Ferritin and Isoferitins

Archives of Physiology and Biochemistry, February 2007; 113(1): 30 – 54
ORIGINAL ARTICLE
Ferritin and ferritin isoforms I: Structure–function relationships,

synthesis, degradation and secretion
A. M. KOORTS & M. VILJOEN

Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by Universitat de Girona on 11/04/14
Department of Physiology, School of Medicine, University of Pretoria, Pretoria, South Africa
Abstract
Ferritin is the intracellular protein responsible for the sequestration, storage and release of iron. Ferritin can accumulate up to
4500 iron atoms as a ferrihydrite mineral in a protein shell and releases these iron atoms when there is an increase in the cell’s
need for bioavailable iron. The ferritin protein shell consists of 24 protein subunits of two types, the H-subunit and the
L-subunit. These ferritin subunits perform different functions in the mineralization process of iron. The ferritin protein shell
can exist as various combinations of these two subunit types, giving rise to heteropolymers or isoferritins. Isoferritins are
functionally distinct and characteristic populations of isoferritins are found depending on the type of cell, the proliferation
status of the cell and the presence of disease. The synthesis of ferritin is regulated both transcriptionally and translationally.
Translation of ferritin subunit mRNA is increased or decreased, depending on the labile iron pool and is controlled by an
iron-responsive element present in the 50 -untranslated region of the ferritin subunit mRNA. The transcription of the genes
for the ferritin subunits is controlled by hormones and cytokines, which can result in a change in the pool of translatable
For personal use only.
mRNA. The levels of intracellular ferritin are determined by the balance between synthesis and degradation. Degradation of
ferritin in the cytosol results in complete release of iron, while degradation in secondary lysosomes results in the formation of
haemosiderin and protection against iron toxicity. The majority of ferritin is found in the cytosol. However, ferritin with
slightly different properties can also be found in organelles such as nuclei and mitochondria. Most of the ferritin produced
intracellularly is harnessed for the regulation of iron bioavailability; however, some of the ferritin is secreted and internalized
by other cells. In addition to the regulation of iron bioavailability ferritin may contribute to the control of myelopoiesis and
immunological responses.
Key words: Ferritin, haemosiderin, H-subunit, L-subunit, isoferritins.
vides a reserve for cytochromes, nitrogenases, ribo-

Introduction
nucleotide reductases, haemoglobin, and myoglobin
Ferritin is the major intracellular protein involved in (Theil, 1990). Perhaps equally important to its
the storage and release of intracellular iron and since function in the storage and release of iron is the role
iron is needed in various cellular functions it does not that ferritin plays in the protection of cells against the
come as a surprise that ferritin is expressed in every deleterious effects of iron. Iron exists in two readily
cell type thus far studied (Worwood, 1990). Iron is interconvertible redox states and, at physiological pH
essential for cellular functions such as oxygen and oxygen tension, Fe2þ is readily oxidized to Fe3þ,
transport, electron transfer, nitrogen fixation, DNA followed by the hydrolysis of Fe3þ and the formation
synthesis, and the production of haemoproteins like of insoluble ferric hydroxide and oxyhydroxide
haemoglobin and myoglobin (Ponka et al., 1998). polymers (Finch et al., 1984; Ponka et al., 1998). In
However, not all cell types harness iron for the same addition, Fe2þ can catalyse the production of
purposes or to the same extent and therefore the role harmful oxygen radicals in the Haber – Weiss reac-
of ferritin in the management of intracellular iron tion resulting in peroxidative damage to cellular
differs between the various types of cells. Ferritin can structures (Ponka et al., 1998, Powell, 1998). Within
play a role in specialized functions, e.g. recycling of cells, iron probably exists in a low molecular weight,
iron in macrophages and short- and long-term redox-active form only for short periods—mainly
storage of iron as in hepatocytes, as well as in within the lysosomes. Lysosomes are therefore
intracellular housekeeping functions where it pro- particularly vulnerable to oxidative stress and may
Correspondence: A. M. Koorts, Department of Physiology, PO Box 2034, Pretoria, 0001. Tel: þ27 (0) 12 319 2921. Fax: þ27 (0) 12 321 1679.
E-mail: [email protected]
Received for publication 12 April 2006. Accepted 29 November 2006.

ISSN 1381-3455 print/ISSN 1744-4160 online ª 2007 Informa UK Ltd.
DOI: 10.1080/13813450701318583
Ferritin and ferritin isoforms 31
burst due to intralysosomal Fenton-type chemistry iron is absorbed and lost daily, mostly due to cell
with ensuing peroxidative destabilization of lysoso- desquamation (Ponka et al., 1998), and an efficient
mal membranes. This could result in leaky lysosomes mechanism exists for the shuttling of iron between
and the induction of cellular damage, or even iron absorption or storage sites (including sites for
apoptotic or necrotic death due to the release of a dismantling of spent iron-containing cells such as red
range of powerful hydrolytic enzymes into the cytosol blood cells), and sites of biosynthesis of iron-
(Persson et al., 2001). By sequestering large amounts containing proteins (Siegenberg et al., 1990). Trans-
of iron as a soluble, nontoxic ferrihydrite mineral ferrin is the major transport protein responsible for
within the confines of the ferritin molecule, ferritin this internal shuttling of iron. About 3 mg of iron is
protects the cell against insoluble ferric oxide and found in association with transferrin at any one time
oxyhydroxide formation, as well as against the and the daily exchange of iron by plasma transferrin
production of oxygen radicals (Ford et al., 1984; is about 30 mg (Ponka et al., 1998). Iron is chelated
Torti & Torti, 2002). by transferrin at the cell membrane of the iron-donor
Ferritin is widely distributed in cells and is found cell and the transferrin is subsequently bound by a
in the cytosol, nucleus, mitochondria and lysosomes transferrin receptor on the membrane of the iron-
and appears in plasma as a result of cellular secretion. acceptor cell (Brittenham, 1994). The transferrin–
This specific distribution enables ferritin to supply receptor complex is then internalized by the cell
the particular enzymes and other proteins with enclosed within an endocytic vesicle where iron is
appropriate amounts of iron, and, equally important, released from transferrin by a process that involves
places ferritin in close proximity to sites where large endosomal acidification. The resultant iron-free
amounts of iron are metabolized. Ferritin is actively apotransferrin remains attached to the receptor in
transported into the nucleus to provide, amongst the endosome, and is returned to the cell surface
others, ribonucleotide reductase with iron and to where the apotransferrin is released from the cell
protect DNA from oxidative damage as a result of (Ponka et al., 1998). Cells contain a pool of
inappropriate oxygen radical production due to chelatable or transit iron known as the labile iron
surplus amounts of iron (Thompson et al., 2002). pool bound to low molecular weight intracellular iron
A specific type of ferritin is also found in mitochon- transport compounds, and it is thought that the
dria. Mitochondria is confronted with large amounts endosomal iron released from transferrin transits this
of metabolically active iron and although most cell pool of iron, whereupon iron enters functional
types contain only very small amounts of a mito- compartments, or is stored in ferritin (Jacobs, 1977;
chondrial ferritin, in certain conditions, a specific Thomson et al., 1999). Since the labile pool of iron
ferritin is translocated to the mitochondrium to contains the metabolically and catalytically reactive
sequester unwanted amounts of iron (Levi & Arosio, iron (Piñero et al., 2000), the magnitude of the labile
2004). The regulation of ferritin distribution in pool of iron is maintained by sophisticated control
different parts of the cytosol and in cellular organelles mechanisms that regulate cellular iron uptake and
is controlled, in part, by the various mechanisms storage in a coordinated manner (Ponka et al., 1998).
involved in the uptake of ferritin by the cellular This is achieved by, amongst others, the coordinated
organelles. Furthermore, ferritin binds to microtu- regulation of the expression of the proteins involved
bules and this interaction cannot only contribute to in iron homeostasis such as transferrin, transferrin
transport of ferritin to specific sites and organelles receptor and ferritin. An increase in the intracellular
within the cell, but also to secretion of ferritin from free iron will, for instance, result in up-regulation of
the cell. Depolymerization of microtubules increase ferritin (increase in efficiency of iron sequestration)
ferritin secretion and support the probable role of and down-regulation of transferrin and transferrin
microtubules in regulating the intracellular concen- receptor expression (decrease in iron acquisition
tration, distribution and release of ferritin under potential), whereas a decrease in intracellular free
different physiological circumstances (Hasan et al., iron will result in the opposite (Piñero et al., 2000;
2005). Ponka et al., 1998). This co-ordinated but divergent
regulation of the expression of these proteins is
governed by a single protein namely the iron-
Iron, other iron-regulatory proteins and ferritin
responsive protein (IRP), which can bind to iron-
Humans contain 2 – 5 grams of iron, of which about responsive elements on the mRNA of the iron
two-thirds is present in the red blood cells as homeostasis proteins. This will result in the post-
haemoglobin and about 15 – 20% in storage form as transcriptional regulation of the mRNA of the
ferritin or haemosiderin. Ferritin is the intracellular proteins regulating iron uptake and storage (Piñero
protein responsible for normal iron storage whereas et al., 2000; Thomson et al., 1999).
haemosiderin is a degradation product of ferritin,
which is prominent during conditions of iron over-
Structure of ferritin
load. The remainder of body iron is found mainly in
muscle myoglobin (8%) and in iron-containing Ferritin, like some of the other proteins involved in
enzymes (Brittenham, 1994). Only about 1 mg of the regulation of iron homeostasis in the body,
32 A. M. Koorts & M. Viljoen
including transferrin and lactoferrin, can serve either inner face and the outer face of the subunit and
as an iron donor or iron acceptor (Levay & Viljoen, therefore the subsequent intra-subunit and inter-
1995). However, unlike transferrin and lactoferrin, subunit interactions. The secondary structure of the
ferritin is capable of accepting, storing and donating H-subunit is very similar to that of the L-subunit,
vast amounts of iron. These abilities of ferritin are all despite the fact that they share only 55% amino acid
a function of the structure of ferritin—especially that sequence homology (Worwood, 1990).
of the ferritin protein shell. Ferritin is a protein with a
molecular weight of 450 000 daltons (Theil, 1990) Intra-subunit and inter-subunit amino acid side-chain
and consists of an outer three-dimensional protein interactions of the ferritin protein shell. Highly conserved
shell enclosing an 80 Å diameter inner cavity amino acid residues are involved in intra-subunit and
(Harrison & Arosio, 1996). In this inner cavity inter-subunit interactions (Boyd et al., 1985; Theil,
ferritin is capable of sequestering variable amounts 1990; Worwood, 1990). The different types of intra-
of Fe3þ-atoms as a ferrihydrite mineral. When fully subunit and inter-subunit amino acid side-chain
saturated, ferritin can store up to 4500 Fe3þ-atoms, interactions that are important for ferritin folding
but the usual amount is closer to 2000 Fe3þ-atoms and stability include hydrogen bonds, salt-bridges
(Ford et al., 1984; Harrison & Arosio, 1996). and hydrophobic interactions (Theil, 1990). The
contributions of these types of interactions to the
folding and stability of the H- and L-subunits differ
Structure of the ferritin protein shell
significantly between the two types of subunits. The
The outer three-dimensional protein shell contains a intra-subunit hydrogen bonds are about 50% more
total of 24 protein subunits arranged symmetrically abundant in the H-subunit than in the L-subunit,
(Ford et al., 1984; Harrison & Arosio, 1996). Two whereas the salt bridges are more important in the
types of protein subunits exist, the H-subunit and the stabilization of the L-subunit and accounts for about
L-subunit. The H-subunit (21 kDa) contains 178 30% of the stabilization energy of the L-subunit
amino acids while the L-subunit (19 kDa) contains (Martsev et al., 1998). Due to the large number of
174 amino acids (Ponka et al., 1998; Worwood, intra- and inter-subunit salt bridges the ferritin
1990). Each subunit is folded into four long a-helices molecule is highly stable to thermal and chemical
(A, B, C, D), with a long loop between C and D, and denaturation (Harrison & Arosio, 1996). In addition,
a fifth short helix (E) at the C-terminal (Harrison & the differences in intra-subunit interactions between
Arosio, 1996). Each subunit is roughly cylindrical the H-subunit and L-subunit result in a linear
(5.5 nm long and 2.7 nm wide) (Worwood, 1990). increase in the resistance to denaturation, with
For the L-subunit the amino acid arrangement into L-subunit homopolymers and heteropolymers con-
the various a-helices is as follows: long a-helix A taining a high L-subunit proportion significantly
(amino acid residues 10 – 39), long a-helix B (amino more resistant than H-subunit homopolymers
acid residues 45 – 72), long a-helix C (amino (Martsev et al., 1998; Wade et al., 1991). One
acid residues 92 – 120), long a-helix D (amino acid important difference in intra-chain interactions that
residues 124 – 155) and short a-helix E (amino acid contributes to the L-subunits being more stable than
residues 160 – 169) (Ford et al., 1984). The four long the H-subunits is the salt-bridge lysine 62—glutamic
a-helices are aligned parallel to one another and are acid 107 in the L-subunit which replaces the
tightly packed into a cylindrical subunit bundle (Ford ferroxidase centre of the H-subunit (Wade et al.,
et al., 1984; Worwood, 1990), thus forming the main 1991). However, these differences in intra-chain
subunit axis. Between the four long a-helices the interactions between the H-subunit and L-subunit
inter-helical contact region extends over a length of appear to be largely masked by the presence of strong
35 Å (Ford et al., 1984), while the longest helix, i.e. inter-subunit contacts in assembled molecules con-
the a-helix D, protrudes beyond this main inter- sisting of a combination of H- and L-subunits
helical contact region and folds sharply back so that (Martsev et al., 1998). The interactions between
the a-helix E lies at an angle roughly 60 degrees to subunits responsible for ferritin assembly involve
the main axis of the subunit (Ford et al., 1984; about 50% of the subunit surface and most of
Harrison & Arosio, 1996). The other feature of the the inter-subunit contacts are conserved in H- and
subunit, the long loop L (amino acid residues 73 – L-subunits. The first inter-subunit interaction to take
91), joins the C-terminus of a-helix B to the place in the assemblage of the ferritin protein shell is
N-terminus of a-helix C (Ford et al., 1984). Due to the formation of interactions along the main subunit
the inter-helical contacts and the arrangement of the axis between two subunits creating a dimer pair
subunit in the protein shell, the a-helices B and D (Theil, 1990). One face of the subunit contains
have one face towards the inside of the ferritin hydrophobic residues from a-helix A (valine 20,
protein shell, the a-helices A and C have one face leucine 24, tyrosine 28, leucine 31) and loop L
each towards the outside of the shell, and the loop L (phenylalanine 78, leucine 81, proline 84). By
is displayed on the outermost surface of the ferritin interaction with an equivalent region of a second
protein shell (Ford et al., 1984). This arrangement subunit this hydrophobic patch of some 22 Å in
determines the types of amino acids situated on the length is buried from solvent (Ford et al., 1984).
Another hydrophobic region on the subunit surface is effect on the rate and specificity of the reaction,
that comprising one face of a-helix E (leucine 154, whereas modifications of the carboxyl groups lining
leucine 161, tyrosine 164, leucine 165, leucine 169) the hydrophilic channels reduce the rate of iron
that will also be buried from solvent upon subunit uptake by about twofold (Levi et al., 1996). The
interactions. There are a large number of inter- hydrophilic channel is funnel-shaped, broadening
subunit interactions that form regions of marked out towards the outside surface to give a wide
hydrophobicity and other regions where polar inter- hydrophilic region (Ford et al., 1984; Levi et al.,
actions predominate (Ford et al., 1984). If these 1996). The hydrophilic regions at the outside surface
hydrophobic residues are to be partially buried from of the hydrophilic channels contain negative charges
solvent then further assembly of dimers must occur. surrounded by patches of positive charges creating
Since the assembly of ferritin consisting of 24 electrostatic fields in order to direct Fe2þ-ions
subunits results in the complete concealment of all toward the channel entrance (Chasteen & Harrison,
hydrophobic patches (Ford et al., 1984), the forma- 1999). Once iron has been directed to the opening of
tion of such a molecule is very favourable. the hydrophilic channel, it is bound to hydrophilic
residues located at the outer and inner openings of
Channels present in the ferritin protein shell. For either the channels (Chasteen, 1998). These residues
mineralization or demineralization of the iron core to include cysteine 130 and histidine 118, both of
occur it is important that substances such as Fe2þ/ which face the outer opening of the hydrophilic
Fe3þ, oxidants, reductants and chelators can gain channel (Chasteen, 1998), and aspartic acid 131 and
access to the interior of the ferritin molecule. In order glutamic acid 134 in the narrowest part of the
for these molecules to gain entrance to the interior of channel (Levi et al., 1996; Theil, 1990). Aside from
the protein shell the ferritin protein shell contains the hydrophobic and hydrophilic channels present in
two main types of channels with strikingly different the ferritin protein shell, an H-subunit specific
physical properties. The first type of channel channel exists that connects the ferroxidase site in
comprises six hydrophobic channels with four-fold the centre of the H-subunit to the outer protein
symmetry (12 Å long and 3 – 4 Å wide), which are surface (Boyd et al., 1985). Although transfer of iron
lined by 12 leucine side-chains in the L-subunit through this channel is in general less efficient, the
and 8 leucine plus 4 histidine side-chains in the transfer of Fe3þ-ions to the outside of ferritin may
H-subunit belonging to the a-helix D. The second become important in certain conditions.
type of channel includes eight hydrophilic channels
with threefold symmetry (3 – 4 Å wide), each lined by The ferroxidase catalytic centre of the H-subunit of the
six carboxyl groups, three aspartate residues (on the ferritin protein shell. The first step in the iron
cavity side of the shell) and three glutamate residues sequestration process by ferritin involves the oxida-
(towards the outside of the molecule) belonging to tion of Fe2þ to Fe3þ by oxygen and is facilitated by
the a-helix E (Chiancone & Stefanini, 1984; Ford the ferroxidase centre contained in the H-subunit
et al., 1984; Harrison & Arosio, 1996; Levi et al., (Chasteen, 1998). This ferroxidase centre comprises
1989). The carboxylate groups of the hydrophilic various amino acid side-chains as important iron
threefold channels are essential for rapid iron ligands in the multi-step oxidation of Fe2þ to Fe3þ
transport across the protein shell (Bou-Abdallah and includes a cluster of hydrophilic amino acid
et al., 2002). The hydrophobic character of the six residues (glutamic acid 27, glutamic acid 61,
hydrophobic channels argues against a possible role glutamic acid 62, histidine 65 and glutamic acid
for these channels in the transport of Fe2þ/Fe3þ-ions 107) which are embedded within each of the four
into the interior of the ferritin molecule. These four- a-helix bundles comprising the subunit (Levi et al.,
fold hydrophobic channels are found to be imperme- 1992; Santambrogio et al., 1996; Wade et al., 1991).
able to all cations with the possible exception of This ferroxidase centre is present in the H-subunit,
protons. It is suggested that these fourfold channels but absent in the L-subunit (Wade et al., 1991). The
facilitate proton transfer in and out of ferritin in order L-subunit’s potential ferroxidase activity is lost (Levi
to maintain electroneutrality during iron deposition et al., 1992) due to amino acid changes including
(Takahashi & Kuyucak, 2003). However, the sub- glutamic acid 62 to lysine and histidine 65 to glycine,
stitution of leucine for histidine in the H-subunit may formation of a salt-bridge between lysine 62 and
confer iron transfer properties to the hydrophobic glutamic acid 107 (Lawson et al., 1989) and swinging
channel of the H-subunit since histidine has a strong of glutamic acid 61 into a position facing the cavity
affinity for iron (Boyd et al., 1985). The hydrophilic (Chasteen, 1998).
channels are the most likely routes of iron entry into
the protein shell and are probably functional in both The nucleation site of the L-subunit on the inner iron/
the H-subunit and the L-subunit. This is indicated protein interface of the ferritin protein shell. The second
by a high degree of conservation of the three step in the iron sequestration process by ferritin
glutamates and three aspartates in both subunits involves the formation of Fe3þ-nuclei and the
(Chasteen & Harrison, 1999). Furthermore, altera- subsequent growth of an iron core. In order for
tion of residues of the hydrophobic channels has little ferritin to support Fe3þ-nuclei formation and the
growth of the iron mineral, ferritin must contain the negative charges responsible for the greater efficiency
Fe3þ-atoms in the inner cavity of the protein shell of L-subunits in iron-core nucleation (Levi et al.,
and stabilize subsequently incoming Fe3þ-atoms on 1992; Levi et al., 1994).
the growing iron core. This is accomplished by
amino acid side-chain ligands present on the inner
The iron mineral
surface of the ferritin protein shell. Binding of Fe3þ
to these ligands supports Fe3þ-nuclei formation, as The iron mineral is enclosed in a cavity by a protein
well as the subsequent growth of the iron core. This shell and contains different types of environments in
results in the contact of the ferritin protein shell with which the Fe3þ-ion is located. These different types
the iron core at several points on the inner surface, of environments include:
forming an iron/protein interface. These iron/protein
interfaces, which define the sites of core nucleation, 1. Iron atoms located at nucleation sites—these
probably exist where the protein subunit dimers Fe3þ-ions are co-ordinated by amino acid side
interact (Theil, 1990). In contrast to the first step in chain ligands from the inner protein surface
iron sequestration, i.e. oxidation of iron, which is and inorganic bonds from the mineral surface
accomplished mainly by the H-subunits, this second (Powell, 1998).
step, i.e. Fe3þ-nuclei formation and the growth of the 2. Iron atoms at the surface of the mineral—these
iron-core is mainly a function of the L-subunits, Fe3þ-ions are connected to the bulk mineral, as
which supply the appropriate amino acid side-chains. well as to the Fe3þ-ions within the nucleation
These amino acid side-chains act as ligands for the sites in the protein shell through inorganic oxide/
initially formed Fe3þ or as negatively charged hydroxide linkages and there may be additional
domains that lower the activation energy of iron- linkages to the Fe3þ-ions of the shell through
core formation (Wade et al., 1991). The reason that dinucleating amino acid side-chain ligands
the H-subunits are less efficient in nucleation and (Powell, 1998).
iron-core formation is that only seven of the 3. Iron atoms which are in environments corre-
hydrophilic amino acid side-chains in L-subunits sponding to those of the bulk mineral connected
that line the inner surface, and which are not through inorganic oxide/hydroxide linkages,
involved in the formation of salt bridges or hydrogen where the Fe3þ-ions are located in the interstices
bonds but thought to bind iron, are conserved in between two hexagonally closely packed layers of
H-subunits. Amino acids important in nuclei forma- oxygen (Wixom et al., 1980; Worwood, 1982).
tion and growth of the iron core include histidine 49, The Fe3þ-ions appear to be in predominantly
arginine 52, glutamic acid 53, glutamic acid 56, octahedral environments but up to one-third of
glutamic acid 57, arginine 59, glutamic acid 60, the Fe3þ-ions are in tetrahedral sites, with an
glutamic acid 61, arginine 64 and lysine 67 on a-helix average of six oxygen atoms at a distance of
B, with glutamic acid 136, lysine 139 and lysine 142 approximately 2 Å. This, more or less, corre-
on a-helix D, as well as 3 residues at the C-terminus, sponds to the structure for ferrihydrite (Chasteen
i.e. lysine 172, histidine 173 and aspartic acid 174. & Harrison, 1999; Ford et al., 1984; Powell,
The differences in amino acid side-chains between 1998).
H- and L-subunits result in the loss of negative
charges in the H-subunit on the inner iron/protein The iron core may differ between ferritin mole-
interface so that H-subunits have a lower ability to cules, as well as within the same ferritin molecule,
nucleate iron (Boyd et al., 1985; Ford et al., 1984; and can contain single or multiple crystallites and
Wade et al., 1991). A cluster of three L-subunit amorphous regions (Chasteen & Harrison, 1999).
glutamates has specifically been implicated in Fe3þ- One or more of such electron-dense crystallites are
nuclei formation. This cluster of glutamates, i.e. anchored to the inner surface of the protein shell
glutamic acid 57, glutamic acid 60, and glutamic acid (Wixom et al., 1980). The average diameter of the
61, form a region of negativity for the binding of iron cores range from 2.5 to 9 nm (Chasteen &
Fe3þ. In the H-subunit glutamic acid 57 and Harrison, 1999). The formation of a single crystallite
glutamic acid 60 are substituted for histidine and approaching the inner diameter of the protein shell or
glutamic acid 61 is involved in the ferroxidase centre the formation of various smaller crystallites depends
of the H-subunit. It is suggested that glutamic acid on the availability of Fe2þ-/Fe3þ-ions during the later
61 could also play a role in the nucleation of Fe3þ phases of mineral growth after nucleation has
after oxidation of Fe2þ. However, this putative occurred (Theil, 1990). Striking features of the iron
nucleation site, involving glutamic acid 61, glutamic cores are their variable phosphate contents (Chasteen
acid 64 and glutamic acid 67, does not play a & Harrison, 1999; Harrison & Arosio, 1996).
role in nucleation and growth of the iron core Phosphate ions are found on the surface of the iron
(Bou-Abdallah et al., 2004). In the L-subunit cores where they replace some of the surface
glutamic acid 61 is swung into a position on the hydroxyl groups (Ford et al., 1984). However, recent
inner cavity surface in proximity to glutamic acid 57 observations indicate that phosphate can also be
and glutamic acid 60 to participate in this cluster of found throughout the iron core, that cores can have
ordered and disordered regions, and that the glutamate glutamic acid 27, whereas site B has two
disorder increases when phosphate increases (Theil, carboxylate ligands in addition to the bridging
1990). In vivo the phosphate contents of ferritin iron carboxylate. These are glutamic acid 107 and
cores appear to be in dynamic equilibrium with cell glutamic acid 61. The third site, i.e. site C, lies in
phosphate (Ford et al., 1984) and it has long been the inner surface of the protein shell at a distance of
suggested that phosphate plays a role in iron home- 7 Å from the di-iron site. The first step in Fe2þ
ostasis (Powell, 1998). This assumption has recently oxidation involves the binding of incoming Fe2þ
been supported by the fact that phosphate can atoms with each of sites A and B, followed by the
stimulate the rate of iron uptake by providing binding formation of a m-oxo-bridge (Treffry et al., 1997).
sites on the mineral surface for incoming iron atoms The affinity of site A for Fe2þ is higher than the
and as such may play a role in the oxidation of Fe2þ affinity of site B, resulting in the occupation of site A
on the mineral surface (Polanams et al., 2005). by Fe2þ before site B. Binding of Fe2þ to site B
follows O2 binding and/or oxidation of the first Fe2þ
(Bou-Abdallah et al., 2002). After about an hour of

Mechanism of iron sequestration and release:
intermediate m-oxo-bridged dimer formation, this
The role of the ferritin protein shell in iron
complex splits into highly mobile Fe3þ-monomers
mineralization and demineralization
that can move to the cavity for Fe3þ hydrolysis,
Ferritin concentrates iron in cells by directing the nucleation and growth of the iron core (Cozzi et al.,
formation of a ferrihydrite mineral in the hollow 2000; Santambrogio et al., 1996). An unusually short
cavity enclosed by the ferritin protein shell. This distance of 2.53 Å between the two Fe2þ ions
results in effective cellular iron concentrations of suggests the presence of a unique triply bridged
more than 1011 times the solubility of the Fe3þ-ion structure requiring a small Fe-O-O angle. This
(Takagi et al., 1998). In times of iron need ferritin geometry should favour decay of the peroxodiferric
releases iron by demineralization of the iron core. The complex by the release of m-oxo or m-hydroxo diferric
exact steps involved in iron release are not completely mineral precursors (Hwang et al., 2000; Liu & Theil,
known, but iron release involves the reduction of 2004). This would result in freeing of the ferroxidase
Fe3þ to Fe2þ. The rates of the processes involved in sites for binding of additional Fe2þ and the start of
iron mineralization and demineralization are con- another round of Fe2þ oxidation (Chasteen, 1998). A
trolled by the protein shell (Theil, 1990)—this by by-product produced during the ferroxidase centre
influencing the local pH and redox potentials (Powell, oxidation of Fe2þ is H2O2, which can result in the
1998). The formation of the ferrihydrite mineral by subsequent production of Fenton chemistry-derived
ferritin is a multi-step process governed by the protein radicals (Chasteen, 1998).
shell. Iron enters the ferritin cavity by passage through
the channels situated in the protein shell facilitated by Oxidation of Fe2þ on the surface of the growing iron core.
the presence of local iron binding sites (Harrison & During the initial stages of ferritin iron core miner-
Arosio, 1996). Upon entering the protein shell the alization, oxidation of Fe2þ takes place in the
iron is oxidized followed by hydrolysation, nucleation ferroxidase centres of the H-subunits of the ferritin
and iron core growth (Ford et al., 1984). Each of these protein shell. Once the mineral attains a certain
steps contributes to the formation of a ferrihydrite critical size, oxidation of Fe2þ can additionally, and
mineral core from soluble Fe2þ-ions. perhaps preferentially, occur on the surface of the
growing iron core (Chasteen, 1998; Ford et al., 1984;
Powell, 1998). Therefore, the main function of the
Oxidation of iron
ferroxidase centre may be oxidation of sufficient iron
Oxidation of Fe2þ by the ferroxidase centre of the H- from Fe2þ to Fe3þ for the initial nucleation events,
subunit. Oxidation of Fe2þ is an obligatory first step and once these nuclei attained a sufficient size
in order for an iron atom to finally be deposited in the for oxidation to take place on the mineral surface,
cavity of ferritin. The H-subunit’s ferroxidase centre, the H-subunit’s role as a ferroxidase is superseded by
formed by various amino acid side-chains, enzyma- oxidation on the mineral surface (Lawson et al.,
tically oxidizes Fe2þ to Fe3þ. Enzymatic oxidation of 1989; Treffry et al., 1997).
Fe2þ by the ferroxidase centre results in rates of iron
oxidation several-fold faster than that which would
Hydrolysis and nucleation of the formed Fe3þ
occur during auto-oxidation of iron. This faster rate
of iron oxidation results from the proper placement The subsequent hydrolysis and nucleation of the
of Fe2þ atoms by the ligands of the ferroxidase centre generated Fe3þ is governed by the amino acid side-
of the H-subunit for subsequent oxidation by O2 chains of the L-subunit (Chasteen, 1998). The highly
(Cozzi et al., 2000). X-ray analysis has revealed three mobile Fe3þ-monomers are directed to the inner
iron-binding sites per H-subunit. Sites A and B (3.8 cavity and properly placed on the protein shell/
Å apart) form a di-iron site and include a common mineral interface by these ligands. The subsequent
bridging carboxylic acid residue. Ligands of site A hydrolysis and nucleation involves the hydrolysis and
also include one equivalent histidine and one aggregation of Fe3þ-ions to form nuclei containing
perhaps as few as four or five constituent Fe3þ-ions For the oxidation reaction:
resulting in the production of iron oxyhydroxides and
oxides (Chasteen & Harrison, 1999; Powell, 1998). 4Fe2þ þ O2 þ 4Hþ ! 4Fe3þ þ 2H2 O
Hydrolysis and nucleation of the Fe3þ-ion is
favoured since binding of Fe3þ to the ligands lowers For the subsequent hydrolysis reaction:
the activation energy of nucleation (Harrison &
Arosio, 1996; Powell, 1998). Such nuclei can 4Fe3þ þ 8H2 O ! 4FeOOHcore þ 12Hþ
become the solid phase once some critical nucleus
size has been reached and will sustain iron mineral For the sum of the oxidation and hydrolysis
growth from these sites (Powell, 1998). These initial reactions:
sites of nuclei formation on the inner surface of the
protein shell, involving specific amino acid side- 4Fe2þ þ O2 þ 6H2 O !4FeOOHcore
chains, will maintain contact with the iron core þ 8Hþ ðChasteen 1998Þ:
(Powell, 1998). This interaction with the iron core

seems to be a key factor in the stabilization of the Such differences in iron oxidation kinetics by
crystal by providing a neutral container, or more ferritin are displayed in cells under varying condi-
likely, the interactions with negatively charged and tions. Under cellular conditions where low Fe2þ
neutral amino acid side-chains such as carboxylates, concentrations relative to the amount of apoferritin
alkoxides, phenolates, and imidazoles provide the may be expected, the ferroxidase activity of
necessary charge compensation (Powell, 1998). H-subunits is probably essential to initiate the
formation of iron nuclei and for iron mineralization
to proceed at a significant rate (Lawson et al., 1989).
Different iron oxidation kinetics and the formation of
Autocatalytic Fe2þ oxidation on the surface of the
different reaction products by the ferroxidase centre
growing iron mineral can only be significant once an
oxidation of iron and oxidation of iron on the mineral
initial iron core has been established (Lawson et al.,
surface
1989). However, it has certain advantages above the
Oxidation of Fe2þ by the ferroxidase centre and process that occurs in the ferroxidase centre of
oxidation of Fe2þ on the mineral surface take place the H-subunit. The first biological advantage is that
under different conditions. These processes have the potentially toxic production of hydrogen peroxide
different iron oxidation kinetics and result in the by the oxidation reaction, that takes place in the
formation of different reaction products (Levi et al., ferroxidase centre, is replaced by the harmless
1994). At low Fe2þ concentrations (550 Fe2þ reduction of di-oxygen to water via the oxidation
atoms/ferritin molecule) oxidation of Fe2þ is cata- reaction on the mineral surface (Chasteen, 1998;
lysed by the ferroxidase centre and the reaction Treffry et al., 1997). As ferritin lacks catalase activity,
stoichiometry is as follows: the H2O2 produced during the ferroxidase centre
catalysed oxidation of iron results in some degrada-
For the oxidation reaction: tion of the ferritin protein shell. In effect, the protein
itself acts as an anti-oxidant (Chasteen, 1998).
2Fe2þ þ O2 þ 2Hþ ! 2Fe3þ þ H2 O2 Another advantage of such a change in iron kinetics
is that it enhances the ability of ferritin to increase the
For the subsequent hydrolysis reaction: rate of iron oxidation when challenged with a large
amount of Fe2þ-ions. The ferroxidase centre can be
2Fe3þ þ 4H2 O ! 2FeOOHcore þ 6Hþ re-utilized for another round of Fe2þ oxidation only
once the generated Fe3þ-ion moves from the
For the sum of the oxidation and hydrolysis ferroxidase centre into the iron storage cavity for
reactions: subsequent nucleation. However, this rate of regen-
eration may be too slow to process a large number of
2Fe2þ þ O2 þ 4H2 O !2FeOOHcore Fe2þ-ions added at once, and under such conditions,
þ H2 O2 þ 4Hþ oxidation on the mineral surface may occur even
during earlier stages. These different oxidation
In the presence of higher Fe2þ concentrations abilities of ferritin to mineralize iron result in the
(4250 Fe2þ atoms/ferritin molecule) the H-subunit formation of crystallites of different sizes. Under
ferroxidase site becomes kinetically saturated and the conditions favouring fast iron accumulation the
size of the growing mineral reaches a size sufficient to ‘ferrihydrite’ produced has a relatively large crystal-
sustain oxidation of Fe2þ on the mineral surface. lite size and a nearly all-or-none distribution within
This results in the changing of the dominant ferritin molecules since the mineralization process
mechanism of iron oxidation from the ferroxidase will be favoured by oxidation of Fe2þ on the mineral
centre to the mineral surface catalysed mechanism surface (Ford et al., 1984) and the rate of iron
(Chasteen, 1998). The reaction stoichiometry deposition will be governed by the surface area of the
changes to the following: iron mineral (Worwood, 1982). However, under
conditions of low Fe2þ concentrations oxidation of supply of Fe3þ the subsequent hydrolysis and
Fe2þ will take place on different ferroxidase centres nucleation processes are driven by the L-subunit
resulting in the formation of many small crystallites (Chasteen & Harrison, 1999; Levi et al., 1992). If
(Ford et al., 1984). insufficient quantities of the L-subunit are assembled
into the protein shell non-specific hydrolysis of Fe3þ
can take place on the outside of the protein shell.
Migration of iron between ferritin molecules
This may lead to aggregation and precipitation of
Iron can migrate between ferritin molecules (Wade ferritin molecules (Levi et al., 1994). Although very
et al., 1991), either as Fe2þ or as Fe3þ. Migration of few H-subunits are necessary to initiate the process
iron between ferritin molecules occurs if the move- of iron oxidation, a deficit in H-subunits can result
ment of Fe2þ/Fe3þ to the next ferritin molecule in poor iron sequestration abilities. Furthermore,
results in favouring of the oxidation or hydrolysis/ H- and L-subunits can act co-operatively when in
nucleation processes. Oxidation of Fe2þ results in the separate molecules (Levi et al., 1992).
generation of highly mobile Fe3þ-ions, which subse- The co-operative roles of the subunits of ferritin in
quently have to be incorporated into a growing the mineralization process of iron have been demon-
mineral crystal by a hydrolysis/nucleation process. strated by the formation of recombinant H-subunit
Since L-subunits promote hydrolysis and nucleation or L-subunit homopolymers and recombinant
of Fe3þ by binding of Fe3þ to specific L-subunit H-subunit/L-subunit heteropolymers. In vitro
ligands, insufficient quantities of the L-subunit experiments involving recombinant ferritin mole-
present in the ferritin molecule can result in the cules showed that:
migration of Fe3þ to a ferritin molecule with
sufficient quantities of L-subunit (Levi et al., 1992). a. Both the H-subunit and L-subunit homopoly-
The generated Fe3þ would move to the outer surface mers have the capacity to incorporate iron.
of the ferritin protein shell via an H-subunit specific However, since the recombinant L-subunit
channel linking the ferroxidase centres with the outer homopolymer relies on auto-oxidation of iron
surface of the protein shell (Levi et al., 1996). This for the initial generation of Fe3þ the recombi-
results in competition between ferritin molecules for nant H-subunit homopolymer exhibits iron
iron and a tendency towards all-or-none distribution uptake and ferroxidase kinetics several-fold
(Ford et al., 1984; Harrison & Arosio, 1996; Wade faster than the recombinant L-subunit homo-
et al., 1991). polymer (Levi et al., 1994; Wade et al., 1991;
Worwood, 1990).
b. The rate of iron uptake increases with an
Non-specific Fe3þ hydrolysis on the outer surface
increase in the H-subunit proportion from 0 to
of the ferritin protein shell
35% of the ferritin molecule. A plateau is,
When insufficient quantities of L-subunits exist for however, reached with further increase in the
the entrapment of Fe3þ in the inner cavity Fe3þ can H-subunit content (Wade et al., 1991).
move to the outer surface of the protein shell where c. Ferritin H-subunit homopolymers have a low
non-specific hydrolysis of Fe3þ can take place (Levi ability to nucleate the generated Fe3þ and
et al., 1994). Non-specific iron hydrolysis on the therefore take up and release iron faster since
outer surface of the ferritin molecule can result in the generated Fe3þ is not tightly bound in an
protein aggregation and precipitation (Levi et al., iron mineral (Boyd et al., 1985).
1994). Spontaneous aerobic iron hydrolysis such as d. Ferritin L-subunit homopolymers, when sup-
that which occurs on the outer surface of the ferritin plied with Fe3þ are more efficient in promoting
protein shell appears to be caused by the formation of iron mineralization than the corresponding
transient mono-nuclear hydrated Fe3þ-ions which H-subunit homopolymers (Levi et al., 1994).
have a strong tendency to aggregate and coagulate e. H-subunit/L-subunit heteropolymers are more
(Levi et al., 1994). efficient in taking up iron than the parent
homopolymers (Levi et al., 1994). A high
synergism between the two subunits occurs in
The cooperative roles of the H-subunit and L-subunit
ferritins with low H-subunit content (10 – 30%)
of the ferritin protein shell in iron mineralization
and high L-subunit content (70 – 90%), a few
Since the H-subunit and the L-subunit of the protein ferroxidase centres are sufficient to promote fast
shell have separate roles in the mineralization process iron oxidation, and many L-subunits are needed
of iron by ferritin and the specific properties of the to reduce non-specific iron hydrolysis and
two subunits are complementary and act synergisti- facilitate iron mineralization (Levi et al., 1994).
cally, co-operation between these subunits is para- f. L-subunit homopolymers can incorporate some
mount for the efficient mineralization of iron by of the iron oxidized by H-subunit homopolymers
ferritin. The ratio of H-subunits to L-subunits is (Levi et al., 1994).
therefore important. While oxidation of Fe2þ by the g. The amount of soluble ferritin molecules
ferroxidase centre of the H-subunit accelerates the increases sharply in heteropolymers with an
L-subunit content higher than 70 – 80%, thus 2005; Liu et al., 2003). Various reductants and
preventing the non-specific iron hydrolysis on chelators, including physiological and toxicological
the outside of the protein shell and the sub- substances, can release iron from ferritin (Agrawal
sequent protein aggregation/precipitation (Levi et al., 2001; Gálvez et al., 2005; Hynes &
et al., 1994). Coinceanainn, 2002; Sánchez et al., 2005). With
h. H-subunit/L-subunit heteropolymers with low the first of the two processes that remove iron from
H-subunit content (18 – 30%) incorporate 3 to 4 intact ferritin, i.e. the process involving the reduction
times more iron than H-subunit homopolymers of Fe3þ to Fe2þ, followed by chelation of Fe2þ, the
(Levi et al., 1994). reductant has to gain access to the interior of the
i. The most efficient heteropolymers for in vitro protein shell to reduce the Fe3þ to Fe2þ. However,
iron incorporation are structurally similar to the the formed Fe2þ will only leave the ferritin protein
ferritins found in the tissues which accumulate shell in the presence of a chelator (Jameson et al.,
iron, such as liver and spleen, which typically 2004). With the second of the two processes, i.e.
contain 80 – 95% L-subunit, 5 – 20% H-subunit. the direct chelation of Fe3þ where the Fe3þ is not
Other tissues with lower needs for iron storage reduced, Fe3þ leaves the ferritin protein shell as a
may prefer ferritins with higher H-subunit Fe3þ-complex. The hydrous ferric oxide cores can be
content which, having a higher ferroxidase reduced by one electron per iron atom accompanied
activity, are probably more efficient for iron by an uptake of two protons per electron from the
detoxification (Levi et al., 1992, 1994). surrounding medium (Watt et al., 1985). This is then
j. L-subunit homopolymers containing nuclei will followed by the chelation of Fe2þ and the transport to
develop at the expense of L-subunit homopoly- sites where Fe2þ is needed (Joshi & Clauberg, 1988;
mers without nuclei by autocatalytic growth Watt et al., 1985). Effective reducing agents for the
processes on the surface of the iron mineral release of iron from ferritin include flavins, cysteine,
(Wade et al., 1991). glutathione, ascorbic acid and superoxide (Ponka
et al., 1998).
The respective and synergistic roles of the The initial rate of iron release shows a dependence
H-subunit and L-subunit of ferritin is also indicated on iron content, with maximum rate of release for
in vivo, since in vivo H-subunit homopolymers relatively iron-poor molecules, which are one-third to
are much less efficient than the H-subunit/L-subunit one-half saturated with iron. It also depends on the
heteropolymers in taking up iron (Cozzi et al., surface area of the crystallite. This dependence
2000). resembles that for iron uptake and suggests a direct
interaction between the surface of the iron core and
the reducing agent (Chiancone & Stefanini, 1984;
The release of iron from ferritin
Theil, 1990; Worwood, 1990). Furthermore, as iron
Two mechanisms have been proposed for the release atoms at the surface of the iron core could be
of iron from ferritin. Iron can be either released from expected to be more accessible to reducing agents
the intact ferritin molecule or released upon the than those in the interior, a last-in-first-out principle
degradation of the ferritin molecule (Aisen, 1991; is obeyed (Worwood, 1990). It has also been
Deiss, 1983). However, the relative importance of suggested that the release of iron from the intact
iron release in vivo by the one or the other of these ferritin molecule is sensitive to changes in conserved
mechanisms is not known. Two processes are amino acids near the outside of the ferritin channels
chemically feasible for removing iron from the intact which are likely to be involved in regulating the
ferritin molecule—the first process the reduction of localised unfolding of the protein shell in order to
Fe3þ to Fe2þ followed by chelation of Fe2þ, and the open the channels and release the reduced iron (Jin
second the direct chelation of Fe3þ (Harrison & et al., 2001).
Arosio, 1996; Liu et al., 2003; Gálvez et al., 2005).
The release of iron from ferritin by these two
processes is accomplished with the aid of reductants Isoferritins
and iron chelators that can cross the protein shell.
Different H-subunit/L-subunit compositions of the ferritin
Reductants and chelators gain access to the interior
protein shell
of the ferritin molecule through the threefold
channels of the protein shell. It is suggested that The multiple forms of ferritin have their molecular
the channels of the ferritin protein shell are dynamic basis in the ratio of the two subunit types present, i.e.
and control the access of reductants and chelators, the H-subunit and the L-subunit (Chiancone &
since reductants and chelators too large to pass Stefanini, 1984). The ferritin protein shell exists as
through the channels can under certain conditions heteropolymers of various combinations of these two
gain access to the interior of the ferritin molecule types of subunits (Arosio et al., 1978)—a phenom-
(Liu et al., 2003). Chaotropes can increase the access enon that gives rise to the existence of isoferritins. As
of reductants and chelators to the interior of ferritin the roles of the H-subunit and L-subunit differ in the
by influencing the gating of the channel (Gálvez et al., mineralization process, the subunit composition of
ferritin will influence the metabolic properties of the does not, however, remain constant and the
assembled ferritin molecules (Harrison & Arosio, proportion of the H- and L-subunits present in
1996; Levi et al., 1994). H-subunit rich ferritins have the ferritin shell changes during differentiation
been shown to accumulate and release iron faster and in various pathological states (Arosio et al.,
than do L-subunit rich ferritins (Arosio et al., 1991; 1976; Boyd et al., 1985; Ponka et al., 1998; Theil,
Chiancone & Stefanini, 1984; Wagstaff et al., 1982; 1990).
Worwood, 1990) and it is suggested that the The variations in the type of isoferritins present
H-subunit rich ferritins permit more dynamic in- in erythroid cells are well studied. The presence of
tracellular traffic of iron (Chiancone & Stefanini, different isoferritins with different metabolic prop-
1984; Speyer & Fielding, 1979). L-subunit rich erties reflects the changing iron needs of the
ferritins apparently contain more iron than those erythroid cell. The erythroid cells contain mainly
ferritins rich in H-subunits (Bomford et al., 1981; H-subunit rich ferritins, which play a major role in
Chiancone & Stefanini, 1984) and there are indica- the intracellular transport and donation of iron for
tions that the L-subunit rich ferritins predominate in the active synthesis of heme (Coccia et al., 1992),
cell types that play a role in the storage of iron (Boyd particularly in immature erythroid cell precursors
et al., 1985; Chiancone & Stefanini, 1984; Coccia such as proerythroblasts and basophilic erythro-
et al., 1992; Powell et al., 1975). However, increases blasts (Invernizzi et al., 1990). However, when iron
in ferritins rich in the H-subunit have been shown to accumulates in erythroid tissue due either to an
provide cells with increased resistance to H2O2 increase in the cellular uptake of iron or a decrease
toxicity (Cozzi et al., 2000). It would further appear in iron usage for heme synthesis, the L-subunit
that a specific subunit composition may cater for iron rich ferritins seem to increase and to be closely
storage, and that iron loading would increase the related to the iron status of the cells (Cazzola et al.,
expression of the L-subunit whereupon these L- 1983; Invernizzi et al., 1990). The H-subunit/
subunit rich isoferritins will sequester the bulk of the L-subunit ferritin composition is reported to
surplus iron (Bomford et al., 1981; Invernizzi et al., decrease with erythroblast maturation (Cazzola
1990). In general, L-subunit rich ferritins contain et al., 1983), with H-subunit rich ferritin content
1500 iron atoms or more whereas H-subunit rich higher in the early erythroblast fractions and
ferritins contain less than a 1000 iron atoms decreasing with maturation. The content of
(Harrison & Arosio, 1996). In situations of iron L-subunit rich ferritin, apparently does not show
overload it may be advantageous to the cell to such consistent changes with maturation (Hodgetts
synthesize L-subunit rich ferritins, since these ferri- et al., 1986).
tins are not only able to store more iron but can also The ferritin present in reticuloendothelial cells
retain iron more firmly and turn over iron more (Invernizzi et al., 1990; Ruggeri et al., 1992) and
slowly than H-subunit rich ferritins (White & Munro, other macrophage-like cells are predominantly
1988). The assumed role that the L-subunit rich L-subunit rich (Cheepsunthorn et al., 1998; Connor
ferritins play in the sequestration of the surplus iron et al., 1994). An exception is the ferritin in human
during iron overload is underlined by the fact that peripheral blood monocytes, which seems to be rich
their concentration in liver, serum and cultured in H-subunits (Jones et al., 1983). However, in vitro
cells is related to iron levels, whereas the H-subunit macrophages, which originate from monocytes,
rich ferritins appear either to be non-affected (in would appear to develop an L-subunit predomi-
liver) or negatively affected (in serum) by increases nance. This phenomenon is mainly associated with
in iron concentrations (Ruggeri et al., 1984). the loss of H-subunits (Jones et al., 1983). At
Furthermore, upon iron supplementation of patients present, indications are that the addition of iron can
with functional iron deficiency in the presence of cause an increase in both H- and L-subunits despite
tissue iron overload there would appear to be a the persistence of a very low H-subunit/L-subunit
proportionately greater change in L-subunit rich ratio. The presence of mainly L-subunit rich ferritin
ferritins than in H-subunit rich ferritins (Ruggeri is in keeping with the role of the reticuloendothelial
et al., 1984). Due to the H-subunit rich ferritin’s cell/other macrophage-like cells in the scavenging of
more dynamic ability of iron uptake and release it effete cells and the sequestration and storage of
would appear to be largely found in cells having large amounts of iron. An interesting phenomenon
high iron requirements for metabolic activities and a is the fact that the same type of process may also
non-existent role in iron storage (Cazzola et al., occur in the brain. Microglia, a macrophage-like cell
1983). Cells with a high content of H-subunit rich present in the brain (Cheepsunthorn et al., 1998;
ferritins include erythroid cells, heart cells, pancrea- Connor et al., 1994), is responsible for the
tic cells, kidney cells, lymphocytes and monocytes phagocytosis of cellular debris during axon remo-
(Jones et al., 1983; Worwood, 1982), whereas the deling and naturally occurring cell death in the
L-subunit rich ferritins are found predominantly in developing brain (Cheepsunthorn et al., 1998).
liver and spleen—organs associated with long-term These cells contain predominantly L-subunit rich
iron storage (Powell et al., 1975; Worwood, 1982). ferritins consistent with its role in long-term iron
The H- to L-subunit ratio of a specific type of cell storage (Connor et al., 1994).
ferritin can vary 1000-fold among different cell types

The synthesis of ferritin
(Theil, 1990). Furthermore, the composition of the
The apoferritin molecule, consisting of 24 H- and H- and L-subunits in the ferritin molecule differs
L-subunits, is assembled from a cytosolic pool of between different cell types, resulting in the cellular-
available H- and L-subunits. This cytosolic pool of dependent variation of isoferritin populations
free H- and L-subunits is maintained by the supply (Harrison & Arosio, 1996). This is achieved by the
of H- and L-subunits upon translation of H- and regulation of the expression of the H- and L-subunit
L-subunit mRNA by free polyribosomes and the genes of ferritin. Different mechanisms of regulation
proper folding of the polypeptide chains. While the exist including transcriptional, modulation of tran-
composition of H- and L-subunits in the ferritin script stability, translational depending on the
molecule is determined by the H- and L-subunits metabolically available iron concentration and trans-
available in this pool, the quantity and composition lational irrespective of the metabolically available
of the H- and L-subunits in this pool of available iron concentration. The regulation of the transcrip-
subunits is regulated at both the transcriptional tion of the H- and L-subunit genes occurs mainly
and translational levels of expression of the ferritin irrespective of the metabolically available iron con-
H- and L-subunit genes. centration. However, there are indications that the
transcription of the H- and L-subunit genes can be
influenced by the metabolically available iron con-
Assembly of ferritin from the pool of available H-
centration in specific conditions (Theil, 1990; White
and L-subunits
& Munro, 1988). The transcriptional regulatory
The 24-subunit ferritin protein shell is assembled in the mechanisms and stability of the mRNA determine
cytosol from a pool of free, unassembled, or only partly the mRNA concentrations of the H- and L-subunits,
assembled H- and L-subunits (Harrison & Arosio, whereas the translational regulatory mechanisms
1996). These H- and L-subunits are synthesized by determine the magnitude of mRNA translation and
free polyribosomes and a basal concentration of free the subsequent formation of the H- and L-subunits
H- and L-subunits is maintained in this cytosolic pool of ferritin. Therefore the relative proportion of
of subunits. When this concentration rises as a H- and L-subunits in the final ferritin molecules
consequence of the synthesis of new H- and L-subunits depends mostly on multiple transcriptional regula-
by free polyribosomes, subunits will be assembled into tions (transcription or stability) that affect the
apoferritin in the cytosol near the polyribosomes. Since respective proportion of H- and L-subunit mRNA
H- and L-subunits have the same conformation and in the total pool of translatable ferritin mRNA,
many identical or similar amino acids are involved in whereas the total amount of ferritin produced in the
the inter-subunit contact regions between H-subunit/ cells depends on the magnitude of translation of the
H-subunit, H-subunit/L-subunit or L-subunit/ H- and L-subunit mRNA (Ponka et al., 1998).
L-subunit interactions, a complete range of subunit Ferritin synthesis is stimulated during development,
compositions of homopolymers and heteropolymers is during cell differentiation, by pro-inflammatory
possible (Harrison & Arosio, 1996; Theil, 1990). cytokines, as well as by some hormones (Festa
However, homopolymers of ferritin consisting of either et al., 2000). Furthermore, a preferential increase in
only H-subunits or only L-subunits are poorly repre- a specific subunit is elicited by the differential
sented in cells, suggesting the existence of preferential transcriptional regulation of the H- and L-subunit
interactions between H- and L-subunits. This is in genes. The differentiation of various cells is asso-
agreement with cross-linking experiments showing a ciated with a consistent increase of ferritin mRNA
preferential formation of H-subunit/L-subunit dimers and ferritin levels, and a preferential accumulation of
(Santambrogio et al., 1993). the H-subunit as a consequence of a selective
Iron is incorporated into apoferritin or iron-poor transcriptional regulation of the H-subunit gene
ferritin molecules only once the ferritin shell is needed to produce ferritin with a structure appro-
completely assembled from the available H- and priate to a differentiated cell type (Festa et al., 2000;
L-subunits (Harrison & Arosio, 1996). This fraction of Harrison & Arosio, 1996; Theil, 1990). During
H- and L-subunits synthesized by free polyribosomes inflammation, stimulation by the cytokine tumour
is destined for the intracellular sequestration of iron necrosis factor (TNF), results in transcriptional up-
while a much smaller fraction of H- and L-subunits are regulation of the H-subunit without a change in
synthesized by membrane-bound polyribosomes and L-subunit expression. This gives rise to an increase in
once assembled the ferritin is secreted by the cell to the the H-subunit to L-subunit ratio of the produced
extracellular fluid (Worwood, 1982). ferritins (Festa et al., 2000). Up-regulation of the
transcription of the H-subunit gene has also been
found in cell lines overexpressing c-myc, in the
Regulation of the expression of the H-subunit
pregnant uterus, in denervated skeletal muscle, in the
and L-subunit genes of ferritin
atherosclerotic aorta, in response to other cytokines
The H- and L-subunit genes of ferritin are expressed and after exposure to exogenous heme (Ponka et al.,
in most cells, but the concentration of the assembled 1998). Somewhat surprising, iron itself does not
seem to affect the amount of H-subunit mRNA, sensitive to the amount of metabolically available
although it has a stimulatory effect on the accumu- iron. In order to accomplish a finely tuned system of
lation of L-subunit mRNA (Ponka et al., 1998). ferritin expression as a function of the size of the
L-subunit gene transcription and total cellular metabolically available iron pool (the labile iron pool)
L-subunit mRNA appear to be dramatically in- it is important that the ferritin gene structure
creased by iron, whereas H-subunit transcription contains sequences that sense the size of the labile
rates and H-subunit mRNA levels are only slightly iron pool (Harford et al., 1994). The 50 -untranslated
increased (White & Munro, 1988). region (50 -UTR) of both the H- and L-subunit
mRNA contains a highly conserved 28-base se-
quence known as the iron-responsive element (IRE)
The gene sequences of the H-subunit and the L-subunit
sensitive to the metabolically available active iron
of ferritin
(Worwood 1990). The IREs are comprised of cis-
The genes for the H- and L-subunits are contained on acting nucleotide sequences. These nucleotide se-
different chromosomes—the gene for the H-subunit quences form stem-loop structures that contain a six-
on chromosome 11 and the gene for the L-subunit on membered loop with the sequence CAGUGN
chromosome 19 (Worwood, 1990). The genes for the (Ponka et al., 1998). These stem-loop structures are
H- and L-subunit contain three introns and four recognized by trans-acting cytosolic RNA-binding
exons (Torti & Torti, 2002) and the gene sequences proteins required for the co-ordinated expression of
for these two subunits show extensive homology in the H- and L-subunits (Theil, 1990). These cytosolic
their coding regions with several common stretches of RNA-binding proteins, IRP1 and IRP2, cause a
20 – 30 nucleotides. However, they differ markedly in decrease in H- and L-subunit mRNA translation by
their non-coding regions (Boyd et al., 1985). These binding to the stem-loop structures of the 50 -UTR of
differences are extremely important for differential the respective mRNAs. IRP1 and IRP2 mediate the
regulation of the expression of the genes for these two translational efficiency by obscuring the subsequent
subunits. Functional analysis for the 50 non-coding binding of the 43S translation pre-initiation complex
region for the H-subunit gene but not for the needed for the initiation of translation (Rogers et al.,
L-subunit gene has been reported. The 50 non-coding 1994). IRP1 and IRP2 both sense and homeostati-
region of the H-subunit gene contains three regulatory cally control the metabolically available iron. For
regions. The first of these regions, the B-box, 742 to IRP1 this is accomplished by the existence of two
762 nucleotides upstream from the start codon and conformationally distinct forms. IRP1 is a 90 kDa
closest to the transcription initiation site is responsive iron – sulphur cluster protein. When iron is abundant
to cAMP. This B-box regulatory region is sensitive to it exists as a cytosolic aconitase. When iron is scarce
the initiation of transcription by hormones and second it assumes an open configuration associated with the
messengers and binds a protein complex termed loss of iron atoms from the iron – sulphur cluster and
B-box binding factor (Bbf). The B-box binding factor the subsequent binding to the IRE stem-loop
comprises the transcription factor NFY, the co- structure, acting as a repressor of ferritin translation
activator p300, and the histone acetylase p300/CBP (Torti & Torti, 2002). In contrast, the 105 kDa IRP2
associated factor (PCAF) (Harrison & Arosio, 1996; protein is regulated by degradation: IRP2 protein is
Torti & Torti, 2002). The second regulatory region abundant in iron scarcity, but is degraded rapidly in
identified in the 50 non-coding region of the H-subunit iron excess through targeting of a unique 73 amino
gene includes a region called the A-box at position acid sequence and subsequent oxidation and ubiqui-
7109 to 7132 upstream from the start codon tination (Meyron-Holtz et al., 1999; Torti & Torti,
(transcription initiation site), which contains a con- 2002). This response of ferritin synthesis to the size
sensus sequence for binding the polymerase II of the metabolically available pool of iron endows the
transcription factor SP1 responsible for about 50% cell with an exceptionally rapid system for increasing
of the activation of gene expression in several cell lines ferritin synthesis upon iron influx. Iron influx
(Harrison & Arosio, 1996; Torti & Torti, 2002). The increases the labile iron pool and, via binding to the
third regulatory region consists of a stretch of 10 Gs IRP1 and IRP2, causes a rapid increase in ferritin
which are termed ‘G-fer’ between 7272 and 7291 translation. This rapid response is achieved by a shift
upstream from the start codon. It is suggested that of stored mRNA from the ribonucleoprotein (RNP)
binding of inhibitory factor 1 to this sequence results fraction to polysomes (translational shift) (White &
in the inhibition of H-subunit gene transcription Munro, 1988). The translation of existing ferritin
(Harrison & Arosio, 1996; Torti & Torti, 2002). mRNA is more rapid than additional ferritin gene
transcription followed by translation. The ferritin
response to iron influx can thus be viewed as a
Translational regulation of the H-subunit and L-subunit
protective rapid response system, allowing immediate
mRNA expression via metabolically available iron
formation of additional ferritin in which to store the
Ferritin is the major intracellular protein involved in surplus iron (Truty et al., 2001). Both the H-subunit
storage and detoxification of iron. It is therefore not and L-subunit mRNA shift from the RNP fraction to
surprising that the expression of ferritin is extremely polysomes to the same extent (White & Munro,
1988). Nevertheless, the transcription of the lysosome can result in the entrapment of ferritin iron
L-subunit gene is preferentially stimulated by an (Wixom et al., 1980). Iron-containing ferritin can
increase in metabolically available iron (Worwood, ultrastructurally be identified in the cytosol as either
1990) and results in an increase in the ratio of randomly dispersed ferritin particles or as clusters of
L-subunit to H-subunit mRNA, which appears first ferritin particles. The ferritin clusters in the cytosol
in the RNP fraction and later in the polysomes are accumulations of ferritin in which ferritin
(White & Munro, 1988). This increase in the particles can be individually resolved. The existence
L-subunit to H-subunit mRNA ratio in the poly- of ferritin either as randomly dispersed ferritin
somes accounts for the change in the ratio of particles or as clusters of ferritin particles depends
L-subunit to H-subunit protein synthesis following on the magnitude of iron handling of the different
iron administration (White & Munro, 1988). There- cell types. In cell types handling relatively low
fore, co-ordinated translational control and differ- quantities of iron, iron-containing ferritin occur as
ential transcriptional control exists between these rare, isolated particles whereas in cell types handling
two genes (White & Munro, 1988). greater quantities of iron such as haemopoietic bone
marrow cells and cells of the reticuloendothelial
system, iron-containing ferritin occurs more fre-
Translational regulation of H-subunit and L-subunit
quently as clusters (Iancu, 1992). In most clusters
expression irrespective of metabolically available iron
the particles are of the iron-rich variety and thus
Various factors other than iron may alter the appear larger and more electron dense than the
translational efficiency of the H- and L-subunit dispersed cytosolic ferritin (Iancu 1992). Cluster
mRNA. This may be accomplished by binding of formation prevents access of the proteins involved in
regulatory factors to specific sequences in the the cytosolic degradation of ferritin and in this way
50 -UTR other than the IRE or by changing the protects the ferritin molecule against degradation.
efficiency of the interaction between the IREs and This may be a regulatory step in the pathway of
IRPs. One specific sequence responsible for transla- ferritin degradation and iron release. However, as
tional control is the 20-nucleotide sequence down- long as the ferritin cluster is not enclosed by a
stream from the IRE known as the acute box membrane, degradation of these ferritin clusters can
(Thomson et al., 1999). This sequence responsible result in the release of iron in times of iron shortage.
for the enhancement of translation operates after Various studies indicated that the formation of large
iron-dependent translational initiation and the for- iron-rich ferritin particles, as a result of an increase in
mation of the 43S ferritin mRNA scanning complex intracellular iron, results in the protection of ferritin
(Rogers et al., 1994). Factors that can influence the molecules against degradation (Iancu, 1982; Truty
efficiency of the interaction between the IRE and IRP et al., 2001; Worwood, 1982) and that iron-depleted
include cytokines (Piñero et al., 2000), various ferritin is easily degraded (Halliday & Powell, 1979;
hormones, that changes the phosphorylation status Wixom et al., 1980). The 20S proteasome enzymatic
of the iron-responsive proteins (Thomson et al., system is responsible for the degradation of damaged
1999; Torti & Torti, 2002), oxidative stress—reactive intracellular proteins and can recognize specifically,
oxygen species (Coccia et al., 1992), hemin (Coccia and degrade, oxidized proteins (Mehlhase et al.,
et al., 1992), phosphatases, hypoxia and reoxygena- 2005; Rudeck et al., 2000). The ferritin protein shell
tion (Thomson et al., 1999) and nitric oxide (NO) is confronted by a multitude of possible oxidative
that causes the activation of both IRP1 and IRP2. stressors. The oxidation of Fe2þ by the ferroxidase
Mechanisms hypothesized to underlie NO-mediated centre of the H-subunit results in the production of
induction of IRP binding activity include cluster H2O2 which could oxidize the protein shell, and the
disassembly (IRP1), intracellular iron chelation surrounding Fe2þ can promote oxygen radical
(IRP1 and IRP2), or increased de novo synthesis production by Fenton type chemistry. Oxidation of
(IRP2) (Torti & Torti, 2002). ferritin results in the loss of ferritin function and
targeting of ferritin to the proteasome degradation
system of the cell (Mehlhase et al., 2005; Rudeck
The degradation of ferritin
et al., 2000). Oxidation of ferritin can also result in
Two different processes can result in the degradation aggregation of ferritin molecules. Sulfhydryl groups,
of cytosolic ferritin. The first of these involves the particularly, are oxidized followed by aggregation of
20S proteasome enzymatic system in the cytosol, and ferritin as a result of the formation of disulfide
the second, degradation in the lysosome by proteo- bridges between ferritin molecules (Welch et al.,
lytic enzymes. Depending on the type of cell, the iron 2001). The H-subunit contains a cysteine at position
status and whether ferritin is degraded free in the 90 located on the BC-loop facing the exterior which
cytosol or within a lysosome, different amounts of is extremely susceptible to oxidation (Welch et al.,
iron are made available for metabolic processes. 2002).
Degradation of ferritin in the cytosol results in the Ferritin cluster formation, however, might also
complete release of the iron from ferritin, whereas stimulate the uptake of ferritin into lysosomes
degradation of ferritin in the confinements of a whereupon less iron will be released during ferritin
degradation. The reason for this is that the release of degradation of the ferritin protein shell in secondary
iron during the degradation process relies on the lysosomes (Finch et al., 1984; Fischbach et al., 1971;
accessibility of the iron core to the reducing system of Ford et al., 1984). For instance, haemosiderin
the cell since dissolution of the iron core is generally contains various amounts of degraded ferritin, as
determined by the reduction of Fe3þ to Fe2þ. well as aggregated dense particles of irregular shape
Therefore degradation of the ferritin protein shell in with diameters ranging from 10 to 75 Å, which
the cytosol gives FMNH2 (the reducing system of the ultrastructurally resemble iron cores (Ringeling et al.,
cell) easy access to the Fe3þ-ions and results in the 1989; Wixom et al., 1980), and haemosiderin
complete dissolution of the iron core. The generated granules are recognised by anti-ferritin antibodies
Fe2þ-ions are re-utilized in metabolic processes or (Harrison & Arosio, 1996). Ferritin is frequently
incorporated into new ferritin molecules. If, however, situated in secondary lysosomes and autophago-
ferritin is degraded within a secondary lysosome somes of normal cells, such as hepatocytes and
(charged with proteolytic enzymes) the iron can no macrophages but its quantity in these organelles
longer readily be made available because it has been increases greatly after loading with iron (Richter,
cut off from the FMNH2 reducing system (Wixom 1978)—demonstrating the protective function of
et al., 1980). Instead digestion by lysosomal enzymes haemosiderin formation against the toxicity of iron.
would proceed, and the resulting aggregates of iron Ferritin finds its way into lysosomes by autophago-
oxyhydroxide (ferritin cores) would no longer be cytosis and/or fusion of ferritin clusters with the
provided with a mechanism for mobilizing and lysosomal membrane. Autophagocytosis is responsi-
recycling their iron. This will result in aggregation ble for the turnover of cellular constituents including
of the ferritin cores and the formation of haemosi- cellular proteins and involves the formation of
derin (Wixom et al., 1980). Cytosolic degradation autophagic vacuoles by invagination of intracytoplas-
may therefore be the major iron turnover mechanism mic membranes enclosing a relatively large volume of
providing the cell with easily accessible iron for cytoplasm, together with various cellular constituents
shunting into metabolic pathways, while degradation (Wixom et al., 1980). The autophagic vacuole
within membrane-encapsulated secondary lyso- receives digestive enzymes by fusion with a primary
somes, with subsequent haemosiderin formation, or secondary lysosome and becomes an autophago-
may prevent the uncontrolled release of iron and some (Wixom et al., 1980). It is within this lysosomal
may become prominent when there is iron overload organelle that the ferritin protein shell is degraded by
(Harrison & Arosio, 1996; Wixom et al., 1980). the action of lysosomal proteases (Richter, 1984). It
Nevertheless, degradation of ferritin in lysosomes can is suggested that the polymerization of ferritin
also produce soluble iron, although these larger (formation of oligomers of ferritin), which results in
masses of ferritin/haemosiderin may require more a change in solubility, heat stability and surface
time for the release of their iron contents. The iron so charge, may predispose ferritin to incorporation
released would then be translocated back to the within lysosomes and transformation into haemosi-
cytosol for reutilization in metabolic processes or derin (Chiancone & Stefanini, 1984; Ringeling et al.,
sequestration by ferritin (Deiss, 1983; Ponka et al., 1989). Only once the ferritin protein shell has been
1998; Radisky & Kaplan, 1998). Thus it seems that modified, most probably by denaturation, resulting
the release of iron from lysosomes depends on the in the formation of insoluble ferritin molecules, does
magnitude of aggregate formation and the subse- proteolytic decomposition of the ferritin protein shell
quent deposition of iron as haemosiderin. by lysosomal enzymes take place (Richter, 1984).
However, not all ferritin molecules in these lysosomal
organelles are susceptible to the action of lysosomal
The formation of haemosiderin from ferritin
proteases. Degradation of the ferritin protein shell
The absolute and relative amounts of iron stored in results in the exposure of the iron oxyhydroxide
the form of the two iron reserves, ferritin and mineral cores followed by aggregation of these
haemosiderin, vary with iron loading and cell type oxyhydroxide particles and the formation of insoluble
(Ford et al., 1984). There is slightly more ferritin masses of iron oxyhydroxide (haemosiderin)
than haemosiderin in the liver and spleen when total (Fischbach et al., 1971; Richter, 1978; Weir et al.,
tissue iron content is normal. As total iron content 1985). Although the main purpose of the formation
increases a progressively higher percentage of iron of haemosiderin would appear to be protection
occurs as haemosiderin (Ford et al., 1984; Miyazaki against iron overload, these larger masses of ferritin/
et al., 2002; Ringeling et al., 1989; Wixom et al., haemosiderin can, at a much slower rate, also release
1980; Worwood 1990). With overloading syndromes iron. This iron is then translocated back to the
such as primary and secondary haemochromatosis, cytosol for re-utilization in metabolic processes or
the iron content of haemosiderin can increase up to a sequestration by ferritin (Deiss, 1983; Ponka et al.,
100-fold, whereas that of ferritin only increases 5- to 1998; Radisky & Kaplan, 1998). Haemosiderin is
10-fold (Powell, 1998). not, however, necessarily the end product as massive
There is enough evidence to believe that haemo- quantities of iron oxyhydroxide (haemosiderin) from
siderin is derived from ferritin as a result of these secondary lysosomes, can accumulate to form
cytoplasmic organelles known as siderosomes L-subunit rich ferritins (Arosio et al., 1991). Futher-
(Richter, 1978). The haemosiderin-containing side- more, H-subunit rich ferritins are more susceptible to
rosomes can thus be regarded as the end-product of proteolysis due to less ordered secondary structures
secondary lysosome action in which the wall of the (Bomford et al., 1981). In particular the loop
original secondary lysosome now encapsulates the L becomes more exposed and/or less immobilized
digested ferritin iron cores (Harrison & Arosio, 1996; when the proportion of H-subunits increases and
Wixom et al., 1980)—although clusters of electron- therefore more accessible to lysosomal enzymes
dense material without membranes or only partially (Chiancone & Stefanini, 1984).
enclosed membranes can also occur (Deiss, 1983;
Harrison & Arosio, 1996; Iancu, 1982). Within
The reticulo-endothelial cell and haemosiderin formation
siderosomes, ferritin can be identified as individual
particles, in clusters, in paracrystalline hexagonal The reticulo-endothelial cell responsible for taking
arrays, or forming circular arrangements (Iancu, up and digesting effete red blood cells are confronted
1992). Siderosomes not only contain ferritin and by tremendous amounts of iron as a result of the
haemosiderin, but occasionally also contain electron- breakdown of the heme contained by the red blood
dense amorphous or spicular iron-containing com- cells. These surplus amounts of iron could result in
pounds which have as yet not been identified iron-induced damage to the reticulo-endothelial cell.
biochemically or ultrastructurally. In cells with In order to prevent possible iron-induced damage,
marked iron overload, solitary siderosomes seem to the reticulo-endothelial cell is capable of storing vast
fuse and form larger bodies described as ‘compound amounts of iron as haemosiderin. The reticulo-
siderosomes’ (Iancu, 1992). endothelial cell takes up red blood cells and
incorporates these red cells into phagocytic vacuoles,
where digestion of the red blood cell and degradation
The increased susceptibility of H-subunit rich ferritins
of heme take place. The heme contained by the red
to degradation
blood cell is degraded in 5 hours. However, remnants
It is suggested that H-subunit rich ferritins are of the red blood cells could still be detected in the
turning over more rapidly than L-subunit rich phagocytic vacuoles 24 hours after uptake of red
ferritins (Boyd et al., 1985; Truty et al., 2001; blood cells by the reticulo-endothelial cell. At this
Worwood, 1982). Haemosiderin, which contains time, moderate numbers of free ferritin molecules are
the degraded ferritin molecules as a result of the present in the cytosol. Forty-eight hours after red
lysosomal breakdown of ferritin, shows the predomi- blood cell uptake the number of ferritin molecules in
nance of denatured subunits to be of the H-subunit the cytosol is increased and continue to accumulate
type (Miyazaki et al., 2002). It was shown that ferritin in the cytoplasm up to 96 hours after red blood cell
is more abundant in the iron overloaded liver than in uptake. However, from 48 hours onward ferritin is
the normal liver, and that it is richer in L-subunits translocated into aggregates situated in autophagic
displaying a L-subunit : H-subunit ratio from 5 : 1 to vacuoles by a process of invagination of intracyto-
12 : 1. In contrast, haemosiderin displayed a plasmic membranes followed by the formation of
predominance of denatured H-subunits over dena- haemosiderin (Wixom et al., 1980). The formation of
tured L-subunits (Miyazaki et al., 2002). A mechan- haemosiderin in reticuolendothelial cells and other
ism may therefore exist for preferentially directing macrophage-like cells are influenced by inflamma-
ferritins rich in the H-subunit into lysosomes tory and infectious conditions. Macrophages sub-
resulting in the formation of haemosiderin containing jected to increased oxidative stress also degrade
a high proportion of denatured H-subunits. It was ferritin faster (Mehlhase et al., 2005). It is therefore
shown, in vitro, that a too great proportion of suggested that during inflammatory and infectious
H-subunits in the ferritin protein shell result in conditions the proportion of poorly accessible (non-
ferritin aggregation. This may be due to the chelatable) iron associated with ferritin similarly
inadequacy of the ferritin protein shell to retain the increases, suggesting a pathway from non-ferritin
formed Fe3þ resulting in the loss of Fe3þ and iron, to loosely associated ferritin iron, to a well-
hydrolysis of Fe3þ on the outside of the ferritin sequestered non-chelatable form existing as haemo-
molecule (Harrison & Arosio, 1996). This may be siderin (Fahmy & Young, 1993). Cytokines such as
the signal for ferritin to be incorporated into tumour necrosis factor-a (TNF-a) and interferon-g
lysosomes. Once inside the lysosome the presence (IFN-g) may be responsible for this effect during
of a large number of H-subunits in the ferritin inflammatory and infectious conditions. These cyto-
protein shell increases the chances of degradation kines may increase lysosomal activity resulting in
(Bomford et al., 1981), since H-subunit rich ferritins, increased degradation of intracellular ferritin, leading
in the presence of denaturing conditions, are less to the formation of haemosiderin, from which iron
stable than L-subunit rich ferritins (Kim et al., 2001; would be less easily liberated for subsequent extra-
Miyazaki et al., 2002). The salt-bridge present in the cellular release (Alvarez-Hernández et al., 1989).
L-subunit appears to be important for the differences In vitro incubation of cells with either TNF-a or IFN-g
in stabilities between H-subunit rich ferritins and increases the expression of ferritin H-subunit mRNA
but not L-subunit mRNA (Fahmy & Young,

Mitochondrial ferritin
1993). Such a differential regulation of ferritin
subunit expression might result in increased Mitochondrial ferritin is found in the matrix of the
amounts of haemosiderin formation since H-subunit mitochondria under specific physiological condi-
rich ferritins are more susceptible to lysosomal tions, but is low in most cell types. Mitochondrial
degradation. ferritin is, in general, structurally and functionally
analogous to cytosolic ferritin and its main function,
similar to that of cytosolic ferritin, is to sequester
Ferritin in cellular organelles surplus iron. It does, however, differ from cytosolic
ferritin in various aspects (Levi et al., 2001). Where
Nuclear ferritin
the ferritin present in the cytosol exists mainly as
H-subunit rich ferritins are transiently present in the heteropolymers consisting of different combinations
nucleus where the presence of these H-subunit rich of the H- and the L-subunit, mitochondrial ferritin
ferritins can be controlled by various factors such as consists of only homopolymers of a subunit similar to
abnormal increases in cellular iron levels, develop- the H-subunit of cytosolic ferritins (Levi et al., 2001).
mental status, pro-inflammatory cytokines and oxi- The mitochondrial ferritin subunit is encoded from
dative stress (Thompson et al., 2002). A specific an intronless gene located on chromosome 5q23.1,
pathway has been shown for the translocation of which is different from the H-subunit gene for the
cytoplasmic H-subunit rich ferritins, but not cyto- cytosolic ferritin. Nevertheless, a high degree of
plasmic L-subunit rich ferritins, into the nucleus sequence homology exists between the cytosolic
(Surguladze et al., 2005). This pathway for the H-subunit and the mitochondrial ferritin subunit
transportation of cytoplasmic H-subunit rich ferritins (Bou-Abdallah et al., 2005; Levi et al., 2001). The
into the nucleus involves the nucleur pore complex mitochondrial ferritin subunit has about 80% se-
(NPC) (Thompson et al., 2002), and the transloca- quence identity to cytosolic ferritin H-subunit in its
tion is an active process that requires energy in the coding region and 55% to that of the cytosolic ferritin
form of ATP (Surguladze et al., 2005; Thompson L-subunit (Drysdale et al., 2002), with a structure
et al., 2002). Although many proteins are translo- very similar to H-subunit ferritin (Levi & Arosio,
cated into the nucleus by binding of a nuclear 2004). More important, the mitochondrial ferritin
localization signal to the nuclear pore complex, no subunit has complete conservation of the amino acids
such nuclear localization signal could be identified constituting the ferroxidase centre (Levi & Arosio,
for H-subunit rich ferritins. However, since 2004). The mitochondrial ferritin gene is expressed
H-subunit rich ferritins, as well as the H-subunit in the cytosol as a 30 kDa polypeptide containing an
rich ferritin mutant containing no ferroxidase centre, N-terminal mitochondrial targeting sequence of 60
are translocated to the nucleus, but L-subunit rich amino acids. Once the 30 kDa polypeptide enters the
ferritins are not, specific amino acids on the outside matrix space of the mitochondria the targeting
of the H-subunit rich ferritin molecule are implicated sequence is removed and the polypeptide processed
in this process (Thompson et al., 2002). O-glycosyla- into a subunit of 22 kDa (Levi et al., 2001). Typical
tion is predicted as the cue for the specific transloca- hollow spherical ferritin shells containing 24 subunits
tion of H-subunit rich ferritins. Once the H-subunit are then assembled from these subunits.
rich ferritins are inside the nucleus these ferritins can As mitochondrial ferritin contains ferroxidase
form stable complexes with the DNA (Surguladze activity it can take up large amounts of iron.
et al., 2004). This places the H-subunit rich ferritins However, the ferroxidase activity is significantly
in a strategic position to ward off possible oxidative lower than that of H-subunit rich cytosolic ferritin
onslaughts to DNA or, alternatively, to donate iron (Bou-Abdallah et al., 2005). This is partially due to
for enzyme activity and possibly the nicking of double the fact that only 12 of the 24 ferroxidase centres of
stranded DNA that could result in relaxation of mitochondrial ferritin would appear to be actively
superhelical stress (Surguladze et al., 2004). It was oxidizing Fe2þ to Fe3þ, and this at a reduced rate,
shown that not only does H-subunit rich ferritin form and although an m-peroxodiferric intermediate is
a stable complex with DNA (Surguladze et al., 2004), formed, mitochondrial ferritin does not regenerate its
but that DNA also contains specific iron-binding ferroxidase centre. The underlying reason that only
sites (Thompson et al., 2002). some of the ferroxidase centres are involved in the
Although the precise functions of H-subunit rich oxidation process is that the side-chain of serine, in
ferritins in the nucleus are still somewhat unclear place of alanine at position 144, protrudes toward a
indications are that H-subunit rich ferritin protects channel that connects to the ferroxidase centre—a
DNA and other nuclear constituents against oxida- configuration that may create steric hindrance to the
tive damage (Thompson et al., 2002), that it donates movement of iron accounting for only 12 active
iron for iron-dependent enzyme or transcription ferroxidase centres and a stoichiometry of 24 Fe2þ
activities (Surguladze et al., 2004) and that it may oxidized per ferritin molecule. The oxidized Fe3þ is
play a role in the regulation of the transcription of stabilized followed by nucleation. The negative patch
specific genes (Broyles et al., 2001). of glutamic acid residues near the ferroxidase centre,
similar to that for the L-subunit of cytosolic ferritin, active iron of the cells is processed in the mitochon-
might constitute the nucleation site. Once nucleation dria due to the synthesis of heme and iron–sulphur
has taken place autocatalytic mineral surface oxida- complexes (Bou-Abdallah et al., 2005; Nie et al.,
tion of iron occurs, resulting in slower oxidation of 2005). Not only are mitochondria surrounded by
iron, but less H2O2 production early in the process of iron but mitochondria also produce great amounts of
core formation (Bou-Abdallah et al., 2005). reactive oxygen species during oxidative respiration.
The iron in the cytosol transverses the double This combination of iron and reactive oxygen species
membrane of the mitochondria and is therefore in the mitochondrium calls for the protection
readily taken up by mitochondrial ferritin (Corsi provided by mitochondrial ferritin in times of iron
et al., 2002). Mitochondrial ferritin, in vitro, takes up dyshomeostasis that may result in the oxidative
iron in a similar fashion as cytosolic ferritin, where damage of mitochondrial constituents (Nie et al.,
Fe2þ is initially oxidized to Fe3þ by a ferroxidase 2005).
centre. This is then soon followed by iron-nuclei
formation and autocatalytic Fe2þ oxidation on the

Extracellular ferritin
mineral surface—since mitochondrial ferritin do not
regenerate its ferroxidase centres. This process was Most of the synthesized ferritin remains within the
shown to occur at a slower rate than for cytosolic cell where it sequesters and releases iron in order to
H-subunit rich ferritin. Despite the slower uptake, maintain intracellular iron homeostasis. However,
in vivo mitochondrial ferritin displays a higher avidity the content of ferritin varies between different cell
for iron than cytosolic H-subunit rich ferritin. types and maturation stages. The content of ferritin
Although excess iron, even when processed by in peripheral white blood cells, for instance, is about
mitochondria, is said not to be retained in mitochon- 103 times higher than that of peripheral red blood
drial ferritin, but is sequestered into cytosolic ferritin cells, with monocytes showing the highest values
(Corsi et al., 2002), it would appear that over- (Invernizzi et al., 1990). Aside from the presence of
expression of mitochondrial ferritin can lead to the ferritin in the cytosol of the cell, various quantities
depletion of cytosolic iron. In situations where of ferritin are found in the plasma. It is suggested that
increased amounts of mitochondrial ferritin are ferritin may enter the circulation either via secretion
available cytosolic ferritin and mitochondrial ferritin of ferritin by cells or through the release of ferritin
competes for cytosolic iron and since mitochondrial from damaged cells (Worwood, 1990). Both me-
ferritin has a higher avidity for iron this can lead to chanisms probably contribute to plasma levels.
the accumulation of iron in mitochondrial ferritin Ferritin destined for intracellular iron homeostasis
(Corsi et al., 2002). This depletion of cytosolic iron is synthesized on free polyribosomes whereas a small
dramatically increases IRP binding to IREs, followed amount of ferritin may be synthesized on the rough
by a decrease in cytosol ferritin levels and an increase endoplasmic reticulum for secretion into the plasma
in transferrin receptor expression (Corsi et al., 2002; (Covell & Worwood, 1984; Cragg et al., 1981). The
Nie et al., 2005). In short, it would at present appear range of plasma ferritin in the normal adult varies
that the high avidity of mitochondrial ferritin for iron, from 15 to 300 mg/l (Covell & Worwood, 1984;
together with a low availability of this iron for Worwood, 1990), and consists mainly of glycosylated
chelation, could lower iron bio-availability in the L-subunit rich ferritins containing insignificant
cytosol (Nie et al., 2005). The functional implication amounts of iron, even in conditions of iron overload
of this is as yet not clear. (Covell & Worwood, 1984; Cragg et al., 1981;
Under normal conditions most cell types contain Jacobs & Worwood, 1975; Ponka et al., 1998). While
only low amounts of mitochondrial ferritin and the the iron content of ferritin in the liver and spleen
stimuli for the expression of mitochondrial ferritin is could be more than 0.2 mg Fe/mg protein in condi-
still somewhat unclear. As mitochondrial ferritin tions of iron overload, the iron content of plasma
does not contain an iron responsive element in its 50 ferritin can be as low as 0.02 – 0.07 mg Fe/mg protein
untranslated region, as is the case for the H-subunit (Worwood et al., 1976).
and L-subunit of cytosol ferritin (Levi et al., 2001), The regulation and functions of secreted ferritins
one would presume that the expression of mitochon- in the plasma remain an enigma. However, a
drial ferritin is not regulated by the labile iron pool as quantitative relationship exists between the level of
for cytosolic ferritin. However, when heme synthesis plasma ferritin and the amount of storage iron
fails in patients with sideroblastic anaemia an (Cazzola & Ascari, 1986). In conditions of iron
increase in mitochondrial ferritin occurs together overload there is generally an increase in the
with the occurrence of ring sideroblasts (Cazzola expression of intracellular L-subunit rich ferritins,
et al., 2003). paralleled by an increase in these ferritins in the
Although details of the regulation of mitochondrial plasma (Halliday & Powell, 1979). Although the
ferritin expression are not known the purpose of the specific cellular origin of plasma ferritin is not known
presence of ferritin in the mitochondria is self- (Torti & Torti, 1994), various experiments indicated
evident. The mitochondrium is confronted with a a large contribution made by the reticuloendothelial
great amount of iron since most of the metabolically cell. An increase in plasma ferritin levels is known to
occur in parallel with the increase in reticulo- glycosylated ferritin of tissues and the glycosylated
endothelial cell ferritin after an increase in plasma ferritin (Hershko & Konijn, 1984). These
reticulo-endothelial cell iron during phagocytosis of differences in clearance may result in a significantly
non-viable red blood cells (Finch et al., 1984; longer half-life for the glycosylated, secreted ferritins
Jacobs & Worwood, 1975). However, elevated in the circulation compared to that of the non-
plasma ferritin levels are also seen in patients glycosylated tissue ferritins (Bellotti et al., 1987;
with parenchymal iron overload whose reticulo- Halliday et al., 1979; Hershko & Konijn, 1984;
endothelial cells are virtually devoid of iron (Finch Worwood, 1990).
et al., 1984). Therefore it would appear that the
plasma ferritin reflects storage iron anywhere in the
The internalization of ferritin by cells
body, regardless of the type of cell in which it was
stored (Finch et al., 1984). However, plasma ferritin Iron delivery to cells in general, and to developing
concentration is affected by a number of factors other erythroid cells in particular, is largely attributed to
than the amount of storage iron, including tissue diferric transferrin (Meyron-Holtz et al., 1994).
necrosis, damage to ferritin-rich tissue, inflamma- However, developing erythroid cells possess on their
tion, infections, neoplastic disease, and increased red surface, in addition to transferrin receptors, receptors
blood cell turnover (Cazzola & Ascari, 1986; that bind specifically, and internalize, H-subunit rich
Hershko & Konijn, 1984; Torti & Torti, 1994; ferritin (Meyron-Holtz et al., 1994). This binding
Worwood, 1982). When any of these conditions are and internalization of H-subunit rich ferritins is
present the relationship between plasma ferritin accomplished by means of a specific saturable
concentration and amount of storage iron no longer process and is highly regulated by the iron status of
holds. With tissue necrosis as in hepatocellular injury the cell (Meyron-Holtz et al., 1999). Since extra-
the increase in plasma ferritin is for instance due cellular ferritin, once internalized by the cell, is
to the release of ferritin from the damaged cells indistinguishable from intracellular ferritin, extracel-
since the increase in ferritin is dependent on both the lular ferritin could possibly also function as an iron
magnitude of cellular damage and liver iron stores donor (Meyron-Holtz et al., 1999). It is, for instance,
(Halliday & Powell, 1979). Furthermore, an increase known that internalized ferritin can increase the
in non-glycosylated, iron-rich ferritin has been repor- cellular labile iron pool and decrease the levels of the
ted upon tissue damage, which is indicative of the iron responsive protein, whereas apoferritin (contain-
release of tissue ferritin from the damaged tissue and ing no iron) has the opposite effect. This supports the
not as a result of active secretion (Cragg et al., 1981). notion that internalized ferritin is an iron donor and
In various neoplastic diseases it is suggested that suggests that apoferritin behaves like an iron chelator
the increase in plasma ferritin are related to an (Meyron-Holtz et al., 1999).
increased production of ferritin by the malignant Developing erythroid cells in the bone marrow are
cells. In leukaemia the normal concentration of often found in close proximity to a central ‘mother’
ferritin in circulating leukocytes is increased up to reticuloendothelial cell which ‘feeds’ ferritin to these
six-fold in acute myeloblastic leukaemia, more than developing red cell precursors (Deiss, 1983; Jacobs
20-fold in acute myelomonocytic leukaemia and two- et al., 1984). This process, known as rhopheocytosis,
to three-fold in chronic granulocytic leukaemia is a highly regulated pathway for iron assimilation by
(Jacobs & Worwood, 1975). In the presence of erythroid progenitor cells while cytosolic ferritin
various solid tumours, including tumours of the serves as an intermediate pool for iron for haem
breast, pancreas and liver an increase of H-subunit synthesis (Gelvan et al., 1996; Konijn et al., 1994;
rich ferritins was shown in the cells of the tumour, as Meyron-Holtz et al., 1994). Erythroid progenitor
well as an increase in plasma ferritin. In addition, the cells contain receptors for ferritin and ferritin finds its
plasma ferritins reflected the increase in H-subunit way into the erythroid precursors by receptor-
rich ferritins of the tumour, therefore the tumours mediated endocytosis (Aisen, 1991; Meyron-Holtz
seem to produce and secrete these H-subunit rich et al., 1994). Ferritin binds first to coated invagina-
ferritins (Kew et al., 1978; Niitsu et al., 1984). tions or pits before appearing in coated intracellular
The concentration of ferritin in plasma is a vesicles, followed by joining of the cytosolic pool of
function of the rate of secretion or release on the ferritin (Aisen, 1991).
one hand, and the clearance by other tissues on the Not only developing eryhroid precursors can take
other (Hershko & Konijn, 1984). The major cell type up ferritin, but ferritin is also rapidly internalized by
responsible for the clearance of plasma ferritins is the hepatocytes. This presumably also occurs via a
hepatocyte. A specific receptor for both glycosylated receptor-mediated pathway. Other cells, capable of
and non-glycosylated ferritin has been demonstrated pinocytosis or more generally endocytosis, have also
on the hepatocyte membrane (Hershko & Konijn, been shown to take up ferritin from extracellular
1984). These receptors bind both the H-subunit and fluid. Therefore, ferritin present intracellulary could
the L-subunit of ferritin (Halliday et al., 1979). have either been synthesized by the cell or could
However, a significant difference is indicated have been taken up (Richter, 1978). Internalized
between the rates of clearance for the non- ferritin can subsequently be degraded, similar to
48
A. M. Koorts & M. Viljoen
Figure 1. Heuristic presentation of intracellular ferritin metabolism.
Synthesis of ferritin: Transcription (I) of the H-subunit and L-subunit ferritin genes occurs in the nucleus of the cell. This is followed by (II) the translocation of the H-subunit and L-subunit mRNA to a pool of
translatable ferritin mRNA. Translation of the H-subunit and L-subunit mRNA of ferritin from this pool of translatable ferritin mRNA is largely controlled by iron from the labile iron pool (III) that contains the
metabolically and catalytically reactive iron. In this pool of translatable ferritin mRNA (II) translation of the H-subunit and L-subunit mRNA, respectively, is prevented by binding of the iron-responsive protein
(IRP) to the iron-responsive element (IRE) on the 50 non-coding stretch of H-subunit and L-subunit mRNA (II.1). Displacement of the IRP takes place upon binding of iron to IRP followed by translation (II.2).
Translation of the ferritin mRNA takes place on free polyribosomes in the cytosol (IV). This is followed by folding of the translated H-subunit and L-subunit polypeptides into the a-helix rich tertiary structures of
the H-subunit and L-subunit. These subunits form a pool consisting of H-subunits and L-subunits (V). From this pool of H-subunits and L-subunits the protein shell of ferritin consisting of 24 subunits
symmetrically arranged is assembled (VI). The completely assembled iron-free ferritin (apoferritin) forms a pool of apoferritin containing different combinations of H- and L-subunits (VII). Ferritin can also be
secreted from the cell (VIII).
Sequestration of iron (IX): Sequestration of iron is shown to occur after the ferritin protein shell is fully assembled. (IX.1) Oxidation of Fe2þ is performed by the ferroxidase centre of the H-subunit. This is followed
by nuclei formation and iron core growth facilitated by L-subunits. Once the iron core reaches a sufficient size oxidation of Fe2þ can take place on the surface of the iron core. (IX.2) Oxidation of Fe2þ is
performed by the ferroxidase centre of the H-subunit. However, if ferritin contains insufficient quantities of L-subunit for nuclei formation the formed Fe3þ can leave the ferritin molecule and move to a ferritin
molecule containing sufficient quantities of L-subunit or an already developed iron core, or (IX.3) the formed Fe3þ can leave the ferritin molecule followed by hydrolysis of the Fe3þ on the outer surface of the
ferritin molecule and ferritin aggregation.
Release of iron from ferritin (X): The release of iron from ferritin is shown to occur either by (X.1) Simultaneous entry of a reductant and a chelator to the interior of the ferritin protein shell whereupon Fe3þ is
reduced to Fe2þ in the confinements of the ferritin protein shell by the reductant followed by the release of Fe2þ as a Fe2þ-chelator complex, or (X.2) entry of only a chelator to the interior of the ferritin protein
shell in which case Fe3þ is not reduced and leaves as a Fe3þ-chelator complex.
Distribution of ferritin (XI): Ferritin occurs in the cytosol either as dispersed ferritin particles (XI.1) or as ferritin clusters (XI.2).
Degradation of ferritin (XII): Two different processes can result in the degradation of ferritin. (XII.1) Degradation by the 20s proteasome enzymatic system, which recognizes and degrades oxidized ferritin. (XII.2)
Degradation by lysosome enzymes in a secondary lysosome. Ferritin finds its way into the secondary lysosome by either autophagocytosis (XII.2.1) or by targeting of ferritin to the secondary lysosome (XII.2.2).
The latter can lead to haemosiderin and eventually siderosome formation.
Nuclear ferritin (XIII): Ferritin is also found in the nucleus. The ferritin in the nucleus consists of cytosolic H-subunit rich ferritins that are translocated back to the nucleus from the pool of apoferritin (VII) where
these ferritins can form stable complexes with the DNA.
Mitochondrial ferritin (XV): Mitochondrial ferritin contains 24 identical subunits transcribed from a different gene than that for the cytosolic H-subunit and L-subunit (XIV). Upon transcription and translation
the mitochondrial ferritin subunit polypeptide is translocated into the matrix of the mitochondria (XV.1). This is followed by the cleavage of the signal sequence and folding of the mitochondrial ferritin subunit
(XV.2). Typical hollow spherical ferritin shells containing 24 subunits are then assembled from these subunits (XV.3). Cytosolic iron from the labile iron pool (III) transverses the double membrane of the
mitochondria followed by sequestration by mitochondrial ferritin (XV.4).
Processes involved in cellular iron acquisition (XVI–XVIII): (XVI) The transferrin receptor binds transferrin and is endocytosed. Upon acidification of the endosome iron is released into the labile iron pool (III).
(XVII) The ferritin receptor binds ferritin and is endocytosed. Ferritin is degraded in a secondary lysosome and the released iron joins the labile iron pool (III). (XVIII) Red blood cells are phagocytosed followed
by degradation and the release of heme iron by heme oxygenase. The released iron joins the labile iron pool (III).
Ferritin and ferritin isoforms
49
intracellularly produced ferritin, within the cell T lymphocytes (CD2 is the surface molecule on
(Moss et al., 1994; Siegenberg et al., 1990) and its T-lymphocytes which facilitates binding to sheep
iron contents released into the labile iron pool of the erythrocytes and the formation of so-called
cell (Hulet et al., 2000). E-rosettes), suppress the in vitro responses of
lymphocytes to various mitogens including PHA
and con A, inhibit the mixed-lymphocyte reaction,
Other functions of ferritin
inhibit delayed-type hypersensitivity responses, block
Ferritin seems to have functions beyond the control the access to T-lymphocytes by various regulatory
of iron bioavailability—amongst others, the down- factors by sitting on the surface of the cells (Hie-won
regulation of myelopoiesis and the suppression of et al., 1984; Worwood, 1990) and decrease leukocyte
certain immune responses. H-subunit rich ferritins migration (Worwood, 1990). Receptors for
are present in most biologic fluids, but not, or only in H-subunit rich ferritins have also been found on
low concentrations, in plasma (Morikawa et al., various T-cell lines, CD4 and CD8 T-lymphocytes
1995). The ferritin present in plasma is mostly and on CD19 B-lymphocytes, and the expression of
L-subunit rich. However, during certain disease H-subunit rich ferritin binding sites on these cells
states the concentration of H-subunit rich ferritin is appears to be closely and positively linked to their
increased. At present it would appear that the activation and proliferation status (Meyron-Holtz
H-subunit rich ferritins are derived mostly from et al., 1994; Morikawa et al., 1995). It would
monocytes and macrophages as indicated by the therefore appear that H-subunit rich ferritins may
secretion of H-subunit rich ferritins from many perhaps act as feedback inhibitors of activation of
monocyte-macrophage cell lines, as well as by peripheral blood cells in a way similar to that
monocytes from blood and bone marrow (Broxmeyer suggested for the cells involved in myelopoiesis.
et al., 1984). The release of H-subunit rich ferritins Quiescent circulating lymphocytes, reticulocytes,
from monocytes is controlled by T-cell subsets. erythrocytes, and monocytes show little expression
T-helper cells enhance release and T-suppressor of the H-subunit rich ferritin receptor, but PHA-
cells suppress the release (Worwood, 1990). stimulated lymphocytes, Epo-induced BFU cells
A number of effects have been attributed to these and differentiated macrophages have all been shown
H-subunit rich plasma ferritins including the down- to express above average levels of the receptor
regulation of myelopoiesis and the suppression of (Halliday et al., 1994), which may result in these
various immune functions (Broxmeyer et al., 1984; cells being more susceptible to inhibition by H-
Torti & Torti, 1994). It has specifically been shown subunit rich ferritins. Increased binding of H-subunit
that H-subunit rich ferritins, but not L-subunit rich rich ferritins to peripheral lymphocytes have also
ferritins, down-regulate myelopoiesis (Broxmeyer been shown to occur in patients with malignant
et al., 1986), i.e. the growth and development of disorders and the magnitude of H-subunit rich
granulocytes, macrophages, erythrocytes, and plate- ferritin binding to lymphocytes was shown to be
lets (Broxmeyer, 1992; Joshi & Clauberg, 1988), related to the stage of the malignant process (Ciriello
both in vitro and in vivo. It has been suggested that et al., 1987). It is postulated that two receptor
H-subunit rich ferritins constitute part of a normal systems exist for the binding and execution of H-
inhibitory feed-back mechanism for the proliferation subunit rich ferritin’s effects. The first receptor
of granulocyte–macrophage colony forming units system internalizes the bound ferritin. This system
(CFU-GM), multipotential colony forming units is similar to the receptor system operating in
(CFU-GEMM) and erythroid burst forming erythroid precursors. However, a regulatory effect
units (BFU-E) (Broxmeyer, 1992; Jacobs et al., on cell proliferation and maturation occurs whereas
1984). H-subunit rich ferritin decreases the prolif- in erythroid precursors such a regulatory effect has
eration of cells during myelopoiesis by directly not been observed (Meyron-Holtz et al., 1994,
affecting these progenitor cells (Broxmeyer, 1992). 1999). The second receptor system, with a Kd three
Surface receptors specific for H-subunit rich ferritins orders of magnitude lower, does not result in the
have been shown on these progenitor cells (Fargion internalization of the bound ferritin (Meyron-Holtz
et al., 1988). These effects of H-subunit rich ferritins et al., 1994, 1999). This suggests a mechanism for
are mediated via the ferroxidase activity of the the regulation of cellular proliferation and matura-
H-subunits—most probably by inducing intracellular tion by ferritin not involving iron or the sequestration
iron starvation (Cozzi et al., 2000; Morikawa et al., of iron.
1995), since addition of iron completely counteracts
the inhibitory effects of the H-subunit rich ferritins
In conclusion
(Broxmeyer et al., 1991). Not only does H-subunit
rich ferritins down-regulate the production of cells There can be no doubt that the primary function of
involved in the immune system, but H-subunit rich ferritin is to regulate the bioavailability of iron and
ferritins also suppress various functions of immune that both the H-subunit, which contains the ferrox-
cells. H-subunit rich ferritins can, for instance, idase centre for oxidation of Fe2þ to Fe3þ, and the
exert inhibitory effects on E-rosette formation of L-subunit, which plays a paramount role in the
subsequent nucleation of Fe3þ and growth of sion would appear to have antiapoptotic effects—not
the iron core, are required for optimal control of related to its iron-binding function (Cozzi et al.,
this bioavailability. However, even in the context 2003).
of the regulation of iron bioavailability, and the Although we are still far from understanding the
effects thereof, many questions about the exact exact role of ferritin and its isoforms in health and
mechanisms and about the interplay between the disease new information on the functions of ferritin
H- and L-subunits remain. It would in general and the movement of iron within the protein shell is
appear that an increase in expression of the H- surfacing. Information on the recently identified
subunit, especially during times of cellular stress such mitochondrial ferritin (Drysdale et al., 2002; Levi
as occurs during inflammation, brings about rapid et al., 2001), a ferritin molecule with biochemical
sequestration of iron. The H-subunit with its active properties very similar to H-ferritin provides, for
ferroxidase centre reduces the labile iron pool, which instance, new insight into the movement of iron
would result in increased IRP activity, decreased within the ferritin protein shell rich in H-subunits
cellular proliferation and increased resistance to H2O2- (Langlois d’Estaintot et al., 2004).
induced oxidative stress—effects that are apparently
down-regulated by prolonged iron overload or inhibi-
Acknowledgements
tion of the ferroxidase centre (Cozzi et al., 2002, 2004).
Information on the influence of the degree to which L- We would like to thank the Skye foundation and the
ferritin is expressed is still somewhat contradictory. HF Verwoerd Research Trust for financial support as
While it is generally accepted that the L-subunit assists well as Mrs Sigi Dannheimer for the artwork of the
the H-subunit in enhancing the incorporation of iron figure.
into ferritin by providing the major nucleation sites
the increased expression of the L-subunit or in the L-
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Ferritin and Isoferitins

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Archives of Physiology and Biochemistry, February 2007; 113(1): 30 – 54

Ferritin and ferritin isoforms I: Structure–function relationships,

A. M. KOORTS & M. VILJOEN

Department of Physiology, School of Medicine, University of Pretoria, Pretoria, South Africa

Key words: Ferritin, haemosiderin, H-subunit, L-subunit, isoferritins.

vides a reserve for cytochromes, nitrogenases, ribo-

Received for publication 12 April 2006. Accepted 29 November 2006.

(Bou-Abdallah et al., 2002). After about an hour of

(Powell, 1998). This interaction with the iron core

ferritin can vary 1000-fold among different cell types

but not L-subunit mRNA (Fahmy & Young,

formation and autocatalytic Fe2þ oxidation on the

Figure 1. Heuristic presentation of intracellular ferritin metabolism.

62(5):1078 – 87. Piperno A, Fiorelli G. 1988. Characteristics and expression of

61:52 – 60. Levi S, Luzzago A, Franceschinelli F, Santambrogio P,

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