Identification of TMPRSS6 cleavage sites of hemojuvelin.

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

doi:10.1111/jcmm.12462

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.

Highlights

Hepcidin is the hepatic peptide hormone which controls iron absorption from the enterocytes and iron release from the stores by degrading the only known cellular iron exporter ferroportin [1]
HJV on cell surface (m-HJV) acts as BMP-coreceptor, allowing BMP6 to signal through BMP-SMAD pathway to activate hepcidin transcription [2,3,4,5]
After 36 hrs, cells were lysed in NET/Triton buffer; 500 lg of total lysates were pulled down using the anti-FLAG M2 agarose affinity gel (Sigma-Aldrich) for 2 hrs at 4°C, followed by three washing with NET/Triton buffer

Summary

Introduction

Hepcidin is the hepatic peptide hormone which controls iron absorption from the enterocytes and iron release from the stores by degrading the only known cellular iron exporter ferroportin [1]. A formal proof that in vivo HJV is the TMPRSS6 substrate is still lacking, in vitro the protease cleaves mHJV into soluble fragments [21], a process that might abolish the signalling and repress hepcidin expression [22, 23]. As fragments originating from TMPRSS6 cleavage are multiple [21, 26, 27], we hypothesize that other sites are cleaved in HJV. To verify this hypothesis, we mutagenized HJV R residues to alanine (A) and analysed the mutant proteins processing, their sensitivity to TMPRSS6 cleavage and their hepcidin activating ability. We better characterized the N-terminal fragments deriving from HJV autoproteolytic process

Materials and methods

Results

Discussion

Conflicts of interest