title: Characterization and identification of calmodulin and calmodulin binding proteins in hemocyte of the black tiger shrimp (Penaeus monodon).
abstract: Calmodulin (CaM), a ubiquitous intracellular calcium (Ca(2+)) 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 Ca(2+) 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.
authors: Department of Biochemistry, Faculty of Science, Kasetsart University, 50 Pahonyothin, Bangkok 10900, Thailand.
authors: Panjana
authors: P
authors: Sengprasert
authors: Center of Excellence for Molecular Biology and Genomics of Shrimp, Department of Biochemistry, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Bangkok 10330, Thailand; National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Paholyothin Road, Klong 1, Klong Luang, Pathumthani 12120, Thailand.
authors: Piti
authors: P
authors: Amparyup
authors: Center of Excellence for Molecular Biology and Genomics of Shrimp, Department of Biochemistry, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Bangkok 10330, Thailand.
authors: Anchalee
authors: A
authors: Tassanakajorn
authors: Department of Biochemistry, Faculty of Science, Kasetsart University, 50 Pahonyothin, Bangkok 10900, Thailand. Electronic address: ratree.w@ku.ac.th.
authors: Ratree
authors: R
authors: Wongpanya
mesh_terms: D000199:Actins / Q000276:immunology; D000595:Amino Acid Sequence; D000818:Animals; D060829:Arthropod Proteins / Q000235:genetics / Q000276:immunology; D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002147:Calmodulin / Q000235:genetics* / Q000276:immunology*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002942:Circular Dichroism; D024202:Electrophoretic Mobility Shift Assay; D020868:Gene Silencing; D006434:Hemocytes / Q000276:immunology*; D008969:Molecular Sequence Data; D033561:Penaeidae / Q000276:immunology* / Q000382:microbiology; D020648:Peptide Elongation Factor 1 / Q000276:immunology; D020652:Peptide Elongation Factor 2 / Q000276:immunology; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013050:Spectrometry, Fluorescence; D011503:Transglutaminases / Q000276:immunology; D014733:Vibrio / Q000276:immunology
chemical_list: D000199:Actins; D060829:Arthropod Proteins; D002135:Calcium-Binding Proteins; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D020648:Peptide Elongation Factor 1; D020652:Peptide Elongation Factor 2; D011994:Recombinant Proteins; D011503:Transglutaminases
keywords: Calmodulin; Immune response; Penaeus monodon; Shrimp; Signaling protein