title: Immunotargeting of glucose oxidase: intracellular production of H(2)O(2) and endothelial oxidative stress.
abstract: Extracellular and intracellular reactive oxygen species attack different targets and may, therefore, result in different forms of oxidative stress. To specifically study an oxidative stress induced by a regulated intracellular flux of a defined reactive oxygen species in endothelium, we used immunotargeting of the H(2)O(2)-generating enzyme glucose oxidase (GOX) conjugated with an antibody to platelet-endothelial cell adhesion molecule (PECAM)-1, an endothelial surface antigen. Anti-PECAM-(125)I-GOX conjugates specifically bind to both endothelial and PECAM-transfected cells. Approximately 70% of cell-bound anti-PECAM-(125)I-GOX was internalized. The cell-bound conjugate was enzymatically active and generated H(2)O(2) from glucose. Use of the fluorescent dye dihydrorhodamine 123 revealed that 70% of H(2)O(2) was generated intracellularly, whereas 30% of H(2)O(2) was detected in the cell medium. Catalase added to the cells eliminated H(2)O(2) in the medium but had little effect on the intracellular generation of H(2)O(2) by anti-PECAM-GOX. Both H(2)O(2) added exogenously to the cell medium (extracellular H(2)O(2)) and that generated by anti-PECAM-GOX caused oxidative stress manifested by time- and dose-dependent irreversible plasma membrane damage. Inactivation of cellular catalase by aminotriazole treatment augmented damage caused by either extracellular H(2)O(2) or anti-PECAM-GOX. Catalase added to the medium protected either normal or aminotriazole-treated cells against extracellular H(2)O(2), yet failed to protect cells against injury induced by anti-PECAM-GOX. Therefore, treatment of PECAM-positive cells with anti-PECAM-GOX leads to conjugate internalization, predominantly intracellular H(2)O(2) generation and intracellular oxidative stress. These results indicate that anti-PECAM-GOX 1) provides cell-specific intracellular delivery of an active enzyme and 2) causes intracellular oxidative stress in PECAM-positive cells.
authors.affiliation: Institute for Environmental Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6068, USA.
authors.forename: A J
authors.initials: AJ
authors.lastname: Gow
authors.forename: F
authors.initials: F
authors.lastname: Branco
authors.forename: M
authors.initials: M
authors.lastname: Christofidou-Solomidou
authors.forename: L
authors.initials: L
authors.lastname: Black-Schultz
authors.forename: S M
authors.initials: SM
authors.lastname: Albelda
authors.forename: V R
authors.initials: VR
authors.lastname: Muzykantov
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology; D000941:Antigens / Q000276:immunology; D002374:Catalase / Q000502:physiology; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005110:Extracellular Space / Q000378:metabolism; D005949:Glucose Oxidase / Q000276:immunology; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007425:Intracellular Membranes / Q000201:enzymology / Q000378:metabolism*; D018384:Oxidative Stress / Q000502:physiology*; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000276:immunology / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D000941:Antigens; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D006861:Hydrogen Peroxide; D005949:Glucose Oxidase; D002374:Catalase