Functional analysis and theoretical modeling of ferroportin reveals clustering of mutations according to phenotype

Abstract

Ferroportin disease is a heterogeneous iron release disorder resulting from mutations in the ferroportin gene. Ferroportin protein is a multitransmembrane domain iron transporter, responsible for iron export from cells, which, in turn, is regulated by the peptide hormone hepcidin. Mutations in the ferroportin gene may affect either regulation of the protein's transporter function or the ability of hepcidin to regulate iron efflux. We have used a combination of functional analysis of epitope-tagged ferroportin variants coupled with theoretical modeling to dissect the relationship between ferroportin mutations and their cognate phenotypes. Myc epitope-tagged human ferroportin expression constructs were transfected into Caco-2 intestinal cells and protein localization analyzed by immunofluorescence microscopy and colocalization with organelle markers. The effect of mutations on iron efflux was assessed by costaining with anti-ferritin antibodies and immunoblotting to quantitate cellular expression of ferritin and transferrin receptor 1. Wild-type ferroportin localized mainly to the cell surface and intracellular structures. All ferroportin disease-causing mutations studied had no effect on localization at the cell surface. N144H, N144T, and S338R mutant ferroportin retained the ability to transport iron. In contrast, A77D, V162Delta, and L170F mutants were iron transport defective. Surface staining experiments showed that both ends of the protein were located inside the cell. These data were used as the basis for theoretical modeling of the ferroportin molecule. The model predicted phenotypic clustering of mutations with gain-of-function variants associated with a hypothetical channel through the axis of ferroportin. Conversely, loss-of-function variants were located at the membrane/cytoplasm interface.

Characterisation of hepcidin response to holotransferrin treatment in CHO TRVb-1 cells

“…The position of C326 in relation to other amino acids is also conserved in the proposed hepcidin binding region, lying outside the transmembrane region and therefore appears to be consistent with its role in binding to hepcidin. K253, Y302 and Y303, which are involved in ferroportin internalisation and degradation upon hepcidin binding are conserved in this characterised CHO-slc40a1 sequence and are located in the same position relative to other amino acids as in human, mouse and rat ferroportin [19]. Also, cysteines which form disulphide bonds and usually play a significant role in the folding and stability of proteins [20], are well conserved.…”

Minihepcidins are rationally designed small peptides that mimic hepcidin activity in mice and may be useful for the treatment of iron overload

“…The structure for hepcidin (NP_ 066998, http://www.ncbi.nlm.nih.gov/protein/NP_066998) was modeled by the coordinate set for the lowest energy conformer from the Protein Data Bank (PDB 2KEF) (18). The structure for ferroportin (NP_055400.1, http://www.ncbi.nlm.nih.gov/protein/NP_055400.1) was approximated using the coordinate set provided by Nathan Subramaniam (19). The ferroportin region spanning residues 306-362 and the hepcidin structure were individually minimized in the HyperChem environment to obtain the lowest energy conformer.…”

“…The input structures were the fulllength hepcidin (PDB 2KEF), with the most recent disulfide bond assignment (18), and a region of ferroportin (residues 306-362) encompassing the C326 extracellular loop and flanking transmembrane helices, with structure based on a recently developed theoretical model of ferroportin molecule (19). Ferroportin and hepcidin structures were refined using HyperChem 7.5 and GROMACS before docking studies.…”

Hepcidin and iron regulation, 10 years later

“…21 This simple and attractive model has been contested by multiple investigators who did not detect multimerization or a trafficking defect in a variety of loss-of-function mutants, as recently summarized. 22,23 Alternatively, some mutations may cause an iron transport defect whose effect could be increased if the mutant ferroportin molecules trigger endocytosis that also entrains wild-type ferroportin.…”