title: Endocytosis of extracellular superoxide dismutase into endothelial cells: role of the heparin-binding domain.
abstract: 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.
  
  
  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.
authors.affiliation: Cardiovascular Center and Department of Internal Medicine, University of Iowa, Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA 52242, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Chu
authors.forename: Robert
authors.initials: R
authors.lastname: Piper
authors.forename: Simon
authors.initials: S
authors.lastname: Richardson
authors.forename: Yoshimasa
authors.initials: Y
authors.lastname: Watanabe
authors.forename: Pragnesh
authors.initials: P
authors.lastname: Patel
authors.forename: Donald D
authors.initials: DD
authors.lastname: Heistad
mesh_terms: D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology; D002966:Clathrin / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology*; D042783:Endothelial Cells / Q000378:metabolism*; D005110:Extracellular Space / Q000201:enzymology*; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D051379:Mice; D017434:Protein Structure, Tertiary / Q000502:physiology; D013482:Superoxide Dismutase / Q000235:genetics* / Q000378:metabolism*; D014018:Tissue Distribution
chemical_list: D002966:Clathrin; D006493:Heparin; C441642:SOD3 protein, human; D013482:Superoxide Dismutase