Biochimica et Biophysica Acta (BBA) - General Subjects
Secreted glyceraldehye-3-phosphate dehydrogenase is a multifunctional autocrine transferrin receptor for cellular iron acquisition
Graphical abstract
Introduction
Iron is an essential element for the growth of all organisms [1]. Practically all extracellular iron is transported in the blood by the iron carrier protein transferrin, which facilitates cellular iron uptake via interaction with specific membrane receptors, transferrin receptors 1 and 2 (TfR1 and TfR2). Membrane bound Tf receptors play a key role in the process by which cells acquire iron for growth and other vital functions [2].
After binding to cell surface receptor, the Tf-receptor complex is internalized into endosomes where iron is released and apo-transferrin receptor complex is recycled [3], [4]. Both TfR1 and 2 are transmembrane glycoproteins. TfR1 is a high affinity ubiquitously expressed receptor while expression of TfR2 is restricted to certain cell types and is not regulated by intracellular iron concentrations. Although TfR1 mediated iron uptake is the major pathway for iron acquisition by most cells and especially developing erythrocytes [5], [6], [7], [8], several studies have indicated that the uptake mechanism varies depending upon the cell type. It is also reported that Tf uptake, independent of these TfRs exists although the mechanisms are not well characterized [8], [9], [10], [11], [12], [13].
Glyceraldehyde-3-phosphate dehydrogenase, a ubiquitously present glycolytic enzyme is predominantly localized in the cytosol. In addition to its well reported role for glycolysis, this protein exhibits numerous diverse functions [14].
Earlier we had reported that, GAPDH expressed on the surface of macrophage cells in an iron dependent manner, functions as a transferrin receptor [15]. Recently we have established that, apart from macrophages, GAPDH also functions as an additional transferrin receptor on numerous cell types and upon iron depletion many of them prefer to utilize their surface localized GAPDH for Tf-iron uptake instead of TfR1. The kinetics of transferrin associated iron delivery, by this pathway, have been characterized [16]. Recent investigations by proteomic based analysis of primary murine macrophage cells have confirmed the role of cell surface GAPDH in cellular iron homeostasis [17].
Apart from cytosolic localization, studies have reported the secretion of GAPDH (sGAPDH) by mammalian cells in culture [15], [18]. It also constitutes a normal component of serum [19]. As transferrin is an iron carrier molecule abundantly present in serum along with GAPDH, we decided to investigate the role of sGAPDH in Tf mediated iron uptake.
In the present study, we demonstrate that, upon iron depletion, GAPDH secretion from cells is enhanced. This can be correlated to an increase in the trafficking of transferrin and iron into cells. To understand the significance of this pathway for transferrin acquisition, we evaluated transferrin delivery in cell lines and primary cells that express known membrane bound transferrin receptors. Cell types that play a key role in mammalian iron homeostasis, such as hepatocytes and cells of the reticuloendothelial system were also selected. Enhanced Tf delivery by sGAPDH to mouse peritoneal macrophages and lymphocytes was also confirmed in vivo. The binding, internalization, recycling and degradation kinetics of this soluble receptor were studied in CHO-TRVb cells that lack TfR1 and 2 (a knock out system). Our findings reveal the dual role of GAPDH as a membrane associated and soluble receptor in Tf iron uptake.
Section snippets
Iron depletion in cells
Cells were cultured in RPMI-1640 supplemented with 10% FCS. For iron depletion, cells were cultured for 24 h in either, complete medium supplemented with 100 μM DFO (Sigma) or with Chelex-100 treated medium [15], [16]. Controls were set up in parallel with normal media. Iron depletion had no significant effect on cell viability which was confirmed by five independent methods as described earlier [16], in addition, elevated GAPDH levels in the medium due to leakage of cytosolic protein were ruled
GAPDH secretion is enhanced upon iron depletion
Iron depleted cells secrete more GAPDH into the culture medium as compared to control cells. (Table 1, Fig. S1A). The possibility that this increase of GAPDH in the medium with time is due to leakage from dying cells was ruled out by the fact that: (i) in some cell lines the secretion is decreased and (ii) there was no significant change in cell viability or release of lactate dehydrogenase (LDH) into the culture supernatant (Table 1, Fig. S1B).
Culture supernatant of iron depleted cells enhances transferrin and iron uptake
Fresh cells, incubated with media supernatant of
Discussion
Till date no soluble/secreted receptor cum delivery molecule has been established for transferrin–iron delivery into mammalian cells. Apart from its membrane bound form, TfR1 is also known to occur in human serum as a soluble truncated monomer form of the membrane anchored protein (sTfR). Despite its relevance as a measure of body iron status [25], [26], [27] sTfR does not function as a secreted mobile carrier/delivery vehicle for transferrin–iron delivery into cells and its release from cells
Acknowledgements
Mr. Anil Theophilus and Dr. Subash Pawar are acknowledged for their technical assistance. S.K. and N.S. received CSIR, Junior Research Fellowships, H.M was the recipient of UGC Junior Research Fellowship. The financial support of DST including a project assistantship to V.T is acknowledged. This is IMTECH communication no. 074/2011.
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- 1
Both these authors contributed equally to the manuscript.
- 2
Current address: CLSC 201, 920E, 58th street, University of Chicago (IL)-60637, USA.