Identification and characterization of the cDNAs encoding the two subunits of Chinese mitten crab (Eriocheir sinensis) calcineurin: Their implications in stress and immune response

• cDNAs for the two subunits of Chinese mitten crab calcineurin (EsCN) were identified.
• Ubiquitously expressed EsCN subunits show the highest expression in hepatopancreas.
• Environmental stress significantly up-regulates EsCN-A and EsCN-B gene expression.
• EsCN-A and EsCN-B were significantly up-regulated by PAMPs and bacterial challenges.
• Inhibition of EsCN activities attenuated LPS-induced nitric oxide production.

Calcineurin (CN), the only Ca2+/calmodulin-activated serine/threonine protein phosphatase, is a key effector participating in Ca2+-dependent signal transduction pathways in a number of cellular processes under normal, stress and pathological conditions. However, the expression and the relevance of CN in stress and immune response have not been characterized in crustaceans. Here, we identified the cDNAs that encode the two subunits of CN (termed EsCN-A and EsCN-B, respectively) in Chinese mitten crab Eriocheir sinensis and analysed their expression patterns in response to stress and immune challenges. The catalytic subunit EsCN-A is comprised of 511 amino acids with a theoretical molecular mass of 57.5 kDa and shows 80% sequence identity with human beings CN-A alpha isoform, while the regulatory subunit EsCN-B protein is composed of 170 amino acids with an estimated molecular mass of 19.3 kDa and shares 88% sequence identity with human beings CN-B type 1. Tissue distribution analysis reveals that both EsCN-A and EsCN-B mRNA transcripts are expressed in all tested tissues with the greatest expression in hepatopancreas and the lowest expression in haemocytes. In addition, both EsCN-A and EsCN-B genes could be significantly up-regulated but with different expression patterns by ambient salinity (15‰ and 30‰ salinities) and pH (pH 6 and 8.5) stresses in gill, hepatopancreas, haemocytes, intestine and muscle. Furthermore, EsCN-A and EsCN-B were up-regulated by LPS and Poly(I:C) immune stimulations in E. sinensis haemocytes in vitro. Moreover, EsCN-A and EsCN-B mRNA were significantly up-regulated in haemocytes, gill, hepatopancreas, intestine and muscle in response to Edwardsiella tarda challenge in vivo. Finally, we revealed the importance of EsCN in LPS-induced nitric oxide production in E. sinensis haemocytes. Together our observations suggest that EsCN, the important downstream effector of CaM-mediated signalling pathway(s), may possess vital roles in stress and immune response in the Chinese mitten crab.