The reticuloendothelial system plays a major role in iron metabolism. Despite this, the manner in which macrophages handle iron remains poorly understood. Mammalian cells utilize transferrin-dependent mechanisms to acquire iron via transferrin receptors 1 and 2 (TfR1 and TfR2) by receptor-mediated endocytosis. Here, we show for the first time that the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is localized on human and murine macrophage cell surface. The expression of this surface GAPDH is regulated by the availability of iron in the medium. We further demonstrate that this GAPDH interacts with transferrin and the GAPDHtransferrin complex is subsequently internalized into the early endosomes. Our work sheds new light on the mechanisms involved in regulation of iron, vital for controlling numerous diseases and maintaining normal immune function. Thus, we propose an entirely new avenue for investigation with respect to transferrin uptake and regulation mechanisms in macrophages.Iron is an essential nutrient for all organisms as a constituent of hemoproteins and iron-sulfur proteins. In addition it is also a critical component of functional groups of several proteins involved in vital housekeeping functions. Cells of the immune system require iron for their normal functions such as proliferation, activation, and maturation of lymphocytes (1-5). Iron is also essential for macrophage-mediated cytotoxicity by the production of highly toxic hydroxyl radicals (6, 7). The mononuclear phagocyte system is composed of monocytes, macrophages, and their precursor cells, which play a vital role in iron metabolism by removing effete erythrocytes and recycling iron. These cells also acquire iron via the receptor-mediated uptake of transferrin and the hemoglobin scavenger receptor (8). Practically all extracellular iron circulating in the plasma is bound to transferrin, an abundant protein with high affinity for iron. Two mammalian transferrin receptors TfR1 4 and TfR2 have so far been characterized. Both these receptors are cell surface transmembrane, glycoproteins (9). Unlike TfR1, TfR2 is not regulated by intracellular iron concentrations. This receptor also binds transferrin in manner similar to TfR1, but with a 25-fold lower affinity (10,11,12). Iron uptake from transferrin involves binding to its receptors followed by internalization to the early endosomes. (13, 14). Although TfR-mediated iron uptake is the major pathway for iron acquisition, several studies have indicated that additional mechanisms independent of known TfRs exist; however, these have not been well characterized (15-18). GAPDH was previously considered to be an abundantly present cytosolic protein with a key role in energy metabolism. However, recent evidence has proved that it functions as a moonlighting protein in both prokaryotic and eukaryotic cells, often differentially localized within the cell (19 -21). It is of interest to note that in Staphylococcus aureus, cell wall-associated GAPDH had previously been identified as a trans...

Mycobacterium tuberculosis (M.tb), which requires iron for survival, acquires this element by synthesizing iron-binding molecules known as siderophores and by recruiting a host iron-transport protein, transferrin, to the phagosome. The siderophores extract iron from transferrin and transport it into the bacterium. Here we describe an additional mechanism for iron acquisition, consisting of an M.tb protein that drives transport of human holo-transferrin into M.tb cells. The pathogenic strain M.tb H37Rv expresses several proteins that can bind human holo-transferrin. One of these proteins is the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Rv1436), which is present on the surface of M.tb and its relative Mycobacterium smegmatis. Overexpression of GAPDH results in increased transferrin binding to M.tb cells and iron uptake. Human transferrin is internalized across the mycobacterial cell wall in a GAPDH-dependent manner within infected macrophages.

Due to their abundant ubiquitous presence, rapid uptake and increased requirement in neoplastic tissue, the delivery of the iron carrier macromolecules transferrin (Tf) and lactoferrin (Lf) into mammalian cells is the subject of intense interest for delivery of drugs and other target molecules into cells. Utilizing exosomes obtained from cells of diverse origin we confirmed the presence of the multifunctional protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which has recently been characterized as a Tf and Lf receptor. Using a combination of biochemical, biophysical and imaging based methodologies, we demonstrate that GAPDH present in exosomes captures Tf and Lf and subsequently effectively delivers these proteins into mammalian cells. Exosome vesicles prepared had a size of 51.2 ± 23.7 nm. They were found to be stable in suspension with a zeta potential (ζ-potential) of -28.16 ± 1.15 mV. Loading of Tf/Lf did not significantly affect ζ-potential of the exosomes. The carrier protein loaded exosomes were able to enhance the delivery of Tf/Lf by 2 to 3 fold in a diverse panel of cell types. Ninety percent of the internalized cargo via this route was found to be specifically delivered into late endosome and lysosomes. We also found exosomes to be tunable nano vehicles for cargo delivery by varying the amount of GAPDH associated with exosome. The current study opens a new avenue of research for efficient delivery of these vital iron carriers into cells employing exosomes as a nano delivery vehicle.

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