Secreted ferritin subunits are of two kinds in insects molecular cloning of cDNAs encoding two major subunits of secreted ferritin from Calpodes ethlius

In insects, holoferritin is easily visible in the vacuolar system of tissues that filter the hemolymph and, at least in Lepidoptera, is abundant in the hemolymph. Sequences reported for insect secreted ferritins from Lepidoptera and Diptera have high sequence diversity. We examined the nature of this diversity for the first time by analyzing sequences of cDNAs encoding two ferritin subunits from one species, Calpodes ethlius (Lepidoptera, Hesperiidae). We found that insect secreted ferritin subunits are of two types with little resemblance to each other. Ferritin was isolated from iron loaded hemolymph of C. ethlius fifth instar larvae by differential centrifugation. The N-terminal amino acid sequences for the nonglycosylated subunit with Mr 24,000 (S) and the largest glycosylated subunit with Mr 31,000 (G) were determined. The N-termini of the two subunits were different and were used to construct degenerate PCR primers. The same cDNA products were amplified from cDNA libraries from the midgut which secretes holoferritin and from the fat body which secretes iron-poor apoferritin. The G subunit most closely resembles the glycosylated ferritin subunit from Manduca sexta and the S subunit resembles the Drosophila small subunit. The S and G subunits from Calpodes were dissimilar and distinct from the cytosolic ferritins of vertebrates and invertebrates. Additional sequences were obtained by 5' and 3' RACE from separate fat body and midgut RACE libraries. cDNAs encoding both subunits had a consensus iron responsive element (IRE) in a conserved cap-distal location of their 5' UTR. An integrin-binding RGD motif found in the G subunit and conserved in Manduca may facilitate iron uptake through a calreticulin (mobilferrin)/integrin pathway. Calpodes and other insect ferritins have conserved cysteine residues to which fatty acids can be linked. Dynamic acylation of ferritin may slow but not prevent its passage out of the ER.     

The characterization of ferritin in an insect

In mammals, the iron storage protein ferritin is predominantly synthesized on free polysomes and accumulates in the cytosol but some is secreted and circulates in the blood as serum ferritin. In insect tissues, on the other hand, iron-containing holoferritin accumulates in the vacuolar system and can be secreted through the Golgi complex. The midgut can secrete it to the gut lumen and other tissues to the hemolymph.Ferritin was isolated from the midgut and hemolymph of fifth instar larvae of Calpodes ethlius, Lepidoptera, Hesperiidae. This holoferritin is stable to heat (75°C) or in the presence of SDS, proteinase K, or urea, has an Mr above 600,000, contains iron and resembles mammalian ferritins in appearance by electron microscopy. Calpodes ferritin is a glycoprotein having N-linked high-mannose oligosaccharides. It is not antigenically related to horse ferritin but is related to that from Manduca sexta, Lepidoptera, Sphingidae. In its native form, Calpodes ferritin has only 3 isoforms with a pI 6.5–7 suggesting a more uniform subunit composition than that in vertebrates. It has two principle subunits, with relative Mrs of 24,000 (L) and 31,000 (G) and two minor subunits with Mrs of 26,000 and 28,000 all of which cross-react with antibody to Manduca ferritin. The 24 kDa subunit is the only one that is not glycosylated. Iron injections induce an increase in the proportion of the 24 kDa subunit. We conclude that Calpodes has ferritin and that it is glycosylated like mammalian serum ferritin.     

Stoichiometry of Fe(II) oxidation during ceruloplasmin-catalyzed loading of ferritin.

Ceruloplasmin catalyzed the incorporation of iron into apoferritin with a stoichiometry of 3.8 Fe(II)/O2. This value remained the same when ferritin containing varying amounts of iron was used. Contrary to the "crystal growth" model for ferritin formation, no iron incorporation into holoferritin was observed in the absence of ceruloplasmin. Fe(II)/O2 ratios close to 2 were obtained for iron incorporation into apo- and holoferritin in Hepes buffer, in the absence of ceruloplasmin, indicating the formation of reduced oxygen species. Sequential loading of ferritin in this buffer resulted in increasing oxidation of the protein as measured by carbonyl formation. Sequential loading of ferritin using ceruloplasmin did not result in protein oxidation and a maximum of about 2300 atoms of iron were incorporated into rat liver ferritin. This corresponded to the maximum amount of iron found in rat liver ferritin in vivo after injection with iron. These results provide evidence for ceruloplasmin as an effective catalyst for the incorporation of iron into both apo- and holoferritin. The possibility that these findings may have physiological significance is discussed.     

Apolipoprotein B binds ferritin by hemin-mediated binding: evidence of direct binding of apolipoprotein B and ferritin to hemin

Apolipoprotein B (apoB) is known to be a ferritin-binding protein. Here we show that apoB binds to ferritin through hemin-mediated binding. Human apoB bound to bovine spleen, horse spleen, and canine liver ferritins, but did not bind to bovine apoferritin, even after incorporation of iron into it. Incubation of apoferritin with hemin resulted in apoB binding with apoferritin at the same level as with holoferritin. In contrast, hemin inhibited binding of apoB to ferritin. Bovine spleen apoferritin bound biotinylated hemin, and hemin inhibited the binding between the apoferritin and biotinylated hemin, suggesting that ferritin binds hemin directly. ApoB and LDL containing apoB bound biotinylated hemin, and their bindings were also inhibited by hemin, but not protoporphyrin IX. These data demonstrate that binding of apoB to ferritin is mediated through ferritin’s binding to hemin, and also that apoB binds hemin directly.     

Musca domestica hemolymph ferritin

We describe a method for the purification of ferritin from Musca domestica larval hemolymph. Musca ferritin occurs in hemolymph predominantly as a native protein with molecular weight equal to 550,000 and subunits of 26,000. The average iron content of purified ferritin was determined to be 3,000 ± 600 iron atoms per molecule. The iron contents of ferritin was heterogeneous; both fully iron loaded molecules and apoferritin are probably present in the Musca hemolymph. The anti-ferritin serum raised in rabbit was able to recognize native ferritin but was not reactive with the protein subunits isolated by SDS-PAGE. The ferritin concentration in hemolymph attains a maximum of 0.28 mg/ml in the wandering stage larvae, decreasing to 0.13 mg/ml at the middle of pupal stadium. The ferritin contents of midgut and fat bodies were also determined. Fat body ferritin content is greatly reduced when the feeding larva passes into wandering stage. © 1996 Wiley-Liss, Inc.     

Ferritin—A general metal detoxicant

Binding of nonferrous metal ions to ferritin was compared to that of the phosphate-free or phosphate containing synthetic iron cores. The Scatchard plots for the synthetic cores reveal a high affinity site for Cd, Zn, Be, and Al, with K_D in the range 10^?5–10^?7 M. Preloading the cores with phosphate increased the number of metal ions bound without altering the K_D. The metal ions with smaller ionic radii (Be, Al) were bound in larger numbers than those with larger ionic radii (Cd, Zn). Ferritin isolated from soybean (Glycina max), horse spleen, and rat liver bound the metal ions in amounts larger than predicted from their iron core. Whereas the iron cores and their nonferrous metal ion complexes were insoluble, those in the protein shell remained in solution. Thus apoferritin precipitated with lower concentrations of aluminum than did holoferritin. Also, Al bound to apoferritin reduced the rate of iron loading into the protein.     

On the Cytoprotective Role of Ferritin in Macrophages and its Ability to Enhance Lysosomal Stability

Macrophages have a great capacity to take up (eg. by endocytosis and phagocytosis) exogenous sources of iron which could potentially become cytotoxic, particularly following the intralysosomal formation of low-molecular weight, redox active iron, and under conditions of oxidative stress. Following autophago-cytosis of endogenous ferritin/apoferritin, these compounds may serve as chelators of such lysosomal iron and counteract the occurrence of iron-mediated intralysosomal oxidative reactions. Such redox-reactions have been shown to lead to destabilisation of lysosomal membranes and result in leakage of damaging lysosomal contents to the cytosol. In this study we have shown: (i) human monocyte-derived macrophages to accumulate ferritin in response to iron exposure; (ii) iron to destabilise macrophage secondary lysosomes when the cells are exposed to H₂O₂; and (iii) endocytosed apoferritin to act as a stabiliser of the acidic vacuolar compartment of iron-loaded macrophages. While the endogenous ferritin accumulation which was induced by iron exposure was not sufficient to protect cells from the damaging effects of H₂O₂, exogenously added apoferritin, as well as the potent iron chelator desferrioxamine, afforded significant protection. It is suggested that intralysosomal formation of haemosiderin, from partially degraded ferritin, is a protective strategy to suppress intralysosomal iron-catalysed redox reactions. However, under conditions of severe macrophage lysosomal iron-overload, induction of ferritin synthesis is not enough to completely prevent the enhanced cytotoxic effects of H₂O₂.     

In vitro loading of apoferritin.

This study compared the effect of loading apoferritin either with ferrous ammonium sulfate in various buffers or with ceruloplasmin and chelated ferrous iron. It was shown that loading of apoferritin with ferrous ammonium sulfate was dependent on buffer and pH, and was directly related to the rate of iron autoxidation. The ceruloplasmin-dependent loading of apoferritin, however, was unaffected by these factors. Isoelectric focusing and amino acid analysis of the differently loaded ferritins showed that ferrous ammonium sulfate loading of apoferritin resulted in the depletion of the basic amino acids, lysine and histidine, probably as a result of protein oxidation. No significant differences in amino acid composition was noted for ceruloplasmin-loaded ferritin. Furthermore, ferritin loaded with ferrous ammonium sulfate released more iron than either native or ceruloplasmin-loaded ferritin when either paraquat or EDTA was used as an iron mobilizing agent. We suggest that the loading of apoferritin with ferrous ammonium sulfate occurred as a result of iron autoxidation and may result in oxidation of amino acids and loss of integrity of the protein, and that ceruloplasmin may act as a catalyst for the incorporation of iron into apoferritin in a manner more closely related to that occurring in vivo.     

Fe2+ binding to apo and holo mammalian ferritin

The binding of Fe2+ to both apo and holo mammalian ferritin has been investigated under anaerobic conditions as a function of pH. In the pH range 6.0-7.5, 8.0 +/- 0.5 Fe2+ ions bind to each apoferritin molecule, but above pH 7.5, a pH-dependent Fe2+ binding profile is observed with up to 80 Fe2+ ions binding at pH 10.0. This Fe2+ binding is reversible and is accompanied by up to two H+ being released per Fe2+ bound at pH 10.0. The Fe2+ binding to apoferritin probably occurs in the 3-fold channels. A much larger and more complex pH-dependent Fe2+ binding stoichiometry was observed for holoferritin with up to 300 Fe2+ ions binding at pH 10.0. This pH-dependent Fe2+ binding was interpreted as Fe2+ interaction at the FeOOH mineral surface with displacement of H+ from -OH or phosphate surface groups by the incoming Fe2+ ions. Mossbauer spectroscopic measurements using 57Fe-labeled Fe2+ under anaerobic conditions showed that 57Fe2+ binding to holoferritin was accompanied by electron transfer to the core, yielding 57Fe3+, presumably bound to the mineral surface. Removal of added iron by Fe2+-specific chelating agents yielded 57Fe2+, demonstrating the reversibility of this electron-transfer process. The Fe2+ bound to apo- and holoferritin is readily converted to Fe3+ by exposure to O2 and strongly retained by the respective ferritin species.     

Kinetic analysis of bovine spleen apoferritin and recombinant H and L chain homopolymers: Iron uptake suggests early stage H chain ferroxidase activity and second stage L chain cooperation

Ferritin utilizes ferroxidase activity to incorporate iron. Iron uptake kinetics of bovine spleen apoferritin (H: L = 1 : 1.1) were compared with those of recombinant H chain ferritin and L chain ferritin homopolymers. H chain ferritin homopolymer showed an iron uptake rate identical to bovine spleen apoferritin (0.19 and 0.21 mmol/min/micromol of protein, respectively), and both showed iron concentration-dependent uptake. In contrast, the L chain homopolymer, which lacks ferroxidase, did not incorporate iron and showed the same level of iron autoxidation in the absence of ferritin. Bovine spleen apoferritin was shown to have two iron concentration-dependent uptake pathways over a range of 0.02-0.25 mM ferrous ammonium sulfate (FAS) by an Eadie-Scatchard plot (v/[FAS] versus v), whereas the H chain ferritin homopolymer was found to have only one pathway. Of the two Km values found in bovine spleen apoferritin, the lower mean Km value was 9.0 microM, while that of the H chain homopolymer was 11.0 microM. H chain ferritin homopolymer reached a saturating iron uptake rate at 0.1 mM FAS, while bovine spleen apoferritin incorporated more iron even at 0.25 mM FAS. These results suggest that the intrinsic ferroxidase of ferritin plays a significant role in iron uptake, and the L chain cooperates with the H chain to increase iron uptake.     

Site of hemolymph lectin production and its activation in vitro by 20-hydroxyecdysone

To identify the tissues which produce hemolymph lectin in larvae of Bombyx mori, ovary, testis, fat body, and hemocytes from 5th-instar larvae were cultured in vitro and the culture medium was partially purified and assayed for hemagglutinating activity. Among the tissues tested, hemocytes appeared to be a major source of the hemolymph lectins. Ovary produced lectins to about one-tenth of the amount observed for the hemocytes, whereas testis and fat body were not productive. To study the hormonal control of hemolymph lectin production by hemocytes, hemocytes from 4th-instar larvae were cultured in vitro. Hemagglutinating activity in the hemolymph of 4th-instar larvae was immunostainable with the monoclonal antibody raised against 350,000 dalton lectin found in the 5th-instar hemolymph, but their molecular sizes were larger than the 5th-instar hemolymph lectins. When 20-hydroxyecdysone was added into the medium, production of the lectin by the hemocytes was remarkably enhanced, depending upon the hormone concentration.     

Prophenoloxidase binds to the surface of hemocytes and is involved in hemocyte melanization in Manduca sexta

In insects, melanotic encapsulation is an important innate immune response against large pathogens or parasites, and phenoloxidase (PO) is a key enzyme in this process. Activation of prophenoloxidase (proPO) to PO is mediated by a serine proteinase cascade. PO has a tendency to adhere to foreign surfaces including hemocyte surfaces. In this study, we showed that in the naïve larvae of the tobacco hornworm Manduca sexta, hemolymph proPO bound to the surface of granulocytes and spherule cells but not to oenocytoids, and about 10% hemocytes had proPO on their surfaces. When larvae were injected with water (injury) or microsphere beads (immune-challenge), hemolymph proPO was activated, and the number of hemocytes with surface proPO/PO increased at 12 h post-injection, but dropped to the normal level at 24 h. Hemocyte surface proPO can be activated in vitro, leading to melanization of these hemocytes. The number of melanized hemocytes from the larvae injected with water or microsphere beads significantly increased. We also showed that neither hemocytes nor cell-free plasma alone triggered melanization of immulectin-2-coated agarose beads in vitro. However, agarose beads were effectively melanized by isolated hemocytes in the presence of cell-free plasma. Our results suggest that activation of hemocyte surface proPO may initiate melanization, leading to the systemic melanization of hemocyte capsules.     

Isolation, purification and characterization of bovine spleen ferritin

Ferritin was isolated from bovine spleen and used to prepare apoferritin and reconstituted ferritin. The mol. wt of bovine ferritin was 464,000 with monomer subunits about 18,000-19,500. Gel electrophoresis showed three bands each for ferritin, apoferritin and reconstituted ferritin; all stained for protein and carbohydrate. Only apoferritin failed to stain for iron. Bovine ferritin had higher concentrations of proline, threonine, and valine than equine or human ferritin. The iron:protein ratio of bovine ferritin was 0.161 and of equine ferritin was 0.192. After iron uptake by the apoferritins the iron:protein ratios were 0.186 and 0.278 for the bovine and equine ferritins, respectively.     

Uptake of iron by apoferritin from a ferric dihydrolipoate complex

A study was made on the uptake of iron by horse spleen apoferritin, by using as an iron source the same ferric dihydrolipoate complex which represents the major product in the anaerobic removal of ferritin-bound iron by dihydrolipoate at neutral pH. The ferric dihydrolipoate complex was chemically synthesized and used as an iron donor to apoferritin. Iron uptake was studied, at slightly alkaline pH and in anaerobic conditions, as a function of the concentration of both the iron donor and apoferritin. Isolation of ferritin from mixtures of ferric dihydrolipoate and apoferritin, and subsequent identification of the oxidation state of ferritin-bound iron, showed that the first metal atoms were taken up in the ferrous form and that this early step was accompanied by accumulation of ferric iron. Total iron uptake increased with the molar ratio of complex to apoprotein and ranged over 25-40% of the iron being supplied. The amount of ferrous iron found inside the protein did not exceed 50-60 mol iron/mol ferritin after a 48-h incubation. At this time, ferric iron represented a significant fraction of the iron found in the isolated ferritin. Analytical and spectroscopic data indicated that fractional rates and equilibria for disassembly of the ferric complex in the presence of apoferritin were independent of the concentration of the protein and of the complex itself.     

Biosynthesis of ferritin in rat hepatoma cells and rat livers. II. Binding of iron by ferritin protein.

Ferritin and its protein subunits in rat hepatoma cell clone M-5123-C1 were biosynthetically labeled with [14C]leucine and 59Fe. Radioimmunoassays of ferritin/apoferritin and of protein subunits in the free polyribosome, membrane-bound polyribosome, smooth membrane, and cytosol fractions were done with ferritin-specific and subunit-specific rabbit IgG antibodies at various time intervals after pulsing. Much more 59Fe was bound by ferritin/apoferritin than by subunits in all of the cell fractions. Binding of iron to subunits may have been a random process. When hepatoma cells were simultaneously pulse-labeled with 59Fe and [14C]leucine, uptake of much of the 59Fe by ferritin occurred relatively early, in comparison to incorporation of [14C]leucine, in all of the cell fractions examined. Thus, 59Fe was readily incorporated into pre-existing ferritin. We conclude that most, if not nearly all, of the iron is incorporated after assembly of protein subunits.     

Molecular size heterogeneity of ferritin in mouse liver

As much as 4% of the total protein in pure liver ferritin from mice with short-term parenteral iron overload produces a minor band migrating anodally to the major (alpha) band of holoferritin with non-denaturing polyacrylamide gel electrophoresis. The components in this minor band and the alpha band have been isolated to purity by preparative electrophoretic fractionation. The protein in the minor band is ferritin, since it contains ferric iron and fulfills defining criteria at the level of biochemistry, immunology and ultrastructure. Native polyacrylamide electrophoresis with pore-size-gradient gels shows that the ferritin molecules in the minor band have a slightly smaller diameter than the holoferritin in the alpha band. Isoelectric focusing reveals that the smaller ferritin has an identical number and range of charge isomers (pI 4.9-5.3) as the larger ferritin, but the relative amount of each size class within some isoferritin bands differs. The smaller ferritin molecules are structurally intact and are made from polypeptide subunits with Mr 18 000; the larger ferritin molecules have subunits with Mr 22 000. The minor species of hepatic ferritin thus has a smaller molecular size because it is made mainly from smaller subunits. No minor electrophoretic band can be detected in liver ferritin obtained from mice with normal iron levels. These results demonstrate that siderosis induces the formation of molecular size polymorphism (macroheterogeneity) in mouse liver ferritin. The new smaller hepatic ferritin could serve to redistribute excess iron into the main storage organs during the early response to iron overload, since it appears to be identical to one of the two types of serum ferritin molecules present in these siderotic mice.     

Iron (III) can be transferred between ferritin molecules.

The iron-storage molecule ferritin can sequester up to 4500 Fe atoms as the mineral ferrihydrite. The iron-core is gradually built up when FeII is added to apoferritin and allowed to oxidize. Here we present evidence, from M?ssbauer spectroscopic measurements, for the surprising result that iron atoms that are not incorporated into mature ferrihydrite particles, can be transferred between molecules. Experiments were done with both horse spleen ferritin and recombinant human ferritin. M?ssbauer spectroscopy responds only to 57Fe and not to 56Fe and can distinguish chemically different species of iron. In our experiments a small number of 57FeII atoms were added to two equivalent apoferritin solutions and allowed to oxidize (1-5 min or 6 h). Either ferritin containing a small iron-core composed of 56Fe, or an equal volume of NaCl solution, was added and the mixture frozen in liquid nitrogen to stop the reaction at a chosen time. Spectra of the ferritin solution to which only NaCl was added showed a mixture of species including 57FeIII in solitary and dinuclear sites. In the samples to which 150 56FeIII-ferritin had been added the spectra showed that all, or almost all, of the 57FeIII was in large clusters. In these solutions 57FeIII initially present as intermediate species must have migrated to molecules containing large clusters. Such migration must now be taken into account in any model of ferritin iron-core formation.     

Siderosomal ferritin. The missing link between ferritin and haemosiderin?

A minor electrophoretically fast component was found in ferritin from iron-loaded rat liver in addition to a major electrophoretically slow ferritin similar to that observed in control rats. The electrophoretically fast ferritin showed immunological identity with the slow component, but on electrophoresis in SDS it gave a peptide of 17.3 kDa, in contrast with the electrophoretically slow ferritin, which gave a major band corresponding to the L-subunit (20.7 kDa). Thus the electrophoretically fast ferritin resembles that reported by Massover [(1985) Biochim. Biophys. Acta 829, 377-386] in livers of mice with short-term parenteral iron overload. The electrophoretically fast ferritin had a lower iron content (2000 Fe atoms/molecule) than the electrophoretically slow ferritin (3000 Fe atoms/molecule). Removal and re-incorporation of iron was possible without effect on the electrophoretic mobility of either ferritin species. On subcellular fractionation the electrophoretically fast ferritin was enriched in pellet fractions and was the sole soluble ferritin isolated from iron-laden secondary lysosomes (siderosomes). The amount and relative proportion of the electrophoretically fast species increased with iron loading. Haemosiderin isolated from siderosomes was found to contain a peptide reactive to anti-ferritin serum and corresponding to the 17.3 kDa peptide of the electrophoretically fast ferritin species. Unlike the electrophoretically slow ferritin, the electrophoretically fast ferritin did not become significantly radioactive in a 1 h biosynthetic labelling experiment. We conclude that the minor ferritin is not, as has been suggested for mouse liver ferritin, 'a completely new species of smaller holoferritin that represents a shift in the ferritin phenotype' in response to siderosis, but a precursor of haemosiderin, in agreement with the proposal by Richter [(1984) Lab. Invest. 50, 26-35] concerning siderosomal ferritin.