title: Caspase-mediated apoptosis in crustaceans: cloning and functional characterization of EsCaspase-3-like protein from Eriocheir.
abstract: The caspase-3-like gene was cloned from Eriocheir sinensis, and its properties were characterized to identify the biological implications of this caspase in apoptosis in crab. Its deduced full-length protein sequence consists of 462 amino acid residues, including the prodomain and the large and small subunits. Moreover, several residues known to be critical in the caspase-3 catalytic center and binding pocket, as well as the active site pentapeptide motif Q(220)ACRG(224), were identically present in the deduced EsCaspase-3-like protein. Subsequently, the recombinant EsCaspase-3-like (rEsCaspase-3-like) protein was expressed from Escherichia coli and obtained via affinity purification. Results of the in vitro enzymatic activity assays indicated that the rEsCaspase-3-like protein is capable of hydrolyzing the substrate Ac-DEVD-pNA, suggesting a functional role in physiology. EsCaspase-3-like gene transcripts were found to be widely distributed in all tissues as detected by quantitative RT-PCR, being especially abundant in hemocytes and comparatively rare in muscles. Furthermore, EsCaspase-3-like, at both the mRNA and protein levels, was demonstrated to participate in the apoptotic process after stimulation by different pathogen-associated molecular patterns (PAMPs) in hemocytes. In conclusion, our findings suggest that the EsCaspase-3-like protein functions as an effector caspase and contributes to immune responses against pathogens.
authors.affiliation: Laboratory of Immunological Defense & Reproduction Biology, School of Life Science, East China Normal University, NO 500 Dong-Chuan Road, Shanghai, China.
authors.forename: Min-Hao
authors.initials: MH
authors.lastname: Wu
authors.forename: Xing-Kun
authors.initials: XK
authors.lastname: Jin
authors.forename: Ai-Qing
authors.initials: AQ
authors.lastname: Yu
authors.forename: You-Ting
authors.initials: YT
authors.lastname: Zhu
authors.forename: Dan
authors.initials: D
authors.lastname: Li
authors.forename: Wei-Wei
authors.initials: WW
authors.lastname: Li
authors.forename: Qun
authors.initials: Q
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D000704:Analysis of Variance; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D001483:Base Sequence; D003386:Brachyura / Q000235:genetics*; D020169:Caspases / Q000235:genetics* / Q000378:metabolism; D002681:China; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli; D006434:Hemocytes / Q000378:metabolism; D008969:Molecular Sequence Data; D011487:Protein Conformation; D060888:Real-Time Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; D020169:Caspases