Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China.
Fish Shellfish Immunol. 2012 Aug;33(2):375-81. doi: 10.1016/j.fsi.2012.05.022. Epub 2012 May 30.
Tyrosinase (TYR), also known as monophenol monooxygenase, is a ubiquitous binuclear copper-containing enzyme which catalyzes the hydroxylation of phenols to catechols and the oxidation of catechols to quinones. In the present study, the cDNA of a tyrosinase (CfTYR) was identified from scallop Chlamys farreri, which encoded a polypeptide of 486 amino acids. The CfTYR mRNA transcripts were expressed in all the tested tissues, including haemocytes, adductor muscle, kidney, hepatopancreas, gill, gonad and mantle, with the highest level in mantle. The expression level of CfTYR mRNA in haemocytes decreased significantly during 3-6 h after LPS stimulation, and reached the lowest level at 6 h (0.05-fold, P < 0.05). Then, it began to increase at 12 h (0.32-fold, P > 0.05), and reached the highest level at 24 h (2.91-fold, P < 0.05). At 3 h after LPS stimulation, the phenoloxidase activity catalyzing L-dopa and dopamine in haemolymph increased significantly to 53.13 and 40.36 U mg(-1) respectively, but it decreased to 10.82 U mg(-1) and even undetectable level after CfTYR activity was inhibited. Furthermore, the antibacterial activity of haemolymph against Escherichia coli was also increased significantly at 3 h after LPS stimulation, but it decreased significantly when the haemolymph was treated by TYR inhibitor. The recombinant protein of the mature CfTYR peptide expressed in the in vitro Glycoprotein Expression Kit displayed phenoloxidase activity of 64.36 ± 5.51 U mg(-1) in the present of trypsinase and Cu(2+). These results collectively suggested that CfTYR was a homologue of tyrosinase in scallop C. farreri with the copper-dependence phenoloxidase activity, and it could be induced after immune stimulation and mediate immune response for the elimination of invasive pathogens in scallop.
酪氨酸酶（TYR），也称为单酚单加氧酶，是一种普遍存在的双核含铜酶，可催化酚类羟化为儿茶酚，儿茶酚氧化为醌。本研究从扇贝 Chlamys farreri 中鉴定出一种酪氨酸酶（CfTYR）的 cDNA，该基因编码一个 486 个氨基酸的多肽。CfTYR mRNA 转录本在所有检测组织中均有表达，包括血细胞、闭壳肌、肾脏、肝胰腺、鳃、性腺和套膜，在套膜中表达水平最高。在 LPS 刺激后 3-6 小时，血细胞中 CfTYR mRNA 的表达水平显著下降，在 6 小时时达到最低水平（0.05 倍，P < 0.05）。然后，它在 12 小时时开始增加（0.32 倍，P > 0.05），并在 24 小时时达到最高水平（2.91 倍，P < 0.05）。在 LPS 刺激后 3 小时，血淋巴中 L-多巴和多巴胺催化的酚氧化酶活性分别显著增加至 53.13 和 40.36 U mg(-1)，但在 CfTYR 活性被抑制后，其活性降低至 10.82 U mg(-1)，甚至无法检测。此外，在 LPS 刺激后 3 小时，血淋巴对大肠杆菌的抗菌活性也显著增加，但当血淋巴用 TYR 抑制剂处理时，其抗菌活性显著降低。在 Glycoprotein Expression Kit 体外表达的成熟 CfTYR 肽的重组蛋白在胰蛋白酶和 Cu(2+)存在下表现出 64.36 ± 5.51 U mg(-1)的酚氧化酶活性。这些结果共同表明，CfTYR 是扇贝 C. farreri 中酪氨酸酶的同源物，具有铜依赖性酚氧化酶活性，可在免疫刺激后被诱导，并介导免疫反应，以消除扇贝中的入侵病原体。
