Liaoning Key Lab of Marine Fishery Molecular Biology, Liaoning Ocean and Fisheries Science Research Institute, Dalian, Liaoning 116023, PR China.
Liaoning Key Lab of Marine Fishery Molecular Biology, Liaoning Ocean and Fisheries Science Research Institute, Dalian, Liaoning 116023, PR China.
Fish Shellfish Immunol. 2014 Feb;36(2):344-51. doi: 10.1016/j.fsi.2013.12.001. Epub 2013 Dec 17.
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.
酚氧化酶（PO）是棘皮动物免疫系统的重要组成部分。本研究采用 3' 和 5' 快速扩增 cDNA 末端（RACE）PCR 方法，从海参体腔细胞中克隆出全长 cDNA 的 PO（AjPO），其长度为 2508bp，开放阅读框（ORF）为 2040bp，编码 679 个氨基酸。AjPO 含有一个跨膜结构域和三个 Cu-氧化酶结构域，铜结合中心分别由 10 个组氨酸、1 个半胱氨酸和 1 个蛋氨酸组成。系统发育分析表明，AjPO 与无脊椎动物的漆酶型 PO 聚类在一起。用分离的膜蛋白作为粗 AjPO，该酶可以催化儿茶酚、L-3,4-二羟苯丙氨酸（L-DOPA）、多巴胺和对苯二酚等底物，但不能氧化酪氨酸。上述结果共同证明 AjPO 是一种膜结合的漆酶型 PO。定量实时 PCR（qRT-PCR）分析显示，AjPO mRNA 在肌肉、体壁、体腔细胞、管足、呼吸树和肠中表达，在体腔细胞中表达水平最高。AjPO 可被脂多糖（LPS）、肽聚糖（PGN）、Zymosan A 和聚肌苷酸-聚胞苷酸（PolyI:C）显著诱导，表明 AjPO 密切参与了对细菌、真菌和双链 RNA 病毒感染的防御。
