title: Phenoloxidase from the sea cucumber Apostichopus japonicus: cDNA cloning, expression and substrate specificity analysis.
abstract: Phenoloxidase (PO) is a crucial component of the immune system of echinoderms. In the present study, the full-length cDNA of PO (AjPO) was cloned from coelomocytes of the sea cucumber Apostichopus japonicus using 3'- and 5'-rapid amplification of cDNA ends (RACE) PCR method, which is 2508 bp, with an open reading frame (ORF) of 2040 bp encoding 679 amino acids. AjPO contains a transmembrane domain, and three Cu-oxidase domains with copper binding centers formed by 10 histidines, one cysteine and one methionine respectively. Phylogenetic analysis revealed that AjPO was clustered with laccase-type POs of invertebrates. Using the isolated membrane proteins as crude AjPO, the enzyme could catalyze the substrates catechol, L-3,4-dihydroxyphenylalanine (L-DOPA), dopamine and hydroquinone, but failed to oxidize tyrosine. The results described above collectively proved that AjPO was a membrane-binding laccase-type PO. The quantitative real-time PCR (qRT-PCR) analysis revealed that AjPO mRNA was expressed in muscle, body wall, coelomocytes, tube feet, respiratory tree and intestine with the highest expression level in coelomocytes. AjPO could be significantly induced by lipopolysaccharide (LPS), peptidoglycan (PGN), Zymosan A and polyinosinic-polycytidylic acid (PolyI:C), suggesting AjPO is closely involved in the defense against the infection of bacteria, fungi and double-stranded RNA viruses.
authors.affiliation: Liaoning Key Lab of Marine Fishery Molecular Biology, Liaoning Ocean and Fisheries Science Research Institute, Dalian, Liaoning 116023, PR China.
authors.forename: Jingwei
authors.initials: J
authors.lastname: Jiang
authors.affiliation: Liaoning Key Lab of Marine Fishery Molecular Biology, Liaoning Ocean and Fisheries Science Research Institute, Dalian, Liaoning 116023, PR China. Electronic address: zunchunz@hotmail.com.
authors.forename: Zunchun
authors.initials: Z
authors.lastname: Zhou
authors.affiliation: Liaoning Key Lab of Marine Fishery Molecular Biology, Liaoning Ocean and Fisheries Science Research Institute, Dalian, Liaoning 116023, PR China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Dong
authors.affiliation: Liaoning Key Lab of Marine Fishery Molecular Biology, Liaoning Ocean and Fisheries Science Research Institute, Dalian, Liaoning 116023, PR China.
authors.forename: Hongjuan
authors.initials: H
authors.lastname: Sun
authors.affiliation: Liaoning Key Lab of Marine Fishery Molecular Biology, Liaoning Ocean and Fisheries Science Research Institute, Dalian, Liaoning 116023, PR China.
authors.forename: Zhong
authors.initials: Z
authors.lastname: Chen
authors.affiliation: Liaoning Key Lab of Marine Fishery Molecular Biology, Liaoning Ocean and Fisheries Science Research Institute, Dalian, Liaoning 116023, PR China.
authors.forename: Aifu
authors.initials: A
authors.lastname: Yang
authors.affiliation: Liaoning Key Lab of Marine Fishery Molecular Biology, Liaoning Ocean and Fisheries Science Research Institute, Dalian, Liaoning 116023, PR China.
authors.forename: Shan
authors.initials: S
authors.lastname: Gao
authors.affiliation: Liaoning Key Lab of Marine Fishery Molecular Biology, Liaoning Ocean and Fisheries Science Research Institute, Dalian, Liaoning 116023, PR China.
authors.forename: Bai
authors.initials: B
authors.lastname: Wang
authors.affiliation: Liaoning Key Lab of Marine Fishery Molecular Biology, Liaoning Ocean and Fisheries Science Research Institute, Dalian, Liaoning 116023, PR China.
authors.forename: Bei
authors.initials: B
authors.lastname: Jiang
authors.affiliation: Liaoning Key Lab of Marine Fishery Molecular Biology, Liaoning Ocean and Fisheries Science Research Institute, Dalian, Liaoning 116023, PR China.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Guan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D007113:Immunity, Innate / Q000235:genetics; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D016415:Sequence Alignment; D047528:Stichopus / Q000145:classification / Q000201:enzymology* / Q000235:genetics* / Q000276:immunology; D013379:Substrate Specificity
chemical_list: D018076:DNA, Complementary; D012333:RNA, Messenger; D014442:Monophenol Monooxygenase
keywords: Phenoloxidase; Sea cucumber (Apostichopus japonicus); Substrate specificity; cDNA cloning; mRNA expression