Expression of four new ferritins from grooved carpet shell clam Ruditapes decussatus challenged with Perkinsus olseni and metals (Cd, Cu and Zn).

Iron has a fundamental role in life and in its biochemical reactions but, when in excess, it can promote the formation of free radicals which can lead to cell death. Therefore, managing the levels of iron is essential to regulate the production of oxidative stress related to iron, and ferritins are one of the main protein families involved in this process. Ferritins are ≈480 kDa multimeric proteins composed by 24 subunits, each with 19-26 kDa, which can accumulate up to 4500 iron atoms. Besides their role in managing iron bioavailability, they have also developed a role in organism immunity and defence present throughout evolution. In this work, we identified and characterized, for the first time, four different ferritin subunits in the clam Ruditapes decussatus, a bivalve commercially and ecologically important along the south Atlantic coast and in the Mediterranean basin, which is a major target of the parasitic protozoa Perkinsus olseni, considered one of the main causes of high levels of clam mortality. Following phylogenetic annotation, the four ferritins subunits identified were subdivided into two cytosolic and two secreted forms. All four subunits maintain the canonical ferritin structure with four main helices α (A-D) and a small helix (E), but the secreted ferritins present an additional helix in their N-terminal region (F), located after the signal peptide and with possible antimicrobial properties. Additionally, we identified in ferritin 4 an extra helix α (G) located between helices B and C. These alpha helix domains revealed high degree of similarity with antimicrobial peptides associated with antibacterial and antifungal activities. Analysis of the expression of these subunits showed that ferritins 1 and 2 are ubiquitously expressed while ferritins 3 and 4 are present mainly in visceral mass. Ferritin 1 lacked a putative functional iron response element (IRE) and appeared to be under a tight regulation. Ferritins 2 and 3 showed a strong response to infection by parasite Perkinsus olseni in contrast to ferritin 4, whose main response was related to exposure to a combination of metals. The synergistic effect between metals and infection promoted a general upregulation of the four ferritins. In conclusion, our results suggest that ferritins, besides their function in iron and metals detoxification, may play a determinant role in clam immune response.