Ferroportin Q248H Mutation Prevents Its Ubiquitination

Ferroportin Q248H mutation is prevalent in African populations and leads to increased serum ferritin. Our recent study shows that ferroportin Q248H protein is resistant to physiologic hepcidin concentrations¹. Also sickle cell disease patients with ferroportin Q248H heterozygote had lower serum ferritin concentration suggesting that the enhanced iron release by macrophages. Ferroportin glutamine 248 is located within the intracellular loop (residues 228-307), which is likely to be located in the cytoplasm. Recently ferroportin internalization was shown to be driven by ubiquitination of lysines lying within residues 229-269 including K229, K240, and K247². The proximity of the K240 and especially to K247 to the Q248 residue suggests that a positively charged histidine in position 248 might change the overall negative charge of the ²⁴⁰eeetelkqlnlhk²⁵³sequence toward a more positive charge, which might affect ubiquitination and subsequent degradation of ferroportin. Here we analyzed and compared ubiquitination of WT and Q248H mutant ferroportin.

WT ferroportin and Q248H mutant were expressed as EGFP-fusions in 293T cells and also combined with the expression of ubiquitin. Ferroportin was immunoprecipitated with anti-EGFP antibodies and analyzed by high resolution mass spectrometry using LTQ-Orbitrap. Phosphorylation and ubiquitination was determined using Proteome Discover and quantified using SIEVE 2.1 software.

WT ferroportin but not the Q248H mutant ferroportin was found to be ubquitinated on lysines 247 and 253 and also phosphorylated on Thr 144. Also WT ferroportin was found to associate with ubiquitine-conjugating enzyme E2 and ubiquitine protein ligase NEDD4. Thus hepcidin resistance of ferroportin Q248H could be due to its inability to undergo ubiquitination.

This project was supported by NIH Research Grants 8G12MD007597 and P30HL107253.

References

1. Nekhai S, Xu M, Foster A, et al. Reduced sensitivity of the ferroportin Q248H mutant to physiological concentrations of hepcidin. Haematologica. 2013;98(3):455-463.

2. Qiao B, Sugianto P, Fung E, et al. Hepcidin-induced endocytosis of ferroportin is dependent on ferroportin ubiquitination. Cell Metab. 2012;15(6):918-924.

No relevant conflicts of interest to declare.