Reduced sensitivity of the ferroportin Q248H mutant to physiological concentrations of hepcidin

Abstract

ciency or ferroportin Q248H had lower circulating tumor necrosis factor-α concentrations than iron-replete children with WT ferroportin, 25 led us to hypothesize that ferroportin Q248H has reduced sensitivity to hepcidin but that this property would be observed at lower concentrations of hepcidin than those used in previous studies. 23 Here, we analyzed the sensitivity of ferroportin Q248H to hepcidin at various concentrations by determining the levels of ferroportin transiently expressed in cultured cells and in human primary monocytes derived from humans with different ferroportin genotypes. We also measured ferritin concentrations in these cells, levels that are indicative of cellular iron status. Finally, we examined the effect of Q248H on serum iron measures in patients with sickle cell anemia who have markedly increased macrophage iron export because of chronic hemolysis. Design and Methods Predictive analysis and worldwide allele frequenciesMinor allele frequencies for missense mutations in the single nucleotide polymorphism database (dbSNP) were determined using Kaviar and sequence data (http://db.systemsbiology.net/kaviar). 26Mutations were analyzed for effects on protein function using a 571-residue ferroportin protein (UniProtKB/Swiss-Prot Q9NP59) and the predictive analysis tools: SIFT (Sorting Intolerant From Tolerant; http://blocks.fhcrc.org/sift/SIFT.html) 27 and PolyPhen2 (Polymorphism Phenotyping version 2; using HumDiv model; http://genetics.bwh.harvard.edu/pph2/index.shtml). 28 Cells and media293T cells were purchased from ATCC (Manassas, VA, USA) and cultured in Dulbecco's modified Eagles medium (DMEM) containing 10% fetal bovine serum (FBS) (Life Technologies) and 1% glutamine (Invitrogen, Carlsbad, CA, USA) at 37°C in the presence of 5% CO 2 . Ferroportin expression constructsHuman WT ferroportin, ferroportin Q248H and ferroportin C326Y mutants cloned with c-Myc and histidine tags in a pcDNA3.1 expression vector were kindly provided by Dr. Hal Drakesmith. 23 To generate enhanced green fluorescent protein (EGFP)-tagged human ferroportin, the ferroportin coding sequences from WT ferroportin, ferroportin Q248H and ferroportin C326Y mutants were amplified by polymerase chain reaction (PCR) with the following primers that included XhoI and Kpn1 restriction sites (shown in italics): forward primer, GCCTC-GAGATGACCAGGGCGGGAGATCAC and reverse primer, GCGGTACCGTAACAACAGATGTATTTGCTTGATTTTC. The PCR products were purified on agarose gel, digested with XhoI and Kpn1 (BioLabs, Ipswich, MA) and ligated into the pEGFP-N1 vector (Clontech, Mountain View, CA, USA) which was also digested with XhoI and Kpn1 and ligated. The ligation products were transformed into E. coli DH5α cells (Invitrogen) and kanamycin-resistant colonies were selected. WT ferroportin-EGFP, C326Y and Q248H ferroportin -GFP-expressing plasmids were purified using a Qiagen (Valencia, CA, USA) purification kit and sequenced using the Macrogen service (Rockville, MD, USA). Transfection and treatment293T cells were seeded in 6-well ...

Effect of ferroportin polymorphism on iron homeostasis and infection

“…However, other investigators recently reported reduced sensitivity to physiologic hepcidin concentrations by FPN Q248H [65]. The researchers also reported that following incubation of primary cells with iron followed by treatment with hepcidin, macrophages recovered from Q248H subjects had lower intracellular iron content compared to macrophages obtained from FPN wild type subjects [65]. Further studies are needed to determine whether Q248H mutation alters FPN interaction with hepcidin.…”

“…Drakesmith and co-workers using supraphysiologic hepcidin concentrations reported that Q248H is a neutral polymorphism and does not confer resistance to hepcidin and the sensitivity of FPN Q248H to hepcidin was identical to FPN wild-type [64]. However, other investigators recently reported reduced sensitivity to physiologic hepcidin concentrations by FPN Q248H [65]. The researchers also reported that following incubation of primary cells with iron followed by treatment with hepcidin, macrophages recovered from Q248H subjects had lower intracellular iron content compared to macrophages obtained from FPN wild type subjects [65].…”

“…Y64N, N144D, N144H and C326Y [64] and Q248H [65], and iron export by FPN can be described as being permanently 'turned on' [48]. The loss of regulatory control of FPN by hepcidin leads to inappropriately high iron uptake at the duodenal enterocyte and increased release of iron by macrophages causing body iron overload.…”

“…Subjects heterozygous for FPN Q248H had significantly lower TNF-α and MIF concentrations compared to FPN wild type subjects. This could be attributed to lower intracellular iron concentration in macrophages of FPN Q248H subjects due to increased cellular iron egress [65].…”

Serum ferritin concentration is affected by ferroportin Q248H mutation in Africans

“…Ferroportin Q248H mutation is present in African descendants but the frequency of the mutation varies across populations (reviewed in [7]). The effect of the ferroportin Q248H mutation on iron homeostasis is inconclusive [8,9].…”