidase in melanoma cells that is a unique endo-beta-glucuronidase (heparanase) capable of specifically cleaving HS at intrachain sites (M. Nakajima, T. Irimura, N. DiFerrante, and G. L. Nicolson, 1984, J. Biol. Chem. 259, 2283-2290). To perform rapid and microscale quantitative assays of heparanase we developed a solid-phase HS substrate by crosslinking radiolabeled HS onto agarose gel beads using one covalent linkage. The HS from bovine lung was partially N-desulfated and labeled with [14C]acetic anhydride. Free HS amino groups were completely acetylated, and reducing terminal saccharides were reductively aminated. The HS derivatives with amino groups at their reducing termini were coupled to amino-reactive agarose beads. Incubation of the solid-phase HS substrates with B16 melanoma cell extracts in the presence of D-saccharic acid 1,4-lactone (a potent exo-beta-glucuronidase inhibitor) resulted in the time- and dose-dependent release of [14C]HS fragments. Human melanoma cell lines were tested for HS-degrading endoglycosidase using the newly developed solid-phase HS substrates. The human malignant melanoma cells tested had high levels of HS-degrading activity that were comparable to those of highly metastatic murine B16-F10 melanoma cells.
D005966:Glucuronidase; D006026:Glycoside Hydrolases / Q000032:analysis*; D006493:Heparin; D006497:Heparan Sulfate; D006801:Humans; D008545:Melanoma / Q000201:enzymology*; D008970:Molecular Weight; D012685:Sepharose; D013379:Substrate Specificity; D000099175:Heparanase
D005966:Glucuronidase; D006026:Glycoside Hydrolases; D006493:Heparin; D006497:Heparan Sulfate; D012685:Sepharose; D000099175:Heparanase