title: Molecular cloning and characterization of caspase-3 in large yellow croaker (Pseudosciaena crocea).
abstract: Caspases-3, a member of the cysteine-aspartic acid protease (caspase) family, plays critical roles in the execution of apoptotic pathway. In this study, a caspase-3 homologue was cloned and characterized from large yellow croaker (Pseudosciaena crocea). The full-length cDNA of large yellow croaker caspase-3 (Lyccasp3) is 2222bp with an open reading frame of 858 bp encoding a polypeptide of 285 amino acids (aa). Lyccasp3 exhibited a conserved caspase-3 architecture including a prodomain, a large subunit and a small subunit. Moreover, several residues known to be critical in the caspase-3 catalytic centre and binding pocket, as well as the active-site pentapeptide motif Q(172)ACRG(176) were present in the deduced Lyccasp3. Recombinant Lyccasp3 (rLyccasp3) produced in Escherichia coli exhibited obvious hydrolyzing activity against synthetic peptide substrate Ac-DEVD-pNA. The Lyccasp3 was constitutively expressed in all the tissues examined, although the expression levels varied from tissue to tissue. Real-time PCR analysis revealed that Lyccasp3 transcript in spleen and kidney was quickly increased after stimulation with either poly (I:C) or inactivated trivalent bacterial vaccine. Enzyme activities of Lyccasp3 were also up-regulated in these two tissues post-stimulation when analyzed by hydrolyzing activity assay. Since the activity of large yellow croaker caspase-9 (Lyccasp9) in the spleen and kidney also increased when the fish was stimulated with the poly(I:C) or bacterial vaccine [1], we therefore proposed that the intrinsic apoptotic pathway, which is initiated by caspase-9 and executed by caspase-3, was activated during the immune response induced by poly(I:C) or bacterial vaccine in large yellow croaker.
authors.affiliation: Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, State Oceanic Administration, Xiamen 361005, PR China.
authors.forename: Mingyu
authors.initials: M
authors.lastname: Li
authors.forename: Yang
authors.initials: Y
authors.lastname: Ding
authors.forename: Yinnan
authors.initials: Y
authors.lastname: Mu
authors.forename: Jingqun
authors.initials: J
authors.lastname: Ao
authors.forename: Xinhua
authors.initials: X
authors.lastname: Chen
mesh_terms: D000276:Adjuvants, Immunologic / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001428:Bacterial Vaccines / Q000276:immunology; D001483:Base Sequence; D053148:Caspase 3 / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D003001:Cloning, Molecular; D005393:Fish Diseases / Q000201:enzymology / Q000276:immunology; D020869:Gene Expression Profiling; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006090:Gram-Negative Bacteria / Q000276:immunology; D016905:Gram-Negative Bacterial Infections / Q000201:enzymology / Q000276:immunology / Q000662:veterinary; D006868:Hydrolysis; D007668:Kidney / Q000187:drug effects / Q000201:enzymology; D008969:Molecular Sequence Data; D010473:Perciformes / Q000145:classification / Q000235:genetics* / Q000276:immunology*; D010802:Phylogeny; D011070:Poly I-C / Q000494:pharmacology; D016415:Sequence Alignment; D013154:Spleen / Q000187:drug effects / Q000201:enzymology
chemical_list: D000276:Adjuvants, Immunologic; D001428:Bacterial Vaccines; D053148:Caspase 3; D011070:Poly I-C