Characterization and identification of calmodulin and calmodulin binding proteins in hemocyte of the black tiger shrimp (Penaeus monodon)

• Nucleotide sequence of PmCaM and CaM from other organisms was very conserved especially in the four EF-hand motifs.
• The PmCaM transcript was expressed in all examined tissues.
• The PmCaM silencing increased a cumulative mortality in V. harveyi injected shrimps in comparison to the control groups.
• The recombinant PmCaM was produced as a Ca2+-bound protein and could interact with TG, EF1α, EF2 and actin.

Calmodulin (CaM), a ubiquitous intracellular calcium (Ca2+) sensor in all eukaryotic cells, is one of the well-known signaling proteins. Previously, CaM gene has shown a high transcriptional level in hemocyte of the pathogen infected shrimp, suggesting that shrimp CaM does not only regulate Ca2+ metabolism, but is also involved in immune response cascade. In the present study, the CaM gene of shrimp Penaeus monodon was identified and the recombinant P. monodon CaM (rPmCaM) was produced and biochemically characterized. The identification of CaM-binding proteins was also performed. The PmCaM cDNA consisted of an open reading frame of 447 bp encoding for 149 amino acid residues with a calculated mass of 16,810 Da and an isoelectric point of 4.09. Tissue distribution showed that the PmCaM transcript was expressed in all examined tissues. The results of gel mobility shift assay, circular dichroism spectroscopy and fluorescence spectroscopy all confirmed that the conformational changes of the rPmCaM were observed after the calcium binding. According to the gene silencing of PmCaM transcript levels, the shrimp's susceptibility to pathogenic Vibrio harveyi infection increased in comparison with that of the control groups. Protein pull-down assay and LC–MS/MS analysis were performed to identify rPmCaM-binding proteins involved in shrimp immune responses and transglutaminase, elongation factor 1-alpha, elongation factor 2 and actin were found. However, by computational analysis, only the first three proteins contained CaM-binding domain. These findings suggested that PmCaM may play an important role in regulation of shrimp immune system.