Characterization of the turbot Scophthalmus maximus (L.) myeloperoxidase. An insight into the evolution of vertebrate peroxidases

Characterization of the turbot Scophthalmus maximus (L.) 1 myeloperoxidase. An insight into the evolution of 2 vertebrate peroxidases 3 4 5 Manuel Noia1, Francisco Fontenla1, Alejandra Valle1, Verónica Blanco-Abad1, José 6 Manuel Leiro2, Jesús Lamas1* 7 1Department of Fundamental Biology, Institute of Aquaculture, Campus Vida, University of 8 Santiago de Compostela, E-15782, Santiago de Compostela, Spain 9 2Department of Microbiology and Parasitology, Laboratory of Parasitology, Institute of 10 Research on Chemical and Biological Analysis, Campus Vida, University of Santiago de 11 Compostela, E-15782, Santiago de Compostela, Spain. 12 13 *Corresponding author: [email protected]; +34881816951 14 15 Submitted to: Developmental and Comparative Immunology 16 17 ABSTRACT 18 We have completed the characterization of the turbot (Scophthalmus maximus) 19 myeloperoxidase (mpx) gene and protein, which we partially described in a previous study. 20 The turbot mpx gene has 15 exons that encode a protein of 767 aa, with a signal peptide, 21 propeptide and light and heavy chains, and also with haem cavities, a Ca+2-binding motif and 22 several N- and O-glycosylation sites. The mature protein forms homodimers of about 150 kDa 23 and is very abundant in turbot neutrophils. In addition to the mpx (epx2a) gene, another three 24 peroxidase genes, named epx1, epx2b1 and epx2b2, were identified in the turbot genome. 25 Epx1, Epx2b1 and Epx2b2 proteins also have signal peptides and many structural 26 characteristics of mammalian MPO and eosinophil peroxidase (EPX). Mpx was strongly 27 expressed in head kidney, while epx2b1 and epx2b2 were strongly expressed in the gills, and 28 epx1 was not expressed in any of the tissues or organs analysed. In vitro stimulation of head 29 kidney leucocytes with the parasite Philasterides dicentrarchi caused a decrease in mpx 30 expression and an increase in epx2b1 expression over time. In turbot infected experimentally 31 with P. dicentrarchi a significant increase in mpx expression in the head kidney was observed 32 on day 7 postinfection, while the other genes were not regulated. However, mpx, epx2b1 and 33 epx2b2 were downregulated in the gills of infected fish, and epx1 expression was not affected. 34 These results suggest that the four genes responded differently to the same stimuli. 35 Interestingly, BLAST analysis revealed that Epx1 and Mpx showed greater similarity to 36 mammalian EPX than to MPO. Considering the phylogenetic and synteny data obtained, we 37 concluded that the epx/mpx genes of Gnathostomes can be divided into three main clades: 38 EPX1, which contains turbot epx1, EPX2, which contains turbot mpx (epx2a) and epx2b1 and 39 epx2b2 genes, and a clade containing mammalian EPX and MPO (EPX/MPO). EPX/MPO and 40 EPX2 clades share a common ancestor with the chondrichthyan elephant shark (Callorhinchus 41 milii) and the coelacanth (Latimeria chalumnae) peroxidases. EPX2 was only found in fish 42 and includes two sister groups. One of the groups includes turbot mpx and was only found in 43 teleosts. Finally, the other group contains epx2b1 and epx2b2 genes, and epx2b1-2b2 loci share 44 orthologous genes with other teleosts and also with holosteans, suggesting that these genes 45 appeared earlier on than the mpx gene. 46 47 KEYWORDS: Fish; turbot; myeloperoxidase; eosinophil peroxidase 48 49 50 1. Introduction 51 The mammalian myeloperoxidase (MPO) belongs to a family of haem peroxidases 52 (Singh et al., 2018), which are involved in innate immunity (MPO, eosinophil peroxidase 53 [EPX] and lactoperoxidase [LPO]), hormone biosynthesis (thyroid peroxidase) and 54 extracellular matrix consolidation (peroxidasin 1) (Hawkins and Obinger, 2018). This family 55 of peroxidases, named chordate peroxidases, belongs to the peroxidase-cyclooxygenase 56 superfamily, whose members are distributed across all domains of life (Zamocky et al., 2008, 57 Nicolussi et al., 2018). The three major mammalian haem peroxidases, MPO, EPX and LPO, 58 can use H2O2 to oxidize Cl-, Br- and the pseudohalide SCN- to generate their respective 59 hypohalous acids and hypothiocyanite, which are considered strong oxidants (Davies et al., 60 2008). All three compounds have antimicrobial activity (Klebanoff, 2005, Malik and Batra, 61 2012, Magacz et al., 2019). 62 Mammalian MPO is mainly found in neutrophils, monocytes and macrophages 63 (Vanhamme et al., 2018), and to a lesser extent in other cells types such as certain subsets of 64 human peripheral B, CD4(+) and CD8(+) T lymphocytes (Okada et al., 2016). The enzyme is 65 particularly abundant in neutrophils, where it accumulates in azurophil granules (Olsson et al., 66 2004). MPO expression occurs in myeloid precursors in the bone marrow, mainly at the 67 promyelocyte stage, but not in mature phagocytes (Austin et al., 1996, Grishkovskaya et al., 68 2017). The protein is formed as a pre-proMPO that undergoes several modifications, including 69 glycosylation, haem acquisition, proteolytic cleavage and dimerization (Andersson et al., 70 1998, Grishkovskaya et al., 2017, Nauseef, 2018). The mature enzyme is inactive until the 71 neutrophils are stimulated, whereby the MPO is released into the phagosome and H2O2 is 72 produced (Klebanoff, 2005). As mentioned above, MPO has been reported to be involved in 73 pathogen killing; however, it has been suggested that the main role of this enzyme is not to 74 defend organisms against infections, as MPO- deficient individuals do not suffer any increase 75 in microbial infections (Vanhamme et al., 2018). In this respect, this enzyme has been found 76 to participate in several physiological and pathological conditions, including regulation of the 77 inflammatory responses (Davies et al., 2008). 78 Eosinophil peroxidase (EPX) is a major protein in mammalian eosinophils (Davies et 79 al., 2008). Human MPO, EPX and LPO genes are located on the same chromosome, forming 80 a cluster, and it has been suggested that they were generated by gene amplification at this 81 locus, from a common ancestral gene (Furtmüller et al., 2006). The genomic organization of 82 human MPO and EPX is similar and in both cases the protein contains a heavy chain and a 83 light chain (Sakamaki et al., 1989). However, the mature EPX protein is a monomer (Carlson 84 et al., 1985). Similarly to MPO, EPX uses H2O2 as an oxidizing substrate to generate potent 85 oxidizing compounds (Acharya and Ackerman, 2014). 86 Knowledge about MPO in non-mammalian vertebrates is scarce. The enzyme has not 87 been found in heterophils of most avian species (Genovese et al., 2013, Salakij et al., 2019, 88 Fingerhut et al., 2020), but it has been reported in heterophils of some reptilian species (Chen 89 et al., 2018) and in amphibian neutrophils (Bricker et al., 2012). In fish, peroxidase activity 90 has been described in neutrophils of many teleost species (Hine and Wain, 1998). However, it 91 was not found in granulocytes of the chondrostean shovelnose sturgeon (Scaphirhynchus 92 platorynchus) (Paliĉ et al., 2011); it has not been conclusively demonstrated in elasmobranch 93 neutrophils or eosinophils (Hine and Wain, 1987), and it has not been found in lamprey 94 granulocytes (Kelényi and Larsen, 1976). The myeloperoxidase gene has been identified and 95 cloned in zebrafish (Danio rerio) (Bennett et al., 2001), turbot (Scophthalmus maximus) 96 (Castro et al., 2008a), crucian carp (Carassius auratus gibelio) (Podok et al., 2014), rock 97 bream (Oplegnathus fasciatus) (Elvitigala et al., 2015) and orange-spotted grouper 98 (Epinephelus coioides) (Wang et al., 2018). The gene is mainly expressed in head kidney cells 99 (Elvitigala et al., 2015, Wang et al., 2018), and fish myeloperoxidase has a signal peptide, 100 propeptide and heavy and light chains, as in mammals (Elvitigala et al., 2015). Functional 101 studies of fish myeloperoxidase are very scarce. The enzyme activity decreases after 102 glycosylation (Castro et al., 2008a) and after treatment with resveratrol (Castro et al., 2008b). 103 Apart from peroxidase positive eosinophils that have been identified in some fish species by 104 cytochemical methods, nothing is known about fish eosinophil peroxidase. There is also a lack 105 of information about the evolutionary history of this chordate peroxidase family in relation to 106 innate immunity. Interestingly, the components of peroxidases such as MPO are very abundant 107 in the granulocytes of some vertebrates but are absent in others. 108 In a previous study, we partially characterized turbot myeloperoxidase (Mpx) cDNA 109 and purified the enzyme by chromatography affinity (Castro et al., 2008a). In the present 110 study, we completed the characterization of the turbot mpx gene and analysed the protein by 111 western blotting (WB) and immunofluorescence. Here we compare and discuss the possible 112 origin of this and other granulocyte peroxidases in fish and other vertebrates. Fish and 113 mammalian peroxidases are referred to as Mpx/Epx and MPO/EPX, respectively. 114 2. Material and methods 115 2.1 Animals and ethical statement 116 All experimental protocols carried out in the present study followed the European 117 legislation (Directive 2010/63/EU) and the Spanish legislative requirements relating to the use 118 of animals for experimentation (RD 53/2013), and they were approved by the Institutional 119 Animal Care and Use Committee of the University of Santiago de Compostela (Spain). 120 Healthy turbot, Scophthalmus maximus (L.), weighing about 10 or 100 g, were obtained from 121 a local fish farm, maintained in 250 l tanks, with recirculating, aerated seawater (16 oC), and 122 fed daily with commercial pellets. Fish of about 10 g body weight were used for the 123 experimental infections and for gene expression analysis in the gills and head kidney of control 124 and infected turbot. Fish were anaesthetized by immersion in a 100 mg/L solution of MS‐222 125 (tricaine methane sulfonate; Sigma-Aldrich, Spain) in sea water before being euthanized by 126 pithing. Female CD-1 mice (Charles River Laboratories, USA) (25 g) were supplied by the 127 Central Animal Husbandry Unit of the University of Santiago de Compostela (Spain). When 128 required during all experiments, mice were anaesthetized with isoflurane and euthanized by 129 decapitation. 130 2.2 Parasites 131 For the in vitro experiments, parasites (Philasterides dicentrarchi, isolate I1) were 132 cultured at 18 °C in L-15 Leibovitz medium containing 10% heat-inactivated foetal bovine 133 serum (FBS), lipids (lecithin and Tween 80), nucleosides and glucose (Sigma-Aldrich, 134 Madrid), as described by Valle et al. (2020). Once the appropriate density was reached, the 135 parasites were collected, centrifuged at 700 × g for 5 min, washed twice in L-15 medium with 136 2% FBS and resuspended in the same medium. For experimental infections, parasites were 137 cultured at 18 °C in sterile seawater containing 5% FBS and 2% ATCC® medium 1651 MA 138 (LGC Standards, Spain). For use in the experimental infections, the parasites were collected 139 from the flasks, centrifuged at 700 × g for 5 min, washed twice and resuspended in seawater. 140 2.3 Experimental infection with Philasterides dicentrarchi 141 Turbot (about 10 g body weight) were experimentally infected with the ciliate parasite 142 Philasterides dicentrarchi, by bath treatment. The parasites were added to a tank containing 143 seawater at a temperature of 18 oC, with aeration. The fish were exposed to parasites (6 x 104 144 cells/mL, final concentration) for 30 min, before being transferred to larger tanks for 7 days, 145 at the same temperature. Control fish were handled in the same way as fish exposed to the 146 parasites. Gills and head kidney from eight fish per group were collected at each sampling 147 time (0, 6, 12, 24, 75 and 168 h post-infection) and processed for qPCR analysis. 148 2.4 Tissue collection and processing 149 Unless indicated otherwise, samples of five fish were used in each experiment. For 150 gene expression analysis, blood was obtained from the caudal vein, diluted in Hanks' Balanced 151 Salt solution containing heparin (10 U/ml), washed with the same solution and frozen in liquid 152 nitrogen. Head kidney, spleen, gills, pyloric caeca, anterior and posterior intestine, skin, 153 muscle, liver, heart and brain samples were immediately submerged in liquid nitrogen and 154 stored at -80 ºC until being processed. 155 For immunofluorescence, blood smears and kidney and spleen samples were fixed with 156 4% paraformaldehyde in 0.1 M phosphate buffer (pH: 7.2) at 4 oC for 20 min and overnight, 157 respectively. Kidney and spleen samples were then rinsed twice in buffer, cryoprotected with 158 30% sucrose in PBS, embedded in OCT compound (Tissue Tek, USA) and frozen with liquid 159 nitrogen-cooled isopentane. Sections (4 μm thick) were cut on a cryostat and mounted on 160 StarFrost slides (Waldemar Knittel, Germany), which were then stored at -20 ºC. 161 2.5 Head kidney cell cultures 162 Head kidney cells were obtained following the method described by Castro et al. 163 (1999). Briefly, the head kidney was aseptically removed and placed in a Petri dish containing 164 Leibovitz's L-15 medium, with 2% foetal bovine serum (FBS) and 0.02% EDTA. Small pieces 165 of head kidney were pushed through a 100 μm nylon mesh with a glass rod, and the resultant 166 cell suspension was layered onto a 34%/49% v/v Percoll gradient. After centrifugation of the 167 gradient at 400 x g for 30 min at 4 oC, the interface cells were collected, washed twice with L-168 15 containing 2% heat-inactivated FBS and 50 μL of penicillin/streptomycin solution (Sigma) 169 and suspended at a concentration of 5 x 107 viable cells/ml in the same medium. 170 For the in vitro experiments, a 500 μL aliquot of head kidney leucocytes (1 x 107 cells 171 in L-15 medium) was added to a 500 μL aliquot of parasites (1 x 104 cells in L-15 medium) 172 and the mixture was incubated in 12-well cell cultured plates for 0, 1, 2, 4 or 6 h (in triplicate). 173 To determine any effect of the plate on gene expression, turbot leucocytes were previously 174 cultured for 0 to 6 h and then mixed with ciliates. The mixtures of leucocytes and parasites 175 were collected, centrifuged at 700 × g for 5 min, and the resulting pellet was prepared for 176 qPCR analysis. 177 2.6 RNA extraction 178 DNA and total RNA were extracted from head kidney leucocytes (107cells/mL), and 179 RNA was extracted from blood, head kidney, spleen, gills, pyloric caeca, anterior and posterior 180 intestine, skin, muscle, liver, heart and brain with a Genomic DNA purification kit or with a 181 GeneJET RNA purification kit (Thermo Scientific), in accordance with the manufacturer’s 182 instructions. The nucleic acid concentration and purity were determined by spectrophotometry 183 (ND-1000, NanoDrop Technologies, Wilmington, USA). Prior to use, the total RNA was 184 treated with DNase I (Thermo Scientific, Surrey, UK), in accordance with the manufacturer's 185 instructions. 186 2.7 Full-length cDNA clone of myeloperoxidase 187 Based on the sequence of turbot myeloperoxidase deposited in the GenBank database 188 of the National Center for Biotechnology Information (NCBI), with accession number 189 EF112175.1, we designed gene specific primers (Supplementary Table S1) for 5′- and 3′-rapid 190 amplification of cDNA ends (RACE) reactions in accordance with the instructions provided 191 with the 5’/3’ RACE kit (Roche). Amplification products of the expected size were cut from 192 1% agarose gel, purified by Ultrafree-DA (Millipore), subcloned into the pGEM-T Easy vector 193 (Promega) and finally transformed into competent cells of Escherichia coli strain DH5α. 194 White colonies were amplified in LB medium, and plasmid DNA was purified with the 195 NucleoSpin Plasmid kit (Machery-Nagel) in accordance with the manufacturer's instructions. 196 Positive plasmids detected by electrophoresis were sent to Sistemas Genómicos (Valencia) for 197 sequencing. 198 2.8 Identification of myeloperoxidase gene introns by PCR amplification 199 The whole myeloperoxidase cDNA sequence was used to design four primer sets to 200 identify introns in the gene (Supplementary Table S1). The PCR was carried out with 40 ng 201 genomic DNA from head kidney leucocytes. The PCR conditions were as follows: 5 min 202 94 °C for initial denaturation, 35 cycles of 30 s denaturation at 94 °C, annealing for 45 s at 60 203 oC, extension for 90 s at 72 °C and a final extension for 7 min at 72 °C. PCR products were 204 cloned and sequenced as described above. The full-length sequence of the myeloperoxidase 205 gene was spliced manually. 206 2.9 Bioinformatics analysis. 207 The sequence of the cDNA fragments was manually assembled to produce the 208 sequence for full-length cDNA, and its open reading frame was identified. The BLAST 209 program (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to analyse the nucleotide 210 sequences and search for protein sequences from other species in the database. The sequences 211 obtained for the myeloperoxidase gene were aligned with CLUSTAL W software and edited 212 with the Jalview Multiple Alignment Editor V1.8. Sites containing gaps were excluded. 213 Multiple sequence alignments were performed using the Clustal Omega program 214 (https://www.ebi.ac.uk/Tools/msa/clustalo/). Identification of protein domains and protein 215 functional analysis were performed using the InterProScan tool (version 4.8) from the 216 European Bioinformatics Institute (EMBL-EBI), available at 217 http://www.ebi.ac.uk/Tools/pfa/iprscan/. The physicochemical properties of the protein were 218 determined from the amino acid sequence by using the ProtParam bioinformatics tool of the 219 ExPASy server, available at https://web.expasy.org/protparam/ The presence and location of 220 signal peptide cleavage sites in amino acid sequences were predicted using the Signal-3L 221 server available at http://www.csbio.sjtu.edu.cn/bioinf/Signal-3L/. The NetNglyc server was 222 used to predict N-glycosylation sites (http://www.cbs.dtu.dk/services/NetNGlyc/), and the 223 NetOGlyc 4.0 Server (http://www.cbs.dtu.dk/services/NetOGlyc/) was used to predict O-224 glycosylation. Phylogenetic analysis software, i.e. BEAST package v2.6.2 and MEGA-X, 225 were used to build Bayesian inference, Maximum-likelihood (ML) and Neighbor-Joining (NJ) 226 trees, which were used to analyse the genetic relationships between epx/mpx genes from 227 different species. For the BEAST analysis, a BEAST2 XML file was generated with BEAUti, 228 using a Gamma site model and the Gamma Category Count was set to 4. The length of the 229 MCMC chain was set to 107. After BEAST2 analysis, the phylogenetic tree was generated 230 with FigTree v1.4.4, and the time scale was set to 450 million years. In the case of ML and NJ 231 analysis, 10000 bootstrap replicates were used. The synteny of mpx/epx loci was analysed 232 using Ensembl Genome Browser (https://www.ensembl.org/index.html) and Genomicus 233 (database version v98.01) (https://www.genomicus.biologie.ens.fr/genomicus-98.01/cgi-234 bin/search.pl). 235 2.10 Purification of turbot Mpx and generation of an Mpx antiserum in mice 236 Purification of turbot Mpx and the generation of mice Mpx antiserum followed the 237 same protocol as described in previous studies by our group (Castro et al., 2008a). Briefly, 238 turbot Mpx was purified from head kidney leucocytes in a chromatography column containing 239 acidic sulphated polysaccharides (ASP) obtained from U. rigida and epoxy-activated 240 Sepharose 6B (Amersham Biosciences, Uppsala, Sweden). The eluted fractions were tested 241 for peroxidase activity by using the chromogen ortho-phenylenediamine (Sigma). The 242 fractions containing Mpx were analysed by sodium dodecyl sulphate-polyacrylamide gel 243 electrophoresis (SDS-PAGE), and the acrylamide gel band obtained was used to immunize 8-244 week-old BALB/c mice. Antiserum specificity was checked by immunodiffusion test and by 245 immunoblotting. The band recognised by mouse antibodies has been demonstrated (by mass 246 spectrometry) to contain turbot Mpx (Castro et al., 2008a). 247 2.11 Immunofluorescence 248 Antigen was retrieved by heating the slides in 0.01 M citrate buffer (pH 6.0) in a water 249 bath at 95 ºC for 30 min and then cooling the slides for 40 min. Glycine (0.1 M; pH 2.3; Sigma-250 Aldrich) was added for 10 min at room temperature to quench autofluorescence, as described 251 by Zhang et al. (2010). The background was reduced by incubating the slides with normal goat 252 serum diluted in Tris-buffered saline (TBS) with 10% bovine serum albumin (BSA; Sigma-253 Aldrich) for 30 min at room temperature. The sections were then incubated overnight at 4 ºC 254 with the polyclonal anti-turbot myeloperoxidase antibody diluted 1:200 in antibody diluent 255 (Dako). The sections were washed three times with TBS-Tween 0.05% and another three times 256 with TBS. The secondary antibody Alexa FluorTM 488 goat anti-mouse (Life Technologies) 257 was added at 1:200 and incubated for 1 h at RT. The sections were washed three times (5 min 258 each) with TBS-T2 and three times (5 min each) with TBS, counterstained for 5 min with 259 DAPI (1 µg/ml in TBS; Sigma-Aldrich), washed three times (10 min each) with TBS, three 260 times with distilled H2O, allowed to dry for 2 hours in a 37 ºC incubator and finally mounted 261 with Mowiol® (Sigma-Aldrich). Images were then acquired with a TCS‐SP2 spectral confocal 262 microscope (Leica, Wetzlar, Germany). 263 2.12 Immunoblotting 264 Total protein was extracted from turbot white blood cells (WBC), serum and peritoneal 265 fluid by using Cell Extraction Buffer (Life Technologies) and was then resolved in a 4–20% 266 Mini-PROTEAN® TGX™ gel (Bio-Rad) under non-reducing and reducing conditions. 267 Lyophilized myeloperoxidase (dissolved in distilled water) was also separated under reducing 268 conditions. Proteins were transferred with a Trans-Blot SD semi-dry transfer cell (BioRad) 269 onto a 0.45 µm pore size nitrocellulose membrane (Bio-Rad). 270 The membrane was blocked with blocking buffer and incubated with anti-turbot 271 myeloperoxidase mouse serum antibodies (1:400), overnight at 4 oC. The membrane was then 272 incubated with peroxidase-labelled rabbit anti-mouse Ig (1:1000) (Dako) for 1 h at room 273 temperature, and, finally, with 0.06% 3,3’-diaminobenzidine tetrahydrochloride-nickel 274 chloride (Sigma) and 0.003% H2O2 for 10 min. Negative samples included lanes without 275 protein sample or without primary antibody. Lanes were recorded photographically with a 276 Panasonic camera DMC-TZ4. 277 White blood cell collection, purification of myeloperoxidase and preparation of 278 myeloperoxidase antiserum were carried out as previously described (by respectively Couso 279 et al., 2001 and Castro et al., 2008a). 280 2.13 cDNA synthesis and real time quantitative PCR (qPCR) analysis. 281 cDNA was synthesized using the cDNA synthesis kit (NZYTech, Lisbon, Portugal), 282 with 1 μg of sample RNA. cDNA amplification was achieved with a qPCR reaction mixture 283 (NZYTech) and 0.3 μM of each specific primer (Supplementary Table S1). As previously 284 described (Blanco-Abad et al., 2018), three potential reference genes were evaluated in order 285 2018), which was also found in turbot MPO. The turbot mpx cDNA encodes a polypeptide 483 containing a signal peptide, a propeptide and light and heavy chains containing the haem 484 cavities, similarly to previous findings in other teleost species (Elvitigala et al., 2015, Wang 485 et al., 2018) and in humans (Hansson et al., 2006, Nauseef, 2018). The signal peptide and the 486 propeptide are eliminated during MPO maturation (Nauseef, 2018); this process also seems to 487 occur in turbot Mpx, as indicated by the MW of the mature protein. Cleavage of the MPO 488 propeptide occurs at “RKLRSLWR” in humans (Grishkovskaya et al., 2017). This sequence 489 does not exist in fish; however, the ProP 1.0 Server program predicted that the cleavage site 490 of turbot Mpx was VHHRHKR-SL. This sequence is not particularly well conserved in the 491 Mpx of other teleosts analysed, but some aa are well conserved (-HH---KRS-). Based on the 492 results obtained in glycosylation prediction programs, turbot Mpx is N- and O-glycosylated in 493 several residues, as in other fish species (Wang et al., 2018, Yeh and Klesius, 2010) and in 494 humans (Van Antwerpen et al., 2010). Partial deglycosylation affects the activity of the 495 enzyme in fish (Castro et al., 2008a) and mammals (Van Antwerpen et al., 2010), and it has 496 been suggested that glycosylation is important for optimal enzyme activity (Van Antwerpen 497 et al., 2010). In addition, turbot Mpx also possesses the distal and proximal catalytic histidines 498 (His-261 and His-502 in human MPO), fourteen cysteine residues, haem linkage residues and 499 Ca+2-binding sites that are present in other teleosts (Elvitigala et al., 2015, Wang et al., 2018) 500 and in mammalian MPO (Booth et al., 1989, Zeng et al., 1992, Grishkovskaya et al., 2017, 501 Nauseef, 2018). Together these findings show that fish and mammalian myeloperoxidase 502 share many structural and (probably) functional features. However, the turbot Mpx lacks the 503 linker peptide ASFVTG, which is cleaved during human MPO maturation to produce the light 504 and heavy subunits of mature MPO, of molecular weight, 14 and 64 kDa, which are linked by 505 a disulphide bond (Andersson et al., 1998, McCormick et al., 2012, Nauseef, 2018). The 506 absence of the linker peptide and the WB results, which identified only a polypeptide of about 507 75 kDa under reduced conditions, suggests that turbot mature Mpx is formed by a single 508 polypeptide, which can form a homodimer, as in mature mammalian MPO (Nauseef, 2018). 509 In this respect, turbot Mpx shows similarities to mammalian eosinophil peroxidase and 510 lactoperoxidase, which share a common phylogenetic origin with MPO, and which are formed 511 by a single-chain glycoprotein (Carlson et al., 1985, Furtmüller et al., 2006). 512 We assume that only turbot Mpx was demonstrated in turbot cells in the 513 immunofluorescence assay, because turbot Mpx/Epx proteins have different molecular 514 weights and the mouse antibody recognises only a single band in the Western blot. As 515 expected, turbot Mpx was very abundant in neutrophils and their precursors. The proportion 516 of Mpx positive cells was low in the spleen but very high in the head kidney, which is a 517 hematopoietic organ, rich in neutrophils and their precursors. Turbot mpx was strongly 518 expressed in head kidney, showed much lower expression in gills and spleen, and it was not 519 expressed in the blood and other organs. Mpx was also strongly expressed in kidney and 520 weakly expressed in blood of orange-spotted grouper (Epinephelus coioides) (Wang et al., 521 2018); however, it was strongly expressed in the blood and weakly expressed in other organs 522 of rock bream (Oplegnathus fasciatus) (Elvitigala et al., 2015). Myeloperoxidase gene 523 transcription occurs only in early myeloid precursors in the bone marrow of humans and is 524 absent in mature neutrophils (Lin and Austin, 2002). Mpx expression was very low in turbot 525 circulating neutrophils, suggesting that the enzyme is also mainly produced in neutrophil 526 precursors. These results are supported by the absence of additional bands to that generated 527 by the mature enzyme in WB analysis of turbot blood. Human MPO is also found in 528 monocytes in mammals (Olsson et al., 2004). However, this does not seem to be the case in 529 turbot, as monocytes were described as peroxidase negative (Chi et al., 2017). However, 530 because myeloperoxidase has been reported in macrophages of some fish species (Meseguer 531 et al., 1994; Neumann et al., 2000), the presence of Mpx in other turbot cell types cannot be 532 ruled out, as the sensitivity of the cytochemical methods is relatively low. 533 An intron of 134 nucleotides was identified in the 5´UTR of turbot mpx gene. As far 534 as we know, this intron has not been reported in the myeloperoxidase of other species. The 535 presence of introns in the 5´UTR seems to be common in human genes, as 35% of human 536 genes have introns within this region (Cenik et al., 2010). It has been suggested that genes 537 regulated by introns are often expressed in most tissues and are among the most highly 538 expressed in the genome (Rose, 2019). In particular, 5´UTR introns have an important effect 539 in increasing gene expression (Hoshida et al., 2017). Because Mpx is very abundant in 540 neutrophils, the presence of an intron in this region may help to increase its expression in 541 myeloid cells. 542 We found that turbot contains four genes related to myeloperoxidase and eosinophil 543 peroxidases, even though this fish species lacks eosinophils (Burrows et al., 2001). The genes 544 included turbot epx1, mpx (epx2a), epx2b1 and epx2b. All of these possess haem peroxidase, 545 animal type domains and were identified by BLAST analysis as eosinophil peroxidases. 546 The results of the phylogenetic analysis suggest that turbot epx1 shares an ancestral 547 precursor with the other eosinophil peroxidase and myeloperoxidase genes in vertebrates. This 548 ancestral gene would generate epx1 and other myelo- and eosinophil peroxidases in 549 Gnathostomes by duplication. The sequence similarity of this gene with orthologous genes in 550 other teleost fish species was intermediate to high (79% aa identity with Oryzias latipes epx), 551 but the similarity was low with epx1 orthologous of non-teleost fish species (≤50 % aa 552 identity). The synteny analysis suggested that epx1 loci has orthologous genes in all groups of 553 jawed fish. The sequence similarity with the turbot paralogous mpx (epx2a), epx2b1 and 554 epx2b2 genes was low (lower than 43 % aa identity), although not exceptionally low as many 555 other teleost paralogous genes showed lower identity (Okamura et al., 2020). The high 556 differences in aa sequence and in gene expression between epx1 gene and the other three genes 557 suggests functional diversification. Three outcomes have been described for duplicate genes, 558 including neofunctionalization, subfunctionalization and conservation of function (Hahn, 559 2009, David et al., 2020). Basal epx1 expression in turbot was very low to undetectable in all 560 organs analysed, suggesting that its expression is regulated or that it is a non-functional gene. 561 However, its presence in all teleost species analysed, as well as the synteny and intermediate 562 to high similarity between them suggest that this gene has important functions in fish that 563 should be investigated. 564 The turbot paralogous mpx (epx2a), epx2b1 and epx2b genes share the same precursor 565 as mammalian MPO and EPX, although they are located are in a different clade and appear to 566 have diverged more recently. Epx2a and epx2b genes have a different genomic location and 567 probably appeared as a consequence of the 3R whole-genome duplication that occurred in the 568 teleost fish after their divergence from mammals (Jaillon et al., 2004, Meyer et al., 2005, 569 Glasauer and Neuhauss, 2014). By contrast, epx2b1 and epx2b2 genes would have been 570 generated more recently by tandem gene duplication, an event that had occurred in a large 571 number of genes in teleosts (Lu et al., 2012). The phylogenetic and synteny analysis showed 572 that turbot epx2b loci share orthologous genes with other teleosts and the holostean spotted 573 gar and that both are in the same clade. However, the mpx loci only share orthologous genes 574 with other teleosts, suggesting lineage-specific evolution. 575 Turbot mpx (epx2a) showed moderate similarity to the epx2b genes, which are located 576 on different chromosomes and have different numbers of exons/introns and differences in gene 577 expression, which suggests that they have evolved independently. Mpx was strongly expressed 578 in the kidney, which contains neutrophil precursor cells, but epx2b1 and epx2b2 were weakly 579 expressed in this organ, suggesting that they are not expressed or are expressed at very low 580 levels in those cell types. The gene expression findings, together with the WB and 581 immunofluorescence assay findings, indicate that turbot Mpx is a highly abundant protein in 582 neutrophils and is probably functionally equivalent to mammalian MPO. To obtain more 583 information about turbot mpx/epx gene function, we analysed the expression of the mpx/epx 584 genes in turbot head kidney leucocyte cultures stimulated with the parasite P. dicentrarchi, 585 observing downregulation of the epx2a (mpx) gene and upregulation of the epx2b1 gene. P. 586 dicentrarchi causes a potent inflammatory response in turbot (Valle et al., 2020), and 587 leucocytes become highly stimulated after being in contact with the parasite (Piazzon et al., 588 2011). If turbot mpx behaves like human MPO, the decrease in expression could be a 589 consequence of myeloid cell differentiation induced indirectly by the parasites. MPO 590 expression decreases in HL-60 cells after in vitro exposure to some chemical substances (Weil 591 et al., 1987), and MPO is turned off when neutrophil precursors are differentiated (Lin and 592 Austin, 2002). However, expression of turbot epx2b1, epx2b2 and epx1 seems to be regulated 593 in a different way. Experimental infection of turbot with the parasite only induced an increase 594 in mpx expression in the kidney at 7 dpi. An increase in mpx expression has also been observed 595 in the kidney of other fish species during infection (Elvitigala et al., 2015; Wang et al., 2018). 596 However, the other epx genes were not regulated in the kidney of infected turbot. Interestingly, 597 epx2a (mpx), epx2b1 and epx2b2 gene expression decreased in the gills of infected fish, 598 supporting the existence of some functional similarities between these genes. It is not known 599 whether the changes in gene expression involve modulation of gene expression or changes in 600 the number of cells. Further studies are needed to identify the cells in which the genes are 601 being expressed and to establish a relationship between them, including the epx1 gene. In this 602 respect, the use of in situ hybridisation, immunohistochemistry, flow cytometry or single cell 603 transcriptomics might help in discovering which cells produce the proteins and to clarify their 604 function. 605 Interestingly BLAST analysis of Epx1 and Mpx revealed higher similarity to 606 mammalian EPX than to MPO, suggesting that the common ancestor of MPO and EPX was 607 more similar to the present EPX gene than to MPO gene. By contrast, the size of turbot Mpx 608 is more similar to human MPO (73 kDa) than to EPX (69.8 kDa), and mature Mpx and MPO 609 form dimers, while the mature EPX is a monomer (Carlson, 1985, Furtmüller et al. 2006). 610 These differences are probably less evident at the functional level, as EPX, MPO and 611 lactoperoxidase are highly conserved in mammals (Sakamaki et al., 2000, 2002, Loughran et 612 al., 2008), particularly at catalytic sites, showing high functional similarities (Furtmüller et al., 613 2006). However, the structural characteristics and the abundance of Mpx in turbot neutrophils 614 indicate that the enzyme is probably the functional equivalent of mammalian MPO. In relation 615 to the other genes, epx1, epx2b1 and epx2b2, further studies are required to determine which 616 the cells they are expressed in and their function. Some of these genes may be equivalent to 617 mammalian EPX, although eosinophils have not been identified in turbot by cytochemical 618 staining. It is possible that other methods of identifying eosinophils are needed, as the absence 619 of eosinophil granules may merely be due to the absence of some basic material in the granule. 620 Acknowledgements 621 This study was financially supported by grant AGL2017-83577-R awarded by the 622 Ministerio de Economía y Competitividad (Spain) and the European Regional Development 623 Fund (ERDF) (European Union), by grant ED431C2017/31 from the Xunta de Galicia (Spain) 624 and by the PARAFISHCONTROL project, which received funding from the European 625 Union’s Horizon 2020 research and innovation programme under grant agreement no. 634429. 626 This publication only reflects the views of the authors, and the European Commission cannot 627 be held responsible for any use which may be made of the information contained herein. FF-I 628 was contracted by a grant from the Xunta de Galicia (Plan I2C). 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Dis. 57, 13-14. 792 964 Scleropages formosus RPFILSSVEEAKRIVDEAYKYSRERTLAQVNS-NTASHSDILRLLKQPSRDARSAVRSAE 86 965 Scophthalmus maximus SSLLQNCFEQAKKIVDDAYLYSRAESLRRVRK-EVVRPHDALRLLKQPRGETRSAVRSAD 85 966 Ictalurus punctatus RPFILDAIQEAKRLVDDSYLYSRRTSLERVRKNDYIKPSDVLRLMKQPKRKAREAVRAAD 89 967 Carassius auratus RRFILDSFEEAKKIVDAAYKYSRDESLARVRR-DVIKPSDKLRLLKQPARKTREAVRAAD 85 968 Danio rerio RPFILDSIEEAKKIVDAAYKYSRDESLARVRK-DVIKPSDKLRLLKQPARKTREAVRAAD 85 969 Xenopus laevis NDIILNSVQEAKKLVDAAYKRTRDVLKARLKQ-KDVRPSDVMAFFKQPVATSRTHIRAAD 91 970 Homo sapiens TSLVLSSMEEAKQLVDKAYKERRESIKQRLRS-GSASPMELLSYFKQPVAATRTAVRAAD 117 971 Mus musculus NSVVLSCMEEAKQLVDRAYKERRESIKRSLQS-GSASPTELLFYFKQPVAGTRTAVRAAD 91 972 Podarcis muralis ---------------------MPLGAKENLRN-PHLTPSDFLRHLKEPVAGTRTAIRAAS 38 973 Gallus gallus ------------------------------------------------------------ 0 974 975 976 Scleropages formosus YTETTLRLIKEKTHHAYKSSINATDMLSPEDMDTIARLTGCKARILPPSCRTIIMLSQYH 146 977 Scophthalmus maximus YMAQTLRLLQERVHHRHKRSLNATDLLSEEDLTNLSKITGCAARVRVPECRTTANVNKYR 145 978 Ictalurus punctatus YLEQTLRIISEKTHHAHKRSINATALLSHEELRTIEDVTGCAGQTLPPPCRTLPDLNRYR 149 979 Carassius auratus YMVQTLRLISEKAHHVHKRSINATNLLTQDELNTIQHLTGCTAQTRPPSCRTTPLINKYR 145 980 Danio rerio YMAQTLRLISEKAHHVHKRSINATNLLTQDELQTITRMTGCEAQTRPPSCRTTPLINKYR 145 981 Xenopus laevis YMGTTLELLSEKVQSVYSRPFNITDVLTANQLDTIYKVSGCAFLHIQRPC---P-NTPYR 147 982 Homo sapiens YLHVALDLLERKLRSLWRRPFNVTDVLTPAQLNVLSKSSGCAYQDVGVTC---PEQDKYR 174 983 Mus musculus YLHVALDLLKRKLQPLWPRPFNVTDVLTPAQLNLLSVSSGCAYQDVRVTC---PPNDKYR 148 984 Podarcis muralis YMETALDLLRQKLQKVWHRRFNITDVLSIQQKDMITRATGCDYLIRPVRC---PQNSPYR 95 985 Gallus gallus ------------------MRFNITDLLDRKQKEMISKETGCDYQIRSIKC---PEDDTYR 39 986 :* * :* : : :** * *: 987 988 Scleropages formosus TATGICNNLKNPRLGASNMPFVRWLPAQYEDGISQPLGWDTKKQHNGFLLPLVREVSNRI 206 989 Scophthalmus maximus TITSLCNNLKNPRLGASNTPFSRLLPAEYDDGISQPKGWNNRTL-NNFLLPLVRQVSNNI 204 990 Ictalurus punctatus TANSVCNNHINPLQGASNTAFTRWLPPVYEDNVSEPRGWNPERLYNGAALPLVRLVSNRI 209 991 Carassius auratus TIANVCNNRRNPLLGASNTAFTRWLPAQYEDGISQPKGWDPKKMHNGALLPLVRLVSNRI 205 992 Danio rerio TASGVCNNRKNPLLGASNTPFARWLPAQYEDAVSQPKGWDPNKLYNGAALPMVRLVSNRI 205 993 Xenopus laevis SFTGECNNRRFPNFGVSNRPYTRLLPAQYEDSRSLPKGWTENRRINGFMLPLARAVSNQI 207 994 Homo sapiens TITGMCNNRRSPTLGASNRAFVRWLPAEYEDGFSLPYGWTPGVKRNGFPVALARAVSNEI 234 995 Mus musculus TITGHCNNRRSPTLGASNRAFVRWLPAEYEDGVSMPFGWTPGVNRNGFKVPLARQVSNAI 208 996 Podarcis muralis TITGECNNRKHPHFGMSNHGYARWLPAEYEDGVSLPKGLIQGQLYHGHPLPLVRQVSNEI 155 997 Gallus gallus TITGECNNRKEPHLGVSNHAFARWLPAAYEDGVSVPRGASEGKLYNAFPLPLVRKVSNEI 99 998 : . *** * * ** : * ** *:* * * * : : :.* *** * 999 1000 Scleropages formosus LRTANKDVENDIQYSHLVTMFGQWIDHDLTFTPSSPSIRSFSNGINCAQSCERTNPCFPM 266 1001 Scophthalmus maximus LNTTDAAVVSDREFTHMVTLFGQWNDHDLTFTPFSPSIRSFSNGVNCDESCEKTEPCIPI 264 1002 Ictalurus punctatus FSTQDQNVQGDTEYTLLITFFGQWNDHDLSFTPFSPSIRSFSNGINCDDSCERSEPCFPI 269 1003 Carassius auratus LSTADSDIESDREFTFMLTIFGQWVDHDLTFTPFSPSIRSFSSGLDCDQSCERSEPCFPI 265 1004 Danio rerio LATADADIESDHDFTFMLTIFGQWVDHDLTFTPFSPSIRSFSNGIDCENSCERSEPCFPI 265 1005 Xenopus laevis LRFPEREQTLDNQRSLMFMQWGQWIDHDLDLAPETPARSSFLKGIDCDHSCARELPCFPL 267 1006 Homo sapiens VRFPTDQLTPDQERSLMFMQWGQLLDHDLDFTPEPAARASFVTGVNCETSCVQQPPCFPL 294 1007 Mus musculus VRFPNDQLTKDQERALMFMQWGQFLDHDITLTPEPATRFSFFTGLNCETSCLQQPPCFPL 268 1008 Podarcis muralis IGTPNEKITDDQERSLAFMHWGQWVDHDMDLAPMTETSI-QNQDVHCDTSCNYTPPCFPI 214 1009 Gallus gallus AHTANENITQDQELSLFFMQWGQWVNHDIDLAPSSGMG--ANPELHCDADCTFRSPCFPI 157 1010 * : : . :** :**: ::* :.* .* **:*: 1011 1012 Scleropages formosus KIPPNDPRFKDKPQVKCLPTPRSAPSCGTGEGGYMFGAV-NVRQQMNTLTAFLDVGQVYG 325 1013 Scophthalmus maximus PIPPGDPRLPSRPD-SCIPAFRSAPACGTGYSAYNFGGEPNKREQINALTAFLDLSQVYG 323 1014 Ictalurus punctatus QIPRSDPRLPLRSN-SCLPVFRSAPACGSGNSPYMFGGVPRLREQLNTLTSYLDAGQLYG 328 1015 Carassius auratus PAPPGDPRL--KPG-TCLPVFRSSPACGSGNTAYMFGGNPNVREQINSLTAFLDGGQVYG 322 1016 Danio rerio SAPPGDQRL--RPN-TCLPVFRSAPTCGSGHTAYMFGEVPNVREQINTLTAYLDAGQVYG 322 1017 Xenopus laevis RIPPNDPRIQNR--SDCIPLFRSSPACMQGP----------VLEQINVLTSYVDASQVYG 315 1018 Homo sapiens KIPPNDPRIKNQ--ADCIPFFRSCPACPGSN--------ITIRNQINALTSFVDASMVYG 344 1019 Mus musculus KIPPNDPRIKNQ--KDCIPFFRSCPACTRNN--------ITIRNQINALTSFVDASGVYG 318 1020 Podarcis muralis KIPPGDPRITKP--GVCMPFIRTTPVCNPTT---------FIREQLNSITSFLDASMVYG 263 1021 Gallus gallus KFPPDDPRMLRS--NSCMPFIQSASVCNPRT---------FTREQINAVTSFIDASMVYG 206 1022 * .* *: *:* :: * :*:* :*:::* . :** 1023 1024 Scleropages formosus SEDALSRNLRDL-TNDGGLLRVNHRFFDNGRELLPFVNMTFNFCATRQKITGNKSLKEVP 384 1025 Scophthalmus maximus SEEKLALNLRNL-SNDGGLLRVNTEFNDNGRELLPFSTLQAQMCATRKRVTNDTNAKEVP 382 1026 Ictalurus punctatus SEEGLAHELRDL-TTDGGLMRINDRFRDNGRELLPFTKVPSKMCATRNRILNTTGLEEVP 387 1027 Carassius auratus SEDGLAKELRDL-TNDGGLLRVNNKFLDNGRELLPFTNVESKMCATRQKILNDTTLTEVP 381 1028 Danio rerio SEDGLAKELRDL-TNDGGLLRVNNRFKDNGRELLPFTSVNTNLCATRQKILNDSTLTEVP 381 1029 Xenopus laevis STNDLARMLRNNTATQLGLMAVNTRFTDGGLPYLPFGTMKEDFCLLTNMS------SGLP 369 1030 Homo sapiens SEEPLARNLRNM-SNQLGLLAVNQRFQDNGRALLPFDNLHDDPCLLTNRS------ARIP 397 1031 Mus musculus SEDPLARKLRNL-TNQLGLLAINTRFQDNGRALMPFDSLHDDPCLLTNRS------ARIP 371 1032 Podarcis muralis SEEPLARSLRNQ-SNSLGLMALNQNFTDAGLGLLPFESNSNSICLYTNRT------ANIP 316 1033 Gallus gallus SEESVAKSLRNQ-TNQLGLMAVNQNFTDAGLELLPFENKTKSVCVLTNKS------TNIP 259 1034 * : :: **: :.. **: :* .* * * :** . . * : :* 1035 1036 Scleropages formosus CSLAGDLRVDENTGLTSLHTLMLREHNRLARVLHQLNPHWESERLYQEARKIMVAYHQVI 444 1037 Scophthalmus maximus CFIAGDVRVDENVALTSIHTLFMREHNRLARALKRLNPHWDSETLYQESRKIMGAYTQIF 442 1038 Ictalurus punctatus CFIAGDERVDENIALTSMHTLFLREHNRLARALRRLNPNWTSEQLYQEARKIVGAYLQNI 447 1039 Carassius auratus CFIAGDIRVDENTALTSVHTLFVREHNRLARALHVLNPTWSSETLYQEARKIVGAYNQIL 441 1040 Danio rerio CFIAGDARVDENPALNSLHTLFVREHNRLARALHVLNPTWSSETLYQEARKIVGAFNQIL 441 1041 Xenopus laevis CFLAGDARVSEQPGLTAFHTIFVREHNRIARELRRLNPTWTGEVLFQEARKIVGAIEQKI 429 1042 Homo sapiens CFLAGDTRSSEMPELTSMHTLLLREHNRLATELKSLNPRWDGERLYQEARKIVGAMVQII 457 1043 Mus musculus CFLAGDMRSSEMPELTSMHTLFVREHNRLATQLKRLNPRWNGEKLYQEARKIVGAMVQII 431 1044 Podarcis muralis CFNAGDKRVTENLGLTALHTLFVREHNRLARELKVLNPHWDGEKIYQEARKIIGAVNQVI 376 1045 Gallus gallus CFRAGDKRVTENLGLSALHTVFLREHNRLVTKLGKLNPHWDGEKLYQESRNIIAAMTQII 319 1046 * *** * * *.:.**:::*****:. * *** * .* ::**:*:*: * * : 1047 1048 Scleropages formosus TFRNYLKLIVGPKHMHKHLGKYPGYNKNVDPSISNVFATAAYRFAHLTVQPIIFRLDDNY 504 1049 Scophthalmus maximus VFRDYLPHILGDNAVRTQIGPYSGYNPNVDPSISNVFATAAYRFAHLAIQPFLSRLDANY 502 1050 Ictalurus punctatus VFKDYLPHIVGTDAMNKQLGKYPGYNPNIDPTISNVFATAAYRFAHATIQPVIFRLDENF 507 1051 Distal Heme Cavity I Distal Heme Cavity II Proximal Heme Cavity I Carassius auratus VFKEYLPHIVGPDVYNRQLGQYPGYDENVDPTIANVFATAAFRFAHLAIQPIIFRLDENY 501 1052 Danio rerio VIKEYLPLIVGPDAYNRHLGPYPGYNENVDPTIANVFATAAFRFAHLTIQPFIFRLDENY 501 1053 Xenopus laevis NYKDYLPLLLGS-TMTRVLPRYTSYNDSVNPGAANVFS-LIFRMGHTMIQPFIYRLVDGY 487 1054 Homo sapiens TYRDYLPLVLGPTAMRKYLPTYRSYNDSVDPRIANVFT-NAFRYGHTLIQPFMFRLDNRY 516 1055 Mus musculus TYRDYLPLVLGPAAMKKYLPQYRSYNDSVDPRIANVFT-NAFRYGHTLIQPFMFRLNNQY 490 1056 Podarcis muralis TFRDYLPLVLGN-EMNKQLPLYKGYNDSEDPTVSNVFS-LAFRFGHGSVPPFVPRLDQNF 434 1057 Gallus gallus TYRDYLPLLLAE-ETSKWIPLYSGYHETVDPTVSNVFS-LAFRFGHTSVQPFVSRLDDSF 377 1058 ::** ::. : * .*. . :* :***: :* .* : *.: ** : 1059 1060 Scleropages formosus NEHPSFRSVMLHNTFFAPWRIIFEGGVDPVMRGLIGRPAKLNSQKHMMHDELRERLFEFS 564 1061 Scophthalmus maximus RENNRFPSVSLYRAFFTPWRIVFEGGVDSLLRGLIGRPAKLNTQDHMMVDALRERLFQFV 562 1062 Ictalurus punctatus QENRQFPTVPLYEAFFTPWRIIFEGGIDPQIRGLISRPAKLNRQDGIMVDAVRERLFAFN 567 1063 Carassius auratus QNHPKFPSVPLFEAFFSPWRVIFEGGIDPLLRGLIGRPAKLNTQDHMMVNALRERLFAFT 561 1064 Danio rerio KNHPQFPSVPLYEAFFSPWRVIFEGGIDPVLRGLIGRPAKLNTQDHMLVNALRERLFAFT 561 1065 Xenopus laevis RTSASLPPIPIHLTFFNTWRVILEGGVDPLLRGLMGNQAKLNRQNQILVDELREHLFELF 547 1066 Homo sapiens QPMEPNPRVPLSRVFFASWRVVLEGGIDPILRGLMATPAKLNRQNQIAVDEIRERLFEQV 576 1067 Mus musculus RPTGPNPRVPLSKVFFASWRVVLEGGIDPILRGLMATPAKLNRQNQIVVDEIRERLFEQV 550 1068 Podarcis muralis KPLVPYSNVLLHLTFSASWRIIMEGGIDPLLRGLLADHAKLMKQNQMMVEELQERLFEQL 494 1069 Gallus gallus QPMGSLPHVPLHLTFCASWRIIMEGGIDPLIRGMVVDHAKLMKQNQMLIEELQNHLFEQT 437 1070 . : : .* **:::***:* :**:: *** *. : : ::::** 1071 1072 Scleropages formosus NSIALDLASLNMQRGRDHALPGYNAWRKFCGLSQPKTLEELAVVMQNKTLAKELMKMYGT 624 1073 Scophthalmus maximus QHLALDLGSLNMQRGRDHGLPGYNAYRRVCGLSQPRNQAELGRVLNNNNLARRLLQLYGS 622 1074 Ictalurus punctatus SKISQDLGSLNLQRGRDHALPGYNEWWRFCGLSAPRNVAELGRVLNNATLAQRILQLYGR 627 1075 Carassius auratus SHIALDLASLNMQRSRDHSIPGYNAWRRFCGLSAPKNEQELGVVMNNTKLARRLIELYGT 621 1076 Danio rerio SHIALDLASLNMQRGRDHAIPGYNAWRRFCGLSAPQNEQELAVVMNNTKLARKLIELYGT 621 1077 Xenopus laevis KRLGLDLGAINMQRGRDHGLPGYNAFRRFCGLSQPRNETELATVLRNRQLAQRLTSLYGT 607 1078 Homo sapiens MRIGLDLPALNMQRSRDHGLPGYNAWRRFCGLPQPETVGQLGTVLRNLKLARKLMEQYGT 636 1079 Mus musculus MRIGLDLPALNMQRSRDHGLPGYNAWRRFCGLPQPSTVGELGTVLKNLELARKLMAQYGT 610 1080 Podarcis muralis ELIGLDLASLNLQRGRDHGLPGYNAWRRFCGLSEPSDEAELAAVMGNSQLAKKFIDLYGT 554 1081 Gallus gallus EIMGLDLAALNLQRGRDHGLPGYNAWRRFCGLSQPQTVDELSEVLGNTELTKKLMDLYGT 497 1082 :. ** ::*:**.***.:**** : :.*** * :*. *: * *::.: ** 1083 1084 Scleropages formosus PDNIDVWLGGVAEPFVKGGRVGPLFACLIATQFQKIRQGDRLWWENNGVFSPAQRASLAC 684 1085 Scophthalmus maximus PDNIDVWMGGVAEPFVQGGRVGPLFACLIGRQFQRIRQGDRLWHEKPGVFTRRQKAALSS 682 1086 Ictalurus punctatus TDNIDLWVGGIAEPFVPGGRVGPLFSCIIATQFQRIRQGDRLWWENMGVFTAAQKASLSR 687 1087 Carassius auratus PENIDIWLGGVAEPFVPGGRVGPLFACLISTQFKRIRQGDRLWYENHGVFTTKQKASLAS 681 1088 Danio rerio PENIDVWLGGVAEPFAPGGRVGSLFACLISRQFQKIRDGDRLWFESNGVFTTKQKTALAS 681 1089 Xenopus laevis PQNIDIWLGAVAEPLVTNGRVGELLACLIGDQFRRSRDGDRFYYERPSIFTPAQLSSIER 667 1090 Homo sapiens PNNIDIWMGGVSEPLKRKGRVGPLLACIIGTQFRKLRDGDRFWWENEGVFSMQQRQALAQ 696 1091 Mus musculus PNNIDIWMGGVSEPLEPNGRVGQLLACLIGTQFRKLRDGDRFWWENPGVFSKQQRQALAS 670 1092 Podarcis muralis PENIDIWIGALAEPFVPYGRVGPLLACLIGTQFRKTRDGDRFWWENPGVFTPWQRRILSG 614 1093 Gallus gallus PDNIDLWIGAIAEPLIPRGRVGPLLACIIGTQFRNLRDGDRFWWENPGVFTPQQLEELTK 557 1094 :***:*:*.::**: **** *::*:*. **:. *:***:: * .:*: * : 1095 1096 Scleropages formosus VSMARIICDNTGITRVPK-NPFLLNPKQLDLVRCKTIPTLNLKPWLERPGNRNVPTENA- 742 1097 Scophthalmus maximus ATLSKIICDNTGITSVPH-NAFNVISGKNRLVRCNNIRGVDLTAWRERICSDNSGPSDSC 741 1098 Ictalurus punctatus VSLASIICDNTGITRVPR-NPFLFSRNQADFVNCGSIQGLDLNAWRETDDSSSETASDEN 746 1099 Carassius auratus VSLARIICDNTGISRVPN-NPFRFT-SPGRFVNCGDIPAFDLTPWIETKNSSIHHSDIGN 739 1100 Danio rerio VSMARIICDNTGILKVPR-DPFRFR-SPASFVNCGNIPAFDLEPWKETDRDSIPGPPGPR 739 1101 Xenopus laevis VTLARMVCDNTRITEVPR-DVFKANQYPANFVRCASIPALDLTPWRRRKSGAELEDPPM- 725 1102 Homo sapiens ISLPRIICDNTGITTVSKNNIFMSNSYPRDFVNCSTLPALNLASWREAS----------- 745 1103 Mus musculus ISLPRIICDNTGITTVSKNNIFMSNTYPRDFVSCNTLPKLNLTSWKET------------ 718 1104 Podarcis muralis ASLSRIICDNTHIQEVPH-DVFKMNHYPSDFMKCSEIGGLDLSAWKE------------- 660 1105 Gallus gallus ISMSRVICDNTRIKKLPR-DMFRAS-SPENFVDCHEIDMLDLSAWKDEPERGSKGT---- 611 1106 :: ::**** * : . : * :: * : .:* * 1107 1108 Scleropages formosus ---------NSKGIEEPVEIPPPTMN-DTQYSAFSMRLGNNPPKPGQVIIFGEATDEGQG 792 1109 Scophthalmus maximus NEVNGTENVTQLSPQHQQSLQDNEVQ---------------------------------- 767 1110 Ictalurus punctatus EIPKEKESNDLQDLMDLLDK------QVGQK----------------------------- 771 1111 Carassius auratus EI--QSPSKDLLDPEDPQD-------------NLVHKV---------------------- 762 1112 Danio rerio GPPGERGPQGVAGPPGPPGIPGPPINTTGQQSAFFASVNSILPATAKVVVFGQVLYNGQN 799 1113 Xenopus laevis ------------------------------------------------------------ 725 1114 Homo sapiens ------------------------------------------------------------ 745 1115 Mus musculus ------------------------------------------------------------ 718 1116 Podarcis muralis ------------------------------------------------------------ 660 1117 Gallus gallus ------------------------------------------------------------ 611 1118 1119 1120 Scleropages formosus HYSTETGMFTCMVSGMYQFHFHCILPRDAGSIHLMRNGELVVPSFLRKQEGFVTASGGAV 852 1121 Scophthalmus maximus ------------------------------------------------------------ 767 1122 Ictalurus punctatus ------------------------------------------------------------ 771 1123 Carassius auratus ------------------------------------------------------------ 762 1124 Danio rerio HYNQTSGMFLCQIPGVYEFEFSCIGTRSLGFVTLKKNNRVELTPETVALNTRSLAEGKAV 859 1125 Xenopus laevis ------------------------------------------------------------ 725 1126 Homo sapiens ------------------------------------------------------------ 745 1127 Mus musculus ------------------------------------------------------------ 718 1128 Podarcis muralis ------------------------------------------------------------ 660 1129 Gallus gallus ------------------------------------------------------------ 611 1130 1131 1132 Scleropages formosus LLLKKEDRVWLQGSHGAKVLSADSTFTGYLLFVM 886 1133 Scophthalmus maximus ---------------------------------- 767 1134 Ictalurus punctatus ---------------------------------- 771 1135 Carassius auratus ---------------------------------- 762 1136 Danio rerio LSLQRGDRVYVEVSRSANGIGFSSYFSGHILFPV 893 1137 Xenopus laevis ---------------------------------- 725 1138 Homo sapiens ---------------------------------- 745 1139 Proximal Heme Cavity II Mus musculus ---------------------------------- 718 1140 Podarcis muralis ---------------------------------- 660 1141 Gallus gallus ---------------------------------- 611 1142 1143 1144 1145 1146 1147 1148 1149 1150 1151 1152 1153 1154 1155 1156 1157 1158 1159 1160 1161 1162 1163 * * * * * * 1164 1165 1166