Introduction

Clonorchiasis, resulted from Clonorchis sinensis infection, is an important zoonosis and public health problem (Qiao et al. 2012; Huang et al. 2012). It mainly distributes in East Asia and Southeast Asia, including China and Korea (Rim 2005). People are infected with C. sinensis by consuming raw or undercooked freshwater fish containing metacercariae of C. sinensis. After entering the duodenum, metacercariae excyst and migrate into bile duct and develop into adults. Excretory/secretory products of C. sinensis adults (CsESPs) were generally considered to play a crucial role in host–parasite interactions (Kang et al. 2010a; Lv et al. 2012). Moreover, adult worms release a number of eggs into the gallbladder which induce the formation of gallbladder stone (Qiao et al. 2012). The infection of C. sinensis can result in serious liver and biliary system damages and, in chronic cases, may induce liver and bile duct cancers (Wu et al. 2012). However, it is essential to search for new therapeutic drugs for clonorchiasis control and to investigate important molecules for development and reproduction of C. sinensis.

Adenylate kinases (ADKs) are key enzymes in the energy metabolism of prokaryotic and eukaryotic cells (Bandlow et al. 1998), and the smallest phosphotransferases (Saint Girons et al. 1987). ADKs catalyze the reversible reaction of phosphoryl exchange between nucleotides ATP + AMP ↔ 2 ADP and are regulated by availability of AMP or ADP (Zeleznikar et al. 1995) that is involved in metabolic energy and signaling pathway (Dzeja and Terzic 2009). Structurally, ADKs are composed of three domains: a core domain, containing a parallel β-sheet surrounded by α-helices; an NMP-binding domain, where the AMP binds; and a domain denominated LID, which is composed of residue segment (Fukami-Kobayashi et al. 1996). LID domain covers the nucleotides and locates in the middle part of the protein (Muller and Schulz 1992; Bouvier et al. 2006).

In vertebrates, there are five ADK isoforms (ADK1-5) with various substrate specificities and tissue distribution that have been found (Van Rompay et al. 2000). They can be divided into short isoform and long isoform with extra 27 amino acid residues in the LID domain (Schulz et al. 1986; Fukami-Kobayashi et al. 1996). Some vertebrate ADK isozymes such as ADK2 and ADK3 belong to the long isoform, whereas other vertebrate ADK isozymes such as ADK1 and ADK5 are the short isoform (Bouvier et al. 2006). AKDs are found in mitochondria, cytosol, and cellular membranes, creating an integrated phosphoryl transfer network (Dzeja et al. 1998; Elvir-Mairena et al. 1996; Tanabe et al. 1993), which is critical for the cell's life cycle. ADK1 localizes to the cytoplasm (Stanojevic et al. 2008). Tissues with great energy demand are particularly rich in ADK1, and ADK1-catalyzed phosphotransfer is essential in the maintenance of cellular energetic economy (Janssen et al. 2000). In Schistosoma mansoni, ADK1 plays a key role in the purine salvage pathway for energy and DNA biosynthesis (Marques Ide et al. 2012). ADK1 of Plasmodium falciparum, located in the cytosol, participates in energy transfer (Ma et al. 2012). ADK3 of C. sinensis was cloned, and its characteristics were analyzed (Yang et al. 2005). However, ADK1 has not been ever identified in C. sinensis. In the current study, CsADK1 gene was cloned, and the biochemical properties of recombinant CsADK1, immunolocalization, and transcriptional level of CsADK1 at different life stages of C. sinensis were characterized.

Materials and methods

Sequence analysis

The cDNA sequence encoding CsADK1 was obtained from a C. sinensis cDNA library according to previous description (Song et al. 2004), and the open reading frame (ORF) was found with ORF finder tool in NCBI web site (http://www.expasy.org/). The web site was also used to predict the physicochemical properties, the function domain in deduced amino acids, and structure modeling. The homology and phylogenetic tree were analyzed by bioinformatics analysis software Vector NTI suit 8.0.

Expression and purification

The ORF of CsADK1 was amplified by polymerase chain reaction (PCR) from the template isolated from the cDNA library of adult C. sinensis. Specific PCR primers were as follows: forward primer 5′-CTACGGATCCTTCCCGATGGCTGAC-3′ and reverse primer 5′-CGTTCTCGAGTGTGGCGTGTTT-3′. The amplification conditions were 94 °C for 45 s, 53 °C for 45 s and 72 °C for 1 min for 35 cycles, and 72 °C for 10 min in GeneAmp PCR System (TProfessional Standard Gradient, Biometra). The PCR product was purified and cloned into a prokaryotic expression vector pET-28a (+) (Novagen, Germany) that predigested with the same restrictive endonucleases. The recombinant plasmid was conformed by DNA sequencing and then transformed into Escherichia coli BL21 (DE3) (Promega, USA).

The bacterial cells containing recombinant pET-28a (+) plasmid was induced by 1 mM isopropyl-1-thio-β-galactopyranoside (IPTG, Sigma, USA) at 37 °C for 4 h. The bacterial cells were collected by centrifugation at 4 °C and sonicated on ice. Supernatant was collected, and recombinant protein was purified by His Bind Purification kit (Novagen, Germany) according to the manufacturer's instructions. The lysate of induced host bacteria and purified protein were subjected to 12 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The concentration of purified recombinant protein was determined by using the BCA protein assay kit (Novagen, Germany).

Preparation for antiserum

To prepare anti-CsADK1 rat serum, recombinant CsADK1 (rCsADK1) was emulsified with complete Freund's adjuvant and subcutaneously immunized with 200 μg of protein for each rat at the first time. Subsequently, each rat was given 100 μg of protein (emulsified with equivalent incomplete Freund's adjuvant) for three booster injections at 2-week intervals. Antiserum was collected at 8 weeks. The antibody titer was determined by enzyme-linked immunosorbent assay (ELISA).

Western blot analysis

The CsESPs were collected from adult worms according to the method previously described (Hu et al. 2007). Adult worms or metacercariae were crushed and cleaned by centrifugation at 10,000×g for 15 min at 4 °C to obtain the proteins in supernatant. The concentration of total proteins was determined using the BCA. The recombinant CsADK1 (3 μg/lane), CsESPs, and the total protein of adult worms and metacercariae (20 μg/lane, respectively) were resolved by 12 % SDS-PAGE and then immobilized onto polyvinylidene difluoride membrane. The membranes were blocked with 5 % (w/v) skim milk at room temperature for 2 h, and then incubated with primary antibody (anti-rCsADK1 rat serum, anti-His mouse serum, rat serum infected with C. sinensis, and naïve rat serum, respectively, 1:100 dilutions in 0.1 % BSA-PBS) overnight at 4 °C. After the washing procedures, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG or HRP-conjugated goat anti-rat IgG (1:3,000 dilutions in 0.1 % BSA–PBS, ProteinTech Group, USA) for 1 h at RT. The results were detected by chemiluminescence (chemiluminescent HRP substrate, Millipore, USA).

Real-time quantitative PCR (qRT-PCR)

Adult worms, excysted metacercaria, metacercariae, and eggs were collected as described previously (Kang et al. 2010b; Na et al. 2008). Total RNA from parasites in four stages were extracted by Trizol methods (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions and quantitated by nucleic acid/protein analyzer (Beckman Coulter, USA) and agarose gel electrophoresis, respectively. Approximately 1 μg of total RNA was reversely transcribed to cDNA with reverse transcriptase kit (TakaRa, Dalian, China). The qRT-PCR based on SYBR-Green I fluorescence (TakaRa) was performed by using Roche LightCycler 480 (Switzerland). The sense and antisense PCR primers of CsADK1 (GenBank accession number: AAN03782.1) were as follows: 5′-AGTCTGTCCATGCCTTTGCG-3′ and 5′-TGTCATCCACCCGACCACTA-3′, respectively. RNA integrity was used.

C. sinensis β-actin as an internal control and primers used for β-actin (GenBank accession number: EU109284) were 5′-ACCGTGAGAAGATGACGCAGA-3′ and 5′-GCCAAGTCCAAACGAAGAATT-3′. Reactions were carried out under the following conditions: 95 °C for 30 s, 40 cycles of 95 °C for 5 s, and 60 °C for 20 s, with an incremental increase of 0.1 °C/s from 60 °C to 95 °C. PCR products were quantified by LC480 Software (Roche Light Cycler 480, Switzerland). The data were analyzed by using the 2−ΔΔСt method (Livak and Schmittgen 2001). All assays were tested in triplicate and repeated three times.

Immunohistochemical localization

Adult worms and metacercariae of C. sinensis were fixed with 4 % paraformaldehyde, embedded with paraffin, and sliced into 3–5 μm in thickness. Sections of worms and metacercariae were deparaffinized with dimethylbenzene and rehydrated with graded ethanol series (100 %, 95 %, 85 %, and 75 %). Sections were blocked with normal goat serum for 2 h at RT and then incubated with the anti-rCsADK1 rat serum and naïve rat serum (1:200 dilutions) overnight at 4 °C. Naïve rat serum was employed as negative control. After washing three times with PBST (phosphate-buffered saline, PBS containing 0.05 % Tween-20), the sections were incubated with Cy3-conjugated AffiniPure goat anti-rat IgG (1:400 dilutions in 0.1 % BSA–PBS, Molecular Probe, USA) for 1 h at RT in dark, then imaged under fluorescence microscope (Axio Imager Z1, ZEISS).

Enzyme activity assays

Activity of rCsADK1 was measured using adenosine diphosphate (ADP, Sigma, USA) as substrate according to previous description (Milagros Camara Mde et al. 2012). The activity was determined spectrophotometrically by monitoring the rate of the reduction of NADP+ to NADPH at 340 nm with a coupled enzyme assay. A sample of 3 μg per milliliter purified protein was added to the reaction mixture containing 100 mM Tris–HCl (pH 7.5, Genebase, China), 20 mM glucose (Sigma, USA), 5 mM MgCl2, 10 mM KCl, 2 mM dithiothreitol, 1 mM NADP+, 5 units/ml hexokinase (Sigma, USA), and 2 units/ml glucose-6-phosphate dehydrogenase (Sigma, USA). After 5 min at 37 °C, the reactions were started by the addition of 10 mM ADP. The activity was calculated by measuring the increase in absorbance at 340 nm that accompanied with the reduction of NADP+. All experiments were adapted to Michaelis–Menten equation V = V max[S]/(K m + [S]).

Statistical analysis

All experiments were repeated at least three times, and the data were presented as mean ± SD and analyzed by one-way ANOVA using SPSS 12.0 software. The p value of less than 0.05 was considered statistically significant.

Results

Sequence analysis and structure modeling of CsADK1

The ORF of CsADK1 contained 594 base pairs (bp) encoding a protein of 197 amino acids (26.7 kDa with 12× His-tag). The deduced amino acid sequence shared 74 %, 73 %, 56 %, 55 %, and 52 % identity with ADK1 from S. mansoni, Schistosoma japonicum, Mus musculus, Rattus norvegicus, and Homo sapiens, respectively (Fig. 1). The amino acids forming the canonic phosphoryl loop (P-loop) were demonstrated with a frame. Comparisons with the AMP and ATP binding sites of ADK1 from mammalian (M. musculus, R. norvegicus, and H. sapiens) showed only one substitution S40T (T39 in mammalian ADK1). Moreover, there were two substitutions of A46S and F154Y in the AMP binding sites (S45 and Y153 in human ADK1, respectively). Furthermore, there was one substitution: H98R (R98 or R97 in other species) in the overlaps by the AMP and ATP binding sites and the AMP binding sites. A phylogenetic tree was constructed based on multiple ADK1 sequences from various species in Fig. 1. CsADK1 was composed of a core domain, an NMP-binding domain, and a LID domain, which was just a small loop (Fig. 2).

Fig. 1
figure 1

a Alignment of the deduced amino acid sequence of Clonorchis sinensis adenylate kinase 1 (C. sinensis, GenBank Accession No. AAN03782.1) with other adenylate kinase 1 sequences: Schistosoma mansoni (S. mansoni, GenBank Accession No. XP_002578326.1), Schistosoma japonicum (S. japonicum, GenBank Accession No. CAX75422.1), Mus musculus (M. musculus, GenBank Accession No. NP_001185719.1), Rattus norvegicus (R. norvegicus, GenBank Accession No. BAA97655.1), and Homo sapiens (H. sapiens, GenBank Accession No. NP000467.1). The conservative substitution was shaded in gray with percent symbol, ampersand, and number sign. Percent symbol: substitution in ATP–AMP binding sites. Ampersand: substitution in AMP binding sites. Number sign: substitution in the overlaps of ATP–AMP binding sites and AMP binding sites. The amino acids forming the canonic phosphoryl loop (P-loop, GxxGxGxxT) were demonstrated with a frame. b The phylogenetic tree of ADK1

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Fig. 2
figure 2

Three-dimensional structure of ADK1 Ribbon presentation. a The three-dimensional structure of CsADK1 Ribbon presentation. b The three-dimensional structure of SmADK1 Ribbon presentation. c The three-dimensional structure of CsADK3 Ribbon presentation. The conserved domains of ADK1 were a core domain, containing a parallel β-sheet surrounded by α-helices; an NMP-binding domain; and a LID domain, which converts the nucleotides. Compared with the LID domain of CsADK3, CsADK1 has a small loop

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Expression and purification of rCsADK1

The soluble rCsADK1 was expressed with 12× His-tag in E. coli BL21 after being induced by 1 mM IPTG at 37 °C for 4 h. The molecular mass of purified protein was about 26.7 kDa (Fig. 3a), and the final protein concentration was 10 mg/L.

Fig. 3
figure 3

a Expression and purification of rCsADK1 by 12 % SDS-PAGE. Protein molecular weight markers (lane M), lysate of Escherichia coli with pET-28a (+) before induction (lane 1) and after induction (lane 2), lysate of E. coli with pET28a (+)-CsADK1 before induction (lane 3) and after induction (lane 4), supernatant (lane 5) and precipitant (lane 6) of lysate of E. coli with pET28a (+)-CsADK1 after induction (lane 7), and the purified recombinant CsADK1 protein. b Identification of CsADK1 as a component of CsESPs. rCsADK1 protein was immobilized and probed with anti-rCsADK1 rat serum, anti-His-tag mouse serum, and Clonorchis sinensis-infected rat serum (lanes 1–3, respectively). The total extracts of adult worms and metacercariae were probed by anti-rCsADK1 rat serum, respectively (lanes 4 and 5, respectively). CsESPs were probed with anti-rCsADK1 rat serum (lane 6). The naïve serum was as control

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Identification of CsADK1 as a component of CsESPs

Purified rCsADK1 could react with anti-rCsAK1 rat serum, anti-His mouse serum, and rat serum infected with C. sinensis (Fig. 3b, lanes 1–3, respectively) at 26.7 kDa. The total extracts of adult worms and metacercariae and CsESPs were probed by anti-rCsADK1 rat serum (Fig. 3b, lanes 4–6). However, no band was detected by naïve rat serum (not shown).

Transcriptional level at different developmental stages

The result was calculated according to the previous methods (Livak and Schmittgen 2001). As shown in Fig. 4, transcriptional level of CsADK1 was detected at the life stages of adult worms, excysted metacercaria, metacercariae, and eggs of C. sinensis. Obviously, it showed that transcriptional level of CsADK1 at the stage of excysted metacercaria was higher than that of adult worm (18.8-folds, P < 0.01), metacercariae (1.5-folds, P < 0.01), and eggs (5.6-folds, P < 0.01).

Fig. 4
figure 4

Real-time quantitative assay of CsADK1 at different life stages of Clonorchis sinensis. y-axis represents the level of quantitative mRNA at life stages. Expression level of CsADK1 at the stage of excysted metacercariae was higher than that of adult worms (18.76-folds, P < 0.01), metacercariae (1.52-folds, P < 0.01), and eggs (5.62-folds, P < 0.01)

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Immunohistochemical localization of CsADK1

Figure 5 showed that CsADK1 was extensively distributed in metacercariae (Fig. 5b) and in the vitellaria and eggs of adult worms (Fig. 5f). No specific fluorescence was detected either in adult worms or in metacercariae when treated with naïve mouse serum (Fig. 5d, h).

Fig. 5
figure 5

Immunolocalization of CsADK1 in Clonorchis sinensis metacercariae and adult worms. CsADK1 was extensively distributes in metacercariae (b). In the adult worms, CsADK1 was localized in the vitellaria and eggs (f). Negative controls were carried out with naïve serum (1:100 v/v dilutions) for metacercariae (d) and for adult worms (h). a, c Corresponding bright fields for metacercariae. e, g Corresponding bright fields for the adult worms. The images were magnified at ×100 for adult worms and ×400 for metacercariae, respectively. v vitellaria, e eggs

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Enzymatic assays

The K m and V max value of CsADK1 was 2.2 mM and 0.9 mM/min when using ADP as substrate in this coupled enzyme assay, respectively (Fig. 6). Enzyme activity of rCsADK1 maintained more than 50 % of maximal activity between 30 °C and 50 °C, and the optimal temperature was 37 °C (not shown). The activity was weak when pH value was under 6.0 and up to 60 % of maximal activity when pH value was 10.0. The optimal pH value was from 7.5 to 8.0 (not shown). The activity was increasing with varied concentrations of Mg2+ from 1 to 5 mM. However, it changed a little when the concentration was up to 10 mM. The optimal concentration of Mg2+ for the enzyme activity was 2 mM (Fig. 7a). The effect of K+ on enzyme activity was detected, and the optimal concentration was 10 mM (Fig. 7b). Table 1 showed that the enzyme activity was increasing in the order Mg2+ > Ca2+ > Mn2+. In the effect of monovalent ions on enzyme activity, it was highly activated by K+ and NH4 +, but slightly by Li+ compared with control.

Fig. 6
figure 6

To determine value of the K m and V max of rCsADK1, the reaction was detected. Lineweaver–Burk double reciprocal plot of rCsADK1

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Fig. 7
figure 7

Enzymatic characteristics of rCsADK1 were detected. a Effects of Mg2+ on rCsADK1 activity was detected. b Effects of K+ on rCsADK1 activity was detected

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Table 1 Effect of metal ions on the enzyme activity
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Discussion

Our genome and transcriptome data of C. sinensis showed enzymes involved in purine metabolism and pyrimidine metabolism (Wang et al. 2011a). However, enzymes which participated in de novo purine nucleotide synthesis were absent in the parasite. On the contrary, enzymes involved in salvage pathway were expressed, such as adenine phosphoribosyltransferase and hypoxanthine phosphoribosyltransferase. It suggested that salvage pathway was present in C. sinensis. In the S. mansoni, it totally depends on the purine salvage pathway in order to supply large quantities of purine precursors for its energy and DNA biosynthesis (Marques Ide et al. 2012), while ADK1 has participated in salvage pathway to provide purine nucleotides for S. mansoni (Marques Ide et al. 2012). It is possible that C. sinensis may obtain purine precursors for energy and DNA biosynthesis by ADK1 because ADK1 is involved in the homeostasis of adenine nucleotides by interconversion of constituents of the adenine nucleotide pool and duplicating the ATP energetic potential via its synthesis from ADP (Schulz et al. 1986; Janssen et al. 2000; Ma et al. 2012).

In the present study, we first identified an isoform enzyme of ADKs and characterized the structures and biochemical properties of CsADK1. The conservative binding sites of CsADK1 were substituted for other residues. This conservative substitution is observed in other ADK1, such as ADK1 from S. mansoni and S. japonicum (Marques Ide et al. 2012). CsADK1 had the P-loop with the conservative residues GxxGxGxxT. CsADK1 was a short isoform, and the LID domain was just a small loop as similar in SmADK1 (Marques Ide et al. 2012). It was obviously different with CsADK3 containing four β-strands (Fig. 2c). In addition, a novel gene ADK1 of C. sinensis was cloned, and the protein was expressed. It is a soluble protein (Fig. 3a) that paves the way for further functional research.

The transcriptional level of CsADK1 was detected in all developmental stages of C. sinensis. CsADK1 were intensively expressed in vitellaria and eggs of adult worms but distributed extensively in metacercariae, indicating it is actively involved in development and reproduction of C. sinensis. In the enzymatic activity assay, the K m and V max value of CsADK1 was 2.2 mM and 0.9 mM/min when using ADP as substrate, respectively. Upon ligand binding, the enzyme undergoes a transition from the inactive open form to the catalytically competent closed structure according to the analysis of the crystal structures (Vonrhein et al. 1995). This transition is mediated by large-scale closure motions of the LID and AMP domains insulating the substrates from the water environment, while occluding some catalytically relevant water molecules (Matsunaga et al. 2012). The kinetic properties of CsADK1 showed that optimal pH value is 7.5 to 8.0, the normal pH scope of bile. However, the enzyme of Tritrichomonas foetus has a broad optimal pH range between 6.0 and 9 (Dinbergs and Lindmark 1989). In other words, CsADK1 was an acid-sensitive enzyme and insensitive to alkaline environment. When metacercariae excyst and migrate into bile duct and develop into adults, CsADK1 maintained its functions in energy supply and the homeostasis of adenine nucleotides. Moreover, Mg2+ was essential for activity of CsADK1 (Bouvier et al. 2006), while Mn2+ had little effects on it. On the contrary, in T. foetus, full enzyme activity requires Mg2+, but Mn2+ can yield half maximal activity (Dinbergs and Lindmark 1989). It demonstrated that CsADK1 was an Mg2+-dependent enzyme. The activity was obviously activated at the low concentration of K+. In Table 1, the effect of NH4+ on activity was the same as K+, but was slightly activated by Li+. Taken together, it could help to explain the slight difference in the kinetic parameters when compared with their mammalian counterpart. Moreover, the presence of different residues in the binding sites opens up the possibility of designing specific competitive inhibitors, which has a lower affinity for the human ortholog, for chemotherapy of clonorchiasis.

Western blotting (Fig. 3b) demonstrated that CsADK1 was one component of total proteins of adult worms and metacercariae of C. sinensis. Moreover, CsADK1 could react with rat serum infected with C. sinensis. It is implied that CsADK1 was also a circulating antigen which might derive from eggs of parasites. Furthermore, CsADK1 was identified as a component of CsESPs (Fig. 3b, lane 6). Interestingly, CsESPs may derive from the excoriation of parasites and excretion through intestine from parasites (Wang et al. 2011b). CsADK1 was relatively highly expressed in eggs of adult worm. It is possible that eggs are released from adult worms into bile duct, and excoriation or excretion of eggs may also be one of the sources of CsESPs. Moreover, qPCR results showed that transcriptional level of CsADK1 at the excysted metacercariae stage was the highest at these life stages. In the process of growth from metacercariae to adult worms, substantial energy and adenine nucleotides requirements must be met during this transformation. Taken together, CsADK1 may play a key role in energy supply and regulation of adenine nucleotides for excysted metacercariae stage of C. sinensis. The results of transcriptional level of CsADK1 were in accordance with immunolocalization at the metacercariae and eggs stages. In many organisms, energetic homeostasis is maintained by remodeling the phosphotransfer network. In mammals, the suppression of the adenylate kinase gene is complemented by glycolytic enzymes (Janssen et al. 2000, 2003; Dzeja et al. 2004). The transcriptional level of CsADK1 was less at adult worms. Moreover, three key enzymes involved in anaerobic glycolysis expressed higher in the stage (unpublished data). It is possible that the downregulation of CsADK1 is complemented by glycolytic enzymes in this stage.

In summary, we have identified a novel CsADK1 gene from the cDNA library of C. sinensis, expressed, and characterized the properties of the protein. In the bioinformatics analysis, CsADK1 had some differences from human ADK1 in conservative binding sites, and enzymatic characteristics were illustrated, indicating that it contributes to designing specific competitive inhibitors for clonorchiasis control. In addition, we demonstrated that it was a component of CsESPs from excoriation or excretion of eggs that was one of the sources of CsESPs. In other organisms, ADK1 is involved in a broad variety of cellular functions; however, in C. sinensis, most of the metabolic processes associated with CsADK1 remain unknown and require further investigation.