Utilization of Intracellular Ferritin Iron for Hemoglobin Synthesis in Developing Human Erythroid Precursors

Abstract

Ferritin (Ft) plays an important role in cellular iron metabolism. It can store substantial amounts of iron in a nontoxic soluble form. However, its ability to donate iron for cellular needs, in particular for hemoglobin (Hb) synthesis in human erythroid cells, is still controversial. We studied the role of intracellular Ft-iron in Hb synthesis and the involvement of lysosomal proteolysis in iron release from Ft. Ft-iron release and its subsequent incorporation into heme was investigated in normal human erythroid precursors developing in culture. Dual staining flow cytometry with antibody (Ab)-specific for Ft and for Hb showed a decrease in cellular Ft content in erythroid cells during their maturation. Cellular Ft-iron participation in heme synthesis was studied by labeling cells with 59Fe. Cells were incubated with 59Fe-labeled human diferric transferrin (Tf), then chased, and intracellular radioiron distribution between Ft and Hb was determined on subsequent days by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and/or Ft immunoprecipitation and heme extraction. On day 6, most of the 59Fe accumulated in Ft. Thereafter, a progressive decrease of radioiron in Ft and a corresponding increase of the label in Hb was observed. Inhibition of heme synthesis with succinylacetone caused radioiron to remain in Ft and prevented its redistribution. Addition of unlabeled diferric Tf to the culture medium did not prevent radioiron from appearing in Hb. Chloroquine repression of lysosomal function prevented radio-iron redistribution between Ft and Hb. Inhibition of proteolysis by chymostatin and/or leupeptin led to Ft-protein accumulation in the cells and also prevented radioiron transfer from Ft to Hb. The results of the present study suggest that intracellular Ft donates iron for heme synthesis and that proteolytic Ft degradation in a lysosmal-like compartment is necessary for iron release and its transfer to heme.