Identification of TMPRSS 6 cleavage sites of hemojuvelin

Hemojuvelin ( HJV ), the coreceptor of the BMP ‐ SMAD pathway that up‐regulates hepcidin transcription, is a repulsive guidance molecule ( RGM c) which undergoes a complex intracellular processing. Following autoproteolysis, it is exported to the cell surface both as a full‐length and a heterodimeric protein. In vitro membrane HJV (m‐ HJV ) is cleaved by the transmembrane serine protease TMPRSS 6 to attenuate signalling and to inhibit hepcidin expression. In this study, we investigated the number and position of HJV cleavage sites by mutagenizing arginine residues (R), potential TMPRSS 6 targets, to alanine (A). We analysed translation and membrane expression of HJV R mutants and the pattern of fragments they release in the culture media in the presence of TMPRSS 6. Abnormal fragments were observed for mutants at arginine 121, 176, 218, 288 and 326. Considering that all variants, except HJV R121A , lack autoproteolytic activity and some (HJV R176A and HJV R288A ) are expressed at reduced levels on cell surface, we identified the fragments originating from either full‐length or heterodimeric proteins and defined the residues 121 and 326 as the TMPRSS 6 cleavage sites in both isoforms. Using the N‐terminal FLAG ‐tagged HJV , we showed that residue 121 is critical also in the rearrangement of the N‐terminal heterodimeric HJV . Exploiting the recently reported RGM b crystallographic structure, we generated a model of HJV that was used as input structure for all‐atoms molecular dynamics simulation in explicit solvent. As assessed by in silico studies, we concluded that some arginines in the von W illebrand domain appear TMPRSS 6 insensitive, likely because of partial protein structure destabilization.