Cloning and expression of 32 kDa ferritin from Galleria mellonella

We have sequenced a cDNA clone encoding 32-kDa ferritin subunit in the Wax Moth, Galleria mellonella. The 32-kDa ferritin subunit cDNA was obtained from PCR using identical primer designed from highly conserved regions of insect ferritins. RACE PCR was used to obtain the complete protein coding sequence. The 32-kDa ferritin subunit encoded a 232 amino acid polypeptide, containing a 19 leader peptide. The iron-responsive element (IRE) sequence with a predicted stem-loop structure was present in the 5'-untranslated region of the wax moth 32-kDa ferritin subunit mRNA. The 32-kDa sequence alignment had 78 and 69% identity with Manduca sexta and Calpodes ethlius (G), respectively. The G. mellonella ferritin subunits showed minimal identity with each other (19%). The glycosylation site (Asn-X-Ser/Thr) was found in the 32-kDa subunit but not in the 26-kDa subunit. Northern blot analysis showed that the mRNA expression of the 32-kDa ferritin was detected in the fat body and midgut. The fat body expression increased after 6 h and the mRNA in midgut dramatically increased about 3-fold the expression level at 12 h after iron feeding. Western blot revealed that a protein level of the 32-kDa subunit is abundant in midgut after 12 and 24 h iron feeding.     

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.     

Sequencing and characterization of a cDNA encoding a ferritin subunit of Colorado potato beetle, Leptinotarsa decemlineata

A differentially expressed cDNA fragment (P311) from Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say), was identified by restriction fragment differential display-polymerase chain reaction (RFDD-PCR) technique, and showed a strong similarity to ferritin heavy chain subunits of other organisms. Based on P311, we constructed specific primers and obtained a 840-bp cDNA fragment spanning the open reading frame of CPB ferritin subunit using the rapid amplification of cDNA ends (RACE) technique. The sequence encodes 213 amino acid residues, including a 19 amino acid signal peptide. The sequence has a conserved cysteine in the N-terminus and has the seven conserved residues that comprise the ferroxidase center, which is the feature of heavy chain ferritins of vertebrates. The CPB ferritin subunit has high amino acid sequence identity with the Apriona germari (69.3%), Galleria mellonela (54.5%), Manduca sexta (54.0%), Drosophila melanogaster (53.2%), Calpodes ethlius (51.4%), and Nilaparvata lugens (47.6%) but lower identity with the Anopheles gambiae (38.7%) and Aedes aegypti (37.8%). Using Northern blot analysis, the subunit mRNA was identified from fat body and midgut of 4th instars with much higher mRNA levels found in midgut than that in fat body (2.5-fold). Nevertheless, only the levels of mRNA in fat body was induced by dexamethasone (1.5-fold).     

Molecular cloning, expression and characterization of cDNAs encoding the ferritin subunits from the beetle, Apriona germari

Insect secreted ferritins are composed of subunits, which resemble heavy and light chains of vertebrate cytosolic ferritins. We describe here the cloning, expression and characterization of cDNAs encoding the ferritin heavy-chain homologue (HCH) and light-chain homologue (LCH) from the mulberry longicorn beetle, Apriona germari (Coleoptera, Cerambycidae). The A. germari ferritin LCH and HCH cDNA sequences were comprised of 672 and 636 bp encoding 224 and 212 amino acid residues, respectively. The A. germari ferritin HCH subunit contained the conserved motifs for the ferroxidase center typical of vertebrate ferritin heavy chains and the iron-responsive element (IRE) sequence with a predicted stem-loop structure was present in the 5′-untranslated region (UTR) of ferritin HCH mRNA. However, the A. germari ferritin LCH subunit had no IRE at its 5′-UTR and ferroxidase center residues. Phylogenetic analysis confirmed the deduced protein sequences of A. germari ferritin HCH and LCH being divided into two types, G type (LCH) and S type (HCH). Southern blot analysis suggested the possible presence of each A. germari ferritin subunit gene as a single copy and Northern blot analysis confirmed a higher expression pattern in midgut than fat body. The cDNAs encoding the A. germari ferritin subunits were expressed as approximately 30 kDa (LCH) and 26 kDa (HCH) polypeptides in baculovirus-infected insect cells. Western blot analysis and iron staining assay confirmed that A. germari ferritin has a native molecular mass of approximately 680 kDa.     

Crassostrea gigas ferritin: cDNA sequence analysis for two heavy chain type subunits and protein purification

Ferritin has been shown as being the principal iron storage in the majority of living organisms. In marine species, ferritin is also involved in high-level accumulation of (210)Po. As part of our work on the investigation of these radionuclides' concentration in natural environment, ferritin was searched at the gene and protein level. Ferritin was purified from the visceral mass of the oyster Crassostrea gigas by ion-exchange chromatography and HPLC. SDS-PAGE revealed one band of 20 kDa. An Expressed Sequence Tag (EST) library was screened and led to the identification of two complementary DNA (cDNA) involved in ferritin subunit expression. The complete coding sequences and the untranslated regions (UTRs) of the two genes were obtained and a 5' Rapid Amplification of cDNA Ends (RACE) was used to obtain the two iron-responsive elements (IREs) with the predicted stem-loop structures usually present in the 5'-UTR of ferritin mRNA. Sequence alignment in amino acid of the two new cDNA showed an identity with Pinctada fucata (85.4-88.3%), Lymnaea stagnalis (79.3-82.2%) and Helix pomatia (79.1-79.1%). The residues responsible for the ferroxidase center, conserved in all vertebrate H-ferritins, are present in the two oyster ferritin subunits. Oyster ferritins do not present the special characteristics of other invertebrate ferritins like insect ferritins but have some functional similarities with the vertebrate H chains ferritin.     

Apoferritin in the vacuolar system of insect hemocytes

Electron microscopy showed no holoferritin in either the cytosol or the vacuolar system of hemocytes (granulocytes) from normal Calpodes ethlius larvae. This does not mean that ferritin is normally absent from hemocytes, since apoferritin lacks contrast and would not be observed. In vitro iron in glycerol treatment of hemocytes from normal larvae caused holoferritin cores to be visible in the rough endoplasmic reticulum, suggesting that hemocytes from normal larvae contain apoferritin. Hemocytes are therefore like the fat body, and could also be a source of hemolymph ferritin. After loading the hemolymph with iron in vivo, many holoferritin cores were resolvable in the vacuolar system of some hemocytes. Ferritin synthesis can therefore be induced by elevated hemolymph iron levels. Iron loading of epidermis and heart showed similar ferritin cores but more rarely. In all tissues they occurred in the secretory pathway and not in the cytosol.     

Characterization of an insect ferritin subunit synthesized in a cell-free system

Ferritin, an iron-sequestering and -binding protein, is localized to the vacuolar system in Calpodes ethlius larvae. The amount of iron-loaded ferritin in intact larval midgut can be increased by pretreatment with iron. When poly(A)+ RNA from control or iron-treated larvae was translated in vitro, a 24 kilodalton (kDa) protein was a major translation product. If the cell-free system was supplemented with dog pancreatic microsomes, the 24-kDa protein was not detectable: the major translation product was 28-30 kDa. The 24-kDa and 28- to 30-kDa proteins were identified as ferritin subunits by immunoprecipitation with anti-Manduca ferritin antibodies. Proteinase K digestion of the translation products showed that the 28- to 30-kDa subunit was targeted into the lumen of, and protected by, the microsomes. The change in molecular mass of the ferritin monomer was attributed to glycosylation of the 24-kDa subunit within the lumen of the microsomes. This was demonstrated by (i) the ability of the 28- to 30-kDa subunit, but not the 24-kDa subunit, to bind concanavalin A on Western blots and (ii) inhibition of the change in molecular mass from 24 to 28-30 kDa if tunicamycin is added to the microsomes. The results indicate that the Calpodes ferritin subunit was synthesized, targeted to microsomes, and glycosylated within their lumen in a rabbit reticulocyte cell-free system primed with midgut poly(A)+ RNA extracted from control or iron-treated larvae.     

Cloning and expression of a ferritin subunit for Galleria mellonella

Ferritin was purified from iron-fed Galleria mellonella hemolymph by ultra centrifugation and FPLC (Superose 6). SDS-PAGE revealed three bands of 26, 30, and 32 kDa. The ferritin 26 kDa subunit cDNA was obtained from RT-PCR using primer designed from N-terminal sequence analysis. 5'-RACE was used to obtain the complete protein coding sequence. The sequence encodes a 211 amino acid polypeptide including a 20 amino acid leader peptide. An IRE (iron-responsive element) sequence with a predicted stem-loop structure was present in the 5'-UTR of ferritin mRNA. Sequence alignment has a sequence identity with Calpodes ethlius (S)(74%), Drosophila melanogaster (50%), and Aedes aegypti (39%). Northern blot analysis indicated that there were 1.5- and 1.75-fold increases in the expression of ferritin mRNA after iron-fed fat body and midgut, respectively. Also, we confirmed that the ferritin mRNA is not expressed in adult ovary and testis. Arch.     

On the origin of the yolk protein ferritin in snails

Summary The iron storage protein, ferritin, is the major yolk protein in freshwater snails. In this report we show by in vitro labelling experiments that yolk ferritin of the snails Lymnaea stagnalis L. and Planorbarius corneus L. is an exogenous protein synthesized in the midgut gland and secreted into the hemolymph. Gonad and mantle tissue are inactive in the synthesis of yolk ferritin, but, together with the midgut gland, they synthesize another ferritin type (soma ferritin) which is not released into the hemolymph and which may be a housekeeping ferritin. Soma ferritin and yolk ferritin are not in a precursor/product relationship since subunits of both ferritins are synthesized as primary translation products in rabbit reticulocyte lysate programmed with poly (A)⁺ RNA from midgut gland and gonad. Results suggest that both ferritins are synthesized on different mRNAs (and possibly on different genes) so they may be regulated in a different way.     

Three ferritin subunits involved in immune defense from bay scallop Argopecten irradians

Ferritin is a ubiquitous protein that plays an important role in iron storage and iron-withholding strategy of innate immunity. In this study, three genes encoding different ferritin subunits were cloned from bay scallop Argopecten irradians (AiFer1, AiFer2 and AiFer3) by rapid amplification of cDNA ends (RACE) approaches based on the known ESTs. The open reading frames of the three ferritins are of 516 bp, 522 bp and 519 bp, encoding 171,173 and 172 amino acids, respectively. All the AiFers contain a putative Iron Regulatory Element (IRE) in their 5′-untranslated regions. The deduced amino acid sequences of AiFers possess both the ferroxidase center of mammalian H ferritin and the iron nucleation site of mammalian L ferritin. Gene structure study revealed two distinct structured genes encoding a ferritin subunit (AiFer3). Quantitative real-time PCR analysis indicated the significant up-regulation of AiFers in hemocytes after challenged with Listonella anguillarum, though the magnitudes of AiFer1 and AiFer2 were much higher than that of AiFer3. Taken together, these results suggest that AiFers are likely to play roles in both iron storage and innate immune defense against microbial infections.     

Structural and functional relationships of human ferritin H and L chains deduced from cDNA clones

We have isolated essentially full-length cDNA clones for human ferritin H and L chains from a human liver cDNA library. This allows the first comparison of H and L nucleotide and amino acid sequences from the same species as well as ferritin L cDNA sequences from different species. We conclude that human H and L ferritins are related proteins which diverged about the time of evolution of birds and mammals. We also deduce the secondary structure of the H and L subunits and compare this with the known structure of horse spleen ferritin. We find that residues involved in subunit interaction in shell assembly are highly conserved in H and L sequences. However, we find several interesting differences in H subunits at the amino acid residues involved in iron transport and deposition. These substitutions could account for known differences in the uptake, storage, and release of iron from isoferritins of different subunit composition.     

Isolation and characterization of mosquito ferritin and cloning of a cDNA that encodes one subunit

Ferritin, an iron storage protein, was isolated from larvae and pupae of Aedes aegypti grown in an iron-rich medium. Mosquito ferritin is a high molecular weight protein composed of several different, relatively small, subunits. Subunits of molecular mass 24, 26, and 28 kDa are equally abundant, while that of 30 kDa is present only in small amounts. The N-terminal sequence of the 24 and 26 kDa subunits are identical for the first 30 amino acids, while that of the 28 kDa subunit differs. Studies using antiserum raised against a subunit mixture showed that the ferritin subunits were present in larvae, pupae, and adult females, and were increased in animals exposed to excess iron. The antiserum also was used to screen a cDNA library from unfed adult female mosquitoes. Nine clones were obtained that differed only in a 27 bp insertion in the 3′ end. Rapid amplification of cDNA ends (RACE) was used to obtain the complete protein coding sequence. A putative iron-responsive element (IRE) is present in the 5′-untranslated region. The deduced amino acid sequence shows a typical leader sequence, consistent with the fact that most insect ferritins are secreted, rather than cytoplasmic proteins. The sequence encodes a mature polypeptide of 20,566 molecular weight, smaller than the estimated size of any of the subunits. However, the sequence exactly matches the N-terminal sequences of the 24 and 26 kDa subunits as determined by Edman degradation. Of the known ferritin sequences, that of the mosquito is most similar to that of somatic cells of a snail. © 1995 Wiley-Liss, Inc.     

Molecular properties and immune defense of two ferritin subunits from freshwater pearl mussel,Hyriopsis schlegelii

Ferritin is a conserved iron-binding protein involved in cellular iron metabolism and host defense. In the present study, two distinct cDNAs for ferritins in the freshwater pearl mussel Hyriopsis schlegelii were identified (designated as HsFer-1 and HsFer-2) by SMART RACE approach and expressed sequence tag (EST) analysis. The full-length cDNAs of HsFer-1 and HsFer-2 were of 760 and 877 bp, respectively. Both of the two cDNAs contained an open reading frame (ORF) of 522 bp encoding for 174 amino acid residues. Sequence characterization and homology alignment indicated that HsFer-1 and HsFer-2 had higher similarity to H-type subunit of vertebrate ferritins than L-type subunit. Analysis of the HsFer-1 and HsFer-2 untranslated regions (UTR) showed that both of them had an iron response element (IRE) in the 5′-UTR, which was considered to be the binding site for iron regulatory protein (IRP). Quantitative real-time PCR (qPCR) assays were employed to examine the mRNA expression profiles. Under normal physiological conditions, the expression level of both HsFer-1 and HsFer-2 mRNA were the highest in hepatopancreas, moderate in gonad, axe foot, intestine, kidney, heart, gill, adductor muscle and mantle, the lowest in hemocytes. After stimulation with bacteria Aeromonas hydrophila, HsFer-1 mRNA experienced a different degree of increase in the tissues of hepatopancreas, gonad and hemocytes, the peak level was 2.47-fold, 9.59-fold and 1.37-fold, respectively. Comparatively, HsFer-2 showed up-regulation in gonad but down-regulation in hepatopancreas and hemocytes. Varying expression patterns indicate that two types of ferritins in H. schlegelii might play different roles in response to bacterial challenge. Further bacteriostatic analysis showed that both the purified recombinant ferritins inhibited the growth of A. hydrophila to a certain degree. Collectively, our results suggest that HsFer-1 and HsFer-2 are likely to be functional proteins involved in immune defense against bacterial infection.     

Heart tissue contains small and large aggregates of ferritin subunits

Ferritin purified from horse heart and applied to nondenaturing polyacrylamide gel electrophoresis migrated as a single band that stained for both iron and protein. This ferritin contained almost equal amounts of fast- and slow-sedimenting components of 58 S and 3-7 S, which could be separated on sucrose density gradients. Iron removal reduced the sedimentation coefficient of the fast-sedimenting ferritin to 18 S, and sedimentation equilibrium gave a molecular weight 650,000, with some preparations containing ferritin of 500,000 molecular weight as well. Sedimentation rates of the 3 S and 7 S ferritins were not affected by iron removal, and sedimentation equilibrium data were consistent with Mr's 40,000 and 180,000, respectively. Preparations of ferritin extracted from horse spleen contained only 67 S (holo) or 16 S (apo) ferritin and no slow-sedimenting species. When examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, all of the ferritins contained the usual H and L subunits (23 and 20 kDa, respectively), but the slow-sedimenting (3 S and 7 S) heart apoferritins also contained appreciable quantities (ca 25%) of three larger subunits of 42, 55, and 65 kDa. All the subunits reacted positively in Western blots to polyclonal antibodies made against specially purified large heart or spleen ferritins containing only 20- and 23-kDa subunits. Similar results were obtained for ferritins from rat heart. The results indicate that mammalian heart tissue is peculiar not just in having an abnormally large iron-rich ferritin but also in having iron-poor ferritins of much lower molecular weight, partly composed of larger subunits.     

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.     

Drosophila melanogaster ferritin: cDNA encoding a light chain homologue, temporal and tissue specific expression of both subunit types.

Drosophila melanogaster secreted ferritin like the cytosolic ferritins of other organisms is composed of two subunits, a heavy chain homologue (HCH) and a light chain homologue (LCH). We report the cloning of a cDNA encoding the ferritin LCH of this insect. As predicted from the gene sequence, it contains no iron responsive element (IRE). Northern blot analysis reveals two mRNAs that differ in length due to the choice of polyadenylation signals. Message levels vary through the life cycle of the fly and are markedly increased by high levels of dietary iron. The gut is the main site of increased message synthesis and iron preferentially increases the amount of shorter messages. Western blotting reveals that LCH is the predominant ferritin subunit in all life stages. The amount of LCH protein corresponds well with the message levels in control animals, while in iron-fed animals LCH does not increase proportionally with the message levels. In contrast, the amount of HCH is less than that would be predicted from message levels in control animals, but corresponds well in iron-fed animals. Ferritin is abundant in gut and hemolymph of larvae and adults and in ovaries of adult flies. At pupariation, ferritin becomes more abundant in hemolymph than in other tissues.