Processing of heparanase is mediated by syndecan-1 cytoplasmic domain and involves syntenin and α-actinin

Abstract

Heparanase activity plays a decisive role in cell dissemination associated with cancer metastasis. Cellular uptake of heparanase 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. Heparan sulfate proteoglycans, and particularly syndecan, are instrumental for heparanase uptake and activation, through a process that has been shown to occur independent of rafts. Nevertheless, the molecular mechanism underlying syndecan mediated internalization outside of rafts is unclear. Here, we examined the role of syndecan-1 cytoplasmic domain in heparanase processing, utilizing deletion constructs lacking the entire cytoplasmic domain (delta), the conserved (C1 or C2) or variable (V) regions. Heparanase processing was markedly increased following syndecan-1 over expression; 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 revealed that conserved domain 2 (C2) and variable (V) regions of syndecan-1 cytoplasmic tail mediate heparanase processing. Furthermore, we found that syntenin, known to interact with syndecan C2 domain, and α actinin are essential for heparanase processing.

Heparanase Upregulation Contributes to Porcine Reproductive and Respiratory Syndrome Virus Release

“…Processing and activation of heparanase occur after proteolytic processing by cathepsin L, a characteristic lysosomal cysteine proteinase of the papain superfamily of peptidases that is implicated in multiple physiological and pathological processes (23)(24)(25). To investigate whether PRRSV infection activates heparanase via cathepsin L, cathepsin L activity in Marc-145 cells was initially assessed at various times or MOIs after PRRSV infection using a Magic Red cathepsin L detection kit.…”

Heparanase Enhances Tumor Growth and Chemoresistance by Promoting Autophagy

“…Preparation of cell and tissue extracts and immunoblotting were carried out essentially as described (10). Following induction of autophagy, the cytosolic form of LC3 (LC3-I) is conjugated to phosphatidylethanolamine (LC3-II) which is recruited to autophagosomal membranes, serving as a most acceptable mean to measure autophagy levels.…”

“…Immunofluorescent staining of methanol-fixed cells and analysis by confocal microscopy was carried out essentially as described (10). In order to better detect LC3 by immunofluorescence or electron microscopy (EM), autophagy was initiated by depriving the cells of amino acids (AA) for 3 h, in the presence of chloroquine (Chl, 50 μg/ml; Sigma).…”

“…In addition, the multitude of polypeptides sequestered and regulated by HS and the ability of heparanase to convert these into bio-available molecules (5, 6) require that these activities will be kept tightly regulated. We have shown previously that following secretion as a latent 65 kDa enzyme, heparanase rapidly interacts with cell membrane HS proteoglycans (HSPG; i.e., syndecans) (7–10), followed by internalization and processing into a highly active 50 kDa enzyme (7, 11). Notably, heparanase was shown to reside primarily within endocytic vesicles, assuming a polar, peri-nuclear localization and co-localizing with lysosomal markers (7, 8, 10, 12).…”

“…We have shown previously that following secretion as a latent 65 kDa enzyme, heparanase rapidly interacts with cell membrane HS proteoglycans (HSPG; i.e., syndecans) (7–10), followed by internalization and processing into a highly active 50 kDa enzyme (7, 11). Notably, heparanase was shown to reside primarily within endocytic vesicles, assuming a polar, peri-nuclear localization and co-localizing with lysosomal markers (7, 8, 10, 12). In addition, it has been demonstrated that incubation with endosomal/lysosomal fraction, but not membrane or cytosolic preparations, leads to heparanase processing and activation (13).…”