Uptake of Iron from N‐Terminal Half‐Transferrin by Isolated Rat Hepatocytes Evidence of Transferrin‐Receptor‐Independent Iron Uptake

Abstract

The aim of the present study was to determine if human N-terminal half-transfenin (N-fragment), prepared by thermolysin cleavage of difemc transferrin, would bind to the rat hepatocyte transfenin receptor and donate iron to the cell. Competition experiments between 'Z51-labelled N-fragment and diferric transferrin revealed no receptor binding of the half-transferrin. Still, the N-fragment delivered iron to the cells in amounts approximately 30-fold above what could be accounted for by uptake of the fragment itself. The rate of celluar iron uptake from the fragment was comparable to what is seen with the intact transferrin. The uptake of '251-labelled N-fragment was not inhibited by excess non-radioactive diferric transferrin. By comparison, the uptake of 59Fe from the N-fragment was inhibited 70% by excess nonradioactive diferric transferrin. This suggests that iron derived from diferric transferrin competes with the iron derived from the N-fragment for a common transport pathway. Although some cellular degradation of the N-fragment occurred, the extent of degradation was too low to explain the amount of iron accumulated by the cells. The results show that the hepatocyte has an effective transferrin-receptor-independent mechanism for accumulation of iron from transfenin.Keywords: half-transferrin; hepatocyte; iron; transferrin receptor.For the majority of cell types, the mechanism of cellular uptake of iron from transferrin is receptor-mediated endocytosis. However, at least for the hepatocyte, alternative mechanisms appear to exist [I]. Non-specific mechanisms such as adsorptive endocytosis or pinocytosis were long since suggested [2-71, and recently pinocytosis was shown to possibly be a major factor in hepatocyte transferrin iron uptake [S]. Recently, studies with cell types other than hepatocytes, e.g. fibroblasts, melanoma cells, bone marrow macrophages, reticulocytes, or transferrin-receptor-depleted Chinese hamster ovary (CHO) cells, have also suggested the presence of a transferrin-receptor-independent route of iron uptake from transfemn 19-13]. In addition, a plasma membrane process driven by an oxidoreductase has been described, but remains uncertain and controversial 114, 151. Thus, receptor-mediated endocytosis may not be the exclusive mechanism by which cells acquire iron from transferrin.To investigate a receptor-independent mechanism for transferrin iron uptake without interfering with the cells by use of inhibitors, one would need either a cell type without any transferrin receptors, or a transferrin that does not bind to the cellular receptors. The fact that transferrin may be proteolytically cleaved to yield N-terminal or C-terminal half-transferrins (N-or C-fragments) that have retained the characteristics of the intact protein with regard to iron binding [16, 171, The present study has examined the interaction of the Nterminal half-transferrin with isolated hepatocytes in terms of receptor binding, and the cell's ability to take up iron from the fragment. We show that the isolated hepatocyt...

Nontransferrin-bound iron uptake by hepatocytes is increased in the Hfe knockout mouse model of hereditary hemochromatosis

“…32 The mechanisms responsible for the low-affinity processes are unclear but evidence suggests there may be 2 pathways, one involving the endocytosis of Tf-Fe and the other, the release of Fe from transferrin at the cell surface. [33][34][35] Using hepatoma cells, we have shown previously that NTBI had no effect on the uptake of Tf-Fe by a high-affinity transferrin receptor-1-mediated process. However, NTBI competitively inhibited the uptake of Tf-Fe released at the surface by the low-affinity process, suggesting a common Fe carrier mediates the uptake of both NTBI and Tf-Fe at the cell surface.…”

Thechiantizebrafish mutant provides a model for erythroid-specific disruption oftransferrin receptor 1

“…Furthermore, while alternative mechanisms to Tf/Tfr1-mediated iron acquisition have been characterized in a variety of mammalian cell lines, their in-vivo roles remain to be elucidated. These include direct uptake of non-Tf-bound iron (NTBI) (Baker et al, 1998;Goto et al, 1983;Hodgson et al, 1995;Inman and WesslingResnick, 1993;Kaplan et al, 1991;Sturrock et al, 1990), Tfr1-independent uptake of Tf-bound iron (Chan et al, 1992;Thorstensen et al, 1995), and receptor-mediated uptake of ferritin (Gelvan et al, 1996;Konijn et al, 1994;Leimberg et al, 2003;Meyron-Holtz et al, 1999). Recently, the soluble protein 24p3/neutrophil gelatinase-associated lipocalin (Ngal) was found to deliver iron to developing mammalian kidney epithelial cells, with a pattern of cell binding and intracellular trafficking independent from that of the Tf/Tfr1, and may present one avenue for the cellular distribution of NTBI (Yang et al, 2002).…”

The surface of rat hepatocytes can transfer iron from stable chelates to external acceptors

“…Previous evidence suggests the existence of a plasma-membrane site for release of iron from stable chelate complexes and transfer to the cell interior, [6][7][8][9] which might also be used by transferrin bound to non-receptor-binding sites. 3 This hypothesis is strengthened in this study, in which a competition between various iron-binding and chelating agents and the hepatocyte surface is demonstrated. A putative iron-binding or -mobilizing activity is able to mediate the transfer of iron from an extracellular chelate to another chelator or into the cell, provided that one of these is present in sufficient concentration.…”

“…Several mechanisms have been proposed to explain the uptake of iron by hepatocytes and other cells from transferrin without receptor-mediated endocytosis, [1][2][3][4]19 or from other sources of ferric iron (non-transferrin-bound iron or NTBI uptake). 6,7,[20][21][22][23][24][25] The most preferred one assumes activity of a surface transmembrane oxidoreductase that reduces ferric iron and thereby creates the soluble ferrous iron, which binds only loosely to transferrin or other chelators specific for the trivalent form.…”

“…The parenchymal cells of the liver, among others, have been shown to possess additional pathways for extracting iron from the plasma protein transferrin without the need of specific receptor binding. [1][2][3][4] The mechanism of this uptake is largely unknown, although binding to low-affinity sites and adsorptive endocytosis may explain iron accumulation. However, it is not known why and how the iron is released from transferrin without the aid of the receptor.…”