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We very much appreciate the kind comments made by Schwienbacher et al. regarding our article on the implication of PC7 as the sheddase of human transferrin receptor 1 (TfR1).[1] We read with great interest their observation of the lack of involvement of PC7 in hemojuvelin (HJV) shedding. Herein, we tested the hypothesis that PC7 could also regulate iron homeostasis by way of shedding of the human hemochromatosis protein (HFE).

HFE is a membrane protein that binds the two receptors TfR1 and TfR2. HFE and holotransferrin compete for binding to TfR1. When the levels of holotransferrin are high, HFE is displaced from TfR1 and binds TfR2 to signal for hepcidin production.[2] A recent study indicated the existence of a soluble HFE in human serum that binds shed TfR1.[3] Analysis of the sequence of HFE revealed single basic amino acid cleavage sites that could potentially be recognized by a proprotein convertase. The most probable ones are in the sequence ….RAAEP RAWPTKLEWERHKIRARQNRAYLERDCPAQ190….

Accordingly, in human hepatoma HuH7 cells we coexpressed PC7 or Furin with HFE carrying a Protein C-tag at its N-terminus and a V5-tag at its C-terminus (Fig. 1A). The data revealed that overexpression of PC7 or Furin did not result in enhanced HFE shedding over background (Fig. 1B). This is likely due to a high helical secondary structure around the potential basic amino acid cleavage sites (Fig. 1A). Moreover, in HuH7 cells coexpression of TfR1 with HFE in the absence or presence of PC7 showed that HFE did not affect the ability of PC7 to shed TfR1 (Fig. 1C).

image

Figure 1. (A) Schematic representation of the human HFE. Depicted are the signal peptide (SP), the transmembrane domain (TM), the potential PC-processing site(s), the N-terminal Protein C-tag (ProtC), and the C-terminal V5-tag. The secondary structures are emphasized below the amino acid sequence (h, helix; c, coil). An unknown sheddase (?) cleaves HFE at a potential shedding site (arrow) predicted from the close similarity of the molecular sizes of intracellular and extracellular HFE. (B) Western blot analysis of lysates and 20-hour conditioned media of HuH7 cells coexpressing (ProtC)-HFE-V5 and either empty vector, PC7, or Furin. (C) Cell lysates and 20-hour conditioned media from HuH7 cells expressing TfR1-V5 and either empty vector, (ProtC)-HFE-V5, and/or PC7 were analyzed by western blotting. The separated proteins were revealed using monoclonal antibody (mAb) V5-HRP or ProtC-Ab and a rabbit horseradish peroxidase (HRP)-conjugated secondary Ab.

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We conclude that, so far, PC7 regulates iron homeostasis only through TfR1 shedding.[1] The available data show that Furin, but not PC7, can activate hepcidin and shed HJV, and that neither Furin nor PC7 can cleave HFE.

  • JOHANN GUILLEMOT, Ph.D.NABIL G. SEIDAH, Ph.D.

  • Laboratory of Biochemical NeuroendocrinologyClinical Research Institute of Montreal (IRCM)Affiliated with the University of Montreal Montreal Quebec, Canada

References

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  2. References
  • 1
    Guillemot J, Canuel M, Essalmani R, Prat A, Seidah NG. Implication of the proprotein convertases in iron homeostasis: PC7 sheds human transferrin receptor 1 and furin activates hepcidin. Hepatology 2013;57:2514-2524.
  • 2
    Gkouvatsos K, Papanikolaou G, Pantopoulos K. Regulation of iron transport and the role of transferrin. Biochim Biophys Acta 2012;1820:188-202.
  • 3
    Ohnishi K, Torimoto Y, Ikuta K, Tanaka H, Hosoki T, Tanaka S, et al. Detection of soluble HFE associated with soluble transferrin receptor in human serum. Int J Mol Med 2011;27:435-439.