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Mutational analysis of the four alpha-helix bundle iron-loading channel of rat liver ferritin
1Biotechnology Center, Utah State University, Logan, Utah 84322-4705, USA.
Insights
The heavy chain of ferritin (rH-Ft) facilitates iron loading via ceruloplasmin, requiring an intact alpha-helix bundle channel and a site that stimulates ferroxidase activity.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Structure-Function Relationships
Background:
- Ferritin heavy chain (H-Ft) is crucial for iron loading with ceruloplasmin.
- The exact mechanism of iron uptake and ceruloplasmin interaction with ferritin remains incompletely understood.
Purpose of the Study:
- To investigate the role of the proposed iron-loading channel in ferritin's heavy and light chains.
- To determine if the ferritin channel and ceruloplasmin ferroxidase activity are interdependent.
Main Methods:
- Site-directed mutagenesis was used to alter putative iron-loading channels in recombinant rat liver H-chain ferritin (rH-Ft) and L-chain ferritin (rL-Ft).
- Mutant ferritins were expressed in insect cells, purified, and characterized for solubility and polymer formation.
- Iron loading assays were performed using ceruloplasmin as the ferroxidase, and ferroxidase activity was measured.
Main Results:
- Mutating the rH-Ft channel prevented iron loading, while mutating the rL-Ft channel allowed some iron loading.
- Iron loading rates were significantly higher for rH-Ft compared to the rL-Ft mutant.
- Ceruloplasmin ferroxidase activity was enhanced by rH-Ft and its mutant, but not by rL-Ft or its mutant.
Conclusions:
- The alpha-helix bundle channel in ferritin is essential for efficient iron loading.
- An additional site on ferritin, likely on the H-chain, is required to stimulate ceruloplasmin's ferroxidase activity for optimal iron uptake.
Abstract:
We previously reported that the heavy chain of ferritin was required for loading it with iron using ceruloplasmin as a ferroxidase [J.-H. Guo, M. Abedi, and S. D. Aust (1996) Arch. Biochem. Biophys. 335, 197-204]. Site-directed mutagenesis, K58E and G61H, on recombinant rat liver L chain ferritin (rL-Ft) was performed to construct a proposed iron-loading channel in the alpha-helix bundle similar to rat liver H chain ferritin (rH-Ft). Conversely, the channel in rH-Ft was closed by mutations E62K and H65G to form a K62 to E107 salt bridge, which is believed to exist in the L chain. Both variants were expressed in insect cells and were soluble and able to form multi-subunit homopolymers. The rH-Ft mutant homopolymer could not be loaded, whereas the rL-Ft mutant homopolymer could be loaded with iron by ceruloplasmin. However, we found that the initial rate of iron loading into the rL-Ft mutant homopolymer by ceruloplasmin was less than that into the rH-Ft homopolymer. When 500 atoms of iron per ferritin were used for loading, 98% was loaded into the rH-Ft homopolymer by ceruloplasmin in 15 min, but only 30% was loaded into the rL-Ft mutant homopolymer in the same time. Moreover, the ferroxidase activity of ceruloplasmin was enhanced in the presence of the rH-Ft and the rH-Ft mutant homopolymers, but not in the presence of the rL-Ft or the rL-Ft mutant homopolymers. These observations suggested that the four alpha-helix bundle channel of ferritin is required for iron loading, but an additional factor, i.e. , a site which stimulate the ferroxidase activity of ceruloplasmin, is also essential.