Purification and cDNA Cloning of Ferritin from the Hepatopancreas of the Freshwater Crayfish Pacifastacus leniusculus

Abstract

hepato pancreasFerritin was purified from the hepatopancreas of the freshwater crayfish Pacifastacus leniusculus after injection of iron. It has the same size as horse spleen ferritin (440 kDa) and migrates as two bands, 19 kDa and 20 kDa, respectively, in SDSPAGE under reducing conditions. Crayfish ferritin (20 kDa) was cloned from a hepatopancreas cDNA library. The deduced amino acid sequence of the crayfish ferritin shows a closer relationship to vertebrate ferritin heavy chains than to insect ferritin and contains the conserved H-specific residues for the ferroxidase centre found in vertebrate femtin heavy chain. An IRE(iron-responsive element)-like sequence with a predicted stem-loop structure was present in the 5' untranslated region of the crayfish ferritin mRNA. Crayfish ferritin does not share the atypical properties of insect ferritins, such as high molecular mass of intact protein, abundance in hemolymph, and export into vacuoles. We suggest that there are two different types of ferritins distributed in different species: insect-type or secretory ferritins which are predominant in the snail oocyte and insects, and vertebrate (crustacean)-type or cytosolic ferritins which are predominant in vertebrates and crustacea.

Ferritin and hemocyanin: 210Po molecular traps in marine fish, oyster and lobster

“…Thus, these chromatographic purifications (Sephacryl S300 and TSK G4000SW, successively) did not allow a complete separation of ferritin and hemocyanin, as shown by the SDS-PAGE results. Nevertheless, our experiments demonstrated a double protein band at 18.5 to 21 kDa corresponding to ferritin subunits, an observation that corroborates ferritin subunit heterogeneity reported for other crustaceans species (Huang et al 1996).…”

Isolation and Properties of Drosophila Melanogaster Ferritin — Molecular Cloning of a cDNA that Encodes One Subunit, and Localization of the Gene on the Third Chromosome

“…Unlike most animal ferritins which are cytosolic, including those of vertebrates (reviewed by Andrews et al, 1992) and the crayfish (Huang et al, 1996), and unlike plant ferritins which are targeted to plastids by N-terminal transit peptides (Ragland et al, 1990), insect femtins (Dunkov et al, 1995;Pham et al, 1996) have acquired a novel role and are secreted into the hemolymph. The sequence indicates that this is also true for the D. rnelanogaster ferritin subunit, which is synthesized with a signal peptide (Fig.…”

“…Using a double-stranded cDNA from D. melanogaster ovary as a template (prepared from Cantons flies, and kindly provided by Dr James D. Sutherland, EMBL) and the two oligonucleotides as primers, a product was obtained by PCR (30 cycles, conditons as reported by Huang et al, 1996). On agarose gel electrophoresis, a very faint band appeared at the expected position corresponding to about 350 bp.…”

“…This mode of regulation has so far been explored extensively only in vertebrates, but has also been demonstrated experimentally in the mRNA encoding a subunit of Drosophila succinic dehydrogenase (Kohler et al, 1995;Gray et al, 1996;Melefors, 1996). Sequences that closely resemble IRES have been found in ferritin messages from two insects, Aedes aegypti (Dunkov et (Pham et al, 1996), and a crustacean (Huang et al, 1996). The investigation reported here shows that D. melanogaster ferritin message also contains an IRE-like element, and thus it seems likely that the regulation of the synthesis of proteins important for the uptake, storage, and utilization of iron in cells of this organism is similar to that shown to operate in vertebrate cells.…”

Molecular characterization of iron binding proteins from Glossina morsitans morsitans (Diptera: Glossinidae)

“…When cellular iron is low, the an iron-regulatory protein (IRP) binds to the IREs and inhibits translation of the mRNAs. The IRE motif consists of two elements that are both required for the interaction with the IRP, a hexanucleotide loop (5'-CAGUGN-3') and a stem interrupted by a bulge with an unpaired C nucleotide (Hentze and Kuhn, 1996, Huang et al, 1996, Pham, 2002, Theil, 1994. GmmFer1HCH contains an IRE that is highly similar in sequence to the stem loop structure in the Ae.…”