title: Cathepsin L is an immune-related protein in Pacific abalone (Haliotis discus hannai)--Purification and characterization.
abstract: Cathepsin L, an immune-related protein, was purified from the hepatopancreas of Pacific abalone (Haliotis discus hannai) by ammonium sulfate precipitation and column chromatographies of SP-Sepharose and Sephacryl S-200 HR. Purified cathepsin L appeared as two bands with molecular masses of 28.0 and 28.5 kDa (namely cathepsin La and Lb) on SDS-PAGE under reducing conditions, suggesting that it is a glycoprotein. Peptide mass fingerprinting (PMF) analysis revealed that peptide fragments of 95 amino acid residues was high similarity to cathepsin L of pearl oyster (Pinctada fucata). The optimal temperature and pH of cathepsin L were 35 °C and pH 5.5. Cathepsin L was particularly inhibited by cysteine proteinase inhibitors of E-64 and leupeptin, while it was activated by metalloproteinase inhibitors EDTA and EGTA. The full-length cathepsin L cDNA was further cloned from the hepatopancreas by rapid PCR amplification of cDNA ends (RACE). The open reading frame of the enzyme was 981 bp, encoding 327 amino acid residues, with a conserved catalytic triad (Cys134, His273 and Asn293), a potential N-glycosylation site and conserved ERFNIN, GNYD, and GCGG motifs, which are characteristics of cathepsin L. Western blot and proteinase activity analysis revealed that the expression and enzyme activity of cathepsin L were significantly up-regulated in hepatopancreas at 8 h following Vibrio parahaemolyticus infection, demonstrating that cathepsin L is involved in the innate immune system of abalone. Our present study for the first time reported the purification, characterization, molecular cloning, and tissue expression of cathepsin L in abalone.
authors.affiliation: College of Food and Biological Engineering, Jimei University, Xiamen, 361021, China.
authors.forename: Jian-Dong
authors.initials: JD
authors.lastname: Shen
authors.affiliation: College of Food and Biological Engineering, Jimei University, Xiamen, 361021, China; Fujian Collaborative Innovation Center for Exploitation and Utilization of Marine Biological Resources, Xiamen, Fujian Province, 361102, China.
authors.forename: Qiu-Feng
authors.initials: QF
authors.lastname: Cai
authors.affiliation: College of Food and Biological Engineering, Jimei University, Xiamen, 361021, China.
authors.forename: Long-Jie
authors.initials: LJ
authors.lastname: Yan
authors.affiliation: College of Food and Biological Engineering, Jimei University, Xiamen, 361021, China; Fujian Collaborative Innovation Center for Exploitation and Utilization of Marine Biological Resources, Xiamen, Fujian Province, 361102, China.
authors.forename: Cui-Hong
authors.initials: CH
authors.lastname: Du
authors.affiliation: College of Food and Biological Engineering, Jimei University, Xiamen, 361021, China; Fujian Collaborative Innovation Center for Exploitation and Utilization of Marine Biological Resources, Xiamen, Fujian Province, 361102, China.
authors.forename: Guang-Ming
authors.initials: GM
authors.lastname: Liu
authors.affiliation: College of Food and Biological Engineering, Jimei University, Xiamen, 361021, China; Fujian Collaborative Innovation Center for Exploitation and Utilization of Marine Biological Resources, Xiamen, Fujian Province, 361102, China.
authors.forename: Wen-Jin
authors.initials: WJ
authors.lastname: Su
authors.affiliation: College of Ocean and Earth Sciences, Xiamen University, Xiamen, 361102, China.
authors.forename: Caihuan
authors.initials: C
authors.lastname: Ke
authors.affiliation: College of Food and Biological Engineering, Jimei University, Xiamen, 361021, China; Fujian Collaborative Innovation Center for Exploitation and Utilization of Marine Biological Resources, Xiamen, Fujian Province, 361102, China. Electronic address: mjcao@jmu.edu.cn.
authors.forename: Min-Jie
authors.initials: MJ
authors.lastname: Cao
mesh_terms: 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; D049851:Gastropoda / Q000201:enzymology / Q000235:genetics* / Q000276:immunology*; D043143:Hepatopancreas / Q000201:enzymology / Q000276:immunology; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D018076:DNA, Complementary; D012333:RNA, Messenger; D056668:Cathepsin L
keywords: Cathepsin L; Immune responses; Molecular cloning; Pacific abalone; Purification