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.
Levi
S
S
Dipartimento di Scienze e Tecnologie Biomediche, Università di Milano, Ospedale San Raffaele, Italy.
Luzzago
A
A
Franceschinelli
F
F
Santambrogio
P
P
Cesareni
G
G
Arosio
P
P
D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005293:Ferritins / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005796:Genes; D006801:Humans; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D046911:Macromolecular Substances; D009154:Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D005293:Ferritins; D007501:Iron