Immunotargeting of glucose oxidase: Intracellular production of H2O2 and endothelial oxidative stress

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 H₂O₂-generating enzyme glucose oxidase (GOX) conjugated with an antibody to platelet-endothelial cell adhesion molecule (PECAM)-1, an endothelial surface antigen. Anti-PECAM-¹²⁵I-GOX conjugates specifically bind to both endothelial and PECAM-transfected cells. Approximately 70% of cell-bound anti-PECAM-¹²⁵I-GOX was internalized. The cell-bound conjugate was enzymatically active and generated H₂O₂ from glucose. Use of the fluorescent dye dihydrorhodamine 123 revealed that 70% of H₂O₂ was generated intracellularly, whereas 30% of H₂O₂ was detected in the cell medium. Catalase added to the cells eliminated H₂O₂ in the medium but had little effect on the intracellular generation of H₂O₂ by anti- PECAM-GOX. Both H₂O₂ added exogenously to the cell medium (extracellular H₂O₂) 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 H₂O₂ or anti-PECAM-GOX. Catalase added to the medium protected either normal or aminotriazole-treated cells against extracellular H₂O₂, 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 H₂O₂ 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.