Association of the transferrin receptor in human placenta with HFE, the protein defective in hereditary hemochromatosis

Abstract

Hereditary hemochromatosis (HH) is a common autosomal recessive disease associated with loss of regulation of dietary iron absorption and excessive iron deposition in major organs of the body. Recently, a candidate gene for HH (also called HFE) was identified that encodes a novel MHC class I-like protein. Most patients with HH are homozygous for the same mutation in the HFE gene, resulting in a C282Y change in the HFE protein. Studies in cultured cells show that the C282Y mutation abrogates the binding of the recombinant HFE protein to beta2-microglobulin (beta2M) and disrupts its transport to the cell surface. The HFE protein was shown by immunohistochemistry to be expressed in certain epithelial cells throughout the human alimentary tract and to have a unique localization in the cryptal cells of small intestine, where signals to regulate iron absorption are received from the body. In the studies presented here, we demonstrate by immunohistochemistry that the HFE protein is expressed in human placenta in the apical plasma membrane of the syncytiotrophoblasts, where the transferrin-bound iron is normally transported to the fetus via receptor-mediated endocytosis. Western blot analyses show that the HFE protein is associated with beta2M in placental membranes. Unexpectedly, the transferrin receptor was also found to be associated with the HFE protein/beta2M complex. These studies place the normal HFE protein at the site of contact with the maternal circulation where its association with transferrin receptor raises the possibility that the HFE protein plays some role in determining maternal/fetal iron homeostasis. These findings also raise the question of whether mutations in the HFE gene can disrupt this association and thereby contribute to some forms of neonatal iron overload.

Figure 1

Immunohistochemical localization of HFE protein in full-term human placental villi. Positive immunoreaction is confined to the apical plasma membrane of the syncytiotrophoblasts (A). The positive reaction was blocked by addition of the CT16 peptide (B). Magnifications, ×1,000 (A); ×400 (B).

Figure 2

Western blot of HFE protein and β₂M in human placenta. Total placental cell homogenate containing 25 μg equivalent protein was analyzed by SDS/PAGE in nonreducing (−) or reducing (+) conditions followed by immunostaining using the mixture of CT16 and anti-human β₂M antibodies. In the first lane (under nonreducing conditions), the β₂M runs at the M_r expected for free β₂M while most of the HFE protein is in high molecular weight complexes too large to enter the separating gel (arrow). In the second lane (under reducing conditions), the polypeptides for monomeric HFE protein and β₂M are evident in addition to polypeptides of 30 and 18 kDa (asterisks) that represent proteolytic products of HFE protein.

Figure 3

Sequential affinity purification of placental hFcRn and HFE protein complexes and characterization of HFE-associated proteins. Lanes 1 and 3 illustrate blots of biotinylated hFcRn and associated proteins eluted from a human IgG Affigel-10 column. The flow-through from the human IgG Affigel-10 column was subjected to a second immunoaffinity purification step using immobilized CT16 antibodies. Lanes 2 and 4 contain blots of biotinylated proteins that were eluted from the CT16 IgG Affigel-10 column. On Western blots, the CT16 antibody did not cross-react with any polypeptide present in the hFcRn preparation (lane 1), but did identify a 48-kDa polypeptide band corresponding to the molecular weight of monomeric HFE protein in the fraction eluted from the CT16 IgG Affigel-10 column (lane 2). Western blot analysis using anti-human β₂M antibody (lanes 3 and 4) identified a 12-kDa polypeptide in both the hFcRn (lane 3) and HFE protein preparations (lane 4).

Figure 4

Immunoprecipitation of transferrin receptor from human placental membrane proteins coisolated with the HFE protein. The biotinylated proteins eluted from the human IgG Affigel-10 (lane 1) and CT16 IgG Affigel-10 columns (lane 2) were immunoprecipitated using monoclonal anti-human transferrin receptor antibody and analyzed using a streptavidin-peroxidase complex. A polypeptide of 93 kDa was present only in the protein preparation isolated by the second column, the CT16 IgG Affigel-10 column (lane 2).

Figure 5

Association of the transferrin receptor with the HFE protein and β₂M. Proteins in the membrane suspension from placenta were chemically cross-linked with a reversible crosslinker. The cross-linked complexes of HFE protein and associated proteins were isolated by immunoaffinity using a CT16 IgG Affigel-10 column (lane 1). The transferrin receptor-associated proteins were isolated by immunoprecipitation using anti-human transferrin receptor antibody (lane 2). The proteins in the complexes isolated by the two methods were dissociated and separated by SDS/PAGE under reducing conditions followed by Western blot analysis. Both the complexes isolated by CT16 IgG Affigel-10 column and by immunoprecipitation with the anti-human transferrin receptor antibody contained polypeptides reacting with CT16 and anti-human β₂M antibodies. Lane 1 shows a 48-kDa band of the HFE protein and the 12-kDa β₂M in addition to the 30- and 18-kDa HFE-species. Lane 2 shows a 45/48-kDa doublet of HFE proteins and the 12-kDa β₂M.