Moonlighting Protein Glyceraldehyde-3-Phosphate Dehydrogenase: A Cellular Rapid-Response Molecule for Maintenance of Iron Homeostasis in Hypoxia.

Hypoxia triggers a rapid increase in iron demand to meet the requirements of enhanced erythropoiesis. The mobilization of iron stores from macrophage to plasma as holo-transferrin (Tf) from where it is accessible to erythroid precursor cells impacts iron homeostasis. Despite the immediate need for enhanced iron uptake by bone marrow cells, numerous studies have shown that transferrin receptor levels do not rise until more than 24 hours after the onset of hypoxia, suggesting the existence of heretofore unknown rapid response cellular machinery for iron acquisition in the early stages of cellular hypoxia.We performed flow cytometry to measure cell surface levels of TfR1, GAPDH, and Tf binding after hypoxia treatment. We utilized FRET analysis and co-immunoprecipitation methods to establish the interaction between Tf and GAPDH.In the current study, we demonstrated that hypoxia induces K562 cells to translocate the cytosolic moonlighting protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) onto cell surfaces and into the extracellular milieu to acquire transferrin-bound iron, even while levels of the classical transferrin receptor TfR1 (CD71) remain suppressed. GAPDH knockdown confirmed this protein's role in transferrin acquisition. Interestingly, macrophages did not show enhanced levels of extracellular GAPDH under hypoxia.Our results suggest the role of GAPDH-mediated Tf uptake as a rapid response mechanism by which cells acquire iron during the early stages of hypoxia. This is a tissue-specific phenomenon for the distinct requirements of cells that are consumers of iron versus cells that play a role in iron storage and recycling. This rapid deployment of an abundantly available multipurpose molecule allows hypoxic cells to internalize more Tf and maintain enhanced iron supplies in the early stages of hypoxia before specialized receptors can be synthesized and deployed to the cell membrane.