Involvement of disulfide bond formation in the activation of heparanase

Heparanase is overexpressed in many solid tumor cells and is capable of specifically cleaving heparan sulfate, and this activity is associated with the metastatic potential of tumor cells; however, the activation mechanism of heparanase has remained unknown. In this study, we investigated the link between disulfide bond formation and the activation of heparanase in human tumor cells. Mass spectrometry analysis of heparanase purified from a conditioned medium of human fibrosarcoma cells revealed two disulfide bonds, Cys(127)-Cys(179) and Cys(437)-Cys(542), and one S-cysteinylation at the Cys(211) residue. It was shown that, although the formation of the Cys127-Cys(179) bond and S-cysteinylation at Cys(11) have little effect on heparanase function, the disulfide bond between Cys(437) and Cys(542) is necessary for the secretion and activation of heparanase. Thus, the present findings will provide a basis for the further refinement of heparanase structural studies and for the development of novel heparanase inhibitors.