A novel TMPRSS6 mutation that prevents protease auto-activation causes IRIDA

Abstract

IRIDA (iron-refractory iron-deficiency anaemia) is a rare autosomal-recessive disorder hallmarked by hypochromic microcytic anaemia, low transferrin saturation and high levels of the iron-regulated hormone hepcidin. The disease is caused by mutations in the transmembrane serine protease TMPRSS6 (transmembrane protease serine 6) that prevent inactivation of HJV (haemojuvelin), an activator of hepcidin transcription. In the present paper, we describe a patient with IRIDA who carries a novel mutation (Y141C) in the SEA domain of the TMPRSS6 gene. Functional characterization of the TMPRSS6(Y141C) mutant protein in cultured cells showed that it localizes to similar subcellular compartments as wild-type TMPRSS6 and binds HJV, but fails to auto-catalytically activate itself. As a consequence, hepcidin mRNA expression is increased, causing the clinical symptoms observed in this IRIDA patient. The present study provides important mechanistic insight into how TMPRSS6 is activated.

Figure 1. Elevated urinary hepcidin levels in the IRIDA patient

Urine was analysed from the IRIDA patient and four healthy age-matched male volunteers (Ctrl) by SELDI–TOF MS applying CM10 ProteinChips. Hepcidin arbitrary intensity units were normalized to creatinine.

Figure 2. Family pedigree

Sequence analysis of the TMPRSS6 gene indicates heterozygosity for the novel c.442A>G mutation in both parents, and homozygosity for the same mutation in the affected proband (marked by an arrow).

Figure 3. c.442A>G base substitution does not affect splicing

A minigene spanning exons 3 to 5 of the TMPRSS6 gene was transfected in HeLa cells. Correct splicing between exons 4 and 5 was detected by sequence analysis of the resulting cDNA. The c.442A>G mutation is indicated by an asterisk. The sequence in capital letters indicates the sequence of the minigene. The sequence in lower case represents the sequence of the wild-type TMPRSS6 cDNA (GenBank® accession number NM_153609).

Figure 4. The tyrosine residue at position 141 is highly phylogenetically conserved

(A) Alignment of the TMPRSS6 SEA domain. Tyr¹⁴¹, shown in bold red, is highly evolutionarily conserved. (B) Ribbon structure of the human SEA domain of TMPRSS6 generated by adaptation of the crystal structure of the SEA domain of the murine TMPRSS11d transmembrane protease (PDB ID 2E7V). The localization of Tyr¹⁴¹ within the second β-sheet of this domain is indicated.

Figure 5. TMPRSS6 and TMPRSS6(Y141C) localize to similar subcellular regions

(A) Localization of transfected TMPRSS6–EGFP and TMPRSS6(Y141C)–EGFP in HeLa cells by live confocal microscopy. (B) Cell-surface expression of TMPRSS6–FLAG and TMPRSS6(Y141C)–FLAG in transiently transfected HeLa cells. Cells were fixed without permeabilization, and membrane proteins were detected using an anti-FLAG antibody. Signal detection was by confocal microscopy.

Figure 6. TMPRSS6(Y141C) binds HJV, but fails to auto-catalytically activate itself

(A) HeLa cells were transfected with plasmids expressing HJV-Myc or pcDNA3.1-FLAG (mock), TMPRSS6-FLAG or TMPRSS6(Y141C)-FLAG as indicated. After TMPRSS6 immunoprecipitation using an anti-Flag antibody (IPαFLAG), TMPRSS6 and the TMPRSS6-bound HJV were immunorecognized by Western blotting using an anti-FLAG antibody (WBαFLAG) or an anti-Myc antibody (WBαMyc) respectively. (B) HeLa cells were transfected with pcDNA3.1-FLAG (mock), TMPRSS6-FLAG or TMPRSS6(Y141C)-FLAG. After 24 in serum-free medium, the conditioned medium (CM) was collected, concentrated and analysed for the presence of the proteolytic TMPRSS6 fragments by Western blotting. (C) HeLa cells were transfected with TMPRSS6-FLAG or TMPRSS6(Y141C)-FLAG, washed and incubated with serum-free OPTImem (Gibco) with or without 0.1 mM 2-mercaptoethanol (β-ME) for 12 h. The conditioned medium was collected, concentrated and analysed by Western blotting for cdTMPRSS6. Effective TMPRSS6 transfection was assessed in the cell lysate (CL).

Figure 7. TMPRSS6(Y141C) expression fails to efficiently suppress hepcidin promoter activity

(A) Luciferase assay. The reporter plasmid pGL3-hepcidin(WT_2.7kb) was transfected with either HJV alone or together with TMPRSS6 or TMPRSS6(Y141C) in Hep3B cells. At 24 h later, luciferase activity was measured and normalized against Renilla luciferase. Results are mean±S.D. fold changes of transfected cells compared with cells transfected with the reporter construct (WT_2.7kb) only. (B) qRT-PCR assay. Hep3B cells were transfected with either HJV alone or together with TMPRSS6 or TMPRSS6(Y141C). At 24 h later, total RNA was extracted. Hepcidin mRNA expression was analysed by qRT-PCR and was normalized to GAPDH. Results are mean±S.D. fold changes.