Co-trafficking of HFE, a nonclassical major histocompatibility complex class I protein, with the transferrin receptor implies a role in intracellular iron regulation.

The mechanism by which a novel major histocompatibility complex class I protein, HFE, regulates iron uptake into the body is not known. HFE is the product of the gene that is mutated in >80% of hereditary hemochromatosis patients. It was recently found to coprecipitate with the transferrin receptor (Feder, J. N., Penny, D. M., Irrinki, A., Lee, V. K., Lebron, J. A., Watson, N., Tsuchihashi, Z., Sigal, E., Bjorkman, P. J., and Schatzman, R. C. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 1472-1477; Parkkila, S., Waheed, A., Britton, R. S., Bacon, B. R., Zhou, X. Y., Tomatsu, S., Fleming, R.E. , and Sly, W. S. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 13198-13202) and to decrease the affinity of transferrin for the transferrin receptor (Feder et al.). In this study, HeLa cells were transfected with HFE under the control of the tetracycline-repressible promoter. We demonstrate that HFE and the transferrin receptor are capable of associating with each other within 30 min of their synthesis with pulse-chase experiments. HFE and the transferrin receptor co-immunoprecipitate throughout the biosynthetic pathway. Excess HFE is rapidly degraded, whereas the HFE-transferrin receptor complex is stable. Immunofluorescence experiments indicate that they also endocytose into transferrin-positive compartments. Combined, these results suggest a role for the transferrin receptor in HFE trafficking. Cells expressing HFE have modestly increased levels of transferrin receptor and drastically reduced levels of ferritin. These results implicate HFE further in the modulation of iron levels in the cell.

The hemochromatosis gene product complexes with the transferrin receptor and lowers its affinity for ligand binding.

We recently reported the positional cloning of a candidate gene for hereditary hemochromatosis called HFE. The gene product, a member of the major histocompatibility complex class I-like family, was found to have a mutation, Cys-282 --> Tyr (C282Y), in 85% of patient chromosomes. This mutation eliminates the ability of HFE to associate with beta2-microglobulin (beta2m) and prevents cell-surface expression. A second mutation that has no effect on beta2m association, H63D, was found in eight out of nine patients heterozygous for the C282Y mutant. In this report, we demonstrate in cultured 293 cells overexpressing wild-type or mutant HFE proteins that both the wild-type and H63D HFE proteins form stable complexes with the transferrin receptor (TfR). The C282Y mutation nearly completely prevents the association of the mutant HFE protein with the TfR. Studies on cell-associated transferrin at 37 degrees C suggest that the overexpressed wild-type HFE protein decreases the affinity of the TfR for transferrin. The overexpressed H63D protein does not have this effect, providing the first direct evidence for a functional consequence of the H63D mutation. Addition of soluble wild-type HFE/beta2m heterodimers to cultured cells also decreased the apparent affinity of the TfR for its ligand under steady-state conditions, both in 293 cells and in HeLa cells. Furthermore, at 4 degrees C, the added soluble complex of HFE/beta2m inhibited binding of transferrin to HeLa cell TfR in a concentration-dependent manner. Scatchard plots of these data indicate that the added heterodimer substantially reduced the affinity of TfR for transferrin. These results establish a molecular link between HFE and a key protein involved in iron transport, the TfR, and raise the possibility that alterations in this regulatory mechanism may play a role in the pathogenesis of hereditary hemochromatosis.

The hemochromatosis founder mutation in HLA-H disrupts beta2-microglobulin interaction and cell surface expression.

We recently reported the positional cloning of a candidate gene for hereditary hemochromatosis (HH), called HLA-H, which is a novel member of the major histocompatibility complex class I family. A mutation in this gene, cysteine 282 --> tyrosine (C282Y), was found to be present in 83% of HH patient DNAs, while a second variant, histidine 63 --> aspartate (H63D), was enriched in patients heterozygous for C282Y. The functional relevance of either mutation has not been described. Co-immunoprecipitation studies of cell lysates from human embryonic kidney cells transfected with wild-type or mutant HLA-H cDNA demonstrate that wild-type HLA-H binds beta2-microglobulin and that the C282Y mutation, but not the H63D mutation, completely abrogates this interaction. Immunofluorescence labeling and subcellular fractionations demonstrate that while the wild-type and H63D HLA-H proteins are expressed on the cell surface, the C282Y mutant protein is localized exclusively intracellularly. This report describes the first functional significance of the C282Y mutation by suggesting that an abnormality in protein trafficking and/or cell-surface expression of HLA-H leads to HH disease.

A 1.1-Mb transcript map of the hereditary hemochromatosis locus.

In the process of positionally cloning a candidate gene responsible for hereditary hemochromatosis (HH), we constructed a 1.1-Mb transcript map of the region of human chromosome 6p that lies 4.5 Mb telomeric to HLA-A. A combination of three gene-finding techniques, direct cDNA selection, exon trapping, and sample sequencing, were used initially for a saturation screening of the 1.1-Mb region for expressed sequence fragments. As genetic analysis further narrowed the HH candidate locus, we sequenced completely 0.25 Mb of genomic DNA as a final measure to identify all genes. Besides the novel MHC class 1-like HH candidate gene HLA-H, we identified a family of five butyrophilin-related sequences, two genes with structural similarity to a type 1 sodium phosphate transporter, 12 novel histone genes, and a gene we named RoRet based on its strong similarity to the 52-kD Ro/SSA lupus and Sjogren's syndrome auto-antigen and the RET finger protein. Several members of the butyrophilin family and the RoRet gene share an exon of common evolutionary origin called B30-2. The B30-2 exon was originally isolated from the HLA class 1 region, yet has apparently "shuffled" into several genes along the chromosome telomeric to the MHC. The conservation of the B30-2 exon in several novel genes and the previously described amino acid homology of HLA-H to MHC class 1 molecules provide further support that this gene-rich region of 6p21.3 is related to the MHC. Finally, we performed an analysis of the four approaches for gene finding and conclude that direct selection provides the most effective probes for cDNA screening, and that as much as 30% of ESTs in this 1.1-Mb region may be derived from noncoding genomic DNA.