Abstract
Deficiency in ferrochelatase (FECH), the last enzyme in the heme biosynthetic pathway, leads to an accumulation of protoporphyrin IX (PPIX) that causes a severely painful phototoxic reaction of the skin in patients with erythropoietic protoporphyria (EPP). Besides phototoxicity of the skin, EPP patients often present with symptoms of iron deficiency in form of a microcytic and hypochromic anemia with low serum iron and ferritin. In addition, elevated aminolevulinic acid synthase 2 (ALAS2) both at the mRNA and protein levels have been observed among EPP patients. ALAS is the first enzyme in the pathway and exists in two isoforms, whereby the isoform 2 (ALAS2) is expressed exclusively in erythropoiesis. The mRNA of ALAS2 contains an iron response element (IRE) at its 5′UTR. When iron is limited, iron response element binding protein 2 (IRP2) binds to the IRE of ALAS2 mRNA and suppresses its translation. In this study, we demonstrated that iron deprivation increased the amount of ALAS2 mRNA as well as the ratio of ALAS2 to FECH mRNAs in cultured erythroleukemic K562 cells. At the protein level, however, iron deprivation in the cell line caused reductions in both enzymes as shown by the Western blot analysis. A comparable increase in the ratio of ALAS2 to FECH mRNAs was also found in EPP patients indicating an imbalance in heme biosynthesis. As iron cannot be completely missing from an organism, we assume that in EPP patients, a certain amount of ALAS2 mRNA is translated despite a partial deficiency of FECH. The increase in ALAS2 enzyme contributes to the accumulation in PPIX in the patients. Targeted inhibition of ALAS2 could therefore be a treatment option for EPP.
Highlights
The heme biosynthetic pathway consists of eight sequential enzymatic steps
The results of all four experiments, each in duplicates, were combined (n = 8) and showed that the amount of aminolevulinic acid synthase 2 (ALAS2) mRNA increased from 28.8 ± 11.3 a.u. in cells with no DFO added to 38.5 ± 19.9, 91.3 ± 43.7 and 136.8 ± 61.3 a.u. in cells treated with 50 μM, 100 μM and 200 μM DFO, respectively (p < .0001)
We demonstrated that iron deprivation induced by iron chelator DFO led to a dose-dependent increase in ALAS2 mRNA in erythroleukemic K562 cells
Summary
The heme biosynthetic pathway consists of eight sequential enzymatic steps. In the last step of the pathway, ferrochelatase (FECH; EC4.99.1.1) inserts a ferrous iron (Fe++) into protoporphyrin IX (PPIX) to give rise to the final product heme. The cause of EPP in the majority of the patients are loss-of-function mutations in the FECH gene together with a splice modulating single nucleotide polymorphism (SNP) c.31548. T and C alleles produce an aberrantly spliced FECH mRNA that contains a 63 bp long intron 3 sequence. Since there is a premature termination codon (PTC) present in the intronic sequence, the aberrant splice product is subjected to nonsense mediated decay (NMD). EPP patients with c.315-48C in trans to a FECH mutation exhibit residual enzyme activities below a threshold of approximately 35% of normal. Very few patients have two separate mutations in the FECH gene [2]
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