Genomic structure, expression and functional characterization of arginine kinase (EcAK) from Exopalaemon carinicauda

Abstract

Arginine kinase (AK, EC 2.7.3.3) plays an important role in cells with high, fluctuating energy requirements. In invertebrates, AK is the major phosphagen kinase that modulates the energy metabolism. Here, the full-length cDNA sequence encoding arginine kinase (EcAK) was obtained from the Exopalaemon carinicauda. The com- plete nucleotide sequence of EcAK contained a 1068 bp open reading frame (ORF) encoding EcAK precursor of 355 amino acids. The genomic DNA fragment of EcAK with the corresponding cDNA sequence is composed of 4 exons and 3 introns. The domain architecture of the deduced EcAK protein contained an ATP-gua_PtransN domain and an ATP-gua_Ptrans domain. EcAK mRNA was predominantly expressed in the muscle. The expres- sion of EcAK in the prawns challenged with Vibrio parahaemolyticus and Aeromonas hydrophila changed in a time- dependent manner. Then, EcAK was recombinantly expressed in Pichia pastoris and the purified recombinant EcAK had the same enzymatic characterization as AK from the muscle of Euphausia superba. In conclusion, EcAK may play the same biological activity in E. carinicauda as those from other crustaceans.

1. Introduction

Arginine kinase (AK, EC 2.7.3.3) plays an important role in cells with high, fluctuating energy requirements [1]. In invertebrates, AK is the major phosphagen kinase that modulate the energy metabolism and catalyzes the reversible phosphorylation of arginine to regenerate ATP during bursts of cellular activity [2,3].

At present, there are a lot of study about AK in crustaceans. In 2003, Kinsey & Lee [4] firstly reported that neither acclimation salinity nor acute salinity change had an effect on high-energy phosphate concen- trations, AK fluX rates, or AK activity in the juvenile blue crab, Callinectes sapidus. Since then, there were a lot of reports about AK in Crustaceans. Yao et al. [5] identified one AK protein in the plasma of Fenneropenaeus chinensis using two-dimensional electrophoresis and electrospray ioni- zation mass spectrometry. Garcia-Orozco et al. [6] purified and identi- fied one AK from Litopenaeus vannamei, which could be recognized by IgE in serum from shrimp-allergic individuals. Rattanarojpong et al. [7] performed the proteomic analysis of gills from yellow head virus (YHV)-infected L. vannamei and identified 13 spots with up-regulated protein expression levels, in which AK was an upregulated protein. Abe et al. [8] found that AK of Marsupenaeus japonicus was up-regulation under hypoXia, which might indicate a provision for oXygen re-supply after anaerobiosis. Xu et al. [9] found that AK of M. japonicus (MjAK) could enhance the replication of WSSV in shrimp by interacting with the envelope protein VP26 of WSSV.

As mentioned in our previous research [10], there is no model animal in Crustacean to be used in basic research. The ridgetail white prawns, Exopalaemon carincauda can be maintained with reproductive capacity all the year round in the laboratory environment with an about 60-day reproduction cycle [11]. In addition, the low-coverage sequencing and de novo assembly of the E. carinicauda genome has been performed, which covers more than 95% of coding regions [12]. In addition, genome editing approach based on CRISPR/Cas9 technology was first successfully applied to the embryos of E. carinicauda in our group [13]. Therefore, E. carinicauda exhibited the potential to be used as an experimental animal in the research of Crustacean.
In this research, an arginine kinase gene (EcAK) was firstly obtained from E. carinicauda. The expression profile of EcAK in different tissues and its immune function against Vibrio parahaemolyticus and Aeromonas hydrophila was analyzed. Furthermore, the recombinant EcAK (rEcAK) was prepared in Pichia pastoris and the partial enzymatic characteriza- tion of rEcAK was also analyzed.

2. Materials and methods

2.1. Experimental animals, bacterial culture and immune challenge

E. carinicauda, length of 5.5 0.5 cm, were bred in our laboratory. Nine tissues (eyestalk, intestine, muscle, cuticle, hepatopancreas, nerve cord, heart, stomach, and gill) were separated from 15 healthy adult E. carinicauda and used for RNA extraction [14].The prawns were challenged with Vibrio parahaemolyticus or Aeromonas hydrophila according to the method [14]. The prawns in experi- mental group were intramuscularly injected individually with 10 μL phosphate saline (PSS) buffer containing V. parahaemolyticus or A. hydrophila (107 CFU mL—1). At the same time, the prawns in control group were injected with 10 μL sterile PSS. Two hundred prawns were sampled from each group. The muscle of five prawns from each group was collected at 0, 12, 24, 48, 72, 96, and 120 h, and used for RNA extraction.

2.2. RNA isolation, cDNA synthesis and bioinformatic analysis

Total RNA of the collected samples was extracted with Trizol® reagent (Thermo, USA) and treated with RQI RNase-Free DNase (Promega, USA). Then, 2 μg total RNA and 0.2 μM random hexamer primers were added to synthesize cDNA using M-MLV reverse transcriptase (Promega, USA) at 42 ◦C for 1 h according to the manufacture’s protocol (9PIM170).

Based on the transcriptomic and genomic data of E. carinicauda [12], one full-length AK sequence of E. carinicauda (EcAK) was cloned by reverse transcription-polymerase chain reaction. The cloned sequence was analyzed for the identity and similarity by BLAST on-line. The multiple sequence alignment was performed using CLUSTAL W [15]. The domain architecture of the predicted amino acid sequence was predicted using SMART (http://smart.embl-heidelberg.de/).

2.3. Expression profile of EcAK mRNA

Quantitative real-time PCR (qRT-PCR) [16] was used to analyze expression profiles of EcAK mRNA in different tissues and at different sampling time in the muscle of E. carinicauda using Mastercycler ep realplex (Eppendorf). 18S rRNA was used as the internal control. Primers are shown in Table 1. The expected sizes of EcAK and 18S rRNA used to analyze expression profiles were 132 bp and 147 bp, respectively. The qRT-PCR was performed according to the program of 40 cycles of 95 ◦C for 15 s, 55 ◦C for 20 s and 72 ◦C for 20 s, followed by an extension of 72 ◦C for 10 min. The data were analyzed according to comparative CT method and then subjected to one-way ANOVA using SPSS 19.0. The p values less than 0.05 were considered to be statistically significant.

2.4. Recombinant expression and purification of EcAK in P. pastoris

Before the recombinant plasmid was constructed, the sequence GTCGAC representing the restriction site of Sal I was mutated to GTA- GAC using reverse PCR and named EcAK(279C > A). There is no change in the encoding amino acid sequence. Then, based on the information of EcAK(279C > A) and multiple cloning sites (MCS) in the pPIC9K, a pair of primers 9k-EcAKF/9k-EcAKR was designed and used to construct recombinant expression plasmid pPIC9k-EcAK(279C > A) according to our previous research [17]. The PCR product and pPIC9k were doubly digested with EcoR I and Not I separately and the digested PCR product was ligated into digested pPIC9k. The resulting constructs pPIC9k-EcAK (279C > A) was transformed into the E. coli DH5α and verified by sequencing. The recombinant plasmid pPIC9k-EcAK(279C > A) was extracted and linearized with Sal I followed by transformation with
P. pastoris KM71 using PEG1000. Genomic DNA of the transformants was isolated and PCR amplifications were carried out to select positive
clones according to Invitrogen’s recommendations with a pair of primers (5′ AOX1/3′ AOX1) (Table 1) following the Invitrogen’s protocol.

For each positive clone, small-scale expression trials were initially performed to identify the most productive transformants by SDS-PAGE using 15% (w/v) separating gel and 5% (w/v) stacking gel. Once the most productive transformant was selected, a large-scale expression of recombinant EcAK was performed and was used to purify the recombi- nant EcAK by affinity chromatography using Ni-NTA-agarose resin [18].

2.5. Enzymatic assay for recombinant EcAK activities

Enzyme assay: The enzyme activity of purified rEcAK was monitored according to the method described by Li et al. [18]. The reaction miXture, containing 0.1 mL of 1 M Tris-HCl (pH 8.0), 0.1 mL of 100 mM L-arginine, 0.2 mL of 10 mM ATP-Na, 0.7 μL 2-mercaptoethanol and 10 μL diluted enzyme solution added ddH2O up to 1 mL. The rEcAK activity was determined by measuring the absorbance at 660 nm. One unit of AK is that amount of enzyme that catalyzes the formation of 1 μmol inor- ganic phosphate per min.

The optimum pH of purified rEcAK was determined by varying pH from 5.0 to 12.0. The optimum temperature was measured in different temperatures (ranging from 20 to 80 ◦C) at the optimal pH condition. Five metal ions (Ca2+, Mg2+, Cu2+ Mn2+, and Zn2+) were used to identify their effect on enzyme activity. Each metal ion was added into reaction miXture with a final concentration at 1 and 10 mM, and then the enzyme activity was determined immediately following the method described above.

3. Results
3.1. Characterization of EcAK

Based on the transcriptomic and genomic data of E. carinicauda [12], one full-length cDNA sequence of EcAK was obtained with 1592 bp (GenBank accession no. MW073459). As shown in Fig. 1, the sequence of EcAK contained a 1068 bp open reading frame (ORF) encoding EcAK precursor of 355 amino acids with a predicted molecular weight (MW) about 39489.55 Da and theoretical isoelectric point (pI) of 6.07. No putative signal peptide was found. The domain architecture of deduced EcAK analyzed by SMART on-line showed that there were an ATP-gua_PtransN domain (residues 9–76) and an ATP-gua_Ptrans domain (residues 133–349) (Fig. 2). In addition, the genomic DNA fragment of EcAK with the corresponding cDNA sequence was obtained, which showed that it was composed of 4 exons and 3 introns (Fig. 3). All intron-exon boundaries are consistent with the consensus splicing junctions at both the 5′ splice donor site (GT) and the 3’ splice acceptor sites (AG) of each intron. A multiple sequence alignment showed that EcAK displayed high identities with arginine kinase 1 of Neocaridina denticulata (NdAK, 98%) and Macrobrachium rosenbergii (MrAK, 98%) (Fig. 4).

3.2. Tissue distribution of EcAK mRNA

EXpression profile of EcAK in nine tissues of E. carinicauda was examined by qRT-PCR (Fig. 5). It was predominantly expressed in muscle, then in hepatopancreas, gill, and stomach. Therefore, muscle is served as the target tissue to study the expression profile after the prawns were challenged with bacteria.

3.3. Expression profiles of EcAK in the muscle of prawns challenged with V. parahaemolyticus or A. hydrophila

The expression of EcAK in the muscle of E. carinicauda was detected using quantitative RT-PCR method. The results showed that the expression of EcAK in muscle of prawns challenged with V. parahaemolyticus or A. hydrophila changed at a time-dependent manner (Fig. 6A and B). As shown in Fig. 6A, the expression of EcAK in muscle of prawns challenged with V. parahaemolyticus was up- regulated at 12 h (p < 0.05), and significantly up-regulated at 24 h (p < 0.01), and then returned to the control levels at 120 h post-challenge (p > 0.05). At the same time, the expression in Aeromonas-challenged group was up-regulated at 12 h (p < 0.05), and significantly up- regulated at 24 h (p < 0.01), and then returned to the control levels at 96 h post-challenge (p > 0.05) (Fig. 6B).

Fig. 1. The nucleotide sequence and deduced amino acid sequence of EcAK. ATP-gua_PtransN domain is underlined in pink, and the ATP-gua_Ptrans domain is underlined in blue. The double underlined sequence GTCGAC was the re- striction site of Sal I. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2. The domain architecture of EcAK.

Fig. 3. The genomic structure of EcAK.

Fig. 4. Alignment of the amino acid sequence of EcAK with NdAK and MrAK. The identical residues are shown in solid boXes. Sequences start at the first methionine residue. N. denticulata NdAK (GenBank accession number, BAH56608.1); M. rosenbergii MrAK (ADN88091.1); Exopalaemon carinicauda EcAK (MW073459, in this study).

Fig. 5. Detection of EcAK transcripts in different tissues of E. carinicauda detected. Tissues were shown in the abscissa. The amount of EcAK mRNA was normalized to the 18S rRNA transcript level. Data are shown as means ± SD (standard deviation) of three separate individuals in the tissues.

3.4. Enzymatic characterization of recombinant EcAK (rEcAK)

Based on the sequence information of pPIC9K and EcAK, GTCGAC in the ORF of EcAK (Fig. 1), which is the restriction site of Sal I, was mutated to GTAGAC using reverse PCR before the recombinant plasmid was constructed. There is no change in the encoding amino acid sequence. The constructed recombinant plasmid, pPIC9k-EcAK(279C > A), was linearized with Sal I and transformed into P. pastoris KM71 using PEG1000 method. The positive yeast cells integrating linearized pPIC9k-EcAK(279C > A) were selected for cultivation and induction using 1% (v/v) methanol at 28 ◦C. Then, EcAK was expressed with the intent to secrete it into the culture media and contained an N-terminal His-Tag for rapid purification at the native condition using affinity chromograph. The purified rEcAK was used to study its enzymatic characterization, including the optimal pH and temperature, the effects of metal ions.

Fig. 6. EXpression profiles of EcAK in the muscle after the prawns were chal- lenged with Vibrio parahaemolyticus (A) or Aeromonas hydrophila (B) and equal volume of PSS at 0, 12, 24, 48, 72, 96, and 120 h. The expression of EcAK mRNA was normalized to the 18S rRNA. Data are shown as means ± SD
(standard deviation) of three separate individuals in the muscle.

Fig. 7. Influence of pH and temperature on enzymatic activity of rEcAK. (A) pH.

The pH dependency of rEcAK is described by an unusual curve with an increase of activity over the pH range 5.0–8.0, followed by a decrease of activity over the pH range 8.0–12.0. (Fig. 7A). Then, the temperature optimum was at pH 8.0 as shown in Fig. 7B, revealing an optimal tem- perature of 30 ◦C. Under the optimal pH and temperature conditions, five metal ions (Ca2+, Mg2+, Cu2+, Mn2+, and Zn2+) were used to identify their effect on enzyme activity. The results showed that rEcAK activity was partially inhibited by the presence of 1.0 mM or 10 mM Cu2+ and Zn2+. In addition, the rEcAK activity was increased by the presence of 1.0 mM or 10 mM Mg2+ and Mn2+. However, there is no effect by the presence of 1.0 mM or 10 mM the Ca2+ (Table 2).

4. Discussion

Arginine kinase plays an important role in both temporal and spatial ATP buffering in cells with high, fluctuating energy requirements [1]. In crustaceans, several AK had been reported, including M. rosenbergii [1], Callinectes sapidus [4], M. japonicus [8], L. vannamei [6,19,20], F. chinensis [21], Metapenaeus ensis [22], Portunus pelagicus [23], Pro- cambarus clarkii [24], Scylla paramamosain [25], Euphausia superba [26], S. serrata [27], N. denticulata [28], etc.Herein, one arginine kinase gene in E. carinicauda was obtained and optimum. (B) Temperature optimum.
named it EcAK. The deduced amino acid sequence of EcAK revealed a typical ATP-gua_PtransN domain (residues 9–76) and an ATP- gua_Ptrans domain (residues 133–349). The domain structure suggested that EcAK has arginine kinase activity. Comparison of the deduced amino acid sequence of EcAK indicated that it was high similar to NdAK-1 from N. denticulata (98%) and MrAK-1 from M. rosenbergii (98%). It was reported that AK evolved independently at least three times, however, Iwanami et al. [28] presented a possible fourth AK lineage by analyzing three AKs (AK1, AK2 and AK3) in the shrimp N. denticulata. The genomic organization of EcAK gene analyzed by the published genome sequence showed that EcAK gene contains four exons and three introns. This is the first report about intron/exon structure characteristics of prawn AK gene. In contrast, the SpAK gene from
S. paramamosain only contains two exons and one intron [25].

In addition, the expression of EcAK was highest in muscle, which was similar with MrAK-1 from M. rosenbergii [1] and LvAK from L. vannamei [20]. Previously, researchers reported that AK might be involved in immune response of the mud crab, S. paramamosain after challenge with V. alginolyticus [23]. In this study, the immune function of EcAK against bacteria was clarified by challenging the prawns with
V. parahaemolyticus or A. hydrophila. Analyzing by real time RT-PCR method, the expression of EcAK in muscle was significantly upregu- lated after V. parahaemolyticus or A. hydrophila challenge, which indicated that EcAK might play a key role in immune defense against bacteria. It had been reported that AK was related to the immune de- fense in crustaceans, such as M. japonicus [9], L. vannamei [20], P. clarkii [24], and S. paramamosain [25], etc.

P. pastoris is one of the most important host organisms for the re- combinant production of proteins [29]. No previous work was reported concerning the heterologous expression of AK in P. pastoris. In this research, the recombinant EcAK (rEcAK) was obtained using P. pastoris because it does not produce endogenous AK. An N-terminal His-Tag containing in the rEcAK facilitates the purification using affinity chro- mograph at the native condition. The partial enzymatic characterization of the purified rEcAK was also confirmed and the optimal temperature and pH was similar to that from other crustaceans. The pH and tem- perature optima of rEcAK was 8.0 and 30 ◦C, which was consistent with the native AK purified from the muscle of E. superba [26]. In conclusion,AK 7 EcAK may play the same biological activity in E. carinicauda as that from other crustaceans.