As a food-borne trematode, F. gigantica is responsible for the hepatobiliary disease fascioliasis. The pathogenicity of F. gigantica is closed related to the massive proliferation of parasites, which brings mechanical stimulation and toxin to the host [1]. In terms of parasite growth and signal transduction, protein phosphorylation or dephosphorylation catalyzed by specialized protein kinases or phosphatases of F. gigantica played crucial roles in corresponding cellular processes, which helps maintain the cell homeostasis in parasites [17]. However, the phosphoproteomic profile of F. gigantica remains unclear. As such, to study the profile is of great significance for exploring the roles of these proteins in the growth and development of parasite, which could also help to screen new drug targets against F. gigantica.
In this work, to the best of our knowledge, a global phosphoproteomic profile of F. gigantica were performed by shotgun proteomics. In total, we identified 635 phosphoproteins of F. gigantica representing 1030 phosphopeptides with 1005 phosphosites (Fig. 1A). A number of phosphorylated enzymes were identified, including metabolic enzymes and protein kinases as well as phosphatases. Therein, as drug candidates, cathepsin L1 (CL1) and glutathione S-transferase (GST) of F. gigantica could be efficiently inhibited by chalcones and two antiparasitic agents (thymoquinone and curcumin), respectively [32, 33], and both of them had been confirmed as single or combination vaccines against both F. hepatica and F. gigantica with good efficacy [3]. Wherefore, concentrating on phosphorylated enzymes recognized in this study, we can find more effective targets and open new avenues to treat fascioliasis. In addition, phosphorylated effectors such us 14-3-3 and thioredoxin with good immuno-reactivity were also identified [34, 35]. However, the phosphorylation processes catalyzed by gelsolin and calreticulin as well as tropomyosin had never been reported before, and BN1106_s378B000167 in particular deserved to mention as a result of hyper-phosphorylation (14 identified phosphopeptides) despite without any annotation, showing that BN1106_s378B000167 is unique in F. gigantica and can be used for subsequent functional studies and drug design in the future. The identified 1005 phosphosites consisted of 858 (85.4%) serine phosphorylation, 138 (13.7%) threonine phosphorylation and 9 (0.9%) tyrosine phosphorylation. Previous studies had found the mutations in important phosphosites might result in the termination of the phosphorylation, leading to disorders of biological processes [18]. Moreover, the modification ratios found in this work seemed to be consistent with previously studies in other organisms [11, 24, 25], so these phosphosites can provide references for future research on phosphorylation process in F. gigantica.
Subsequently, GO functional annotations (level 2) containing BP, MF, and CC categories were conducted to characterize the identified phosphoproteins. The top 3 in the BP category were involved in cellular process and metabolic process as well as single-organism process, highlighting the correlation between phosphorylation process and bioenergetics in F. gigantica. The largest percentage of CC terms were involved in cell and cell part such as membranes, organelles and nuclei, showing intracellular components were predominantly regulated by phosphorylation. Based on the localization, the MF were involved in binding and catalytic activities, indicating critical roles of phosphorylation in the structural and functional regulation of F. gigantica [36].
According to KEGG annotation, tight junction, spliceosome and RNA transport were found to be the most enriched pathways of the phosphoproteins. In addition, signaling pathways (PI3K-akt and oxytocin), protein processing and the regulation of actin cytoskeleton, as well as carbon metabolism were ranked in the top 10 of KEGG pathway annotation (each one contains ≥ 10 identified proteins), which seemed to be roughly consistent with the phosphoproteomic annotation in other parasites [11, 12]. For phosphoproteins, nearly 7% of them indicating multiple roles in different biological processes were predicted to be annotated in more than 5 KEGG pathways. Therein, malate dehydrogenase (MDH) of F. gigantica was a single cytosolic enzyme to catalyze the reversible oxidation of malate to oxaloacetate using NAD+. Been as an isoenzyme, the pattern of MDH in F. gigantica and F. hepatica were the same due to similar relative mobilities [37]. Further study found the superimposition structure model of FgMDH and human MDH showed overall structural similarity in the active site loop region, however, the conformation of the residues was different [38]. Combining with our annotations on the functions of FgMDH in metabolic pathways such us pyruvate metabolism, cysteine and methionine metabolism, glyoxylate and dicarboxylate metabolism, the FgMDH can be used in future drug design for the treatment of fascioliasis. P21-activated protein kinase (PAKs) had long been established to play important roles in vital cellular functions such as proliferation, survival and motility. Emerging evidence showed host PAK1 increased cell survival during the stage of virus infections [39], and recent study found PAK1 enhanced macrophage activation, resulting in promoting of Th17 cell response during Schistosoma japonicum infection [40], both of which guide the future research on the immunomodulation roles of host PAK1 and parasitic PAK1 in Fasciola infection. Camodulins (CaMs) were involved in fundamental processes including the phosphorylation of protein kinases, gene transcription and calcium transport [41]. In Schistosoma mansoni, calmodulins had been implicated in egg hatching, miracidial transformation and larval development. While in F. hepatica, the FhCaMs functioned as a Ca2+ modulator was proved to be important for the growth and movement of juvenile fluke [42, 43]. Recent research clarified calmodulin of Caenorhabditis elegans was implicated in the plasticity impairment of high-activity neurons with age, indicating a different but novel role in neuronal activity [44]. Furthermore, others phosphoproteins of F. gigantica annotated in more than 10 KEGG pathways were compared with other parasites (Table 1).