Mutational analysis of the channel and loop sequences of human ferritin H-chain.

Human ferritin H-chain mutants were obtained by engineering the recombinant protein expressed by Escherichia coli. The mutagenesis were directed to the C-terminal sequence forming the hydrophobic channel, to the hydrophilic channel and to the loop sequence. The mutants were analysed for extent of expression, for stability, for capacity to incorporate iron and for kinetics of iron uptake and iron oxidation. Of the 22 mutants analysed only two with deletions of single residues in the loop sequence and one with deletion of the last 28 amino acid residues did not assemble into ferritin-like proteins. The other mutants assembled correctly and showed similar chemical/physical properties to the wild-type; they included duplication of an 18-amino acid-residue stretch, deletion of the last 22 and the last seven residues and various mutations of single amino acid residues. Two mutants with extensive alteration in the C-terminal sequence had a diminished thermostability associated with incapability to incorporate iron though they still catalysed iron oxidation. The mutants with alterations of the sequence around the hydrophilic channel showed diminished iron uptake and oxidation kinetics, together with a slightly larger apparent molecular size. The results indicate (i) that two of the sequences are important for ferritin assembly/stability, (ii) that the presence of the hydrophobic channel is essential for formation of the iron core and (iii) that the sites of iron interaction and the path of iron penetration into ferritin remain unidentified.

Expression and structural and functional properties of human ferritin L-chain from Escherichia coli.

The human ferritin L-chain cDNA was cloned into a vector for overproduction in Escherichia coli, under the regulation of a lambda promoter. The plasmid obtained contains the full L-chain coding region modified at the first two codons. It is able to direct the synthesis of the L-chain which can constitute up to 15% of the total soluble protein of bacterial extract. The L-chains assemble to form a ferritin homopolymer with electrophoretic mobility, molecular weight, thermal stability, spectroscopic, and immunological properties analogous to natural ferritin from human liver (95% L-chain). This recombinant L-ferritin is able to incorporate and retain iron in solution at physiological pH values. At variance with the H-ferritin, the L form does not uptake iron at acidic pH values and does not show detectable ferroxidase activity. It is concluded that ferritin L-chain lacks the ferroxidase site present in the H-chain and that the two chains may have specialized functions in intracellular iron metabolism.

Mechanism of ferritin iron uptake: activity of the H-chain and deletion mapping of the ferro-oxidase site. A study of iron uptake and ferro-oxidase activity of human liver, recombinant H-chain ferritins, and of two H-chain deletion mutants.

To study the functional differences between human ferritin H- and L-chains and the role of the protein shell in the formation and growth of the ferritin iron core, we have compared the kinetics of iron oxidation and uptake of ferritin purified from human liver (90% L) and of the H-chain homopolymer overproduced in Escherichia coli (100% H). As a control for iron autocatalytic activity, we analyzed the effect of Fe(III) on the iron uptake reaction. The results show that the H-chain homopolymer has faster rates of iron uptake and iron oxidation than liver ferritin in all the conditions analyzed and that the difference is reduced in the conditions in which iron autocatalysis in high: i.e. at pH 7 and in presence of iron core. We have also analyzed the properties of two engineered H-chains, one lacking the last 22 amino acids at the carboxyl terminus and the other missing the first 13 residues at the amino terminus. These mutant proteins assemble in ferritin-like proteins and maintain the ability to catalyze iron oxidation. The deletion at the carboxyl terminus, however, prevents the formation of a stable iron core. It is concluded that the ferritin H-chain has an iron oxidation site which is separated from the sites of iron transfer and hydrolysis and that either the integrity of the molecule or the presence of the amino acid sequences forming the hydrophobic channel is necessary for iron core formation.