Identification and characterization of a phospholipid scramblase encoded by planarian Dugesia japonica

Accepted Manuscript Identification and characterization of a phospholipid scramblase encoded by planarian Dugesia japonica

Yu Han, Ao Li, Lili Gao, Weiwei Wu, Hongkuan Deng, Wenjing Hu, Na Li, Shimin Sun, Xiufang Zhang, Bosheng Zhao, Baohua Liu, Qiuxiang Pang PII: DOI: Reference:

S0378-1119(16)30928-3 doi: 10.1016/j.gene.2016.11.029 GENE 41681

To appear in:

Gene

Received date: Revised date: Accepted date:

24 August 2016 27 October 2016 15 November 2016

Please cite this article as: Yu Han, Ao Li, Lili Gao, Weiwei Wu, Hongkuan Deng, Wenjing Hu, Na Li, Shimin Sun, Xiufang Zhang, Bosheng Zhao, Baohua Liu, Qiuxiang Pang , Identification and characterization of a phospholipid scramblase encoded by planarian Dugesia japonica. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Gene(2016), doi: 10.1016/j.gene.2016.11.029

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ACCEPTED MANUSCRIPT Identification and Characterization of a Phospholipid Scramblase Encoded by Planarian Dugesia japonica Yu Han1,2, Ao Li1, 2¶, Lili Gao1, 2¶, Weiwei Wu1, 2, Hongkuan Deng1,2, Wenjing Hu1,2, Na Li1,2, Shimin Sun1,2, Xiufang Zhang1, Bosheng Zhao1,2§, Baohua Liu1,2,3§, Qiuxiang Pang1,2* Laboratory of Developmental and Evolutionary Biology, School of Life Sciences, Shandong

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Anti-aging ＆ Regenerative Medicine Research Institution, School of Life Sciences,

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University of Technology, Zibo, Shandong, 255049, China.

Shandong University of Technology, Zibo, Shandong, 255049, China. Shenzhen University of Health Science Center, Shenzhen, Guangdong, 518060, China

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Co-first author: Ao Li¶, Lili Gao¶

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¶

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Corresponding author: [email protected]

Co-corresponding author: [email protected]; [email protected]

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Highlights

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japonica.

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A phospholipid scramblase gene (DjPLSCR) is isolated and characterized from planarian D.

mRNAs of DjPLSCR are predominantly expressed in the pharynx of intact adult and regenerating planarians. DjPLSCR may participate in the immune response upon pathogen invasion.

ACCEPTED MANUSCRIPT Abstract Phospholipid scramblases (PLSCRs) are the conserved calcium-binding, type II transmembrane proteins synthesized in all eukaryotic organisms. In mammals, these proteins play essential roles in various physiological processes, especially in the immune responses.

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However, the existence of PLSCRs and their biological functions in planarian are still

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unknown at present. In this study, a new member of PLSCRs was identified in planarian

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Dugesia japonica (D. japonica), named DjPLSCR. The sequence analysis revealed that it contains an opening reading frame consisting of 897 bp encoding a putative protein of 241

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amino acids with a predicted molecular mass of ~28.7 kDa and an isoelectric point of 6.21.

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Whole-mount in situ hybridization showed that mRNAs of DjPLSCR are predominantly expressed in adult and regenerative pharynx which is an important organ of immune system

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in planarians. Importantly, we found that the transcription level of DjPLSCR was significantly

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upregulated when planarians were stimulated with the pathogen-associated molecular patterns [polyinosinic-polycytidylic acid, lipopolysaccharide, peptidoglycan and β-glucan],

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suggesting that DjPLSCR is involved in the immune response upon pathogen invasion. Our

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findings provide the first experimental insights into the characteristics and potential functions of PLSCR in planarians. Keywords: Dugesia japonica; Phospholipid scramblase; Expression pattern; Immunity

ACCEPTED MANUSCRIPT 1. Introduction Phospholipid scramblase (PLSCR) is a group of homologues, ATP-independent, lipid-raft-associated plasma membrane proteins, which involve in the Ca2+-dependent movement of phospholipid between membrane leaflets (Wiedmer et al., 2000; Kodigepalli et

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al., 2015). It is a conserved protein that is found in all eukaryotic organisms. PLSCR has

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several potential functional domains, including a proline-rich N-terminal region, a

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cysteine-rich region, a conserved calcium ion binding motif (EF-hand-like), a putative transmembrane region, a nonclassical “nuclear localization signal” (NLS) and a DNA binding

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motif, among which the NLS and DNA binding motif are always identified in PLSCR1,

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which participates in the gene transcriptional regulation (Zhou et al., 2005; Chen et al., 2005; Ben-Efraim et al., 2004; Sahu et al.,2007).

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The PLSCR orthologs appear to be conserved from Caenorhabditis elegans (C. elegans)

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to humans, suggesting a pivotal role for them in various physiological processes. Previous studies have shown that PLSCR involves in destroying plasma membrane phospholipid

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asymmetry (Wiedmer et al., 2000), lipid metabolism (Wiedmer et al., 2004), transcriptional

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regulation (Chen et al., 2005), cell signaling (Sun et al., 2001), cell differentiation and proliferation (Chen et al. 2013). Recently, PLSCR1 mRNA level was found to upregulate with the response to the stimulation of lipopolysaccharide (LPS), zymosan and turpentine in mice (Lu et al., 2007); overexpression of PLSCR1 could protect cell from infection of Staphylococcus aureus a-toxin (Lizak et al., 2012). Moreover, PLSCR1 can suppress vesicular stomatitis virus proliferation via inhibiting the accumulation of primary virus transcripts (Dong et al., 2004). These results indicate that PLSCR plays an essential role in

ACCEPTED MANUSCRIPT the immune responses in mammals. However, in invertebrates, the characterization of PLSCR and its biological functions have been rarely studied. Planarian Dugesia japonica (D. japonica) has traditionally been a favored animal model in regeneration, development and immunity (Tejada-Romero et al., 2015; Liu et al., 2013;

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Abnave et al., 2014). Our previous studies reported that the humoral fluid in planarians

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contains phenoloxidase (Pang et al., 2010), lectin (Pang et al., 2012) and trypsin-like serine

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protease (Zhou et al., 2012). Recently, immune related genes and the RIG-I-like receptor signaling pathway in the freshwater planarian D. japonica has also been reported (Pang et al.,

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2016). However, there is still little information on the immune defense system in this

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evolutionarily important organism.

In this paper, we firstly identified a PLSCR gene from the D. japonica using rapid

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amplification of cDNA ends (RACE) technology. Using whole-mount in situ hybridization,

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we found that mRNAs of DjPLSCR were predominantly expressed in the pharynx of adult and regenerating planarians. By analyzing the DjPLSCR transcription alteration, we found

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that the transcription level of DjPLSCR was significantly upregulated when planarians were

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stimulated with different pathogen components, suggesting that DjPLSCR is an immune-related gene and involved in the immune response upon pathogen invasion.

2. Materials and methods 2.1. Animals D. japonica used in this study were collected from a spring water in Tumen, Yiyuan, Shandong province, China. The animals were cultured in Lushan fountain at 19°C and fed with beef liver every three days. After 24 h feeding, the fresh fountain was replaced.

ACCEPTED MANUSCRIPT Planarians were starved for one week before the start of the experiments. 2.2. Rapid Amplification of cDNA Ends (RACE) Total RNA of adult planarians was extracted using a Trizol reagent (Invitrogen, California, USA) and was quantified by optical density measurements at 260 nm.

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5'/3'-RACE-Ready cDNA was synthesized from 1 μg total RNA using the ClontechSMARTer

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RACE cDNA Amplification Kit (Clontech, Japan) according to the manufacturer’s protocol.

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The gene-specific primers (DjPLSCR-5 and DjPLSCR-3) were designed on the basis of transcriptome sequencing of D. japonica (Pang et al., 2016). The corresponding transcripts of

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DjPLSCR were PCR amplified from 5'/3'-RACE-Ready cDNA using primer pairs

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DjPLSCR-5/5'-universal primer provided in the RACE Kit and DjPLSCR-3/3'-universal primer (all primers and probes are listed in Table 1). The PCR parameters were as the

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following: 94°C for 2 min, followed by 35 cycles of 30 s at 94°C, 30 s at 55°C and 45 s at

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72°C, with a final step at 72°C for 10 min. The PCR products were purified with a gel extraction kit (Omega, Beijing) and were cloned into the pMD18-T vector (TaKaRa, Janpan)

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sequences.

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for sequencing. Finally, the full-length of DjPLSCR was obtained from the overlappling

2.3. Sequence analysis and phylogenetic tree construction The full-length cDNA and deduced protein sequences of DjPLSCR were analyzed with the DNA Tools program. Protein conserved domains were predicted using Conserved Domains network server at the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). Sequence homology analysis of protein was performed using Megalign. The phylogenetic tree was constructed by

ACCEPTED MANUSCRIPT neighbor-joining method with 1000 bootstrap replications using the MAGE 4 software (Saitou et al., 1987). Multiple protein sequences alignment was performed using Megalign program. 2.4. Whole-mount in situ hybridization

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The intact planarians, approximately 2-4 mm, were selected for in situ hybridization as

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previously described (Pearson et al., 2009), with some modifications. Briefly, planarians were

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exposed to 5% N-Acetyl-L-cysteine for 5 min, and fixed in 4% paraformaldehyde for 20 min at room temperature. Subsequently, they were washed with phosphate-buffered saline

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containing 0.1% Triton X-100 (PBST) and incubated in Reduction buffer (the constituents of

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solution were listed in Table 2) for 15 min at 37°C. After the incubation, the samples were dehydrated with a 100% and 50% methanol, followed bleached in 6% H2O2 overnight under

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direct light irradiation. Samples were permeabilized with 10 μg/mL protease K solution

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(Sigma, USA) for 10 min, and then fixed again in 4% paraformaldehyde at room temperature. After being washed with PBST, samples were incubated in pre-hybridization and

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hybridization solution containing probes (Table 1) for 2 and 16 h at 56°C, respectively.

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Samples were washed with preheated solutions I, II, III, IV (Table 2) successively and performed with an anti-DIG antibody (1:1000, Roche), followed being washed by MABT washing buffer (Table 2) at least 6 times to avoid the nonspecific signal. Finally, samples were stained with NBT, eliminated nonspecific background signal with ethanol, and visualized with a Nikon SMZ 1500 stereomicroscope (Nikon, Japan). 2.5. Quantitative reverse transcription-PCR (qRT-PCR) Planarians were exposed to 10 μg/mL polyinosinic-polycytidylic acid (poly(I:C)), LPS,

ACCEPTED MANUSCRIPT peptidoglycan (PGN) or β-glucan (β-Glu). The untreated planarians being used as negative control were randomly selected. At different time points (0, 1, 5, 9, 12, 24 h) post treatment, total RNA was isolated as described above. Total cDNA was synthesized using the reverse transcription system (Thermo, USA). The RNA extracted from untreated planarians was used

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to determine the background level of DjPLSCR expression. qRT-PCR was performed using

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Fast Start Universal SYBR Green Master (Rox) (Roche, Switzerland) according to the

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manufacturer’s protocol. The qPCR data of gene were normalized to β-actin, and the relative ct

expression was calculated using the 2－△△ method (Schmittgen et al., 2008).

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3. Results

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3.1. Isolation and characterization of DjPLSCR cDNA

The full-length cDNA of DjPLSCR of D. japonica was achieved by RACE and

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deposited the nucleotide sequences in GenBank under Accession KX765181. As shown in

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Fig. 1, DjPLSCR contains 897 bp including a 5′-untranslated region (UTR) of 47 bp, a putative open reading frame (ORF) of 658 bp and a 3′-UTR of 124 bp. The 3′-UTR is found

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containing a canonical polyadenylation signal sequence (AATAA) and a poly (A) tail. The

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ORF encodes a putative protein of 241 amino acids with a predicted molecular mass of ~28.7 kDa and an isoelectric point of 6.21.

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Fig. 1. General features of the nucleotide sequence of DjPLSCR. The initiation codon and the termination codon were labeled with shade. Polyadenylation signal (AATAA) was indicated by rectangle. Poly(A) tail

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was underlined.

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3.2. Homology analysis and phylogenetic analysis To understand the characteristics of the DjPLSCR, protein conserved domains analysis

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were performed. Sequence analysis identified that DjPLSCR is a member of phospholipid

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scramblases because it contains all the conserved motifs of this family except for a proline-rich N-terminal region and a cysteine rich region (Fig. 2). Subsequently, amino acid sequences of DjPLSCR were aligned with other members of PLSCR family selected from invertebrates and vertebrates to analyze their evolutionary conservation. As shown in Fig. 2, four conservation motifs, including a DNA binding motif, a nonclassical NLS, a conserved Ca2+-binding EF-hand-like motif and a transmembrane motif (Sahu et al., 2007; Kodigepalli et al., 2015) were found in DjPLSCR.

ACCEPTED MANUSCRIPT To investigate the evolutionary relationships of DjPLSCR, a phylogenetic tree was inferred using the neighbor-joining method, and rooted into H. vulgaris PLSCR1. As shown in Fig. 3, all the PLSCRs were found clustering into two clades. Vertebrate PLSCR1s clustered into one clade, including HsPLSCR1, HsPLSCR2, MmPLSCR1, MmPLSCR2,

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GgPLSCR1, AcPLSCR2, XlPLSCR1, XlPLSCR2, DrPLSCR1, DrPLSCR3. DjPLSCR and

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the other five PLSCRs (CiPLSCR1, DmPLSCR1, DmPLSCR2, Cescrm1 and Cescrm4)

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clustered into one clade, among which DjPLSCR, Cescrm1 and Cescrm4 were clustered into an independent sub-clade, which was at the base of the tree. Our results indicated that the

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branch of PLSCR is classified according to the evolutionary position of species of cnidaria,

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platyhelminthes, nematoda, arthropoda, urochordata and chordata, and the phylogenetic

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position of DjPLSCR is consistent with the location of D. japonica.

ACCEPTED MANUSCRIPT Fig. 2. Multiple alignment of PLSCR sequences of D. japonica and 8 other members. GenBank accession numbers of PLSCR homologs used in this paper were listed as following: HsPLSCR1 (Homo sapiens phospholipid scramblase, NP_066928.1, 29.5%), HsPLSCR2 (NC_000003.12, 27.5%), HsPLSCR3 (NP_001188505.1, 26.2%), HsPLSCR4 (NP_001121776.1, 24.4%), CiPLSCR2(Ciona intestinalis,

vulgaris,

XP_002163447.2,

28.8%)

and

AqPLSCR2

(Amphimedon

queenslandica,

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(Hydra

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XP_002131364.1, 30.1%), Cescrm1 (Caenorhabditis elegans, NP_001251705.1, 26.7%)，HvPLSCR1

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XP_003382485.1, 29.0%). Highly conserved amino acids were labeled with shade. Five blocks of functionally crucial regions were labeled with roman numerals. The positions of the conserved functional

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motif were indicated being numbered I to V. (I) DNA binding motif; (II) cysteine palmitoylation motif; (III)

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NLS; (IV) Ca2+ binding EF-hand-like motif; and (V) transmembrane region.

Fig. 3. Phylogenetic tree of DjPLSCR proteins. The phylogenetic tree was constructed with the method of

ACCEPTED MANUSCRIPT the neighbor-joining building by MEGA4 software. The numbers refer to bootstrap values 1,000 replicates. HvPLSCR1 was selected as the outer group. DjPLSCR was marked with shade. GenBank accession numbers used in this paper were listed as following: HsPLSCR1 (NP_066928.1), HsPLSCR2 (NC_000003.12),

MmPLSCR1

(Mus

musculus,

NP_035766.2),

MmPLSCR2(NP_001182013.1),

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GgPLSCR1 (Gallus gallus, XP_001231237.1), AcPLSCR2 (Anolis carolinensis, XP_003230373.2),

DrPLSCR3

(NP_001098583.1),

CiPLSCR1

(XP_002121993.3),

DmPLSCR1

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XP_003201533.3),

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XlPLSCR1 (Xenopus laevis, NP_001089425.1), XlPLSCR2 (NP_001090508.1), DrPLSCR1 (Danio rerio,

(Drosophila melanogaster, AAF50165.3), DmPLSCR2 (AAF47705.1), Cescrm1 (NP_001251705.1),

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Cescrm4 (NP_492975.3) and HvPLSCR1 (XP_002163447.2).

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3.3. Localization of DjPLSCR mRNA in intact adult and regenerating planarians To investigate the spatial and temporal expression pattern of the DjPLSCR mRNAs in

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intact adult and regenerating planarians, whole-mount in situ hybridization was performed.

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As shown in Fig. 4A-B, mRNAs of DjPLSCR were mainly detected in the pharynx of adult intact. qRT-PCR also was performed to confirm the localization of DjPLSCR. Consistent with

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the whole-mount in situ hybridization mentioned above, the transcription levels of DjPLSCR

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in the middle fragment contained pharynx were significant higher than that in the head and tail fragments (Fig. 4C). Planarians were cut into two pieces (head and tail) and the localization of DjPLSCR mRNAs in regenerating fragments was reexamined. None of the hybridization signals in head or tail blastema was identified in the initial regeneration stages (Fig. 4D-G). But, mRNAs of DjPLSCR firstly emerged with newly pharynx formation at 5 days of regeneration (Fig. 4H-I). At 10 days of regeneration, mRNAs of DjPLSCR were located in the pharynx of regeneration

ACCEPTED MANUSCRIPT planarians, which had a similar distribution pattern with that in adult intact (Fig. 4L-M). Moreover, this localization pattern remained at 12 days of regeneration (Fig. 4N-O). These results indicated that mRNAs of DjPLSCR are mainly expressed in the pharynx of intact adult

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and regenerating planarians.

Fig. 4. The spatial and temporal expression pattern of DjPLSCR in intact adult and regenerating planarians.

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Whole-mount in situ hybridization was performed with DjPLSCR probe to detect the localization of the DjPLSCR mRNAs. (A) Planarian without adding DjPLSCR probe as the negative control. (B) The distribution of DjPLSCR mRNAs in the adult intact planarians was shown as the blue foci. (C) The transcription levels of DjPLSCR in the head, tail or middle fragments. Planarians were cut into three fragments and pharynx was located in the middle fragments. The transcription levels were normalized to that of β-actin transcripts and were shown as the percentages of the corresponding genes in the tail. Data were analyzed by Student’s t test. *, p< 0.05, **, p < 0.01. Error bars indicate standard deviations from

ACCEPTED MANUSCRIPT three independent experiments. (D-O) Whole-mount in situ hybridization showing the distribution of DjPLSCR mRNAs in regenerating planarians at different time points (1, 3, 5, 7, 10, 12 days) regeneration. White bars represent 260 μm.

3.4. Expression pattern of DjPLSCR upon pathogen components stimulation

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Considering that PLSCR plays essential roles in immune responses in mammals and

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DjPLSCR mRNAs mainly located in the pharynx, a crucial immune organ in planarians, we

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speculated that DjPLSCR performs the same function. To investigate the potential involvement of DjPLSCR in immune responses, qRT-PCR was performed to analyze the kinetics

of

DjPLSCR

in

planarians

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transcription

stimulated

with

the

different

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pathogen-associated molecular patterns including poly(I:C), LPS, PGN and β-Glu, respectively. Untreated planarians were used as control. As shown in Fig. 5A, at 1 and 5 h

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after stimulated with poly(I:C), the transcription level of DjPLSCR was increased by 8- and

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5-fold compared to that in control. The transcription of this gene was then gradually downregulated and returned to the normal level at 9 h post treatment. Similar to the

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expression pattern in planarians stimulated with poly(I:C), the transcription level of DjPLSCR

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reached to the peak at 1 h after stimulated with LPS, and it was also gradually downregulated from 5 h to 9 h after stimulation (Fig. 5B). However, a slight recovery was observed at 12 h after stimulation. As shown in Fig. 5C, the transcription level of DjPLSCR in planarians stimulated with PGN displayed an ambiguous variation tendency. The transcripts of DjPLSCR were rapidly increased after stimulation for 5 h, and then they were downregulated to the normal at 9 h post treatment. However, the transcription level reached to the peak after stimulated with PGN for 24 h. In Fig. 5D, the highest transcription level of DjPLSCR was

ACCEPTED MANUSCRIPT observed at 1 h post treatment with β-Glu. It was then severely downregulated, and at 5 h post treatment, the transcription levels in stimulated planarians or control were comparable. Although DjPLSCR had multifarious expression patterns when planarians were stimulated with different pathogen-associated molecular patterns, the transcription level of it was

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significantly upregulated at early phase in the mass, suggesting that DjPLSCR participates in

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the immune response.

Fig. 5. qRT-PCR analysis of DjPLSCR transcription in planarians stimulated with the pathogen-associated molecular patterns. Planarians were stimulated with 10 μg/mL poly(I:C), LPS, PGN or β-Glu. At different time points (0, 1, 5, 9, 12, 24 h) post treatment, transcription patterns of DjPLSCR were measured by qRT-PCR. The transcription levels were normalized to that of β-actin transcripts and are shown as the percentages of the corresponding genes in untreated planarians. Data were analyzed by Student’s t test. *,

ACCEPTED MANUSCRIPT p< 0.05, **, p < 0.01. Error bars indicate standard deviations from three independent experiments.

4. Discussion Planarian are naturally exposed to various pathogens but typically survive because of its powerful innate immune system (Peiri et al., 2014). However, the immunity and

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immune-related genes of planarian remain largely unexamined at present. Recently, PLSCRs

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are found playing an essential role in the immune responses in mammals (Dong et al., 2004;

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Lizak et al., 2012). In planarian, the existence of PLSCRs and their biological functions have not been determined unambiguously.

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In this study, we first cloned the full-length cDNA of PLSCR from the D. japonica,

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named DjPLSCR. Sequence analysis identified that it is a member of scramblases, because DjPLSCR containes all the conserved domains of this family except for a proline-rich

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N-terminal region and a cysteine rich region. As shown in Fig. 2, the region comprising

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residues N23–D54 (I in Fig. 2) of DjPLSCR is the DNA-binding motif but it shows a low conservation with the others. Like the other PLSCRs, DjPLSCR has a nonclassical NLS

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(G191-Y199, III in Fig. 2) and a transmembrane motif (K222-E240, V in Fig. 2), which are highly

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conserved in all the sequences. Moreover, a proposed Ca2+ binding EF-hand-like motif exists in DjPLSCR (K207-D218, IV in Fig. 2) as well. Amino acids at position 1 (K207), 3 (K209), 5 (F211), 7 (V213), 9 (F215) and 12 (D218) are supposed to form a octahedrally loop to bind calcium ion, among which two D residues in 1st and 3th position of DjPLSCR have been replaced by K residue compared to that of human PLSCR1. Because mutation of amino acid residues at positions corresponding to 1, 3, 5, 7, 9 and 12 of EF-hand like motif in human PLSCR1 would result in a marked reduction in phospholipid scrambling activity (Zhou et al.,

ACCEPTED MANUSCRIPT 1998), DjPLSCR may have a low Ca2+-binding affinity and scramblase activity (Ye et al., 2004). Our results indicate that DjPLSCR has not a highly conserved cysteine motif or a rich proline-rich N-terminal region, unlike the other PLSCRs. The highly conserved cysteine

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(CCCPCC, II in Fig. 2) is an important functional region in scramblases, because this

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sequence is the site of palmitoylation and modification on which can regulate the trafficking

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and subcellular localization of PLSCR (Wiedmer et al., 2003). At present, this motif is conserved in most of the species, other than the yeast and planarian ortholog, implying a

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possible lack of palmitoylation in saccharomyces cerevisiae (S. cerevisiae) and planarian

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PLSCR homologue (Sahu et al., 2007). Another lacking motif is a proline-rich N-terminal region which shows the functional interaction with SH3 and WW domain containing proteins

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(Sahu et al., 2007). The location and the number of proline residues are not conserved in

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PLSCR homologues, and in C. elegans PLSCR (Sahu et al., 2007), S. cerevisiae PLSCR (Sahu et al., 2007), D. melanogaster PLSCR2 (Acharya et al., 2006), D. japonica PLSCR and

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hPLSCR2 (Wiedmer et al., 2000), this motif does not exist.

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Based on our transcription data, we found that the transcription level of DjPLSCR was significantly upregulated when planarians were stimulated with poly(I:C), LPS, PGN and β-Glu. This may result from the activation of immune response in which DjPLSCR plays a role as an immune-related protein. LPS, PGN and β-Glu is the components of cell wall in Gram-positive bacteria, Gram-negative bacteria and fungus respectively (Wiens et al., 2005; Kataoka et al., 2002), and poly(I:C) is the viral analogue (Liu et al., 2015), they have high antigenicity and have been widely used as immune stress reagents to induce immune

ACCEPTED MANUSCRIPT response (Gao et al., 2014; Yang et al., 2011). Previous studies demonstrate that PLSCR participates in the immune response in mammals (Dong et al., 2004; Lizak et al., 2012). For example, PLSCR1 mRNA level is upregulated with the stimulation of LPS in mice (Lu et al., 2007); PLSCR1 can reduce the infection of various pathogens, including Hepatitis B virus

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(Yang et al., 2012), vesicular stomatitis virus (Dong et al., 2004), encephalomyocarditis virus

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(Dong et al., 2004) and Staphylococcus aureus a-toxin (Lizak et al., 2012). In D. japonica,

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DjPLSCR may perform the same function. Moreover, the distribution of DjPLSCR mRNAs may help furtherly explain its immune-related function. Pharynx, the first line resistant to

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pathogens, is the crucial immune organ in planarians. Consistent with the potential function

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of DjPLSCR as a participator in immune response, DjPLSCR mRNAs should highly express at the crucial immune organs of planarians. Our result confirms that DjPLSCR mRNAs

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mainly locate at pharynx and supports the speculated idea that DjPLSCR participates in the

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immune response in invertebrate as in mammals. In summary, our experiments provide the first experimental insights into the

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characteristics and potential functions of PLSCR in planarians. These results provide a

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valuable information for further explorations into the molecular mechanism by which DjPLSCR participates in the immune response of planarians. Future studies will focus on the exact function and molecular mechanism of DjPLSCR in the immune response, which have been rarely studied in invertebrate. Furthermore, because we found that PLSCR family has several members in all species (Kodigepalli et al., 2015; Sahu et al., 2007) and planarian D.japonica may adopt a similar manner to encode several PLSCRs, future studies also should focus on cloning other members of this family from planarian D. japonica, which may help

ACCEPTED MANUSCRIPT us to explore the functions of this family.

Authors' contributions Conceived and designed the experiments: Qiuxiang Pang, Bosheng Zhao and Baohua Liu. Performed the experiments: Yu Han, Lili Gao and Weiwei Wu. Analyzed the data: Yu

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Han, Hongkuan Deng and Wenjing Hu. Contributed reagents/materials/analysis tools:

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Xiufang Zhang, Na Li and Shimin Sun. Contributed to the writing of the manuscript: Yu Han,

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Qiuxiang Pang and Ao Li. All authors read and approved the manuscript.

Acknowledgements

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This study was supported by the National Natural Science Foundation of China

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(31172074; 31572263) and the Natural Science Foundation of Shandong Province, China

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(ZR2014DM015).

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Acharya, U., Edwards, M.B., Jorquera, R.A., Silva, H., Nagashima, K., Labarca, P., et al,

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1 is imported into the nucleus by a receptor-mediated pathway and interacts with DNA.

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ACCEPTED MANUSCRIPT Table 1 Primer sequences used in this study Primer name

Sequence

RACE 5'-GGAGACATAAGTTCAGTCCTTTTCAG-3'

DjPLSCR-3

5'-AAGCATGTAGTATTCTCATTGGACC -3'

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DjPLSCR-5

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Real-time

5'-CAGATGGAGCGACGTTAT-3'

RT-DjPLSCR-3

5'-GGACTGAACTTATGTCTCC-3'

RT-Djβ-actin-F

5'-ACACCGTACCAATCTATG-3'

RT-Djβ-actin-R

5'-GTGAAACTGTAACCTCG-3'

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RT-DjPLSCR-5

5'-GGTCCAATGAGAATACTAC-3'

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T-DjPLSCR-5

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Probe

5'-GATCACTAATACGACTCACTATAGGGGTTGACCTGGTGG TGCTTG-3'

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T-DjPLSCR-3

ACCEPTED MANUSCRIPT Table 2 Formula in in situ hybridization

Formula

Reduction Buffer

50 mM DTT, 1% NP-40, 0.5% SDS in PBS

Protease K solution

2 ug/mL Proetinase-K, 0.1% SDS in PBST

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Name

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50% formamide, 5 × SSC, 1 mg/mL yeast RNA, Pre-Hybridization Solution

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1% Tween-20, 100 μg/mL heparin, 5 mM DTT,

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1 × Denhardt’s in PBST

10% Dextran Sulfate in Pre-Hybridization

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Hybridization Solution

Solution

100 mM maleic acid, 150 mM NaCl, 0.1%

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MABT

20×SSC

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Wash I

tween-20 in H2O, pH 7.5 3 M NaCl, 0.3 M sodium citrate in H2O, pH 7.0 Pre-Hybridization Solution Pre-Hybridization Solution : 2 × SSC=1:1

Wash III

2 × SSC

Wash IV

0.2 × SSC

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Wash II

ACCEPTED MANUSCRIPT List of abbreviations PLSCR – phospholipid scramblase Poly (I:C) – polyinosinic-polycytidylic acid LPS – lipopolysaccharide

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PGN – peptidoglycan

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β-Glu – β-glucan

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NLS – nuclear localization signal RACE – rapid amplification of cDNA ends

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NCBI – National Center for Biotechnology Information

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UTR – untranslated region

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ORF – open reading frame