Abstract
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, Cys127-Cys179 and Cys437-Cys542, and one S-cysteinylation at the Cys211 residue. It was shown that, although the formation of the Cys127-Cys179 bond and S-cysteinylation at Cys211 have little effect on heparanase function, the disulfide bond between Cys437 and Cys542 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.
Highlights
Heparan sulfate and heparan sulfate proteoglycans, which are located in the extracellular matrix and on the external surface of cell membranes, play a major role in cell-cell and cell-extracellular interactions [1,2,3,4]
Overexpression of heparanase has been observed in many human tumor types, such as those in the head and neck [19], pancreatic tumors [20], hepatocellular carcinoma [21], esophageal carcinoma [22], and cultured human tumor cell lines [19, 23]; such associations are thought to indicate the involvement of heparanase in tumor progression
Electrophoresis of heparanase from HT1080-HP-MH cells under nonreducing conditions revealed a more rapidly migrating form than that observed under reducing conditions, as detected by Western blotting (Fig. 1B); these findings suggested that heparanase contains at least one disulfide bond
Summary
Heparan sulfate and heparan sulfate proteoglycans, which are located in the extracellular matrix and on the external surface of cell membranes, play a major role in cell-cell and cell-extracellular interactions [1,2,3,4]. It has been reported that heparanase is translocated into the nucleus, degrades nuclear heparan sulfate, and thereby affects nuclear functions that are thought to be regulated by heparan sulfate [17, 18]. Overexpression of heparanase has been observed in many human tumor types, such as those in the head and neck [19], pancreatic tumors [20], hepatocellular carcinoma [21], esophageal carcinoma [22], and cultured human tumor cell lines [19, 23]; such associations are thought to indicate the involvement of heparanase in tumor progression. We previously showed that Nglycosylation is required for the secretion of heparanase protein in cultured cells [12], other posttranslational modifications responsible for the regulation of heparanase function have yet to be elucidated. It will be necessary to understand the activation mechanisms of heparanase in human tumor cells
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