We have completed the characterization of the turbot (Scophthalmus maximus) myeloperoxidase (mpx) gene and protein, which we partially described in a previous study. The turbot mpx gene has 15 exons that encode a protein of 767 aa, with a signal peptide, propeptide and light and heavy chains, and also with haem cavities, a Ca⁺²-binding motif and several N- and O-glycosylation sites. The mature protein forms homodimers of about 150 kDa and is very abundant in turbot neutrophils. In addition to the mpx (epx2a) gene, another three peroxidase genes, named epx1, epx2b1 and epx2b2, were identified in the turbot genome. Epx1, Epx2b1 and Epx2b2 proteins also have signal peptides and many structural characteristics of mammalian MPO and eosinophil peroxidase (EPX). Mpx was strongly expressed in head kidney, while epx2b1 and epx2b2 were strongly expressed in the gills, and epx1 was not expressed in any of the tissues or organs analysed. In vitro stimulation of head kidney leucocytes with the parasite Philasterides dicentrarchi caused a decrease in mpx expression and an increase in epx2b1 expression over time. In turbot infected experimentally with P. dicentrarchi a significant increase in mpx expression in the head kidney was observed on day 7 postinfection, while the other genes were not regulated. However, mpx, epx2b1 and epx2b2 were downregulated in the gills of infected fish, and epx1 expression was not affected. These results suggest that the four genes responded differently to the same stimuli. Interestingly, BLAST analysis revealed that Epx1 and Mpx showed greater similarity to mammalian EPX than to MPO. Considering the phylogenetic and synteny data obtained, we concluded that the epx/mpx genes of Gnathostomes can be divided into three main clades: EPX1, which contains turbot epx1, EPX2, which contains turbot mpx (epx2a) and epx2b1 and epx2b2 genes, and a clade containing mammalian EPX and MPO (EPX/MPO). EPX/MPO and EPX2 clades share a common ancestor with the chondrichthyan elephant shark (Callorhinchus milii) and the coelacanth (Latimeria chalumnae) peroxidases. EPX2 was only found in fish and includes two sister groups. One of the groups includes turbot mpx and was only found in teleosts. Finally, the other group contains epx2b1 and epx2b2 genes, and epx2b1-2b2 loci share orthologous genes with other teleosts and also with holosteans, suggesting that these genes appeared earlier on than the mpx gene.

我们已经完成了大菱鲆 ( Scophthalmus maximus ) 髓过氧化物酶 ( mpx ) 基因和蛋白质的表征，我们在之前的研究中对其进行了部分描述。turbot mpx基因有 15 个外显子，编码 767 个氨基酸的蛋白质，具有信号肽、前肽和轻链和重链，还具有血红素腔、Ca ⁺²结合基序和几个 N- 和 O- 糖基化位点。成熟蛋白形成约 150 kDa 的同源二聚体，在大菱鲆中性粒细胞中非常丰富。除了mpx ( epx2a ) 基因，另外三个过氧化物酶基因，分别命名为epx1、epx2b1和epx2b2，在大菱鲆基因组中被鉴定。Epx1、Epx2b1 和 Epx2b2 蛋白还具有信号肽和哺乳动物 MPO 和嗜酸性粒细胞过氧化物酶 (EPX) 的许多结构特征。Mpx在头肾中强烈表达，而epx2b1和epx2b2在鳃中强烈表达，而epx1在所分析的任何组织或器官中均不表达。用寄生虫Philasterides dicentrarchi在体外刺激头部肾白细胞导致mpx表达下降和epx2b1表达随时间增加。在实验性感染P. dicentrarchi的大菱鲆中， mpx显着增加在感染后第 7 天观察到头肾中的表达，而其他基因未受到调节。然而，mpx、epx2b1和epx2b2在感染鱼的鳃中被下调，并且epx1表达不受影响。这些结果表明，这四个基因对相同刺激的反应不同。有趣的是，BLAST 分析显示 Epx1 和 Mpx 与哺乳动物 EPX 的相似性高于与 MPO 的相似性。考虑到获得的系统发育和同线性数据，我们得出结论，Gnathostomes 的epx/mpx基因可分为三个主要进化枝：EPX1，包含大菱鲆epx1，EPX2，包含大菱鲆mpx（epx2a) 和epx2b1 和 epx2b2基因，以及包含哺乳动物 EPX 和 MPO (EPX/MPO) 的进化枝。EPX/MPO 和 EPX2 进化枝与软骨鱼类象鲨 ( Callorhinchus milii ) 和腔棘鱼 ( Latimeria chalumnae ) 过氧化物酶具有共同的祖先。EPX2 仅在鱼类中发现，包括两个姐妹群。其中一组包括turbot mpx，仅在硬骨鱼中发现。最后，另一组包含epx2b1和epx2b2基因，并且epx2b1-2b2基因座与其他硬骨鱼类和全骨鱼类共享直系同源基因，这表明这些基因比mpx基因出现得更早。