D-Amino Acid Oxidase as a Chemogenetic Tool for Spatiotemporally
Controlled Hydrogen Peroxide Production: The Oxygen Connection
Abstract
Background and Purpose: D-amino acid oxidase (DAO) has been developed as
a chemogenetic tool to precisely manipulate redox levels in tissues and
cells by generating H2O2 in the presence of D-amino acids. The enzyme’s
significant oxygen consumption during H2O2 production may influence
cellular oxygen levels. We set out to explore if DAO activation results
in hypoxia, which in turn alters the activity of the cellular oxygen
sensors that are prolyl-4-hydroxylase domain enzymes and thus the
hypoxia-inducible factor (HIF) pathway. Experimental Approach: We
analyzed oxygen consumption rate (OCR), pericellular oxygen
concentration, and HIF-1α stabilization in HEK293 cells stably
overexpressing DAO in either the cytoplasm or mitochondrial matrix. The
obtained experimental data were used to test whether a simple
mathematical model can be used to predict oxygen levels. Key Result:
Using HEK293 cells expressing DAO in either the cytoplasm or
mitochondrial matrix, we found a dose-dependent increase in OCR upon
stimulating the DAO reaction, which resulted in a decreased pericellular
O2. Correspondingly, HIF-1α protein levels were stabilized over time,
reflecting the biological cellular response to DAO-induced hypoxia.
Furthermore, we provide a spreadsheet with a simplified diffusion model
that allows users to input experimental parameters and assess the risk
of hypoxic conditions in cell cultures. Conclusion and Implications:
High DAO activity can induce hypoxia due to increased oxygen
consumption. Caution should be taken to prevent hypoxic conditions in
cell culture experiments while using DAO. The provided spreadsheet is
intended to help control oxygen levels in combination with OCR
measurements.