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Sea cucumber Apostichopus japonicus is an important economic species in echinoderms; however, its culture frequently suffered from diseases. Environmental stress was found to be a dominant inducer of A. japonicus diseases, and thus, discovering the characteristics of immune system under environmental stress is of great significance for disease control in A. japonicus culture. Phenoloxidase (PO) is recognized as a crucial immune‐related enzyme in invertebrates. In order to preliminarily illustrate the different roles that A. japonicus PO isoforms play under environmental stresses, the transcriptional expression of three identified A. japonicus PO isoforms (named as AjPOI, AjPOII and AjPOIII respectively) after different in vivo environmental stress simulations was determined with quantitative real‐time PCR method. Meanwhile, the transcriptional expression analysis of other representational immune‐related factors under environmental stresses was also performed for the evaluation of relationships between AjPOs and other immune‐related factors. The correlations between AjPOs and other immune‐related factors were analysed using Pearson correlation coefficient, and the effects of AjPOs on other immune‐related factors were then tested by dsRNA interference method. The results of transcriptional expression analysis indicated that AjPOI was involved in the resistance to low salinity, low temperature and hypoxia; AjPOII and AjPOIII were both required for the resistance to low salinity, high temperature, low temperature and hypoxia; and all three AjPOs were excluded from the response against high salinity. In addition, three AjPOs were found to take part in different periods of resistance to environmental stresses, and this property made A. japonicus PO system capable of outputting resistance activities continuously. The correlation analysis combined with dsRNA interference test revealed that AjPOI was capable of regulating AjCTSD, AjI‐LYZ and AjMTF expression; AjPOII and AjPOIII both played important roles in the regulation of AjC‐LYZ expression; and AjPOIII was also able to regulate AjToll expression. These findings suggested that A. japonicus PO isoforms function in innate immunity not only via catalysing the oxidative reactions, but also by broadly regulating the expression of other immune‐related factors. On the whole, three AjPOs showed obvious differences among each other in the resistance to environmental stresses and the regulation of other immune‐related factors, but comparatively, AjPOII and AjPOIII shared more similarity. [ABSTRACT FROM AUTHOR]

In order to preliminarily illustrate the functional differences of phenoloxidases (POs) in Apostichopus japonicus , the full-length cDNAs of two POs (named as AjPOⅡ and AjPOⅢ, respectively) were cloned from the coelomocytes of A. japonicus using 3′- and 5′-rapid amplification of cDNA ends method, and combined with the previously acquired full-length cDNA of a laccase-type PO from A. japonicus (Accession No. KF040052 , named as AjPOⅠ), the sequence structure and phylogenic status of POs from A. japonicus (AjPOs) were comparatively analyzed, and the transcriptional expression of AjPOs in different tissues, at different developmental stages and after different bacterial challenges was determined with quantitative real-time PCR method. Sequence analysis indicated AjPOⅡ and AjPOⅢ were both laccase-type POs, coincident to the results of phylogenic analysis. Sequence analysis also showed that AjPOⅠ had a transmembrane domain (J. Jiang et al., 2014), AjPOⅡ contained a signal peptide, and AjPOⅢ possessed a signal peptide and a transmembrane domain, implying that three AjPOs might play different roles in immune and physiological processes. Transcriptional expression analysis showed that AjPOⅡ and AjPOⅢ were most abundant in tube feet, while AjPOⅠhad the highest expression level in coelomocytes (J. Jiang et al., 2014), suggesting that AjPOⅠ may be mainly involved in immune response, while AjPOⅡ and AjPOⅢ are probably responsible for other physiological processes in addition to immune response. Besides, three AjPOs were determined to have different expression patterns during organism development and different spectrums of response against bacteria, which further indicated that there might be immune and physiological functional differentiation among three AjPOs. [ABSTRACT FROM AUTHOR]

Abstract: Phenoloxidase (PO) is a crucial immune-related enzyme in invertebrates. In this study, three POs of the sea cucumber Apostichopus japonicus were detected in coelomic fluid using linear-gradient native-PAGE combined with catechol staining and then partially purified by gel excising. The results showed that the three POs had a color of mahogany (AjPO1), yellow (AjPO2) and purple (AjPO3) respectively with molecular weights smaller than 21kDa in native-PAGE after staining with catechol. Enzymatic activities analysis revealed that AjPO1, AjPO2 and AjPO3 had optimal temperature of 45, 95 and 85°C and pH of 5.0, 8.0 and 8.0, respectively. Kinetic analysis showed that the K m values of AjPO1 for catechol, l-DOPA, dopamine and hydroquinone were 3.23, 0.86, 3.98 and 1.20mmol/l, respectively, those of AjPO2 were 0.31, 0.38, 2.05 and 1.30mmol/l, respectively, and those of AjPO3 were 5.95, 1.28, 5.81 and 0.62mmol/l, respectively. These results suggest that the three POs are laccase-type phenoloxidase. The activities of all three A. japonicus POs were significantly promoted by Ca2+, Mg2+ and Mn2+, and strongly inhibited by ethylenediamine tetraacetic acid disodium (EDTA), sodium diethyldithiocarbamate (DETC) and some common antioxidants. The inhibitions by EDTA and DETC suggest that the three A. japonicus POs are copper-containing metalloenzymes. Immune-responsive analysis showed that the total PO activities in coelomocytes (TPAC) increased greatly after lipopolysaccharide (LPS) challenge and declined significantly after polyinosinic–polycytidylic acid (PolyI:C) challenge, implying that A. japonicus PO immune system, which is composed of several isoenzymes with different characteristics, is closely involved in the defense against the infection of Gram-negative bacteria and double-stranded RNA viruses. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]