The course of phosphorylation and non-phosphorylation of protein is a significant molecular mechanism that regulating function protein activity (Hunter, 1995). Cell division cycle 25 (Cdc25), which has important physiological functions, is one of the dual specificity phosphatase (DSP) that can dephosphorylate the phosphate domains on the target protein tyrosine residue and serine/sine residue (Lavecchia et al., 2010). During cell division, Cdc25 activates cyclin-dependent kinase 1 (CDK1) by dephosphorylation that works with cyclin B to influence cell cycle (Lew and Kornbluth, 1996). DNA damage will induce a collection of biochemical reactions, inhibiting the effect of Cdc25C on cell cycle-dependent protein kinases (CDKs) and preventing cell division (Sanchez et al., 1997). Cdc25 plays a critical role in nervous system development in the course of embryonic development (Nakajo et al., 2011). Moreover, some studies prove that Cdc25 participates in heat stress, antioxidation, hyperosmolarity and other stress response (Folch-Mallol et al., 2004).

Sipunculus nudus that distributes in the warm water area, as one of the crucial precious marine products in south china, has high nutritional value and medicinal properties (Li et al., 2017; Zhang et al., 2018). The natural resources of S. nudus are destroyed by deteriorated inshore environment further and increased fishing activity. At present, artificial breeding work has been gradually carried out in China, such as Suikai County, Zhanjiang City, Guangdong Province, Beihai City, Guangxi Province, and Danzhou City, Hainan Province, but the shortage of seed has become one of the factors in restricting the development of the industry. The output of S. nudus is far from enough to meet the market needs, even though the industrial aquaculture and the seedling rearing in earth ponds about S. nudus have been raised by scholars (Chen et al., 2015, Haiyang yuyu ye (Ocean & Fishery), 2015(8): 64-65; Wang et al., 2016, China Patent, ZL201610500667.7). Studying the molecular mechanism of germ cell development is beneficial to the further development and optimization of artificial breeding technology. At the beginning of the study, our research group screened a batch of genes that play an important role in the course of oocytes development from the transcriptome database we built of different developmental stages in S. nudus oocytes (Zhang, 2019). In our study, the full-length cDNA of Cdc25 was cloned from S. nudus (Sn-Cdc25) using RACE technology, and then we analyzed its sequence feature and relative expression of Sn-Cdc25 in oocytes at different developmental stages to explore the function of Sn-Cdc25 in the oocytes development process. The above results have accumulated basic data for further understanding of the developmental mechanism of Sipuncula oocytes and the optimization of artificial breeding techniques.

1 Result and Analysis

1.1 Gene sequence analysis of Sn-Cdc25

The gene sequence of Sn-Cdc25, which has the active site sequence HCX₅R, is 4 130 bp in length, including 3'UTR 1 849 bp and 5'UTR 427 bp and the open reading frame (ORF) is 1 854 bp which encodes 617 amino acids (Figure 1).

Figure 1 The full length and the deduced amino acid sequence of Sn-Cdc25 cDNA sequence Note: The 5' and 3' UTR are indicated with lower case letters; Coding regions and deduced amino acid sequences are shown in uppercase letters; The M-phase inducer phosphatase domain is displayed with a character border; The underlined part is the Rhodanese-like domain; HCX5R, the sequence of the active site of Cdc25 is highlighted with a gray background

1.2 Sequence analysis of Sn-Cdc25 protein

The prediction of physicochemical properties shows that the molecular weight of Sn-Cdc25 protein is 69.58 kD and the theoretical isoelectric point is 6.59. The grand average of hydropathy (GRAVY) is -0.845, which means it belongs to hydrophilic protein.

The prediction of the secondary structure shows that the random coil of Sn-Cdc25 protein is the highest (66.61%), and the alpha helix is the second highest (24.80%) that mostly exist C-terminal (Figure 2). The α-helix region (aa: 549-556) and the active site of Cdc25 are separated by two amino acid and they maybe perform the biological function mutually.

Figure 2 Prediction of secondary structure of Sn-Cdc25 protein Note: The sequence of the active site of Cdc25 on red background, the blue background is the α-helix region adjacent to the active site

1.3 Tertiary structure analysis of Sn-Cdc25 protein

Forecast the protein tertiary structure of S. nudus, Lingula anatine (XP_013383189.1), Octopus vulgaris (XP_029639960.1), Mizuhopecten yessoensis (XP_021352882.1). The result finds that the spatial conformation of Cdc25 homologous protein and their active site is highly conservative, and the invagination of the active site is shallow (Figure 3). Our study speculate that it may be compatible with Cdc25 protein function.

Figure 3 Three dimensional structure of homologous Cdc25 protein in S. nudus (a), L. anatine (b), O. vulgaris (c) and M. yessoensis (d) Note: The yellow part is sequence of the active site, HCX5R

1.4. Homology analysis of Sn-Cdc25

1.4.1 Multiple sequence alignment and Motif analysis of Cdc25

Take the multiple sequence alignment about O. vulgaris, Crassostrea gigas (XP_011440880.1), M. yessoensis, L. anatine, Pomacea canaliculata (XP_025096647.1), Aplysia californica (XP_012937345.1), Biomphalaria glabrata (XP_013070587.1), Danio rerio (NP_001108567.1), Cricetulus griseus (NP_001278987.1), Aquila chrysaetos chrysaetos (XP_029863893.1), three subtypes protein of Homo sapiens (Cdc25A:NP_001780.2; Cdc25B:NP_068659.1; Cdc25C; NP_001781.2) and the protein of Sn-Cdc25. The result shows that the homology and conservation of Cdc25 protein are higher in C-terminal relatively. In N-terminal, the homology is lower and the variation extent is larger relatively (Figure 4).

Figure 4 Multiple sequence alignment of Cdc25 homologous protein Note: Red indicates high conservatism, followed by blue; Consistency values are obtained by sequence comparison

Searching the homologous protein sequences of Sn-Cdc25 to take the Motif analysis. The result shows that five Motifs were found (Figure 5). Four of five Motif exist in C-terminal that further illustrates that the C-terminal of Cdc25 protein has higher conservation relatively. Through prediction and comparison by Tomtom, the result indicates that Motif 1 is Paxillin LD motif (ELME000125) and Motif 2 is the binding motif of the MYND domain (ELME000340). These conservative motifs may be closely related to the biological function of Cdc25.

Figure 5 Motif analysis of Cdc25 homologous protein

1.4.2 Phylogeny and domain analysis of Cdc25

Building phylogenetic trees and forecast conservative domain for 15 homologous protein sequences，which shows that all of them have Rhodanese-like domain, but part of invertebrate (A. californica and P. canaliculata) don't have M-phase inducer phosphatase domain(Figure 6). Phylogenetic tree analysis reveals that Cdc25s are clustered into two branches: invertebrate and vertebrate, Sn-Cdc25 is clustered into the invertebrate branch with L. anatine, O. vulgaris and so on (Figure 6). Sn-Cdc25 protein has two typical Cdc25 protein domains mentioned above (Figure 1, Figure 6).

Figure 6 Phylogenetic trees and domains of homologous Cdc25 Note: The green square is the M-phase induced phosphatase domain; the yellow square is the RHOD domain

1.5 Expression profile analysis of Sn-Cdc25

Test the expression level of Sn-Cdc25 in six different developmental stages of oocytes by qRT-PCR. The result shows that Sn-Cdc25 express in all developmental stages of oocytes and expression trend overall show twin-peak patterns. While the different periods of oocytes in coelomic fluid (O1-O4), the expression of Sn-Cdc25 is lower significantly when the oocytes develop the early stage of yolk formation (O1) than other stages(p<0.05). And then, the expression goes up first and then down which reaches the peak in the late stage of vigorous yolk synthesis (O3). When oocytes enter nephridium the expression of Sn-Cdc25 rises sharply again and significantly higher than in other periods. The expression of Sn-Cdc25 is down obviously when the oocytes discharged outside the body (O6) and is lower than O5 significantly, but the difference with O3 is not significant (Figure 7).

Figure 7 Relative expression of Sn-Cdc25 in oocytes at different developmental stages Note: O1~O4: Different periods oocytes in coelomic fluid; O5: Oocytes in nephridioduct; O6: Oocytes in vitro; There is significant difference between different lowercase letters (p<0.05)

2 Discussion

Phosphorylation of protein by protein kinase and dephosphorylation by protein phosphatase are significant molecular mechanisms that regulate function protein activity (Hunter, 1995). Protein phosphatase is divided into serine/threonine phosphatases, tyrosine phosphatase and DSP. DSP has both the functions of serine/threonine phosphatases phosphorylating serine/threonine and tyrosine phosphatase to dephosphorylate tyrosine (Lavecchia et al., 2010). Now, studies have shown that Cdc25 is an important DSP that participates in regulating cell cycle, responding to DNA damage, early embryonic development, stress response and other biological processes (Lew and Kornbluth, 1996; Sanchez et al., 1997; Folch-Mallol et al., 2004; Nakajo et al., 2011).

The full-length cDNA of Sn-Cdc25 from S. nudus was cloned using RACE technology. The homology analysis finds that the homology of Cdc25 protein is higher in C-terminal relatively (Figure 4), and motifs exist in C-terminal (Figure 5). All Cdc25s have Rhodanese-like domain in C-terminal while M-phase inducer phosphatase domain in N-terminal don't (Figure 6). Above result prove that the C-terminal of Cdc25s is more conservative than N-terminal. Study has shown that the N-terminal of Cdc25s is easy to form variable splicing so that it could form the different protein (Zwergel et al., 2017), which causes the N-terminal of Cdc25s to change.

Our study found that Sn-Cdc25 and homologous protein both have Rhodanese-like domain including the active site sequence HCX5R that exist in protein tyrosine phosphatase (PTPs) (Andersen et al., 2001). Hofmann found that the catalytic domain of Cdc25 is similar to the domain of Rhodanese by series comparison analysis, and thought that all PTPs and DSP have evolve from rhodanese precursor protein and the structural difference between the Cdc25 protein family and ancestor proteins is minimal after repeated spectrogram search and analysis (Hofmann et al., 1998). That is the reason why Cdc25s and PTPs both have the homologous active site sequence HCX5R.

A study pointed out that the active site HCX5R could be combined with the corresponding phosphoamino acids, which catalyze phosphorylation reaction (Zwergel et al., 2017). The high conservation of the active site of Cdc25 protein in different species that is important for Cdc25 protein to perform similar functions. The protein tertiary structure predicted result finds that the spatial conformation of homologous Cdc25 protein in S. nudus, L. anatine, O. vulgaris and M. yessoensis and their active site is highly conservative, the active site has a flat conformation and shallow indentation (Figure 3). The α-helix region (aa: 549-556) and the active site of Cdc25 that are separated by two amino acid. A study points out that the α-helix region adjacent to the active site of Cdc25 is useful for cysteine residue to deprotonate, which promote the beginning of dephosphorylation (Andersen et al., 2001). Based on this, our study speculates that the α-helix region adjacent to the active site of Sn-Cdc25 may be useful for cysteine residue to deprotonate, and then the dephosphorylation of phosphorylated amino acids by Sn-Cdc25 is initiated.

After the analysis of Motif, our study finds that the Motif1 is Paxillin LD motif. Paxillin, as the adhesive adaptor protein, have five Leucine-rich sequences at the end of the sequence that is called LD motif (Tumbarello et al., 2002). The LD motif that has high conservation mainly takes part in the protein recognition about cytoskeletal reorganization (Nikolopoulos and Turner, 2000). Some studies show that after the Cdc25 is suppressed, which will hinder the normal assembly of spindle and the capture of chromosome metaphase (Cazales et al., 2007; Potapova et al., 2011). This blocking effect may be related to the Paxillin LD motif. The motif2 is the binding sequence of the MYND domain. Nuclear receptor corepressor 1 (NCOR1, UniProtKB ID: O75376) and Nuclear receptor corepressor 2 (NCOR2, UniProtKB ID: Q9Y618) both have this motif. Studies point out that the MYND domain exists in many nucleoproteins, such as BS69, Bop and PDCD2. And it is related to transcriptional repression (Gottlieb et al., 2002; Scarr and Sharp, 2002; Spadaccini et al., 2008). NCOR and MYND could interact and suppress cell proliferation and differentiation (Liu et al., 2007). Based on this, speculate that Sn-Cdc25 may have similar functions as NCOR, Sn-Cdc25 may take part in processes such as transcription and cell proliferation by interacting with proteins with MYND domains.

Building phylogenetic trees and forecasting conservative domain for different Cdc25 protein shows that Cdc25 is clustered into two branches: invertebrate and vertebrate, Sn-Cdc25 is clustered into the invertebrate branch with L. anatine, O. vulgaris and so on (Figure 6). The result implies that Cdc25 protein has some differentiation in vertebrate and invertebrate group.

According to the ultrastructure of different size of oocytes, Zhang Jiawei defines the oocytes developmental stages from the point of cell cycle (Zhang, 2019). The result shows that O1-O2 are mainly in the DNA synthesis prior phase (G1 phase), O3-O4 are in from DNA synthesis phase (S phase) to the first meiotic prophase, O5-O6 are in meiotic metaphase. Our study has tested the expression of Sn-Cdc25 in six different developmental stages. The expression tendency is bimodal and two peaks occur in O3 and O5 (Figure 7).

There are three types of Cdc25 protein (Cdc25A, B and C) playing a different role in cell cycle that were found in mammal (Gasparotto et al., 1997). The multiple sequence alignment result shows that Homo sapiens Cdc25B is the most like Sn-Cdc25 (30.77%). A study has shown that Cdc25B dephosphorylates CDK2/CyclinA to promote the cell cycle transition from G1 to S phase, which activates DNA replication (Frazer and Young, 2012). The expression of Sn-Cdc25 goes up rapidly from O1 to O3 is likely to help the oocytes transform from G1 to S phase.

Cdc25 catalyzes the phosphorylation of CDK to activate MPF, which will set off the depolymerization of nuclear lamina and nuclear pore complex then cause the disruption of nuclear membrane during the process of mitosis G2/M-phase or from meiotic prophase MI to metaphase MI (Gabrielli et al., 1996; Margalit et al., 2005; Schmidt et al., 2017). Above process is called Germinal vesicle breakdown (GVBD) in invertebrate. Our study finds that the expression of Sn-Cdc25 rises sharply that is beneficial to activate MPF to promote the GVBD, which could promote stages of meiosis in the stage from O4 to O5 (before and after the oocytes enters the metanephridia). That is why the expression of Sn-Cdc25 changing cyclically could allow the meiosis of oocytes to proceed in an orderly manner.

3 Materials and Methods

3.1 Experimental material

The Sipunculus nudus for experiments was collected in Caotan Town, Zhanjiang City, Guangdong Province, China in October 2018. Sipunculus nudus that could bore through the sand with strength and whose body walls are intact was selected to experiment, their weight is (9.68±1.55) g. Using the sampling scheme that built by the search group (Zhang et al., 2019; Zhou et al., 2019), we separate four developmental stages of oocytes sample from the body fluid cavity. According to the particle size from small to large, these oocytes are named as Oocyte1 (>48 μm, the early stage of yolk formation), Oocyte2 (48~108 μm, the early stage of vigorous yolk synthesis, O2), Oocyte3 (108~150 μm, the late stage of vigorous yolk synthesis, O3) and Oocyte4 (>150 μm, maturity, O4). The oocytes separate from metanephridia named Oocyte5 (O5). The oocytes that discharge outside the body called Oocyte6 (O6).

3.2 Full-length cDNA cloning of Sn-Cdc25

The total RNA extraction and synthesis of the first-strand cDNA can be done in Lai Zhuoxin's way (Lai et al., 2019). Using the part of Sn-Cdc25 sequence from transcriptome database that was got in the early stage of study to design the primers by Primer premier 6.0 (Table 1).

Table 1 Primer sequence used in the cloning and real-time PCR of Sn-Cdc25 gene

Using nested PCR amplification to amplify the 3' UTR and 5' UTR of Sn-Cdc25. Testing the product by 1% agarose gel electrophoresis and combining the product with the carrier of pMD18-T. And then, moving the ligation product into the DH5α competent cell, which is cultured 12 h on solid ampicillin selection medium. Selecting positive monoclonal flora to test by PCR. The last, sending the qualified bacterial solution to the Sangon Biotech (Shanghai) Company for sequencing.

3.3 Sequence analysis of Sn-Cdc25

Splicing the sequencing result and obtain the full-length cDNA of Sn-Cdc25 by the use of DNAMAN software. Predicting the ORF and amino acid sequence of Sn-Cdc25 with ORF Finder (https://www.ncbi.nlm.nih.gov/ orffinder). Using ProtParam (https://web.expasy.org/protparam/) to forecast the physicochemical properties of Sn-Cdc25 protein. Using NCBI Blast Online analysis tools (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to search homologous protein. The multiple sequence alignment by NCBI COBALT (https://www.ncbi.nlm.nih.gov/tools/ cobalt/) and DNAMAN 9. Forecasting the conserved domains of homologous protein by the use of Pfam (https:// pfam.xfam.org/search). Using SOPMA (https://npsa-prabi.ibcp.fr/NPSA/npsa_sopma.html) to predict secondary structure. Forecasting the tertiary structure by use of phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cg- i?id=index). Analyzing Motif by MEME (http://meme-suite.org/index.html) and drawing the phylogenetic tree by Mega X.

3.4 Differential expression analysis of Sn-Cdc25

Take 60S-L7 as the reference gene to test the expression level of Sn-Cdc25 in different developmental stages of oocytes. The reaction system of 10 μL：upstream primer and downstream primer each 0.4 μL，SYBR® Select Master Mix 5 μL，cDNA template 0.4 μL，sterile water 3.8 μL. Run qRT-PCR in RocheLightCycler®96 System，the operation conditions is 95°C, 5 min；95°C, 10 s, 60°C, 15 s and 72°C, 20 s, 40 circulations. Calculate the relative expression of Sn-Cdc25 by the method of 2^-ΔΔCt. Use IBM SPSS Statistics 19 software for significance analysis (confidence level is 95%).

Authors’ contributions

Su Yonglin is the experimental executor and author of the paper of the study; Liu Qi, Ye Jianming, Zeng Yetao and Zhang Wei participated in the research of the experiment, the analysis of the experimental results and the processing of the data; and Wang Qingheng was the designer of the experiment and responsible for the revision of the thesis. All authors read and approved the final manuscript.

Acknowledgments

The study was jointly funded by Science and Technology Planning Project of Guangdong Province, China (2016A020209010; 2017A030303075) and Science and Technology Innovation Project for College Students of Guangdong Province, China (201810566049).

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