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

Andrew J. Gow, Frank Branco, Melpo Christofidou-Solomidou, Linda Black-Schultz, Steven M. Albelda, Vladimir R. Muzykantov

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33 Citations (Scopus)

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 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.

Original languageEnglish
JournalAmerican Journal of Physiology - Lung Cellular and Molecular Physiology
Volume277
Issue number2 21-2
Publication statusPublished - Aug 1999
Externally publishedYes

Keywords

  • Bioconjugation
  • CD31
  • Drug delivery
  • Hydrogen peroxide
  • Platelet-endothelial cell adhesion molecule-1

ASJC Scopus subject areas

  • Pulmonary and Respiratory Medicine
  • Cell Biology
  • Physiology
  • Physiology (medical)

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  • Cite this

    Gow, A. J., Branco, F., Christofidou-Solomidou, M., Black-Schultz, L., Albelda, S. M., & Muzykantov, V. R. (1999). Immunotargeting of glucose oxidase: Intracellular production of H2O2 and endothelial oxidative stress. American Journal of Physiology - Lung Cellular and Molecular Physiology, 277(2 21-2).