Calmodulin (CaM)

Introduction
The first line of invertebrate defense mechanism is believed to rely on innate immunity to limit pathogen infection. The innate immune system consists of physical, humoral and particularly cellular responses, which play a crucial role in phagocytosis, encapsulation and nodule formation. Moreover, the humoral barrier can also synthesize many antimicrobial peptides, pathogen recognition receptors (PRRs), prophenoloxidase and clotting cascade to eradicate pathogens [1-3]. However, all mechanisms have to occur in a concerted fashion for effective response. Accordingly, signaling pathways are an important mechanism to regulate a co-operation of immunity responses.
Calcium ion (Ca2+), one of the most essential intracellular second messengers, plays a crucial role in signaling pathway to activate many biological functions including immune response. The signal transduction is induced by altering of intracellular concentration of Ca2+ which depends on various stresses such as pathogen infection [4-5].
Calmodulin (CaM), a ubiquitous intracellular Ca2+ sensor, is a small signaling protein (~17 kDa) that contains N- and C- terminal lobes linking with a flexible central linker. Each lobe composes of two EF hand motifs, helix-loop-helix structure, with two Ca2+ binding sites [6-7]. Ca2+ binding causes conformational change of CaM by bringing two helices of each lobe to more perpendicular conformation and expose hydrophobic residues on their surface, which makes it able to interact with specific target proteins and regulate their functions [8].
In marine invertebrate, CaM does not only regulate Ca2+ metabolism of intracellular processes, it has been believed to play a crucial role in extracellular processes including biomineralization leading to shell formation in mussel [5]. Moreover, it can regulate shrimp molting in crayfish, Procambarus clarkii [9]. In shrimp, CaM was firstly isolated from Crangon crangon in 1992. Their biological and physiological properties are alike with the other invertebrate CaMs [10]. Furthermore, altering expression level of CaM gene and protein in hemocytes of pathogen infected shrimp was detected in Litopenaeus vannamei and Penaeus monodon, respectively [4, 11-12]. It could be indicated that CaM might also relate to pathogen defense mechanism via signaling pathway. Although, CaM-mediated processes have been extensively study, the signal transduction that involved in pathogen defense mechanism is still limited.
In this study, CaM recombinant protein from black tiger shrimp, Penaeus monodon (PmCaM), was characterized to verify a secondary structure and monitoring a conformational change upon calcium binding and identify CaM binding proteins involved in shrimp hemocytes. Moreover, PmCaM gene was silenced to determine whether it affects to Vibrio harveyi infected shrimp mortality.