Endocytosis of extracellular superoxide dismutase into endothelial cells: role of the heparin-binding domain.

OBJECTIVE Extracellular superoxide dismutase (EC-SOD) is a secreted antioxidant enzyme that binds to the outer plasma membrane and extracellular matrix through its heparin-binding domain (HBD). Carriers of a common genetic variant of EC-SOD (EC-SOD(R213G), within the HBD) have higher plasma concentration of EC-SOD and increased risk for vascular disease. In the present study, we used confocal fluorescence microscopy to examine mechanisms of endocytosis of EC-SOD to determine whether EC-SOD translocates to the nucleus of endothelial cells, and to test the hypothesis that EC-SOD, but not EC-SOD(R213G), is endocytosed into endothelial cells. METHODS AND RESULTS Mouse endothelial cells (MS-1) were incubated with EC-SOD, EC-SOD(R213G), or HBD-deleted EC-SOD (EC-SODdeltaHBD). Binding to MS-1 was observed only with EC-SOD, but not EC-SOD(R213G) or EC-SODdeltaHBD. Endocytosis of EC-SODs was monitored after coincubation of MS-1 cells with EC-SODs and BSA-Texas Red (BSA-TR), which marks endosomes and lysosomes. Only EC-SOD was endocytosed, colocalizing with BSA-TR. EC-SOD also colocalized with early endosome antigen 1 (EEA-1), a specific marker for endocytosis. Endocytosis of EC-SOD was inhibited by chlorpromazine, but not by methyl-beta-cyclodextrin or nystatin, which suggests that endocytosis of EC-SOD is mediated by clathrin but not by caveolae. Minimal or no localization of EC-SOD in the nucleus of MS-1 cells was detected. CONCLUSIONS Our findings indicate that EC-SOD, but not EC-SOD(R213G), is endocytosed into endothelial cells through clathrin-mediated pathway, but does not translocate to the nucleus. We speculate that impairment of endocytosis may contribute to high plasma levels of EC-SOD(R213G) in R213G carriers.