The Developmental Phosphoproteome of Fasciola Gigantica by Shotgun

Background: Protein phosphorylation plays key roles in a variety of essential cellular processes. Fasciola gigantica is a tropical liver uke causing hepatobiliary disease fascioliasis, leading to public health threats and heavy economic losses. Although the genome and protein kinases of F. gigantica provided novel insights to understand the molecular biology and etiology of this parasite, there is scant knowledge of protein phosphorylation events in F. gigantica. Results: In this study, we characterized the phosphoproteome of F. gigantica. A total of 1030 phosphopeptides and 1005 phosphorylation sites were identied in 635 F. gigantica phosphoproteins. The bioinformatic analyses and homologous comparisons of these phosphoproteins identied their features participating in cellular processes including metabolic regulation and signal transduction. Conclusions: To the best of our knowledge, this work performed the global phosphoproteomic analysis in F. gigantica, which provides a valuable resource to facilitate the development of novel interventions targets in F. gigantica and related nematodes in the future.


Background
As a tropical liver uke, Fasciola gigantica (F. gigantica) is a parasitic food-borne trematode causing hepatobiliary disease-fascioliasis, leading to extensive liver tissue damage and cirrhosis [1]. Combining with Fasciola hepatica (F. hepatica) infection, fascioliasis is endemic worldwide infecting 17 million people from 61 countries, and 180 million people are at risk of infection [2,3]. Meanwhile, these trematodes infect approximately 600 million domestic ruminants with a signi cantly negative effect on their live weight gains, causing profound damage to animal productivity and economic losses in excess of US$3 billion per year [4,5]. As the only drug effective against the infection, triclabendazole (TCBZ) was recently approved to be used by the FDA and there are currently no therapeutic alternatives to treat human fascioliasis. However, someone treated with TCBZ still continue excreting eggs through feces despite a standard care regimen [6]. Additionally, emerging hybridization and introgression between F. gigantica and F. hepatica make the prevention and control of fascioliasis more di cult [7].
Post-translational modi cations (PTMs) are biochemical changes to regulate the modi ed proteins, which are indispensable for their functional diversities [8]. Therein, as one of the most important PTMs, reversible phosphorylation at serine, threonine and tyrosine residues plays key roles in a variety of cellular processes including signal transduction, membrane transport and metabolic regulation [9,10]. Protein kinases and protein phosphatases are enzymes that catalyze the protein phosphorylation/ dephosphorylation processes via addition and removal of phosphoryl groups to substrates [11,12]. Particularly, protein kinases from Schistosoma and Leishmania as well as Toxoplasma had been identi ed as potential drug targets against these zoonotic parasites [13][14][15]. For F. gigantica, the draft genome (size: 1.04-1.13 Gb) was assembled with a high degree of genomic polymorphism. Using DNA library construction and sequencing as well as bioinformatics analysis, a total of 20858 genes were predicted and 455 protein kinases were identi ed in 2020 [16,17]. However, due to the limitations in genome annotation and functional study of these kinases, we have scant knowledge of protein phosphorylation events in F. gigantica. Furthermore, recent progress has been made in the global phosphoproteomics of different worms [11,12,18], which made contributions to understanding the complex biology of these organisms and developing new antiparasitic candidates.
In the present study, to obtain a comprehensive understanding of phosphorylation in F. gigantica, we used phosphopeptides enrichment method and LC-MS/MS analysis to de ne the rst global phosphoproteomic of F. gigantica. Here, in total 1005 phosphosites from 635 F. gigantica proteins were discovered. Combining with functional annotations and biological connections, this work provides an overview and discusses current knowledge on phosphorylation of F. gigantica, that enables to develop new therapeutics to control fascioliasis.

Parasite preparation
Fasciola gigantica were isolated from the bile ducts of naturally infected buffaloes, washed thoroughly with phosphate-buffered saline (PBS, pH 7.4), and then incubated in RPMI 1640 medium at 37 ℃ for 1 h.
The ukes were frozen in liquid nitrogen and stored at -80 ℃ until use.

Protein extraction and digestion
Protein extraction was obtained from three biological repeats from the frozen samples. Each sample was ground into powder and suspended in a lysis buffer (8 M urea in 100 mM triethyl ammonium bicarbonate, pH 8.5) with 1% protease inhibitor cocktail (Merck, Denmark). Subsequently, the sample was ultrasonicated on ice at 30 s bursts for 10 min. The lysate debris were eliminated by centrifugation at 12000 g for 30 min at 4 ℃, and individual supernatants were collected and conducted to determine the protein concentration by a bicinchoninic acid (BCA) protein assay kit (ThermoFisher Scienti c, USA).
Protein digestion was carried out as previously described [18,19], with some modi cations. Brie y, 300 µg protein sample was reduced with 20 mM dithiothreitol at 56 ℃ for 30 min, followed by alkylation with 100 mM iodoacetamide at room temperature for 30 min, then dissolved into urea solution with the addition of 25 mM NH 4 HCO 3 . Finally, the digestion process was conducted in a trypsin (Promega, USA) solution (6 µg trypsin in 40 µL 100mM NH 4 HCO 3 ).

Phosphopeptides enrichment
After enzymatic hydrolysis, the peptide solution was lyophilized in vacuum and dissolved in a 2,5dihydroxybenzoic acid (DHB) buffer [20]. Phosphopeptides were enriched using a titanium dioxide (TiO 2 ) enrichment method [11,21], with some modi cations. Brie y, TiO 2 beads (ThermoFisher Scienti c, USA) were mixed with peptides and incubated for 40 min at room temperature, and then eluted with 1% ammonia solution and 30% acetonitrile. Subsequently, phosphopeptides were freeze-dried and then dissolved with 20 µL 0.1% formic acid (FA). Therein, 6 µL sample was analysed for LC-MS/MS analysis.

LC-MS/MS
LC-MS/MS analysis was performed using the Q Exactive Mass spectrometer (ThermoFisher Scienti c, USA) with a nanoliter ow rate HPLC liquid system-EASY-nLC1000 (ThermoFisher Scienti c, USA) according to the manufacturer's instructions.
The tryptic peptides were injected into the enrichment column EASY column SC001 traps 150 µm*20 mm RP-C18 (ThermoFisher Scienti c, USA) at an isocratic ow of 5 µL/min of 2% CH 3 CN containing 0.1% FA (solvent A) for 6 min, and then separated by the analytical column EASY column SC200 150 µm*100 mm RP-C18 (ThermoFisher Scienti c, USA) at an isocratic ow of 400 nL/min. The eluents were abovementioned solvent A and 84% CH 3 CN in 0.1% FA (solvent B). The gradient of solvent B was as follows: (i) 0-110 min, 0%-55% solvent B; (ii) 110-118 min, 55%-100% B; (iii) 118-120 min, maintaining at 100% B. Subsequently, the elution products were analyzed by the Q Exactive Mass spectrometer. The spectra of rst-grade MS (MS1) were acquired during the scan scope of 300-1800 m/z with a 2.0 kV electrospray voltage, a resolution of 70000, an automatic gain control (AGC) target of 3e 6 and a maximum IT time of 50 ms. The spectra of second-grade MS (MS2) were obtained using the following parameter: a resolution of 17500; a maximum injection time of 40 ms; an AGC target of 1e 5 . In addition, the mode of MS2 spectra was high-energy collisional dissociation (HCD) and the normalized collision energy of which was set as 30 eV.

Data analysis
All MS/MS data were analyzed using Maxquant1.3.0.5 implementing a Mascot search engine (Matrix Science, London, UK version 2.6.1). The mascot was set up to search the database P17429_fasciola_hepatica_33454_20170815.FASTA (total number of sequences: 33,454; download link: http://www.uniprot.org). The search parameters were set as follows: (i) enzyme: trypsin; (ii) missed cleavage sites: set to 2; (iii) a xed modi cation: carbamidomethyl cysteine; (iv) variable modi cations: an oxidation of methionine (M), acetylation of protein N-term, phosphorylation of serine (S), threonine (T) and tyrosine (Y). For proteins and peptides, the lter parameter false discovery rate (FDR) was set as ≤ 0.01.

Bioinformatic analysis
BLAST analysis was performed using Blast2GO (http://www.blast2go.com), and the Gene Ontology (GO) analysis was performed to identify functional phosphoproteins as previously described, which could be roughly summarized as Blast, GO mapping, GO annotation (http://www.geneontology.org) and InterProScan (http://www.ebi.ac.uk/inter pro/) [22,23]. The InterPro database was utilized for the inspection of each category of phosphoproteins.
In addition, the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis (http://www.genome.jp/kegg/) were carried out to identify the enriched pathways. The classi ed pathways were shown in the hierarchical categories according to the data from the KEGG website.

GO enrichment analysis
Using the automatic annotation tool Blast2GO online, functional annotations were conducted to characterize the identi ed phosphoproteins in this project. By way of blast, mapping and annotation as well as inspection, the GO enrichment analysis showed that these phosphoproteins involved in a wide variety of biological processes (BP) including cellular, metabolic or single-organism processes and biological regulation, localization, cellular component organization or biogenesis as well as in response to stimulus (Fig. 2, Additional le 4: Table S4), which played important roles in protein phosphorylation [18]. Regarding the subcellular localization of the phosphoproteins, the GO annotations based on level 2 and level 5 inferred that these proteins were found predominantly in cellular components (CC) like membranes, organelles and nuclei. Interestingly, only a small portion of them were predicted to located in extracellular regions (Fig. 2, Additional le 5: Table S5), which made the nding reliable as extracellular components were not typically regulated by phosphorylation. According to the localization, the molecular functions (MF) of phosphoproteins was predominantly (51.3%) associated with term "binding" based on level 2, followed by different activities such as catalytic activity (40.6%) and both transporter activity and structural molecule activity that occupied a small proportion (Fig. 2, Additional le 6: Table S6). In total, we found that half (317/635) of the phosphoproteins were annotated with at least one GO category (BP, CC or MF) and one third of them were annotated with at least two GO categories (Additional le 7: Table  S7).

KEGG pathway annotation
To determine whether the identi ed phosphoproteins function independently or in collaboration with other proteins in related biological or signaling pathways, KEGG database was carried out to annotate these phosphoproteins, which helped us understand their potential functions. The KEGG annotation inferred that tight junction, spliceosome and RNA transport were found to be the most enriched pathways of the phosphoproteins, of which each one contains 15 identi ed proteins (Fig. 3, Additional le 8: Table S8). PI3K-akt signaling pathway, oxytocin signaling pathway, cAMP and cGMP-PKG signaling pathway as well as hippo and insulin signaling pathway were ranked in the top 20 of KEGG pathway annotation.
Additionally, parts of phosphoproteins were also predicted to participate in the focal adhesion and protein processing as well as carbon metabolism (Additional le 8: Table S8). In terms of phosphoproteins, we found that 30.4% (193/635) of them were annotated with at least one KEGG pathway and 16.9% was annotated with at least two KEGG pathways (Additional le 8: Table S8). Therein, metabolic enzymes such as adenylate cyclase 9 (ADCY9) and hexokinase (HK), and protein kinases including p21-activated kinase 1 (PAK1), Rho-associated protein kinase 2 (ROCK2), mitogen-activated protein kinase 1/3 (MAPK1/3) and serine/threonine-protein kinase mTOR, as well as effectors like calmodulin (CALM) and heat shock protein 90 (HSP90) were predicted to be annotated in more than 10 KEGG pathways (Additional le 9: Table S9).

Comparison with phosphoproteins of other parasites
According to KEGG database, we compared abovementioned phosphoproteins of F. gigantica annotated in multiple pathways with other parasites including protozoa and helminth in order to identify possible connections between the biological processes and the phosphoproteins of these parasites. Based on recent reports (Table 1), calcium/calmodulin-dependent protein kinase (CAMK4/CDPK4) and 14-3-3 protein of Plasmodium falciparum participated in controlling the parasite invasion and regulating the assembly of signaling complexes, respectively [26,27]. In Leishmania, mitogen activated protein kinase 1 (MAPK1) and heat shock protein 90 (HSP90) played important roles in stress regulation and nascent protein synthesis [28,29]. Additionally, the metabolic enzymes acted as sensors to regulate the metabolites and metabolic ux, contributing to parasite growth [30,31]. In short, the relationship between these proteins of different parasites (Table 1) will help us better understand the phosphoprotein pro le of F. gigantica.

Discussion
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 pro le of F. gigantica remains unclear. As such, to study the pro le is of great signi cance 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 pro le of F. gigantica were performed by shotgun proteomics. In total, we identi ed 635 phosphoproteins of F. gigantica representing 1030 phosphopeptides with 1005 phosphosites (Fig. 1A). A number of phosphorylated enzymes were identi ed, 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 e ciently inhibited by chalcones and two antiparasitic agents (thymoquinone and curcumin), respectively [32,33], and both of them had been con rmed as single or combination vaccines against both F. hepatica and F. gigantica with good e cacy [3]. Wherefore, concentrating on phosphorylated enzymes recognized in this study, we can nd 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 identi ed [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 identi ed 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 identi ed 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 modi cation 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 identi ed 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 identi ed 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 Ca 2+ modulator was proved to be important for the growth and movement of juvenile uke [42,43]. Recent research clari ed 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).

Conclusions
In this work, we de ned the phosphoproteome of a tropical liver uke by a phosphorylation-speci c enrichment technique. From the mass spectrometry and data analysis, we identi ed several phosphoproteins and predicted their functions, which enhanced our knowledge of key biological processes in F. gigantica. With a view towards future exploration into the phosphoproteomics of this parasite at different life-cycle stages, new effective intervention targets will be developed for the treatment of fascioliasis.

Declarations
Ethics approval and consent to participate The experiments were performed in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of China. All experimental animal procedures and protocols (Approval No. 14567) were approved by the Institutional Animal Care and Use Committee of Yangzhou University.

Consent for publication
Not applicable.

Competing interests
The authors declare that they have no competing interests. Higher Education Institutions Veterinary Medicine ; The funders were not involved in the study design; collection, analysis and interpretation of the data; or in writing the manuscript.
Authors' contributions SYH and MP conceived and designed the study. MP, SYB, JZG, and DDL performed the laboratory analyses. QWJ and JPT analyzed the data. All authors critically appraised and interpreted the results. MP drafted the rst version of the manuscript. All authors provided feedback on the manuscript, and read and approved the nal version.   Gene Ontology (GO) term distribution of F. gigantica phosphoproteins in three categories (biological process, molecular function and cellular component). GO annotation and categorization were performed using Blast2GO.