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Characterization of apoptotic caspase 8 from rock bream, Oplegnathus fasciatus
Abstracts / Fish & Shellfish Immunology 34 (2013) 1692–1752
1149, showing its probiotic effect. In conclusion, we confirmed the ex vivo assay for the probiotic-pathogen-host interactions in tilapia, a warm-water fish model. * Corresponding author. E-mail address: [email protected] (Z. Zhou) xThese authors have contributed equally to this work.
P-103. Characterization of an immune deficiency homolog (IMD) from shrimp (Fenneropenaeus chinensis) and crayfish (Procambarus clarkia) Q. Ren 1, 2, x, J. Lan 1, J. Zhou 1, X. Zhang 1, Z. Wang 1, X.F. Zhao 1, J.X. Wang 1,*, x. 1 School of Life Sciences, Shandong University, Jinan, Shandong 250100, PR China; 2 Jiangsu Key Laboratory for Biodiversity & Biotechnology and Jiangsu Key Laboratory for Aquatic Crustacean Diseases, College of Life Sciences, Nanjing Normal University, 1 Wenyuan Road, Nanjing 210046, PR China
Abstract The IMD signal pathway mainly mediates anti-Gram-negative bacteria infection in invertebrates and IMD pathway is the counterpart of tumor necrosis factor receptors (TNFRs) pathway of vertebrates. In this paper, two IMDs (FcIMD, PcIMD) were identified from Chinese white shrimp, Fenneropenaeus chinensis and red swamp crayfish, Procambarus clarkia. Both proteins have a death domain located at the C-terminal. Phylogenetic analysis showed that FcIMD and PcIMD were clustered with RIPs from vertebrates. FcIMD was mainly expressed in gills and stomach and PcIMD was detected mainly in heart, hepatopancreas and stomach. FcIMD in hemocytes had the highest level at 12 h WSSV challenge and it had the highest level at 6 h WSSV challenge in gills and it was downregulated at 2 h Vibrio challenge. PcIMD in hemocytes was first downregulated post 2 h Vibrio challenge and then up to the highest level at 12 h and it was down-regulated by WSSV challenge. PcIMD was first up to the highest level post 2 h Vibrio challenge and then went down at 6 h and then went up from 12 to 24 h challenge. Western blot showed that FcIMD protein could be detected in all detected tissues and immunohistochemistry analysis indicated that FcIMD was localized in the cell cytoplasm of granular cell and hyaline cell and the positive signal was observed around the nuclei of semi-granular cell. RNAi analysis showed that anti-lipopolysaccharide factor (PcALF1) and lysozymes 2 (PcLyso2) in hemocytes was down-regulated after knock down of IMD in P. clarkia. Our research may indicate its possible roles in shrimp and crayfish innate immunity. * Corresponding author. E-mail address: [email protected] (J.X. Wang) xThese authors have contributed equally to this work.
P-470. Characterization of apoptotic caspase 8 from rock bream, Oplegnathus fasciatus
1733
death effector domains (DED). Rbcasp8 genome has 6215 bp and is organized into 12 exons and 11 introns. Rbcasp8 possessed an open reading frame of 1485 bp coded for a protein of 495 amino acids with molecular mass of 56 kDa and isoelectric point of 5.6. The large (p20) and small (p10) subunits were coded by the exons 9-12, with the active site (QACQG) coded by exon 11, similar to the other fish caspase 8 genes from medaka and sea bass. The phylogenetic analysis revealed the closer identity of Rbcasp8 with the other fish homologues. The pairwise alignment revealed highest identity with the fish homologues with a reasonable percentage of identity with the mammalian homologues. The quantitative RT-PCR analysis revealed constitutive expression of Rbcasp8 in the tissues like liver, muscle, skin, gill, blood, brain, heart, kidney, spleen, head kidney, and intestine. Highest level of expression was observed in blood followed by liver. Upregulation in Rbcasp8 mRNA transcripts were observed in tissues like liver, spleen, and head kidney isolated from lipopolysaccharide and poly I:C challenged rock bream. * Corresponding author. E-mail address: [email protected] (J. Lee) xThese authors have contributed equally to this work.
P-471. Characterization of innate cytosolic viral sensors from rock bream, Oplegnathus fasciatus K.S. Revathy x, J. Lee*, x. Marine Molecular Genetics Lab, Department of Marine Life Sciences, Jeju National University, Republic of Korea Abstract The retinoic acid inducible gene I (RIG-I) like receptors (RLR) comprise three homologues: RIG-I, melanoma differentiation-associated gene 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2). These cytosolic pattern recognition receptors (PRRs) play a vital role in the activation of host interferon system upon recognition of viral RNA pathogen associated molecular patterns (PAMPs) in the cytoplasm. In this study, MDA5 (RbMDA5) and LGP2 (RbLGP2) homologues were identified from rock bream and characterized from genome to proteome level. RbMDA5 genome possessed 16 exons interrupted by 15 introns. The open reading frame (ORF) of RbMDA5 (2979 bp) encoded for a protein of 992 amino acids with molecular mass of 112 kDa and isoelectric point of 5.6. In silico characterization of the RbMDA5 protein revealed three core protein domains including a RNA helicase domain near the C terminus (HELICc), one or more caspase activation and recruitment domains (CARDs) and an ATP dependent DExD/H domain. RbLGP2 genome possessed 11 exons interrupted by 10 introns. The ORF of RbLGP2 (2046 bp) encoded for a protein of molecular mass of 77 kDa with isoelectric point of 6.4. RbLGP2 protein revealed the presence of DExD/ H and HELICc domains. In addition, a conserved ATP binding motif (PTGGGKT), an ATPase motif (DECH), an RNA unwinding motif (TAS), and an RNA binding motif (QASGRARA) were predicted in the RbLGP2 protein. The quantitative RT-PCR analysis revealed the ubiquitous presence of both RbMDA5 and RbLGP2 transcripts in all the examined tissues including heart, brain, liver, spleen, intestine, blood, gill, kidney, head kidney, muscle and skin. Highest level of expression was present in blood and liver. In vivo poly I:C challenge of rock bream induced the expression of RbMDA5 and RbLGP2 mRNA in liver, blood, gill, head kidney and spleen between 6 h to 12 h post challenge. This reveals the involvement of RbMDA5 and RbLGP2 in antiviral defense mechanism of rock bream.
K.S. Revathy x, J. Lee*, x. Marine Molecular Genetics Lab, Department of Marine Life Sciences, Jeju National University, Republic of Korea Abstract Caspases are cysteine proteases, involved in the execution of apoptosis and hence called as killer proteases. Caspases utilize the sulfur atom in cysteine to cleave polypeptide chains. Caspases 8 and 10 are initiator caspases involved in the early steps of apoptosis and once activated can activate the effector caspases like caspase 3 and 7 through proteolytic cleavage. The rock bream caspases 8, Rbcasp8 was characterized from genome to proteome level. Rbcasp8 proteins revealed the presence of two characteristic
* Corresponding author. E-mail address: [email protected] (J. Lee) xThese authors have contributed equally to this work.
P-464. Antioxidant and immunostimulant evaluation of functional feed in Atlantic salmon under overcrowding stress S. Reyes-Cerpa 1, M.J. Gonzalez-Bown 1, A. Carrasco-Malio 1, P. Dantagnan 2, A. Hernández 2, U. Rodríguez 2, A. Bórquez 2, R. Vidal 3, E. Spencer 1, A.M. Sandino 1, 4, *.
1149, showing its probiotic effect. In conclusion, we confirmed the ex vivo assay for the probiotic-pathogen-host interactions in tilapia, a warm-water fish model. * Corresponding author. E-mail address: [email protected] (Z. Zhou) xThese authors have contributed equally to this work.
P-103. Characterization of an immune deficiency homolog (IMD) from shrimp (Fenneropenaeus chinensis) and crayfish (Procambarus clarkia) Q. Ren 1, 2, x, J. Lan 1, J. Zhou 1, X. Zhang 1, Z. Wang 1, X.F. Zhao 1, J.X. Wang 1,*, x. 1 School of Life Sciences, Shandong University, Jinan, Shandong 250100, PR China; 2 Jiangsu Key Laboratory for Biodiversity & Biotechnology and Jiangsu Key Laboratory for Aquatic Crustacean Diseases, College of Life Sciences, Nanjing Normal University, 1 Wenyuan Road, Nanjing 210046, PR China
Abstract The IMD signal pathway mainly mediates anti-Gram-negative bacteria infection in invertebrates and IMD pathway is the counterpart of tumor necrosis factor receptors (TNFRs) pathway of vertebrates. In this paper, two IMDs (FcIMD, PcIMD) were identified from Chinese white shrimp, Fenneropenaeus chinensis and red swamp crayfish, Procambarus clarkia. Both proteins have a death domain located at the C-terminal. Phylogenetic analysis showed that FcIMD and PcIMD were clustered with RIPs from vertebrates. FcIMD was mainly expressed in gills and stomach and PcIMD was detected mainly in heart, hepatopancreas and stomach. FcIMD in hemocytes had the highest level at 12 h WSSV challenge and it had the highest level at 6 h WSSV challenge in gills and it was downregulated at 2 h Vibrio challenge. PcIMD in hemocytes was first downregulated post 2 h Vibrio challenge and then up to the highest level at 12 h and it was down-regulated by WSSV challenge. PcIMD was first up to the highest level post 2 h Vibrio challenge and then went down at 6 h and then went up from 12 to 24 h challenge. Western blot showed that FcIMD protein could be detected in all detected tissues and immunohistochemistry analysis indicated that FcIMD was localized in the cell cytoplasm of granular cell and hyaline cell and the positive signal was observed around the nuclei of semi-granular cell. RNAi analysis showed that anti-lipopolysaccharide factor (PcALF1) and lysozymes 2 (PcLyso2) in hemocytes was down-regulated after knock down of IMD in P. clarkia. Our research may indicate its possible roles in shrimp and crayfish innate immunity. * Corresponding author. E-mail address: [email protected] (J.X. Wang) xThese authors have contributed equally to this work.
P-470. Characterization of apoptotic caspase 8 from rock bream, Oplegnathus fasciatus
1733
death effector domains (DED). Rbcasp8 genome has 6215 bp and is organized into 12 exons and 11 introns. Rbcasp8 possessed an open reading frame of 1485 bp coded for a protein of 495 amino acids with molecular mass of 56 kDa and isoelectric point of 5.6. The large (p20) and small (p10) subunits were coded by the exons 9-12, with the active site (QACQG) coded by exon 11, similar to the other fish caspase 8 genes from medaka and sea bass. The phylogenetic analysis revealed the closer identity of Rbcasp8 with the other fish homologues. The pairwise alignment revealed highest identity with the fish homologues with a reasonable percentage of identity with the mammalian homologues. The quantitative RT-PCR analysis revealed constitutive expression of Rbcasp8 in the tissues like liver, muscle, skin, gill, blood, brain, heart, kidney, spleen, head kidney, and intestine. Highest level of expression was observed in blood followed by liver. Upregulation in Rbcasp8 mRNA transcripts were observed in tissues like liver, spleen, and head kidney isolated from lipopolysaccharide and poly I:C challenged rock bream. * Corresponding author. E-mail address: [email protected] (J. Lee) xThese authors have contributed equally to this work.
P-471. Characterization of innate cytosolic viral sensors from rock bream, Oplegnathus fasciatus K.S. Revathy x, J. Lee*, x. Marine Molecular Genetics Lab, Department of Marine Life Sciences, Jeju National University, Republic of Korea Abstract The retinoic acid inducible gene I (RIG-I) like receptors (RLR) comprise three homologues: RIG-I, melanoma differentiation-associated gene 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2). These cytosolic pattern recognition receptors (PRRs) play a vital role in the activation of host interferon system upon recognition of viral RNA pathogen associated molecular patterns (PAMPs) in the cytoplasm. In this study, MDA5 (RbMDA5) and LGP2 (RbLGP2) homologues were identified from rock bream and characterized from genome to proteome level. RbMDA5 genome possessed 16 exons interrupted by 15 introns. The open reading frame (ORF) of RbMDA5 (2979 bp) encoded for a protein of 992 amino acids with molecular mass of 112 kDa and isoelectric point of 5.6. In silico characterization of the RbMDA5 protein revealed three core protein domains including a RNA helicase domain near the C terminus (HELICc), one or more caspase activation and recruitment domains (CARDs) and an ATP dependent DExD/H domain. RbLGP2 genome possessed 11 exons interrupted by 10 introns. The ORF of RbLGP2 (2046 bp) encoded for a protein of molecular mass of 77 kDa with isoelectric point of 6.4. RbLGP2 protein revealed the presence of DExD/ H and HELICc domains. In addition, a conserved ATP binding motif (PTGGGKT), an ATPase motif (DECH), an RNA unwinding motif (TAS), and an RNA binding motif (QASGRARA) were predicted in the RbLGP2 protein. The quantitative RT-PCR analysis revealed the ubiquitous presence of both RbMDA5 and RbLGP2 transcripts in all the examined tissues including heart, brain, liver, spleen, intestine, blood, gill, kidney, head kidney, muscle and skin. Highest level of expression was present in blood and liver. In vivo poly I:C challenge of rock bream induced the expression of RbMDA5 and RbLGP2 mRNA in liver, blood, gill, head kidney and spleen between 6 h to 12 h post challenge. This reveals the involvement of RbMDA5 and RbLGP2 in antiviral defense mechanism of rock bream.
K.S. Revathy x, J. Lee*, x. Marine Molecular Genetics Lab, Department of Marine Life Sciences, Jeju National University, Republic of Korea Abstract Caspases are cysteine proteases, involved in the execution of apoptosis and hence called as killer proteases. Caspases utilize the sulfur atom in cysteine to cleave polypeptide chains. Caspases 8 and 10 are initiator caspases involved in the early steps of apoptosis and once activated can activate the effector caspases like caspase 3 and 7 through proteolytic cleavage. The rock bream caspases 8, Rbcasp8 was characterized from genome to proteome level. Rbcasp8 proteins revealed the presence of two characteristic
* Corresponding author. E-mail address: [email protected] (J. Lee) xThese authors have contributed equally to this work.
P-464. Antioxidant and immunostimulant evaluation of functional feed in Atlantic salmon under overcrowding stress S. Reyes-Cerpa 1, M.J. Gonzalez-Bown 1, A. Carrasco-Malio 1, P. Dantagnan 2, A. Hernández 2, U. Rodríguez 2, A. Bórquez 2, R. Vidal 3, E. Spencer 1, A.M. Sandino 1, 4, *.