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

Abstract

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.

Evolution of the iron-responsive element

“…A phylogenetic analysis strongly suggests that the order of IREs has been preserved during evolution. Not only are the IREs highly conserved, but also are three (Durand et al 2004), P. leucophryna (Jeong et al 2006), P. leniusculus (Huang et al 1999), D. variabilis (Mulenga et al 2004), and L. vannamei (Hsieh et al 2006). Structural features of this class of IRE include (1) the UGC bulge where the G is able to pair with the C in the opposite strand; (2) a variability of the UGC consensus sequence; and (3) a G-A pair in some of the structures.…”

Identification and characterization of a clam ferritin from Sinonovacula constricta

“…In lower vertebrates, a third subunit type named M subunit has been reported to possess both the ferroxidase center of H subunits and the iron nucleation site of L subunits [12,13]. Most identified invertebrates ferritins shared higher identities with the H-type subunit found in vertebrates [14].…”

A comparative proteomic study on the effects of metal pollution in oysters Crassostrea hongkongensis

“…Ferritin has two functions, iron detoxification and iron storage. This protein plays a vital role in the cellular homeostasis as the physiological source of iron for the cell by storing excess iron (Durand et al, 2004). In the oyster samples from BJ site, the very high concentration of Fe was observed (Table 1).…”