Glutathione transferase (GST) catalyzes the conjugation of glutathione (GSH) thiolate anion with various xenobiotics or intracellularly generated cytotoxic substances. The solubilized GSH conjugates of these toxic molecules are then removed by relevant transporter proteins present in plasma membranes [1]. In addition to the GSH-tagging activity, several GSTs function as peroxidases, maleylacetoacetate isomerases, or thiol transferases [2–4]. Interestingly, the detoxifying enzymes are involved in the modulation of immune responses by mediating prostaglandin (PG) and leukotriene syntheses [5]. Mammalian GSTs are classified into multiple classes including alpha, mu, pi, omega, sigma, theta, and zeta, based on their amino acid (aa) conservation patterns and substrate/inhibitor specificity, while a series of proteins show mosaic patterns in their enzymatic properties [6]. Homologs of the diverse GST-class members have been identified in parasitic trematodes [7–9].

Prostaglandins are oxygenated eicosanoids that participate in several physiological processes, including inflammatory immune responses [10]. Prostaglandins are produced from polyunsaturated fatty acids, mostly arachidonic acids, via cyclooxygenase (formation of PGH₂) and subsequent PG species-specific PG synthase (PGS) activities [10]. Of the multiple PG species, such as PGI₂, PGF_2α, PGD₂, and PGE₂, PGD₂ plays a principal role in regulating inflammatory responses by recruiting Th₂ cells, eosinophils, and basophils and thereby controls the pathogenesis of allergic diseases and vasodilatation [11]. In mammals, generation of PGD₂ from PGH₂ is catalyzed by 2 distinct enzymes, GSH-independent lipocalin-type PGD₂ synthase (L-PGDS) and GSH-dependent hematopoietic PGDS (HPGDS) [12]. The mammalian HPGDS shares structural properties with the sigma-class GSTs [5,13]. Levels of immunomodulatory PGs are frequently elevated in vertebrates during infections with parasitic helminths [14]. Therefore, helminth parasites are likely to express a protein(s) either exhibiting PGS activity or that stimulates host cells to synthesize PGs [14]. Sigma-class GSTs characterized in Onchocerca volvulus, Schistosoma haematobium, and Fasciola hepatica can be considered as the genuine homologs or, at least, functional equivalents of the mammalian HPGDS [15–17].

Proteins secreted into host environments or localized in parasite teguments may constitute the first line of defense to protect invading worms against host immune factors. Various GST-class members are commonly identified in the secretory-excretory products (ESPs) of trematode parasites such as F. hepatica [18], Opisthorchis viverrini [19], and Clonorchis sinensis [9]. Of the secreted proteins, the sigma-class GST proteins comprise the second most abundant component of the C. sinensis ESP [9]. The F. hepatica ESP also contains a protein categorized into this class, although the relative concentration was much lower than that of the C. sinensis orthologs [20,21]. Consequently, we hypothesized that the secreted sigma-class GST plays an important role in relation to the modulation of host immune response by providing PGS activities during trematode infections.

Clonorchis sinensis is a digenean trematode that infects the bile ducts of piscivorous mammals, including humans. Humans are usually infected with the liver fluke by eating raw or undercooked freshwater fish harboring metacercaria (food-borne trematodiasis). Clonorchiasis is highly prevalent in several Asian countries including Korea, China, and Vietnam, and imposes substantial socioeconomic and public health burdens in these endemic areas [22]. Patients with heavy infection exhibit clinical symptoms such as fever, right upper-quadrant pain, and intermittent colic pain, while those with light infection are generally asymptomatic. However, chronic infection with C. sinensis can cause fibrosis of the bile ducts, destruction of surrounding liver parenchyma, and recurrent pyogenic cholangitis. Furthermore, the long-standing clonorchiasis is significantly associated with the development of cholangiocarcinoma, a biliary ductal carcinoma that has a poor prognosis [23].

In addition to the repeated ingestion of C. sinensis metacercariae, transition of host immune response from type 1 to type 2, which can be triggered by the parasite factors, helps establish chronic clonorchiasis by generating parasite-friendly environments [24]. Several Clonorchis proteins with spatiotemporal expression profiles predominantly found in the adult stage have been identified as the plausible immunomodulators [24]. Lipid mediators including PGs and leukotrienes generated by host and/or parasite enzymes also exert a profound effect during the initiation phase of type 2 immune responses [25]. Herein, we investigated the enzymatic properties of sigma- and mu-class GSTs detected in C. sinensis ESP to assess their potential as the PG-generating immunomodulator.

This study comprehensively screened sigma-class GST proteins present in the proteome of C. sinensis. The proteins shared structural properties with HPGDs identified in mammalian vertebrates, including human. The phylogenetic analysis based on the conservation patterns of primary protein structures demonstrated that gene(s) encoding the sigma-class GST proteins had undergone gene duplication events that specifically occurred in an opisthorchiid genome ancestral to C. sinensis, O. viverrini, and several other trematode lineages. Recombinant forms of the sigma-class GSTs, as well as mu-class proteins, exhibited considerable GST and PGS activities. However, the relative binding affinities toward PGH₂ (average K_m of 30.7 and 3.0 μm for the PGDS and PGES activities, respectively) were much greater than those toward CDNB (average K_m of 1,205.1 μm). Comparison of the enzymatic efficiency suggested that the C. sinensis GST proteins function more effectively as a PGS (average K_cat/K_m of 3.1 and 0.7 s⁻¹μm⁻¹ for PGDS and PGES, respectively) than as a GST (average K_cat/K_m of 7.0×10⁻³ s⁻¹μm⁻¹).

Genetic dosages responsible for the expression of sigma-class GST varied among trematode species (Fig. 2). Five paralogous genes were found in the C. sinensis and O. viverrini genomes, whereas 2 and 3 were found in the genomes of Fasciola spp. and P. westermani, respectively. However, only a single homologous gene was identified in each of the schistosome genomes. The phylogenetic analysis based on their encoded aa sequences suggested that the copy number variation resulted from genetic duplication and/or following deletion events that had specifically occurred in the trematode species/lineages (Fig. 2). A difference in the copy number has also been observed with the mu-class GST genes, where the copy numbers were greater in Fasciola spp. than in opisthorchiids [8,26]. Considering that the copy number of functional genes has a profound effect on their phenotypic expression [27], the differential copy numbers of GST genes would reflect the specifically evolved roles in the given trematode species. Clonorchis sinensis and F. hepatica secrete sigma- and mu-class GST proteins into surrounding environments. However, the relative amounts of these GSTs differ between these species; the sigma-class proteins, especially CsGST-σ3, are prominently found in the C. sinensis ESP [9], whereas the mu-class members are more abundant in that of F. hepatica [17,21]. The major component of Opisthorchis felineus ESP is also a sigma-class GST protein [28]. Furthermore, inductive expression profiles either during development or against exogenous stimuli substantially differ among the GST genes in C. sinensis [26]. Thus, the GST proteins, which have previously been known to function as phase II antioxidant enzymes in intracellular spaces, are presumed to have gained additional roles involved in the host-parasite interplays in these liver flukes. Despite the similar tissue tropism toward the biliary tracts of mammalian hosts, C. sinensis/O. viverrini and Fasciola spp. show differences in the routes and periods of penetration of the biliary lumen [29,30]. Factors derived from the differential host environments may have provided a selection pressure for the functional diversification of these trematode GST proteins.

Helminths parasitizing mammalian hosts might have evolved molecular machineries associated with regulating host immune responses and sequestering harmful endogenous/exogenous oxidants. Prostaglandins generated by these tissue-invading helminth parasites are essential for establishing and maintaining infection in their definitive hosts. In the extracellular host spaces, the eicosanoids are intimately involved in modulating host immune responses [19,31]. During chronic infection, these molecules induce pathophysiological changes such as pyrexia, alteration of cellular metabolism, and neoplasia in the affected tissues or hosts [32]. Meanwhile, PGs produced within the parasite seem to play key roles in association with the parasite penetration of host skin [33] and embryogenesis [17]. Considering the spatial expression patterns [9] and enzymatic properties (Table 1), the CsGST-μ and CsGST-σ proteins can provide dual activities for the immunomodulation and antioxidant defense in C. sinensis. Whether the GST proteins directly participate in de novo synthesis of PGs from arachidonic acids in the trematode cells is currently unclear because the parasites do not encode the cyclooxygenase isozymes responsible for the generation of PGH₂ [14]. The C. sinensis ESP stimulates the expression of host genes involved in the generation of immunomodulatory eicosanoids [34] and the protein components penetrate the plasma membranes of surrounding host cells [35]. Therefore, the C. sinensis GSTs in ESP are likely to cooperate with host enzymes to generate PGD₂ and/or PGE₂ from PGH₂ in intracellular spaces. Inflammatory responses accompanying the infiltration and activation of immune cells [24] may provide an opportunity to establish the intracellular PG-generating environments.

Human HPGDS (single sigma-class GST member in mammals) and GSTMs (mu-class GST members) are cytosolic proteins that also exhibit the PGDS and PGES activities, respectively [12,36]. The CsGST-σ proteins share structural and enzymatic properties with the human HPGDS (Figs. 1, 3). Interestingly, the Clonorchis proteins could also catalyze conversion of PGH₂ to PGE₂, although their specific PGES activities and catalytic efficiencies were much lower than those for PGES (Fig. 3; Table 1). The CsGST-μ proteins had enzymatic properties similar to those of the CsGST-σ proteins. The highest expression of human HPGDS mRNA is found in macrophages [5], which are highly activated during C. sinensis infection [24]. Therefore, the eicosanoid molecules may play important roles in immune responses invoked by C. sinensis such as during type 2 inflammation and fibrotic responses [24]. The human HPGDS contains 3 conserved Asp residues forming Ca²⁺ ion-binding sites in the primary structure (Fig. 1). The cytosolic influx of Ca²⁺ ion is commonly observed in activated immune cells, and the interaction between dimeric HPGDS and Ca²⁺ ion increases the PGDS activity up to 150% of the basal level in a concentration-dependent manner [12]. The invariant Asp residues were not detected in the corresponding regions of C. sinensis proteins (Fig. 1). Further investigations on the inductive expressions of PG-related genes in affected host cells may help generate insight into the roles of PG molecules in C. sinensis infection.

In summary, we showed that the sigma- and mu-class GSTs expressed in C. sinensis have variable PGDS and PGES activities in addition to the conventional GSH-conjugating activity. These proteins displayed much greater affinities toward PGH₂ than those toward CDNB. The efficiency of PGS activities, expressed as K_cat/K_m obtained by steady-state kinetic analyses, was also higher than that of the GST activity. Therefore, C. sinensis GST proteins may act as antioxidant enzymes in parasitic parenchyma, where the concentration of the proteins and probable substrates are relatively high. Conversely, these proteins are probably responsible for the generation of PGs such as PGD₂ and PGE₂, after being secreted into the surrounding host environments. The antioxidant and immunoregulatory roles are pivotal for the survival of the parasite and maintenance of the infection in definitive hosts. Therefore, these GST proteins can provide effective targets during investigations of therapeutics or vaccines to control clonorchiasis that is highly prevalent in Asian countries such as Korea.