title: Molecular cloning and characterization of cathepsin L from freshwater mussel, Cristaria plicata.
abstract: Cathepsin L is one of the crucial enzyme superfamilies and involved in the immune responses. The Cathepsin L cDNA and genome of Cristaria plicata（CpCL） was cloned from the hemocytes using degenerate primers by the rapid amplification of cDNA ends (RACE) PCR. The genomic DNA was 9353 bp long and had a total of six introns and seven exons. The full-length cDNA of CpCL was 1144 bp, the cDNA contained a 5' untranslated region (UTR) of 34 nucleotides, the 3' UTR of 108 bp with a canonical polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) of 1002 bp, encoding 333 amino acid residues with 37.65 kDa predicted molecular weight. The theoretical isoelectric point was 8.61. The prepro-cathepsin L was consisted of a typical signal peptide (Met1-Gly20), a pro-region peptide (Leu21-Glu116) and a mature peptide (Tyr117-Val333). Many members of the papain family possessed of a proline residue at position 2 in the mature enzymem, this was also observed in CpCL. The preproprotein included an oxyanion hole (Gln 135), the active center formed by Cys141, His280 and Asn 300, the potential N-glycosylation site (Asn38, Asn 113 and Asn 272) and the conserved GCXGG motifs, which was characteristic of cathepsin, the conserved ERWNIN and GNFD motifs, which were characteristic for cathepsin L. Homology analysis revealed that the CpCL shared 49-87% identity to other known cathepsin L sequences. The phylogenetic tree showed that the CpCL clustered with the invertebrate cathepsin L cysteine proteases, and was closely related to the cathepsin L of Hyriopsis cumingii. The expression of CpCL mRNA was detected in hepatopancreas, hemocytes, mantle, gills and adductor muscle, and the higher expression level was in hepatopancreas. After A. hydrophila stimulation, the expression of the CpCL mRNA was up-regulated in hemocytes and hepatopancreas, and the expression level was significantly lower in gill than one after PBS challenge group.
authors.affiliation: School of Life Sciences and Food Engineering, Institute of Life Science, Nanchang University, Nanchang 330031, China.
authors.forename: Xiaojuan
authors.initials: X
authors.lastname: Hu
authors.affiliation: School of Life Sciences and Food Engineering, Institute of Life Science, Nanchang University, Nanchang 330031, China.
authors.forename: Xiangping
authors.initials: X
authors.lastname: Hu
authors.affiliation: School of Life Sciences and Food Engineering, Institute of Life Science, Nanchang University, Nanchang 330031, China.
authors.forename: Baoqing
authors.initials: B
authors.lastname: Hu
authors.affiliation: School of Life Sciences and Food Engineering, Institute of Life Science, Nanchang University, Nanchang 330031, China. Electronic address: cgwen@ncu.edu.cn.
authors.forename: Chungen
authors.initials: C
authors.lastname: Wen
authors.affiliation: School of Life Sciences and Food Engineering, Institute of Life Science, Nanchang University, Nanchang 330031, China.
authors.forename: Yanhai
authors.initials: Y
authors.lastname: Xie
authors.affiliation: School of Life Sciences and Food Engineering, Institute of Life Science, Nanchang University, Nanchang 330031, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Wu
authors.affiliation: School of Life Sciences and Food Engineering, Institute of Life Science, Nanchang University, Nanchang 330031, China.
authors.forename: Zhiying
authors.initials: Z
authors.lastname: Tao
authors.affiliation: State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei Province 430072, China.
authors.forename: Aihua
authors.initials: A
authors.lastname: Li
authors.affiliation: State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei Province 430072, China. Electronic address: gaoqian@ihb.ac.cn.
authors.forename: Qian
authors.initials: Q
authors.lastname: Gao
mesh_terms: D016980:Aeromonas hydrophila / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D056668:Cathepsin L / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010802:Phylogeny; D060888:Real-Time Polymerase Chain Reaction; D016415:Sequence Alignment; D049876:Unionidae / Q000235:genetics* / Q000378:metabolism / Q000382:microbiology
chemical_list: D018076:DNA, Complementary; D056668:Cathepsin L
keywords: Cathepsin L; Cristaria plicata; Expression; Genome; Molecular clone