Tyrosinases (EC 1.14.18.1) comprise the type-3 copper protein family, which contain an active di-copper center, together with catecholoxidases [1,2]. The enzymes mediate the biochemical conversion of tyrosine into dihydrophenylalnine (DOPA; monophenol oxidase activity) and DOPA into DOPA quinone (diphenol oxidase activity) [3,4]. Tyrosinases are ubiquitously distributed across taxa ranging from bacteria to mammals and engaged in diverse biological events, such as pigmentations, innate immunity, sclerotization, and wound healing, depending on their primary structures [2]. In parasitic trematodes, tyrosinases are produced in the mature vitellocytes and packed within secreting vacuoles termed vitelline droplets, together with eggshell precursor proteins [5]. Following secretion in the ootype, the tyrosinases convert tyrosine residues on the eggshell proteins into DOPA quinones, which are then cross linked to other amino acids, such as tyrosine and lysine, on adjacent proteins to make the sclerotized eggshell [6].

Clonorchis sinensis is the causative agent of clonorchiasis in humans, of which clinical manifestations are represented by abdominal pain, mechanical obstruction of the hepatobiliary ducts, cholangiectasis, and biliary stones [7]. Chronic infection of C. sinensis also appears to lead to high incidences of cholangiocarcinoma [8,9]. Human infection occurs by eating raw or undercooked freshwater fish containing C. sinensis metacercariae. Clonorchiasis is highly endemic in Asian countries, such as China, Vietnam, and Korea, where it causes great socio-economic and public health burdens [7]. In a previous report [10], the structural and biochemical properties of multiple tyrosinases, as well as their spatiotemporal expression patterns, have been investigated in the liver fluke. Expression of these vitellocyte-specific tyrosinase genes was tightly related to the sexual maturation of C. sinensis. However, the relative expression levels of these tyrosinase paralogs were substantially different, although any molecular evidence demonstrating functional diversification could not be detected among them [10].

In general, gene expression level is regulated by multiple factors, such as promoter activity [11], open reading frame (ORF) length and amino acid composition [12], intron length [13], and codon usage pattern [14]. Unequal usage of synonymous codons encoding the same amino acid during translation of a gene, which is known as codon usage bias (CUB), is a commonly observed phenomenon in a wide variety of organisms. The codon usage pattern is a unique property of a genome, and it may vary between genomes and genes from the same genome [15,16]. Many genomic factors, such as gene length and GC-content, have been known to be highly associated with CUB [15]. Comprehensive studies with various organisms further demonstrated that multiple factors, including mutation pressure, natural/translational selection, secondary structure of protein, hydropathic character of protein, and numbers of tRNA gene copies, influence on the codon usage patterns [15,17]. It is also likely that highly expressed genes exhibit significant bias in their codon usage toward those with abundant tRNA gene copy numbers. Therefore, the codon usage patterns of genes may be applied in the prediction of expression level of relevant genes [16,18].

In this study, the CUB of C. sinensis tyrosinase genes and their trematode orthologs was analyzed to understand the process of molecular evolution and the probable factors influencing the codon usage profile, which is ultimately related to the expression level of the corresponding gene.

CUB, which depicts unequal usage of synonymous codons to encode identical amino acid, is a unique property of genomes, and it may differ even between paralogous genes [15,16]. Of the diverse factors associated with CUB, compositional constraints under mutational bias and selection pressures, including translational selection, were considered to be the major determinants of codon usage variation among different organisms [15,25,26]. Translational selection was extrapolated from the number of tRNA copies and inevitably from the expression level of respective genes [25], while the mutational bias was largely associated with the GC content of donor genome. Genes encoded in a GC-rich genome tended to use synonymous codons ending with G and C, whereas those in an AT-rich genome preferred codons with A and T at the third position [27]. The fraction of G- and C-ending codons (34.5±6.6) among the frequently used ones in Clonorchis tyrosinase genes was significantly smaller than the average GC₃ content (47.8±2.4) of corresponding genes (P<0.05), while the average GC₃ content was similar to that of all CDSs encoded in the liver fluke genome (48.0) (Tables 1 and 2). As was in the Clonorchis genes, the average GC₃ content of O. viverrini tyrosinase genes (48.3±2.5) was also similar to that of whole CDSs (47.8). However, the values were greatly reduced in the S. mediterranea (29.6±6.5) and Schistosoma spp. (14.7± 3.3) genes compared to the respective GC contents of whole CDSs (36.0 and 36.5±0.36; P<0.05) (Table 1). In the correspondence analysis of RSCU, the tyrosinase gene positions moved toward the right side of the primary axis, which had strong negative correlation with the GC₃ content, compared to codons (Fig. 3; Table 3). These facts seemed to demonstrate that codon usage pattern, or that of frequently used synonymous codons, was biased to some extent toward A- and T-ending codons in the platyhelminth tyrosinase genes.

The mutation-selection equilibrium in shaping CUB could be estimated by neutrality analysis, which examined relationship between GC₁₂ and GC₃ of respective genes [28]. If the relationship was statistically significant and the slope of the best-fitting line was close to 1.0, mutational bias was taken as the main force that affects CUB. Conversely, selection predominating against mutational bias caused no correlation between GC₁₂ and GC₃ and/or the smaller slope of their regression line [29,30]. The GC₁₂ and GC₃ were significantly correlated in platyhelminth tyrosinase genes, although the slope was much closer to 0.0 than 1.0 (a=0.2168, R=0.8272, P<0.0001; Fig. 1). Interestingly, the relationship was found to be quite different among paralogous gene sets. In the neutrality analyses performed at the intragenome level, the slope of regression line was slightly increased among C. sinensis (a=0.3825; R=0.7693) and O. viverrini (a=0.2284; R=0.4475) genes. However, the value came close to 0.0 among genes encoded in Schistosoma spp. (a=0.0580; R=0.2391) and S. mediterranea (a=0.0508; R=0.1690). Taken together, these data strongly suggested that the selection pressure, such as translational selection rather than mutational bias, was the main force governing bias in nucleotide composition of tyrosinase codons (refer [31] and references therein), even though it was apparent that mutational bias also provided significant force causing the bias at least in the C. sinensis and O. viverrini genes. The predominant impact of selection pressure on CUB of tyrosinase genes was further supported by plots between ENC and GC₃, and between ENC and CAI (Fig. 2) [20,31].

The expression of tyrosinase genes was specific to the vitellocytes and thus, coincided with the sexual maturation in trematodes, including C. sinensis [10,32–34]. Structural and biochemical analyses of the Clonorchis tyrosinases did not show any evidence revealing functional diversification, while the relative expression levels were found to be quite different among them (GAA27975>GAA32069>GAA48883>GAA48882; [10]). CUB-related criteria, such as ENC and CAI, had been known to be reflective of the expression level of corresponding gene [20,35,36]. However, the ENC and CAI values of Clonorchis genes did not have strong relation to their expression levels, which were empirically determined (Table 1). Meanwhile, C. sinensis tyrosinase genes, as well as O. viverrini orthologs, were widely scattered only along the second axis in the correspondence analysis of their RSCU values (Fig. 3A). Correlation analysis between the Clonorchis gene positions along the axis and nucleotide compositions demonstrated that the positions were positively correlated with the GC contents of codons (correlation coefficient=0.971), especially at the third positions (correlation coefficient=0.933). Therefore, the highly expressed tyrosinase genes of C. sinensis, such as GAA27975 and GAA32069, were likely to prefer synonymous codons with the lower GC content, which was also governed by selection pressures, including translational selection [37]. Investigations on the relationship between tRNA gene copy numbers and CUB of tyrosinase genes will be helpful to address the issue in various platyhelminth species.

In conclusion, the codon usage patterns of tyrosinase genes identified in the genomes of diverse platyhelminths, including C. sinensis, were comparatively analyzed to gain insights on the evolutionary process and molecular clues related to the differential expressions of paralogous genes. The CUB detected in the platyhelminth tyrosinase genes was basically governed by selection pressures, rather than mutational bias, although the latter factor provided an additional force in shaping CUB of the C. sinensis and O. viverrini genes. It was also apparent that the equilibrium point between selection pressure and mutational bias was much skewed toward selection pressure in the highly expressed C. sinensis genes than that of poorly expressed paralogs. Further studies on physicochemical properties of tyrosinase proteins, tRNA gene copy numbers, and promoter activity of respective genes will be needed to confirm these results.