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

Extracellular and intracellular reactive oxygen species attack different ta rgets and may, therefore, result in different forms of oxidative stress. To specifically study an oxidative stress induced by a regulated intracellula r flux of a defined reactive oxygen species in endothelium, we used immunot argeting of the H2O2-generating enzyme glucose oxidase (GOX) conjugated wit h an antibody to platelet-endothelial cell adhesion molecule (PECAM)-1, an endothelial surface antigen. Anti-PECAM-I-125-GOX conjugates specifically b ind to both endothelial and PECAM-transfected cells. Approximately 70% of c ell-bound anti-PECAM-I-125-GOX was internalized. The cell-bound conjugate w as enzymatically active and generated H2O2 from glucose. Use of the fluores cent dye dihydrorhodamine 123 revealed that 70% of H2O2 was generated intra cellularly, whereas 30% of H2O2 was detected in the cell medium. Catalase a dded to the cells eliminated H2O2 in the medium but had little effect on th e intracellular generation of H2O2 by anti-PECAM-GOX. Both H2O2 added exoge nously to take cell medium (extracellular H2O2) and that generated by anti- PECAM-GOX caused oxidative stress manifested by time- and dose-dependent ir reversible plasma membrane damage. Inactivation of cellular catalase by ami notriazole treatment augmented damage caused by either extracellular H2O2 o r anti-PECAM-GOX. Catalase added to the medium protected either normal or a minotriazole-treated cells against extracellular H2O2, yet failed to protec t cells against injury induced by anti-PECAM-GOX. Therefore, treatment of P ECAM-positive cells with anti-PECAM-GOX leads to conjugate internalization, predominantly intracellular H2O2 generation and intracellular oxidative st ress. These results indicate that anti-PECAM-GOX 1) provides cell-specific intracellular delivery of an active enzyme and 2) causes intracellular oxid ative stress in PECAM-positive cells.