Page 64 - 2018_11-Haematologica-web

C. Ka et al.
In contrast to all other types of hemochromatosis,
which are characterized by hepcidin deficiency, we found
increased serum hepcidin levels in three affected individu-
als from family 3. This recapitulates previous observations
in seven patients with the recurrent and well-character-
ized p.Val162del loss-of-function ferroportin 1 muta- tion.11,30,31
M. Speletas et al. and S. Cunat et al. failed to detect the p.Arg178Gln missense mutation in the DNA of 253 bone marrow donors from central Greece27 and 50 French con- trols.25 The results presented herein of 730 DNA samples from healthy subjects born in the western part of France (Brittany) were identical. The variant was also absent in GnomAD, which is an extension of the Exome Aggregation Consortium (ExAC) database and includes 123,136 exome sequences and 15,496 whole-genome sequences from unrelated individuals sequenced as part of various disease-specific and population genetic stud- ies. The p.Arg178Gln missense mutation can thus be expected to be very rare; nevertheless, it has now been associated with hyperferritinemia in 25 patients from France, Belgium, Greece and Iraq. This provides another indication of the pathogenicity of the SLC40A1 p.Arg178Gln allele, which is not restricted to European populations.
We and others have previously shown that the p.Arg88Gly, p.Ile152Phe and p.Asn174Ile clinical muta- tions are defective in terms of iron egress while being nor- mally addressed to the cell surface.14,17,32,33 In the present study, we demonstrated that the p.Arg178Gln substitu- tion follows an identical trend (Figure 3). This was not a typical situation of loss of ferroportin 1 function, which is usually associated with protein mislocalization.14,21 This prompted us to look at the 3D structure of human ferro- portin 1 and examine the molecular mechanism responsi- ble for reduced iron export.
Ferroportin 1 is a member of the major facilitator super- family (MFS),17 which is the largest group of secondary active membrane transporters, essential for the movement of a wide range of substrates across biological membranes.34 In recent years, the number of experimental 3D structures has increased dramatically, leading to a bet- ter appreciation of the conformational changes that are needed for effective MFS-mediated transport.35,36 All MFS transporters share a common and characteristic core fold that is organized in two similar domains (N and C lobes), each consisting of six consecutive transmembrane seg- ments (TM1-TM6 and TM7-TM12). They progress through a conformational cycle that involves at least four conformational states: inward open state, the ligand- bound and ligand-free occluded states, and outward open state.36 These conformational changes are orchestrated by
a set of specific residues that mediate interactions between the N and C lobes.35
The recent report of the crystal structures of a putative bacterial homologue of ferroportin 1 (BbFPN) and the description of inter- and intra-domain conformational rearrangements during the transport of iron open up new avenues for predicting the atomic details of the organiza- tion of human ferroportin transmembrane helices and elu- cidating the detailed mechanisms of iron egress.19 Such structural investigation has already been conducted, using more distant 3D structures as a template.14,17,37 In the pres- ent study, we built an outward-open conformation of human ferroportin 1, using the experimental structure of BbFPN as a template. This allowed us to specifically inves- tigate interactions between TM3, TM4 and TM5 of the N lobe and TM8 and TM9 of the C lobe (Figure 5). We demonstrated that Arg178 (TM5) forms a salt bridge with Asp473 (TM8). This bond may act in the same way as those observed between Asn174 (TM5) and Gln481 (TM10), or between Arg88 (TM3) and Glu486 (TM11) and between Asp157 (TM4) and Arg489 (TM11), thus extend- ing the definition of an interaction network on the intra- cellular side of the outward facing structure of BbFPN.19 That the interaction between Arg178 and Asp473 is important in stabilizing human ferroportin 1 in the out- ward facing state is further supported by the results pre- sented in Figure 6, where replacing arginine 178 or aspartic acid 473 by alanine strongly decreased the ability of ferro- portin 1 to export iron. The reason why the Asp473Ala mutant showed a smaller impact on protein trafficking than Arg178Ala, despite its stronger effect on iron egress, remains to be elucidated. One possibility is that the inter- ruption of the charge-helix dipole interaction (Arg178 – Asp 181) may destabilize the local structure of TM3.
To conclude, the present study demonstrates the causali- ty of the p.Arg178Gln missense mutation, which is now considered to be one of the most frequent SLC40A1 loss-of- function mutations. It also reveals a new molecular mecha- nism of disease, involving residues that participate in the stabilization of the different conformational states and thus mediate iron export. These findings can be extended to the functional interpretation of other rare missense mutations that are associated with typical reticuloenthelial iron over- load but do not significantly alter the cell surface expression of ferroportin 1. They also confirm that it is essential to identify so-called “gating residues” in order to fully under- stand the action mechanism of MFS transporters.35
Funding
The authors would like to thank the French Hospital Clinical Research Program (Progamme Hospitalier de Recherche Clinique 2009) for funding; Brest University Hospital UF0857.
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