The Macrophage Cell Surface Glyceraldehyde-3-phosphate Dehydrogenase Is a Novel Transferrin Receptor

Abstract

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 acquires iron by cell-surface sequestration and internalization of human holo-transferrin

“…We selected GAPDH for further analysis because: (i) GAPDH has recently been identified as a transferrin receptor in macrophages and numerous other cell types [26][27][28] , (ii) the highly conserved nature of the protein and (iii) its role as a virulence factor [29][30][31][32] .…”

“…GAPDH has also been reported as a transferrin-binding protein in S. aureus and S. epidermidis 21 . In eukaryotes, numerous functions have been attributed to GAPDH including its role as a receptor for transferrin in different cell types and especially in macrophages [26][27][28]39 . Since this molecule is highly conserved across species (B50% identity with human GAPDH), we initially selected GAPDH to further explore the role of this transferrinbinding protein in M.tb.…”

“…Cells were washed and blocked as for TEM studies and incubated with 20 mg transferrin-Alexa 488 (Tf-A488, Invitrogen) in 100 ml PBS, 1% BSA for 2 h at 4°C. Samples were fixed in 4% buffered paraformaldehyde for 20 min and FRET analysis was carried out by the acceptor photobleaching method on a Nikon A1R confocal microscope as described previously 26 . The intensities of a total of 12 regions of interest from multiple, randomly selected fields were measured before and after photobleaching.…”

“…M.tb H37Ra, GAPDH mCh and mCh transformants were pelleted and washed with neutral buffer 26 . Cells were then resuspended at a concentration of 1 Â 10 8 cells per assay tube in 200 ml assay buffer (pH 8.0) as described earlier.…”

Identification of novel pathways in pathogenesis of ketosis in dairy cows via iTRAQ/MS

“…G3PD, initially well characterised for its role in glycolysis, is now recognised as a novel multifunctional protein exhibiting new functions including the regulation of mRNA stability (4-6, 13, 20, 31), intracellular membrane trafficking (26), iron uptake and transport (17), haeme metabolism (7), maintenance of genomic integrity (3,8,15), regulation of gene expression (10,30), and nuclear tRNA export (23). SORD is a key enzyme in the polyol metabolic pathway.…”