Secreted glyceraldehye-3-phosphate dehydrogenase is a multifunctional autocrine transferrin receptor for cellular iron acquisition

https://doi.org/10.1016/j.bbagen.2013.03.019Get rights and content

Highlights

  • Enhanced secretion of GAPDH upon iron depletion increases Tf–Fe delivery into cells.

  • Tf trafficking by this novel route is more efficient than via classical membrane bound receptors.

  • A long suspected but uncharacterized primitive pathway of cellular iron acquisition uncovered.

  • Implications for diseases involving high glycolysis and iron demand such as infectious diseases and cancer.

Abstract

Background

The long held view is that mammalian cells obtain transferrin (Tf) bound iron utilizing specialized membrane anchored receptors. Here we report that, during increased iron demand, cells secrete the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which enhances cellular uptake of Tf and iron.

Methods

These observations could be mimicked by utilizing purified GAPDH injected into mice as well as when supplemented in culture medium of model cell lines and primary cell types that play a key role in iron metabolism. Transferrin and iron delivery was evaluated by biochemical, biophysical and imaging based assays.

Results

This mode of iron uptake is a saturable, energy dependent pathway, utilizing raft as well as non-raft domains of the cell membrane and also involves the membrane protein CD87 (uPAR). Tf internalized by this mode is also catabolized.

Conclusions

Our research demonstrates that, even in cell types that express the known surface receptor based mechanism for transferrin uptake, more transferrin is delivered by this route which represents a hidden dimension of iron homeostasis.

General significance

Iron is an essential trace metal for practically all living organisms however its acquisition presents major challenges. The current paradigm is that living organisms have developed well orchestrated and evolved mechanisms involving iron carrier molecules and their specific receptors to regulate its absorption, transport, storage and mobilization. Our research uncovers a hidden and primitive pathway of bulk iron trafficking involving a secreted receptor that is a multifunctional glycolytic enzyme that has implications in pathological conditions such as infectious diseases and cancer.

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.

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