title: Molecular cloning and functional characterization of cathepsin B from the sea cucumber Apostichopus japonicus.
abstract: Cathepsin B (CTSB), a member of lysosomal cysteine protease, is involved in multiple levels of physiological and biological processes, and also plays crucial roles in host immune defense against pathogen infection in vertebrates. However, the function of CTSB within the innate immune system of invertebrates, particularly in marine echinoderms, has been poorly documented. In this study, the immune function of CTSB in Apostichopus japonicus (designated as AjCTSB), a commercially important and disease vulnerable aquaculture specie, was investigated by integrated molecular and protein approaches. A 2153 bp cDNA representing the full-length of AjCTSB was cloned via overlapping ESTs and RACE fragments. AjCTSB contained an open reading frame of 999 bp encoding a secreted protein of 332 amino acid residues with a predicted molecular mass of 36.8 kDa. The deduced amino acid of AjCTSB shared a typical activity center containing three conserved amino acid residues (Cys108, His277and Asn297). Phylogenetic tree analysis also supported that AjCTSB was a new member of CTSB family with clustering firstly with invertebrate CTSBs. Quantitative real time PCR analysis revealed that AjCTSB was ubiquitously expressed in all examined tissues with the highest levels in intestine. The Vibrio splendidus challenged sea cucumber and LPS-exposed coelomocytes could both significantly boost the expression of AjCTSB. Moreover, the purified recombinant AjCTSB exhibited dose-dependent CTSB activities at the concentration ranged from 0 to 0.24 μg μL-1. Further functional analysis indicated that coelomocytes apoptosis was significantly inhibited by 0.16-fold in vivo and the apoptosis execution Ajcaspase 3 was extremely reduced in Apostichopus japonicus coelomocytes treated with specific AjCTSB siRNA. Collectively, all these results suggested that AjCTSB was an important immune factor and might be served as apoptosis enhancers in pathogen challenged sea cucumber.
authors.affiliation: School of Marine Sciences, Ningbo University, PR China.
authors.forename: Huahui
authors.initials: H
authors.lastname: Chen
authors.affiliation: School of Marine Sciences, Ningbo University, PR China.
authors.forename: Miao
authors.initials: M
authors.lastname: Lv
authors.affiliation: School of Marine Sciences, Ningbo University, PR China.
authors.forename: Zhimeng
authors.initials: Z
authors.lastname: Lv
authors.affiliation: School of Marine Sciences, Ningbo University, PR China. Electronic address: lichenghua@nbu.edu.cn.
authors.forename: Chenghua
authors.initials: C
authors.lastname: Li
authors.affiliation: Louisiana State University, Agricultural Center, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Xu
authors.affiliation: School of Marine Sciences, Ningbo University, PR China.
authors.forename: Weiwei
authors.initials: W
authors.lastname: Zhang
authors.affiliation: School of Marine Sciences, Ningbo University, PR China.
authors.forename: Xuelin
authors.initials: X
authors.lastname: Zhao
authors.affiliation: School of Marine Sciences, Ningbo University, PR China.
authors.forename: Xuemei
authors.initials: X
authors.lastname: Duan
authors.affiliation: School of Marine Sciences, Ningbo University, PR China.
authors.forename: Chunhua
authors.initials: C
authors.lastname: Jin
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002401:Cathepsin B / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D007113:Immunity, Innate; D008070:Lipopolysaccharides / Q000494:pharmacology; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D016415:Sequence Alignment; D047528:Stichopus / Q000235:genetics* / Q000276:immunology* / Q000382:microbiology; D014733:Vibrio / Q000502:physiology
chemical_list: D018076:DNA, Complementary; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D002401:Cathepsin B
keywords: Apostichopus japonicus; Cathepsin B; Cell apoptosis; Gene expression; Recombinant protein