title: Identification of TMPRSS6 cleavage sites of hemojuvelin.
abstract: Hemojuvelin (HJV), the coreceptor of the BMP-SMAD pathway that up-regulates hepcidin transcription, is a repulsive guidance molecule (RGMc) 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 TMPRSS6 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 TMPRSS6 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 TMPRSS6. 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 TMPRSS6 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 RGMb 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 Willebrand domain appear TMPRSS6 insensitive, likely because of partial protein structure destabilization.
authors.affiliation: Division of Genetics and Cell Biology, IRCCS Ospedale San Raffaele, Milan, Italy; Vita Salute University, Milan, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Rausa
authors.forename: Michela
authors.initials: M
authors.lastname: Ghitti
authors.forename: Alessia
authors.initials: A
authors.lastname: Pagani
authors.forename: Antonella
authors.initials: A
authors.lastname: Nai
authors.forename: Alessandro
authors.initials: A
authors.lastname: Campanella
authors.forename: Giovanna
authors.initials: G
authors.lastname: Musco
authors.forename: Clara
authors.initials: C
authors.lastname: Camaschella
authors.forename: Laura
authors.initials: L
authors.lastname: Silvestri
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D058851:GPI-Linked Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D000071020:Hemochromatosis Protein; D006801:Humans; D006860:Hydrogen Bonding; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D011485:Protein Binding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*
chemical_list: D058851:GPI-Linked Proteins; C481006:HJV protein, human; D000071020:Hemochromatosis Protein; D008565:Membrane Proteins; D050505:Mutant Proteins; D001120:Arginine; D012697:Serine Endopeptidases; C504318:TMPRSS6 protein, human
keywords: HJV; RGM; TMPRSS6; hepcidin; homology modelling; iron; serine protease