Identification of TMPRSS6 cleavage sites of hemojuvelin

Marco Rausa, Michela Ghitti, Alessia Pagani, Antonella Nai, Alessandro Campanella, Giovanna Musco, Clara Camaschella, Laura Silvestri

Research output: Contribution to journalArticle

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 HJVR121A, lack autoproteolytic activity and some (HJVR176A and HJVR288A) 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.

Original languageEnglish
Pages (from-to)879-888
Number of pages10
JournalJournal of Cellular and Molecular Medicine
Volume19
Issue number4
DOIs
Publication statusPublished - Apr 1 2015

Keywords

  • Hepcidin
  • HJV
  • Homology modelling
  • Iron
  • RGM
  • Serine protease
  • TMPRSS6

ASJC Scopus subject areas

  • Cell Biology
  • Molecular Medicine

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  • Cite this

    Rausa, M., Ghitti, M., Pagani, A., Nai, A., Campanella, A., Musco, G., Camaschella, C., & Silvestri, L. (2015). Identification of TMPRSS6 cleavage sites of hemojuvelin. Journal of Cellular and Molecular Medicine, 19(4), 879-888. https://doi.org/10.1111/jcmm.12462