Premium
Ferroportin residue C326 is critical for its interaction with hepcidin
Author(s) -
Nemeth Elizabeta,
Fernandes Augustine,
Ganz Tomas
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.1191.4
Subject(s) - hepcidin , internalization , ferroportin , chemistry , mutant , hereditary hemochromatosis , microbiology and biotechnology , hemochromatosis , biochemistry , cell , biophysics , biology , gene , genetics , immunology , inflammation
The hormone hepcidin binds to the Fe exporter Fpn and induces its internalization and degradation, decreasing Fe efflux from cells into plasma. Fpn mutations that confer resistance to hepcidin cause classical hemochromatosis with parenchymal Fe loading. We analyzed mutations C326S, C326T, N144D and N144T to address the mechanism of Fpn interaction with hepcidin. Human Fpn‐EGFP construct was subjected to site‐directed mutagenesis and analyzed in HEK293T cells. The Fpn mutants localized properly to the cell surface and exported Fe normally. However, they were either completely (C326S/T) or partially (N144D/T) resistant to hepcidin‐mediated internalization, and exported Fe even in the presence of hepcidin. 125I‐hepcidin binding was completely (C236S/T) or partially (N144D/T) abrogated. C326 and C205 were reported to be cell surface thiols. A non‐permeable thiol‐dependent crosslinker linked hepcidin to Fpn but not in C326S/T mutants, and only partly in N144D/T mutants. Alkylation of Fpn‐expressing cells ablated 125I‐hepcidin binding. We next examined whether hepcidin binding depended on a transient SS‐bond formation between C205 and C326. C205S/T substitutions, however, only partially inhibited hepcidin uptake and crosslinking. Bacitracin had no effect on hepcidin uptake or Fpn internalization indicating that isomerisation of a C205‐C326 disulfide bond was not necessary. Thus the C326 thiol is located in or near the binding site of hepcidin and is essential for hepcidin binding and subsequent internalization of ferroportin. Our observations help explain the severity of hemochromatosis in patients with C326X mutations.