Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2002 Mar 5;99(5):3117-22.
doi: 10.1073/pnas.042701499. Epub 2002 Feb 26.

Regulation of transferrin-mediated iron uptake by HFE, the protein defective in hereditary hemochromatosis

Abdul Waheed  1 , Jeffrey H Grubb, Xiao Yan Zhou, Shunji Tomatsu, Robert E Fleming, Mark E Costaldi, Robert S Britton, Bruce R Bacon, William S Sly
Affiliations

Regulation of transferrin-mediated iron uptake by HFE, the protein defective in hereditary hemochromatosis

Abdul Waheed et al. Proc Natl Acad Sci U S A. .

Abstract

The protein defective in hereditary hemochromatosis, called HFE, is similar to MHC class I-type proteins and associates with beta2-microglobulin (beta2M). Its association with beta2M was previously shown to be necessary for its stability, normal intracellular processing, and cell surface expression in transfected COS cells. Here we use stably transfected Chinese hamster ovary cell lines expressing both HFE and beta2M or HFE alone to study the effects of beta2M on the stability and maturation of the HFE protein and on the role of HFE in transferrin receptor 1 (TfR1)-mediated iron uptake. In agreement with prior studies on other cell lines, we found that overexpression of HFE, without overexpressing beta2M, resulted in a decrease in TfR1dependent iron uptake and in lower iron levels in the cells, as evidenced by ferritin and TfR1 levels measured at steady state. However, overexpression of both HFE and beta2M had the reverse effect and resulted in an increase in TfR1-dependent iron uptake and increased iron levels in the cells. The HFE-beta2M complex did not affect the affinity of TfR1 for transferrin or the internalization rate of transferrin-bound TfR1. Instead, HFE-beta2M enhanced the rate of recycling of TfR1 and resulted in an increase in the steady-state level of TfR1 at the cell surface of stably transfected cells. We propose that Chinese hamster ovary cells provide a model to explain the effect of the HFE-beta2M complex in duodenal crypt cells, where the HFE-beta2M complex appears to facilitate the uptake of transferrin-bound iron to sense the level of body iron stores. Impairment of this process in duodenal crypt cells leads them to be iron poor and to signal the differentiating enterocytes to take up iron excessively after they mature into villus cells in the duodenum of hereditary hemochromatosis patients.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Effect of β2M on stability and maturation of HFE protein in TRVb-1 cells. TRVb-1 cells overexpressing HFE only and HFE-β2M were pulse labeled for 30 min with 35S-translabel and chased for 0.5–8 h with nonradioactive methionine and cysteine. The HFE protein was immunoprecipitated and analyzed by SDS/PAGE followed by fluorography. Quantitative analysis of fluorogram is presented (Lower).
Figure 2
Figure 2
Iron accumulation rate in TRVb-1 cells. TRVb-1 cells expressing HFE alone (TRVb-1/HFE only) and HFE-β2M (TRVb-1/HFE-β2M) were incubated with 100 nM Tf-59Fe at 37°C. The cells were washed to remove cell surface-associated radioactivity before intracellular iron radioactivity was determined. The radioactivity accumulated per milligram of cell protein was calculated.
Figure 3
Figure 3
Effect of HFE and HFE-β2M on iron status of TRVb cell derivatives. (Upper) TRVb cells (CHO cells lacking hamster TfR1); TRVb-1 cells (TRVb cells expressing human TfR1); TRVb-1/HFE only (TRVb-1 cells expressing HFE alone); and TRVb-1/HFE-β2M (TRVb-1 cells expressing HFE and β2M) were grown to confluency. The cell homogenates, equivalent to 30 μg of cell protein, were analyzed by SDS/PAGE followed by Western blot analysis by using TfR1 and ferritin (Ferritin) antibodies (Upper). The polypeptide intensities were quantitated and presented as average relative density (Lower).
Figure 4
Figure 4
Cell surface expression of HFE-β2M proteins and association with TfR1. The cell surface proteins of CHO and TRVb-1 cells overexpressing HFE-β2M were biotinylated at 4°C. (1) and (2) refer to independent clones. The TfR1-HFE-β2M complex was immunoprecipitated by using TfR1 monoclonal antibodies. The immunoprecipitates were analyzed by SDS/PAGE followed by Western blot by using streptavidin–peroxidase conjugate. The polypeptides for TfR1, HFE, and β2M are marked. A nonspecific polypeptide of 64 kDa was seen in all lanes (*).
Figure 5
Figure 5
HFE-β2M enhances the number of TfR1 receptors at the cell surface. CHO and TRVb-1 cells alone and overexpressing HFE-β2M were grown to confluency. (A) The cell homogenates containing 30 μg of cell protein were analyzed by SDS/PAGE followed by Western blot by using TfR1 antibodies. The polypeptide corresponding to total TfR1 (cell surface and intracellular) is marked. The relative intensity of the polypeptide was decreased because of overexpression of HFE-β2M (as in Fig. 3). (B) The cell surface receptor-binding activity was determined by incubating the cells with 100 nM 125I-Tf at 4°C for 1 h. After washing the cells, the cell-associated radioactivity was determined and presented as 125I-Tf binding per milligram of cell protein. The cell surface-binding activity was increased by overexpression of HFE-β2M, despite the lower amount of total cellular TfR1.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Cartwright G E, Edwards C Q, Kravitz K, Skolnick M, Amos D B, Johnson A, Buskjaer L. N Engl J Med. 1979;301:175–179. - PubMed
    1. Cox T M, Lord D K. Eur J Haematol. 1989;42:113–125. - PubMed
    1. Bacon B R, Tavill A S. In: Hepatology: A Textbook of Liver Disease. Zakim D, Boyer T D, editors. Philadelphia: Saunders; 1996. pp. 1439–1472.
    1. McLaren G D, Nathanson M H, Jacobs A, Trevett D, Thomson W. J Lab Clin Med. 1991;117:390–401. - PubMed
    1. Feder J N, Gnirke A, Thomas W, Tsuchihashi Z, Ruddy D A, Basava A, Dormishian F, Domingo R, Jr, Ellis M C, Fullan A, et al. Nat Genet. 1996;13:399–408. - PubMed

Publication types

MeSH terms