Heparanase is the only functional endoglycosidase capable of cleaving heparan sulfate (HS)in mammals, activity that is highly implicated in cell dissemination associated with tumor metastasis, inflammation and angiogenesis. Heparanase uptake is considered a pre-requisite for the delivery of latent 65 kDa heparanase to lysosomes and its subsequent proteolytic processing and activation into 8 and 50 kDa protein subunits by cathepsin L. Efficient uptake of heparanase was evident also by GPI-deficient cells (i.e., lack cell surface glypicans), suggesting preferential involvement of syndecans in this process. The molecular mechanism underlying heparanase uptake is still largely obscure. Here, we examined the necessity of syndecan 1 cytoplasmic domain for heparanase internalization and processing. To this end, we transfected cells with full length mouse syndecan 1 or deletion constructs lacking the entire cytoplasmic domain (delta), the conserved (C1 or C2) or variable (V) regions. Heparanase binding, internalization and processing were then evaluated by immunofluorescent staining and immunoblotting. Heparanase uptake was markedly increased following syndecan 1 over expression, thus challenging the notion that HS coat of the cell membrane is at saturation and does not limit ligand binding. In contrast, heparanase was retained at the cell membrane and its processing was impaired in cells over expressing syndecan 1 deleted for the entire cytoplasmic tail. We have next identified that conserved domain 2 (C2) and variable (V) regions of syndecan 1 cytoplasmic tail mediate heparanase internalization and processing. Furthermore, we found that syntenin, known to interact with syndecan C2 domain and α actinin, mediates heparanase uptake. These results further illustrate the tight regulation of heparanase uptake and bioavailability.
