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Iron Transport and Cellular Uptake

Iron kinetics
schematic of iron body iron metabolism
Figure 1. Iron is assiduously conserved and recycled for use in heme and non-heme enzymes. About 1 to 2 mg of iron are lost each day to sloughing of skin and mucosal cells of the gastrointestinal and genitouretal tracts. This obligate iron loss is balanced by iron absorption from the gastrointestinal tract. Only a small fraction of the 4 grams of body iron circulate as part of transferrin at any given time. Body iron is most prominently represented in hemoglobin and in ferritin.
Only a small proportion of total body iron daily enters or leaves the body's stores on a daily basis (Figure1). Consequently, intercellular iron transport, as a part of the iron reutilization process, is quantitatively more important that intestinal absorption. The greatest mass of iron is found in erythroid cells, which contain about 80% of the total body endowment. The reticuloendothelial system recycles a substantial amount of iron from effete red cells, approximating the amount used by the erythron for new hemoglobin production.
 
 

Transferrin

 Of the approximate 3 grams of body iron in the adult male, approximately 3mg or 0.1% circulates in the plasma as an exchangeable pool (Table 1). Essentially all circulating plasma iron normally is bound to transferrin. This chelation serves three purposes: it renders iron soluble under physiologic conditions, it prevents iron-mediated free radical toxicity, and it facilitates transport into cells. Transferrin is the most important physiological source of iron for red cells (Ponka, 1997). The liver synthesizes transferrin and secretes it into the plasma. Transferrins are produced locally in the testes and CNS. These two sites are relatively inaccessible to proteins in the general circulation (blood:testis barrier, blood:brain barrier). The locally synthesized transferrin could play a role in iron metabolism in these tissues. Information on the function of transferrin produced in these localized sites is sparce, however.
 
 

 Plasma transferrin is an 80 kDa glycoprotein with homologous N-terminal and C-terminal iron-binding domains (reviewed in Huebers and Finch, 1987]. The molecule is related to several other proteins, including ovotransferrin in bird and reptile eggs (Williams et al., 1982), lactoferrin in extracellular secretions and neutrophil granules (Mazurier et al., 1983); (Metz-Boutigue et al., 1984) and melanotransferrin (p97), a protein produced by melanoma cells (Brown et al., 1982). Ovotransferrin may help protect the developing embryo in the semi-permeable egg by sequestering  iron that microbes need to grow. Lactoferrin, in secretions such as milk and tears, might have a similar function. One recent report indicates that lactoferrin can act as a site-specific DNA binding protein, and could mediate transcriptional activation. Such a function is, however, at odds with its existence as an extracellular protein (He and Furmanski, 1995).

 X-ray crystal structures exist for human lactoferrin and rabbit transferrin (reviewed by [Baker and Lindley, 1992]. All members of the transferrin protein superfamily have similar polypeptide folding patterns. N-terminal and C-terminal domains are globular moieties of about 330 amino acids; each of these is divided into two sub-domains, with the iron- and anion-binding sites in the intersubdomain cleft. The binding cleft opens with iron release, and closes with iron binding. N- and C-terminal binding sites are highly similar.

Iron binding by Transferrin

 The precise mechanics of iron loading onto transferrin as it leaves intestinal epithelial cells or reticuloendothelial cells is unknown. The copper-dependent ferroxidase, ceruloplasmin, may play a role. Compelling evidence indicates that the protein is involved in mobilizing tissue iron stores to produce diferric transferrin (Osaki and Johnson, 1969); (Osaki et al., 1971); (Yoshida et al., 1995); (Harris et al., 1995).

Transferrin binds iron avidly with a dissociation constant of approximately 1022 M-1 (Aisen and Listowsky, 1980). Ferric iron couples to transferrin only in the company of an anion (usually carbonate) that serves as a bridging ligand between metal and protein, excluding water from two coordination sites (Aisen and Listowsky, 1980); (Harris and Aisen, 1989); (Shongwe et al., 1992). Without the anion cofactor, iron binding to transferrin is negligible. With it, ferric transferrin is resistant to all but the most potent chelators. The remaining four coordination sites are provided by the transferrin protein - a histidine nitrogen, an aspartic acid carboxylate oxygen, and two tyrosine phenolate oxygens (Bailey et al., 1988); (Anderson et al., 1989). Available evidence suggests that anion-binding takes place prior to iron-binding. Iron release from transferrin involves protonation of the carbonate anion, loosening the metal-protein bond.
Table 1. Distribution and Kinetics of Body Iron
Compartment
Iron (grams)
Percent of Total
Hemoglobin
2.7
66
Myoglobin
0.2
3
Heme Enzymes
0.008
0.1
Non-heme Enzymes
< 0.0001
---
Intracellular Storage
(Ferritin)
1.0
30
Intracellular Labile Iron
(Chelatable Iron)
0.07 (?) 
1
Intercellular Transport
(Transferrin)
0.003
0.1 

Transferrin/Iron Physiology

 The sum of all iron binding sites on transferrin constitutes the total iron binding capacity (TIBC) of plasma. Under normal circumstances, about one-third of transferrin iron-binding pockets are filled. Consequently, with the exception of iron overload where all the transferrin binding sites are occupied, non-transferrin-bound iron in the circulation is virtually nonexistent. Distribution of plasma and tissue iron can be traced using 59Fe as a radioactive tag. The subject receives autologous transferrin loaded with radioactive iron that then can be monitored. Blood samples can be analyzed at timed intervals to determine the rate of loss of the radioactive label. Such ferrokinetic studies indicate that the normal half-life of iron in the circulation is about 75 minutes (Huff et al., 1950). The absolute amount of iron released from transferrin per unit time is the plasma iron turnover (PIT).

 Such radioactive tracer studies indicate that at least eighty percent of the iron bound to circulating transferrin is delivered to the bone marrow and incorporated into newly formed erythrocytes (Jandl and Katz, 1963); (Finch et al., 1982); Fig. 1). Other major sites of iron delivery include the liver, which is a primary depot for stored iron, and the spleen. Hepatic iron is found in both reticuloendothelial cells and hepatocytes. Reticuloendothelial cells acquire iron primarily by phagocytosis and breakdown of aging red cells These cells extract the iron from heme and return it to the circulation bound to transferrin. Hepatocytes take up iron by at least two different pathways. The first involves receptor-mediated endocytosis of transferrin. In addition, hepatocytes can take up ionic iron by a process independent of transferrin (Inman and Wesling-Resnick, 1993).

Ferrokinetics and the Bone Marrow

 Given the preeminent role of the bone marrow in the clearance of labeled iron from the circulation, ferrokinetics provide a window on erythropoietic activity. Conditions that augment erythrocyte production increase the PIT. For example, hemolytic anemias such as hereditary spherocytosis and sickle cell disease induce rapid delivery of transferrin-bound iron to the marrow. In contrast, disorders that reduce red cell production prolong the PIT. This picture is seen, for example, with anemia due to Diamond Blackfan anemia.

When erythrocytes are produced and released into the circulation in a normal fashion, the process of erythropoiesis is termed "effective". In patients with certain hemolytic anemias, however, the nascent red cells are so abnormal they are destroyed before leaving the marrow cavity. In this circumstance, the erythropoiesis is "ineffective", meaning simply that the erythropoietic precursors have failed to accomplish their primary task: the delivery of intact erythrocytes to the circulation. The ferrokinetic profiles such cases show rapid removal of iron from transferrin with a delayed entry of label into the pool of circulating red cell hemoglobin. ß+-thalassemia is an important example of this pattern of hemolytic anemia with ineffective erythropoiesis. In ß+-thalassemia, ineffective erythropoiesis is coupled with a markedly enhanced PIT.

Cellular Iron Uptake

 Although transferrin was characterized fifty years ago (Laurell and Ingelman, 1947), its receptor eluded investigators until the early 1980s. In a quest to better understand the behavior of neoplastic cells, investigators prepared monoclonal antibodies against tumor cells. The target of these monoclonal antibodies later was found to be the cell surface transferrin receptor glycoprotein (Sutherland et al., 1980; Seligman et al., 1980).

 A broad body of literature now supports the concept that the iron-transferrin complex is internalized by receptor-mediated endocytosis. The general structure of the transferrin receptor is shown in Figure 2. This disulfide-linked homodimer has subunits containing 760 amino acids each (Kuhn et al., 1984); (Schneider et al., 1983); (Jing and Trowbridge, 1987). Oligosaccharides account for about 5% of the 90 kDa subunit molecular mass (Reckhow and Enns, 1988). Four glycosylation sites (three N-linked and one O-linked) line the protein (Hayes et al., 1992). Glycosylation-defective mutants have fewer disulfide bridges, bind transferrin less efficiently and are expressed less prominently on the surface expression than are normal receptors (Williams and Enns, 1993a); (Williams and Enns, 1993b).
 
Schematic representation of the transferrin receptor
Schematic of transferrin receptor
Figure 2. The molecule is a transmembrane homodimer linked by disulfide bonds. An acyl group attached to the cytoplasmic tail of the molecule anchors the assembly to the plasma membrane.

  The transmembrane domain, between amino acids 62 and 89, functions as an internal signal peptide, as none exits at the N-terminal end (Zerial et al., 1986). A molecule of fatty acid (usually palmitate) covalently links each subunit to the internal edge of the transmembrane domain and could play a role in membrane localization. Interestingly, non-acylated mutants mediate faster iron uptake than normal receptors (Alvarez et al., 1990); (Jing and Trowbridge, 1990). The transferrin binding regions of the protein are unidentified (Williams and Enns, 1993a); (Williams and Enns, 1993b). Efforts to crystallize transferrin receptor protein are underway.

 Iron is taken into cells by receptor-mediated endocytosis of monoferric and diferric transferrin (Karin and Mintz, 1981); (Klausner et al., 1983); (Iacopetta and Morgan, 1983); (Fig. 3). Receptors on the outer face of the plasma membrane bind iron-loaded transferrin with a very high affinity. The C-terminal domain of transferrin appears to mediate receptor binding (Zak et al., 1994). Diferric transferrin binds with higher affinity than monoferric transferrin or apotransferrin (Huebers et al., 1984); (Young et al., 1984). The dissociation constant (Kd) for bound diferric transferrin ranges from 10-7 M to 10-9 M at physiologic pH, depending on the species and tissue assayed (Stein and Sussman, 1983); (Sawyer and Krantz, 1986). The Kd of monoferric transferrin is approximately 10-6 M. The concentration of circulating transferrin is about 25 mM. Therefore, cellular transferrin receptors ordinarily are fully saturated.

 After binding to its receptor on the cell surface, transferrin is rapidly internalized by invagination of clathrin-coated pits with formation of endocytic vesicles (Figure 3). This process requires the short, 61 amino acid intracellular tail of the transferrin receptor molecule (Rothenberger et al., 1987); (Alvarez et al., 1990); (McGraw and Maxfield, 1990); (Girones et al., 1991); (Miller et al., 1991). Receptors with truncated N-terminal cytoplasmic domains do not recycle (Rothenberger et al., 1987). This portion of the molecule contains a conserved tyrosine-threonine-arginine-phenylalanine (YTRF) sequence which functions as a signal for endocytotic internalization (Collawn et al., 1993). Genetically engineered addition of a second YTRF sequence enhances receptor endocytosis (Collawn et al., 1993). A number of stimuli reversibly phosphorylate the serine residue adjacent to the YTRF sequence, at position 24 by the action of protein kinase C (Davis et al., 1986). The role of receptor phosphorylation is unclear. Despite removal of the phosphorylation site by site-directed mutagenesis, the transferrin receptor recycles normally (Rothenberger et al., 1987).
 
Receptor-mediated transferrin endocytosis
Schematic of transferrin receptor-mediated endocytosis
Figure 3. Ferro-transferrin binds to transferrin receptors on the external surface of the cell. The complex is internalized into an endosome, where the pH is lowered to about 5.5. Iron separates from the transferrin molecule, moving into the cell cytoplasm. Here, an iron transport molecule shuttles the iron to various points in the cell, including mitochondria and ferritin. Ferritin molecules accumulate excess iron. Lysosomes engulf aggregates of ferritin molecules in a process termed "autophagy".

  An ATP-dependent proton pump lowers the pH of the endosome to about 5.5 (Van Renswoude et al., 1982); (Dautry-Varsat et al., 1983); (Paterson et al., 1984); (Yamashiro et al., 1984). The acidification of the endosome weakens the association between iron and transferrin. Even at pH 5.5, Fe3+ would not normally dissociate from transferrin in the several minutes between its endocytosis and the return of transferrin apoprotein to the cell surface (Ciechanover et al., 1983). A plasma membrane oxidoreductase reduces transferrin bound iron from the Fe3+ state to Fe2+, directly or indirectly facilitating the removal of iron from the protein (Low et al., 1987); (Thorstensen and Romslo, 1988); (Nunez et al., 1990). Conformational changes in the transferrin receptor also play a role in iron release (Bali et al., 1991); (Sipe and Murphy, 1991).

 Rather than entering lysosomes for degradation, as do ligands in other receptor-mediated endocytosis pathways, intact receptor-bound apotransferrin recycles to the cell surface, where neutral pH promotes detachment into the circulation (Zak and Aisen, 1990). Thus the preservation and re-use of transferrin are accomplished by pH-dependent changes in the affinity of transferrin for its receptor (Van Renswoude et al., 1982); (Klausner et al., 1983); (Dautry-Varsat et al., 1983). Exported apotransferrin binds additional iron and undergoes further rounds of iron delivery to cells. The average transferrin molecule, with a half-life of eight days, may be used up to one hundred times for iron delivery (Harford et al., 1994).

 Topologically, the cell exterior and the endosome interior are equivalent compartments. The primary role of the transferrin-transferrin receptor interaction is to bring iron into the vicinity of the cell surface, thereby increasing the likelihood of iron uptake. Following its release from transferrin within the endosome, iron must traverse the plasma membrane to enter the cytosol proper. The molecules effecting this transport have not been identified, but the process may be carrier-mediated (Egyed, 1988). Two anemic, mutant animals, the Belgrade rat (b/b) and the hemoglobin deficit mouse (hbd/hbd) appear to have lesions at or near this step. Their cells take up ferrotransferrin into endosomes, but fail to release iron into the cytoplasm (Garrick et al., 1987); (Garrick et al., 1993). The molecular basis of the defects in these animals have not been elucidated.

 The endosomal transporter may reside on the plasma membrane of the cell prior to endocytosis (Pollack, 1992). If so, it should be oriented to transport iron directly into the cell, without the assistance of transferrin. Such non-transferrin-bound iron uptake activities have been characterized in tissue culture. This uptake system could function constitutively but inefficiently. Coupling the transferrin cycle to transport across the plasma membrane might augment iron uptake by creating an iron-rich environment for the transporter within the endosome. This same elusive transport molecule could also be involved in intestinal iron uptake. The phenotype of the mk/mk mouse (see above) suggests that red cell iron uptake and intestinal iron uptake share a common component which could be the 'endosomal' transporter.

 Once inside the cell cytoplasm, iron appears to be bound by a low molecular weight carrier molecule, which may assist in delivery to various intracellular locations including mitochondria (for heme biosynthesis) and ferritin (for storage). The identity of the intracellular iron carrier molecule(s) remains unknown. The amount of iron in transit within the cell at any given time is minuscule and defies precise measurement. This minute pool of transit iron, which is believed to be in the Fe2+ oxidation state, is the biologically active form of the element. Metabolically inactive iron, stored in ferritin and hemosiderin, is in equilibrium with exchangeable iron bound to the low molecular weight carrier molecule (Figure 3).

 Both prokaryotes and eukaryotes produce ferritin molecules for iron storage. Ferritins are complex twenty-four subunit heteropolymers of H (for heavy or heart) and L (for light or liver) protein subunits (Theil, 1987). L subunits are 19.7 kDa in mass, with isoelectric points of 4.5-5.0; H subunits are 21 kDa with isoelectric points of 5.0-5.7. The subunits of the ferritin molecule form a sphere with a central cavity in which up to 4500 atoms of crystalline iron is stored in the form of poly-iron-phosphate oxide (Theil, 1987). Eight channels through the sphere are lined by hydrophilic amino acid residues (along the three-fold axes of symmetry) and six more are lined by hydrophobic residues (along the four-fold axes; [Harrison et al., 1986].) Strong interspecies amino acid conservation exists in the residues that line the hydrophilic channels, while marked variation exists in those along the hydrophobic passages. Hydrophilic channels terminate with aspartic acid and glutamic acid residues , and are lined by serine, histidine and cysteine residues (all of which potentially bind metal ligands). The evolutionary conservation of the hydrophilic channels suggests that they provide the route for iron entry and exit from the ferritin shell, but this contention remains unproved. Little is known about how iron is released from ferritin for use.

 Although the two ferritin chains are highly homologous, only H ferritin has ferroxidase activity. A mechanism involving dioxygen converts ferrous to ferric iron, promoting incorporation into ferritin (Levi et al., 1988); (Lawson et al., 1991). The composition of ferritin shells varies from H-subunit homopolymers to L-subunit homopolymers, and includes all possible combinations between the two. Isoelectric focusing of ferritin from a particular tissue reveals multiple bands representing shells with different subunit compositions. These isoferritins, as they are called, show tissue specific variation (Drysdale, 1988). Ferritin from liver, for instance, is rich in L-subunits, as is that from the spleen. In contrast, the heart has ferritin rich in H-subunits. Increased H subunit content correlates with increased iron utilization, while increased L subunit content correlates with increased iron storage (Drysdale, 1988); (Theil, 1987). The H:L ratio rises with activation of heme synthesis or cell proliferation (Pattanapanyasat et al., 1987); (McClarty et al., 1990). Ferritin thus provides a flexible reserve of iron.

 Ferritin molecules aggregate over time to form clusters, which are engulfed by lysosomes and degraded (Iancu et al., 1977); (Bridges, 1987); Figure 3). The end-product of this process, hemosiderin, is an amorphous agglomerate of denatured protein and lipid interspersed with iron oxide molecules (reviewed by (Wixom et al., 1980). In cells overloaded with iron, lysosomes accumulate large amounts of hemosiderin which can be visualized by Prussian blue staining. Although the iron enmeshed in this insoluble compound constitutes an endstage product of cellular iron storage, it remains in equilibrium with soluble ferritin. Ferritin iron, in turn, is in equilibrium with iron complexed to low molecular weight carrier molecules. Therefore the introduction into the cell of an effective chelator captures iron from the low molecular weight "toxic iron" pool, draws iron out of ferritin, and eventually depletes iron from hemosiderin as well, though only very slowly. As might be expected, the bioavailability of hemosiderin iron is much lower than that of iron stored in ferritin.
 

Non-Transferrin-Bound Iron Uptake

 Alhough compelling evidence exists that the transferrin cycle is important for iron acquisition by the erythron (Ponka and Schulman, 1993; Ponka, 1997)), other tissues can import iron by alternative mechanisms. Some patients and mutant mice that have little or no circulating transferrin (Heilmeyer, 1966); (Goya et al., 1972); (Bernstein, 1987); (Huggenvik et al., 1989). Despite severe hypochromic, microcytic anemia, non-erythroid tissues are grossly normal. While the red cells suffer from iron deficiency, serum iron levels (iron not bound to transferrin) are elevated, and excess iron is deposited in the liver. The iron-deprived bone marrow likely signals the gut to increase absorption, exacerbating tissue iron excess. Ponka and Schulman speculate that non-erythroid cells depend less on transferrin because their modest iron needs can be met by turnover of endogenous ferritin and heme iron. Red cells are more vulnerable because of greater iron use to form hemoglobin (Ponka and Schulman, 1993; Ponka, 1997). The transferrin cycle could serve primarily to enhance iron uptake by tissues with a great demand for the element.

 Iron overload produces fully saturated transferrin and non-transferrin bound iron circulating in a chelatable, low molecular weight form (Hershko et al., 1978); (Hershko and Peto, 1978); (Craven et al., 1987); (Grootveld et al., 1989). This iron is weakly complexed to albumin, citrate, amino acids and sugars, and behaves differently from iron associated with transferrin. Non-hematopoietic tissues, particularly the liver, endocrine organs, kidneys and heart preferentially take up this iron.

 Radiolabeled iron administered to mice with and without available transferrin binding capacity has quite different patterns of distribution (Craven et al., 1987). In normal animals, hematopoietic tissues are the prime sites of uptake. When free transferrin sites are absent, however, most iron is deposited in the liver and pancreas, indicating that these organs serve as iron reservoirs in the situation of iron overload. Notably, this pattern of distribution is similar to that seen in idiopathic hemochromatosis. These data support the idea that, while the transferrin pathway is important for meeting the needs of the erythron, it is not essential for iron uptake by all tissues.

 Kaplan and coworkers have studied iron incorporation from FeNH4 citrate (Sturrock et al., 1990); (Kaplan et al., 1991). Intriguingly, they find that transferrin-independent uptake increases in direct proportion to the concentration of this compound, similar to hepatic uptake of non-transferrin-bound iron in patients with saturated transferrin. They speculate that this is a protective alternative pathway that removes the toxic metal from the circulation. Other investigators have described similar uptake in HepG2 cells, and shown that it is reversible by addition of chelating compounds (Randell et al., 1994).

 A non-transferrin iron uptake mechanism with different properties has been described in K562 erythroleukemia cells (Inman and Wessling-Resnick, 1993). In the absence of ferric transferrin, iron uptake into K562 cells is sensitive to treatment with trypsin, suggesting that it requires a protein carrier. Higher ambient iron concentrations do not increase cellular iron uptake. As discussed above, this transport may be accomplished by the same machinery responsible for passage of iron out of transferrin cycle endosomes into the cytoplasm (Pollack, 1992). These two processes accomplish essentially the same task. The putative endosomal iron transporter must be oriented to transport iron from an endocytosed extracellular compartment into the cytoplasm. This transporter may exist on the cell surface prior to receptor-mediated endocytosis, with the capacity to transport iron to a modest extent. This activity is not restricted to erythroid cells. PHA-stimulated human peripheral lymphocytes have a similar transferrin-independent iron uptake mechanism (Hamazaki and Glass, 1992).
 
 

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