Ferritin Apoferritin

Figure 16-3 Structure of the protein shell of ferritin (apoferritin). (A) Ribbon drawing of the 163-residue monomer. From Crichton.62 (B) Stereo drawing of a hexamer composed of three dimers. (C) A tetrad of four subunits drawn as a space-filling diagram and viewed down the four-fold axis from the exterior of the molecule. (D) A half molecule composed of 12 subunits inscribed within a truncated rhombic dodecahedron. B-D from Bourne et al.7i...

Ferritin is another protein that is important in the metabolism of iron. Under normal conditions, it stores iron that can be called upon for use as conditions require. In conditions of excess iron (eg, hemochromatosis), body stores of iron are greatly increased and much more ferritin is present in the tissues, such as the liver and spleen. Ferritin contains approximately 23% iron, and apoferritin (the protein moiety free of iron) has a molecular mass of approximately 440 kDa. Ferritin is composed of 24 subunits of 18.5 kDa, which surround in a micellar form some 3000-4500 ferric atoms. Normally, there is a little ferritin in human plasma. However, in patients with excess iron, the amount of ferritin in plasma is markedly elevated. The amount of ferritin in plasma can be conveniently measured by a sensitive and specific radioimmunoassay and serves as an index of body iron stores. 

Ferritins have been found in a wide range of species, and sequence data - some, as in the first ever sequence of horse spleen apoferritin (Heusterspreute and Crichton, 1981) determined by direct methods, but many now by DNa sequencing 1, have been deposited for more than 70 ferritins. They vary in length from 154-185 residues per subunit. Some ferritins have N-terminal extensions which lie on the outside of the assembled shell and target the ferritin to a specific destination such as plastids in plants and yolk sac in snails (Andrews etah, 1992 Lobreaux etah, 1992). For example, pea ferritin is synthesized with an N-terminal extension of 75 residues, which is missing from the mature protein. The first part of this extension is a chloroplast-targetting sequence of 47 residues, which is lost on entry into the plastid. The second part, an extension peptide, is lost prior to assembly of the... 

Horse-spleen apoferritin crystallizes in a face-centred, close-packed, cubic arrangement, in the space group F432, with molecules at the 432 symmetry points of the crystal lattice (Harrison, 1959). This publication was the logical extension of the DPhil thesis of the Oxford chemist Pauline M. Cowan (as she was before her marriage to Roy Harrison), and represented the first publication in what was to be a long and distinguished series of contributions on ferritin from the undisputed Iron Lady of iron metabolism. ... 

The crystallization of horse spleen apoferritin was in fact a fortuitous coincidence, because, as we mentioned earlier, attempts to crystallize horse-liver ferritin were not successful, whereas the iron-rich ferritin from horse spleen could be crystallized (Laufberger, 1937). This was certainly related to the relatively high content of H subunits (average composition L12H12) in horse liver (something that was only discovered 50 years later). It has generally proved very difficult if not impossible to crystallize heteropolymers, and the best results in crystallographic terms have been obtained with recombinant homopolymers. As will be discussed later in this chapter,... 

When pea seed apoferritin is reconstituted in vitro in the absence of phosphate, the reconstituted mineral core consists of crystalline ferrihydrite (Rohrer et ah,1990 Wade et ah, 1993 Waldo et ah, 1995). Conversely, horse spleen ferritin reconstituted in the presence of phosphate produces an amorphous core (Rohrer et ah,1990 St. Pierre et ah, 1996)... 

Several binding sites for Tb3+ or Cd2+ ions have been identified in the interior of the apoferritin protein shell, some of which may be iron-binding sites (Harrison et ai, 1989 Granier et ah, 1998). In HoSF and HoLF, two sites were identified on the inner surface of the B helix at the subunit dimer interface (Figure 6.15, Plate 11) which bind two Cd2+ ions. One involves Glu-57 and Glu-60 as ligands and the other Glu-61 and Glu-64 (Granier et al., 1998). In H-chain ferritins the first pair of Glu-57 and Glu-60 are both replaced by His and only a single Tb3+ is found bound to Glu-61 and Glu-64 (Lawson et al, 1991). 

Iron oxidized by ferritin must be at or near the outer surface of the apoferritin molecule, since iron appears to be exchanged between ferritin molecules, as shown by Mossbauer spectroscopy (Bauminger et ah, 1991a,b) and by the observation that iron oxidized by ferritin can be taken up directly by apotransferrin (Bakker and Boyer, 1986 Jin and Crichton, 1987). 

We assume that all substances involved in ferritin iron deposition (Fe2+, Fe3+, 02) need to gain access to the interior of the apoferritin protein shell. The most likely pathway is via the three-fold channels, which would involve passing through the 12 A long channel, and then traverse a further distance of 8 A along a hydrophilic pathway from the inside of the... 

In order to deal with a system whose structural characteristics were unaltered by the loading with Gd(III) chelates, we choose Apoferritin because it allows the Imaging Probes to be entrapped inside its inner cavity (60). The exterior of such Gd(III)-loaded Apoferritin is exactly the same as in the parent Ferritin and then, once administered intravenously, it is quickly cleared-up by the proper receptors on hepatocytes (172). The process of loading Apoferritin with [GdHPD03A(H20)] consists first of the dissociation of the protein into subunits at pH 2, followed by its reforming at pH 7, thereby trapping the solution components (e.g., [GdHPD03A(H20)])... 

Ferritin is composed by the arrangement of 24 protein subunits, which results in a hollow shell of 8 nm inner diameter and 13 nm outer diameter (Fig. 13). Ferritin from vertebrates have two types of subunits heavy (H) and light (L). The subunit composition of human ferritins depends on the origin of the protein H2L22 for liver ferritin, H20L4 for muscle ferritin, etc. Access channels are formed by the intersection of subunits. The 8 channels located at the intersection of three subunits are hydrophilic while the 6 channels located at the intersection of 4 subunits are hydrophobic. The empty protein is called apoferritin (30). 

Fig. 15. Longitudinal NMRD profile of ferritin ( ) and apoferritin ( ) aqueous solutions at 37°C. The contribution of the ferrihydrite core to the relaxation ( ) is obtained by the subtraction of the profiles. Ferritin solution has an iron concentration of 100 mM, while the protein concentration of both samples is 0.058 mM. Longitudinal NMRD profile of akaganeite particles (O) with an iron concentration of 100 mM.

Ferritin The longitudinal relaxation rate of ferritin is not significantly influenced by the pH of the aqueous solution (47,48). However, after subtraction of the apoferritin contribution, the effect is more significant (Fig. 18). The transverse relaxation of ferritin solutions is almost pH independent (Pig. 16). 

Since the (Fen05(0H)6> unit is stable, it has been speculated(8b,17b) that it might also be present in the ferritin core. Since the majority of phosphate in ferritin is adventitious, surface bound and the metallic core can be reconstituted in the absence of phosphate groups with no change in the X-ray powder diffraction pattem(l), replacement of bridging phosphate by bridging carboxylate groups should not influence the three dimensional structure of the core. Calculations show that -409 Fell nnits could fill the apoferritin inner cavity. Further details can be found in reference 17. 

Formation of ferritin involves assemblage of the protein subunits to form the apo-ferritin shell which is then filled with the phosphated ferrihydrite core. The mechanism by which ferritin is filled and the iron core built up, has been investigated intensively in vitro. The experiments usually involved incubating apoferritin (from horse spleen) with Fe salts in the presence of an oxidant such as molecular oxygen. They showed that ferritin could be reconstituted from apoferritin and a source of Fe both the iron and the oxygen enter the protein shell, whereupon oxidation of Fe is catalysed by the interior surface of the protein shell (Macara et al., 1972). 

Macara, I.G. HoyT.G. Harrison, P.M. (1972) Formation of ferritin from apoferritin. Kinetics and mechanism of iron uptake. Bio-chem. J. 126 151-152... 

Protein subunits will partially dissociate from crystalline ferritin in dilute salt solutions to yield a non-crystallizable ferritin. The non-crystallizable ferritin, in turn, in the presence of apoferritin appears to pick up protein subunits and by action yield crystalline ferritin molecules. The scheme for this process is shown in Fig. 6. The salient feature of this scheme is the initial formation of an iron micelle from soluble iron chelates which is then stabilized by protein subimits S5uithesized by the tissue (726). Harrison and Gregory 127) have used glacial acetic acid to dissociate apoferritin completely. When the pH is adjusted to 4 in the presence of thiol compoimds, apoferritin is rapidly formed, indicating strong subunit interaction. 

The early work of Fineberg and Greenberg 128) as well as that of Drysdale and Monroe 129) indicate that oral or intramuscular administration of iron enhances the rate of ferritin synthesis in the intestinal mucosa as well as other tissue. Drysdale and Monroe foimd little effect of actinomycin, an inhibitor of RNA synthesis, on iron-induced ferritin synthesis and suggested that the apoferritin was stabilized by being bound to the iron core. Recently Yoshino et al. 130) using higher concentrations of actinomycin were able to inhibit markedly the induced synthesis of ferritin. The mechanism for iron induction of ferritin is certainly not clear at this time. 

The tendency for ferritin to aggregate is shown 135, 136) by its heterogeneity as demonstrated on gel electrophoresis, column chromatography, ultra-centrifugation, and many other techniques. This association is readily reversible. The re-examination of any single ferritin fraction isolated from a mixture will show at least four fractions similar to the original preparation. This association is a function of the protein subunit surface of the molecule. Apoferritin molecules behave in a quite similar fashion 137) the iron core has little if any effect. 

Iron is transported via transferrin. When body stores of iron are high, ferric iron combines with apoferritin to form ferritin. Ferritin is the protein of iron storage. About 80 percent iron in plasma goes to erythroid marrow. The excretion of iron is minimal. Only little amount of iron is lost by exfoliation of intestinal mucosal cells and trace amount is excreted in urine, sweat and bile. 

Absorption, transport, and storage of iron. Intestinal epithelial cells actively absorb inorganic iron and heme iron (H). Ferrous iron that is absorbed or released from absorbed heme iron in the intestine (1) is actively transported into the blood or complexed with apoferritin (AF) and stored as ferritin (F). In the blood, iron is transported by transferrin (Tf) to erythroid precursors in the bone marrow for synthesis of hemoglobin (Hgb) (2) or to hepatocytes for storage as ferritin (3). The transferrin-iron complexes bind to transferrin receptors (TfR) in erythroid precursors and hepatocytes and are internalized. After release of the iron, the TfR-Tf complex is recycled to the plasma membrane and Tf is released. Macrophages that phagocytize senescent erythrocytes (RBC) reclaim the iron from the RBC hemoglobin and either export it or store it as ferritin (4). Hepatocytes use several mechanisms to... 

In addition to the storage of iron in intestinal mucosal cells, iron is also stored, primarily as ferritin, in macrophages in the liver, spleen, and bone, and in parenchymal liver cells (Figure 33-1). Apoferritin synthesis is regulated by the levels of free iron. When these levels are low, apoferritin synthesis is inhibited and the balance of iron binding shifts toward transferrin. When free iron levels are high, more apoferritin is produced to sequester more iron and protect organs from the toxic effects of excess free iron. 

---