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Immunoglobulin superfamily Dscam exhibited molecular diversity by in hemocytes of crustacean, Eriocheir sinensis

ARTICLE in FISH & SHELLFISH IMMUNOLOGY · JULY 2013 Impact Factor: 2.67 · DOI: 10.1016/j.fsi.2013.06.029 · Source: PubMed

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Xing-Kun Jin Wei-Wei Li East China Normal University East China Normal University

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Minhao Wu Qun Wang East China Normal University East China Normal University

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Available from: Qun Wang Retrieved on: 11 October 2015 Fish & Shellfish Immunology 35 (2013) 900e909

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Fish & Shellfish Immunology

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Immunoglobulin superfamily protein Dscam exhibited molecular diversity by alternative splicing in hemocytes of crustacean, Eriocheir sinensis

Xing-Kun Jin 1, Wei-Wei Li 1, Min-Hao Wu, Xiao-Nv Guo, Shuang Li, Ai-Qing Yu, You-Ting Zhu, Lin He, Qun Wang*

Department of Biology, School of Life Science, East China Normal University, No. 3663 North Zhong-Shan Road, Shanghai, China article info abstract

Article history: Be absent of adaptive immunity which have both specificity and memory, invertebrates seem to have Received 16 April 2013 evolved alternative adaptive immune strategies to resist various intruding pathogens. Whereas verte- Received in revised form brates could generate a wide range of immunological receptors with somatic rearrangement, in- 20 June 2013 possibly depend on alternative splicing of pattern-recognition receptors (PRRs). Recently, it Accepted 27 June 2013 has been suggested that a member of the immunoglobulin superfamily (IgSF), cell Available online 13 July 2013 adhesion molecule (Dscam), plays a crucial role in the alternative of in- vertebrates. At present, we successfully isolated and characterized the first crab Dscam from Eriocheir Keywords: Alternative adaptive immune sinensis. EsDscam has typical domain architecture compared with other Dscam orthologs, including one fi Chinese mitten crab signal-peptide, 10 immunoglobulin (Ig) domains, 6 bronectin type III domains (FNIII), one trans- Down syndrome membrane domain (TM) and one cytoplasmic tail. We had detected four hypervariable regions of (Dscam) EsDscam in the N-terminal halves of Ig2 (25) and Ig3 domain (30), the complete Ig7 (18) and also the Immunoglobulin domain transmembrane domain (2), potentially generate 27,000 unique isoforms at least. Transcription of Pathogen associated molecular patterns EsDscam were both a) detected in all tissues, especially in immune system, digestive system and nerve (PAMPs) system; b) significantly induced in hemocytes post lipopolysaccharides (LPS), peptidoglycans (PG) and b-1, 3-glucans (Glu) injection. Importantly, we had detected membrane-bound and secreted Dscam isoforms in E. sinensis, and showed that secreted isoforms were extensively transcribed post different PAMPs challenge respectively. Results from immuno-localization assay revealed that EsDscam evenly distributed in the cell surface of hemocytes. These findings indicated that EsDscam is a hypervariable PRR in the innate immune system of the E. sinensis. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction had successfully survived under the long time evolution pressure from various kinds of pathogens, hence they must have sufficient With the ongoing pressure for survival, animals evolved potential to diversify their immune strategies comparable with refinement of intricate immune systems to distinguish and elimi- those confronting jawed vertebrates [3]. In the last few years, a nate foreign “non-self” to maintain self-owned health, which plenty of studies had indeed observed the phenomenon that some consist of innate immune and adaptive immune [1]. Up to now, only degree of immune specificity existed in invertebrates, such as flea higher vertebrates were found to both have innate and adaptive (Daphnia magna), bumblebee (Bombus terrestris), fruit fly immunity which achieved through the vast diversity of somatically (), beetle (Tribolium castaneum) and shrimp rearranged immunological receptors such as antibodies [2]. (Litopenaeus vannamei) [4e8]. Invertebrates seem to have emerged Invertebrates rely solely on innate immunity as their major different ways to enable challenge-specific protection serving the mechanism to fight against pathogens intruding without any “true” same functionality as adaptive immunity to some extent lymphocytes and functional antibody. Nevertheless, invertebrate [9]. The analogous recognition function of these remarkably different immune systems involved innovative genetic modifica- tion of innate-immune-system components, such as Immuno-

* þ þ globulin Superfamily (IgSF) [10]. The IgSF molecules Corresponding author. Tel.: 86 (0) 21 54345224; fax: 86 (0) 21 62233754. fl E-mail addresses: [email protected], [email protected] (Q. Wang). appear either at the cell surface or in body uid, bind diverse 1 These two authors contribute equally to this work. ligands, and contribute to a variety of cellular activities, including

1050-4648/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsi.2013.06.029 X.-K. Jin et al. / Fish & Shellfish Immunology 35 (2013) 900e909 901 adhesion and immune responses in both vertebrates (also known crabs were injected into the arthrodial membrane of the last pair of as antibodies) and invertebrates [11]. In gastropod, fresh snail IgSF walking legs with approximately 100 ml of lipopolysaccharides (LPS molecules, the Ig domain-containing, fibrinogen-related proteins from E. coli, SigmaeAldrich), 100 ml of peptidoglycan (PG from (FREPs), were found to been capable of generating diverse mole- S. aureus, SigmaeAldrich) and 100 ml of zymosan (Glu from cules involved in internal defense [12]. In , more and S. cerevisiae, SigmaeAldrich) resuspended (500 mg/mL) in ESS more Dscam orthologs were found to have alternative (Eriocheir sinensis saline, 0.2 M NaCl, 5.4 mM KCl, 10.0 mM CaCl2, splicing features [13e19], and even acted as the central immune 2.6 mM MgCl2, 2.0 mM NaHCO3; pH 7.4) [18,23,24]; while one surveillance factors with extensive molecular diversity in inverte- control group with 100 ml ESS (pH 7.4). More than five crabs were brate innate immune system [13,14,16,18,19]. randomly selected at each time interval of 0 (as blank control), 2, 6, DSCAM, one of the largest IgSF members (220 kDa) discovered 12 and 24 h post each kind of PAMPs injection. Hemocytes were containing 10 Ig domains and 6 fibronectin type III (FN) domains, harvested according to methods above, and were stored at 80 C, which was first identified in the human 21q22 asso- after the addition of 1 mL Trizol reagent (Invitrogen, CA, USA) for ciated with Down Syndrome [20]. Invertebrate Dscam was initially subsequent RNA extraction. discovered as an axon guidance in insect nerve system, serving the same function as higher vertebrate [21]. Yet, in the 2.2. Total RNA extraction and first-strand cDNA synthesis N-terminal halves of the Ig2 and Ig3, the full Ig7 and trans- membrane domain, highly variable exons generated by alternative Total RNA was extracted from E. sinensis tissues sampled from Ò splicing were detected, may amazingly yield more than 38,000 Section 2.1 using Trizol reagent (RNA Extraction Kit, Invitrogen, unique isoforms, which were never been found in vertebrate CA, USA) according to the manufacturer’s protocol. The total RNA DSCAM [21]. Subsequently, in fat body cells and hemocytes, which concentration and quality were estimated using spectrophotom- both were major tissues of the insect immune system, Dscam was etry at an absorbance at 260 nm and agarose-gel electrophoresis found extensively expressed. Moreover, the immune function of respectively. Dscam was unveiled for its participation in bacteria recognition and Total RNA (5 mg) isolated from testis and hemocytes was reverse phagocytosis [13,14]. transcribed using the SMARTerÔ RACE cDNA Amplification kit In the present study, we investigate how this novel invertebrate (Clonetech, USA) for full-length cDNA cloning. For Quantitative RT- pattern recognition receptor (PRR) might be involved in the innate PCR expression analysis, total RNA (4 mg) was reverse transcribed immune response in crab. Base on the initial sequences from our using the PrimeScriptÔ Real-time PCR Kit (TaKaRa, Japan). previously constructed Eriocheir sinensis transcriptome profiles of testis [22], and hemocytes (unpublished), we firstly cloned and 2.3. Cloning of full length EsDscam cDNA characterized the EsDscam full-length cDNA sequence, not only identified its domain architecture but also compared its phyloge- Eriocheir sinensis Dscam partial cDNA sequence was extended netic similarity among animal orthologs. Detected the hypervari- using 50 and 30 RACE (SMARTerÔ RACE cDNA Amplification kit, able regions in the N-terminal halves of the Ig2 and the Ig3 Clontech), and designed -specific primers (Table S1, Fig. S1) domains, the entire Ig7 domain, and the transmembrane domain, based on the original transcriptome sequences from testis [22] and by which altogether not less than 27,000 unique isoforms could hemocytes (unpublished). The 30 RACE PCR reaction was carried out potentially generate. Through examination of tissue-dependent in a total volume of 50 ml containing 2.5 ml (800 ng/ml) of the first- and post-PAMPs-challenged mRNA expression patterns, we were strand cDNA reaction as a template, 5 mlof10 Advantage 2 PCR able to report immunological action and diversified inducibility of buffer, 1 ml of 10 mM dNTPs, 5 ml(10mM) gene-specific primer EsDscam post various PAMPs injection in E. sinensis. Moreover, we (Table S1, Fig. S1), 1 ml of Universal Primer A Mix (UPM; Clonetech, discovered membrane-bound and secreted EsDscam isoforms both USA), 34.5 ml of sterile deionized water, and 1 U 50 Advantage 2 at mRNA and protein level. These observations could potentially polymerase mix (Clonetech, USA). For the 50 RACE, UPM was used as provide a vehicle for further invertebrate immunology research. forward primers in PCR reactions in conjunction with the reverse gene-specific primers (Table S1). PCR amplification conditions for 2. Materials and methods both the 30 and 50 RACE were as follows: 5 cycles at 94 C for 30 s, 72 C for 3 min; 5 cycles at 94 C for 30 s, 70 C for 30 s, and 72 C for 2.1. Animal immune challenge and sample collection 3 min; 20 cycles at 94 C for 30 s, 68 C for 30 s, and 72 C for 3 min. PCR amplicons were size separated and visualized on an ethidium Healthy adult Chinese mitten crabs (n ¼ 200; 80 20 g wet bromide stained 1.2% agarose gel. Amplicons of expected sizes were Ò weight) were collected from the Tongchuan aquatic product market purified with Wizard SV Gel and PCR Clean-Up System (Promega, in Shanghai, China. After acclimated for one week at 20e25 C USA), and inserted into a pZeroBack/Blunt Vector (Tiangen, China) in filtered, aerated freshwater, crabs were placed in an ice bath for and transformed into TOP10 E. coli. Positive clones containing in- 1e2 min until each was lightly anesthetized. Hemolymph was draw serts of an expected size were two-way sequenced using 23-mer from the hemocoel in arthrodial membrane of the last pair of and 24-mer primers (Table S1). walking legs using a syringe (approximately 2.0 mL per crab) with an equal volume of anticoagulant solution (0.1 M glucose, 30 mM 2.4. Identification of EsDscam hypervariable regions citrate, 26 mM citric acid, 0.14 M NaCl, 10 mM EDTA) added [23], and centrifuged at 500 g at 4 C to isolate hemocytes. The other Base on the EsDscam full length mRNA sequence, we designed tissues (hepatopancreas, gills, muscle, stomach, intestine, heart, many pairs of primers to identify the potential hypervariable re- testis, ovary, thoracic ganglia and brain) were harvested, snap gions. PCR reactions for each region with gene specific primer pairs Ò frozen in liquid nitrogen, and stored at 80 C prior to nucleic acid (Table S1, Fig. S1) were carried out by TaKaRa ExTaq Hot Start analysis. For cloning and expression analysis, each type of tissues Version (Takara, Japan). PCR amplification conditions was as fol- harvested above from 10 crabs respectively were pooled, and lows: 98 C for 10 min, 94 C for 30 s; 35 cycles at 94 C for 30 s, ground with a mortar and pestle prior to extraction. 58 C for 30 s, and 72 C for 2 min; 72 C for 10 min. The amplicons For PAMPs stimulation, more than 120 crabs were divided of each potential hypervariable regions from testis and hemocytes, equally into four groups (sex ratio 1:1): three experimental groups were cloned into TOP10 E. coli according to methods in Section 2.3, 902 X.-K. Jin et al. / Fish & Shellfish Immunology 35 (2013) 900e909 and 50 individual clones from each tissue were two-way free hemolymph and hemocyte extracts (approximately 15 mg) sequenced. were separated by 12% SDS-PAGE and transferred to PVDF mem- brane. The membrane was blocked with 5% dry skim milk in TBS 2.5. Sequence analysis and phylogenetic analysis (10 mM TriseHCl, pH 7.5, 150 mM NaCl) at room temperature for 2 h and then subjected to immunoblot assay using anti-Dscam EsDscam full-length cDNAs and deduced amino acid sequences rabbit polyclonal antibody (1:500) diluted by 2% dry skim milk in were compared against other vertebrates and invertebrates ortho- TBS overnight. Positive reactivity was detected using HRP- logs, using the BLAST program (http://blast.ncbi.nlm.nih.gov). These conjugated goat anti-rabbit IgG (1:2000) (HuaBio, China), and multiple sequence alignment were analyzed by ClustalX and Clus- was visualized through the instrument ChemiDocXRS (Bio-Rad, talW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/). An unrooted USA) detection. Maximum Likelihood phylogenetic tree was constructed with MEGA5.0. The homologous conserved domains and signal peptides 2.9. Immunofluorescence staining of EsDscam in hemocytes were identified by SMART (Simple Modular Architecture Reseach Tool, http://smart.embl-heidelberg.de) program. The conserved Hemocytes were collected from healthy crab as described pre- motifs of each identified hypervariable regions were derived using viously, then washed with PBS (137 mM NaCl, 2.7 mM KCl, 10 mM WebLogos (http://weblogo.berkeley.edu/). Na2HPO4, 2 mM KH2PO4, pH 7.4) prior to fixation in 4% para- formaldehyde. Cell permeabilization was performed in ice by PBS 2.6. Transcription analysis by real-time quantitative RT-PCR (1% Triton-X100 solution). After blocking with PBS (5% BSA and 0.2% Triton-X100) in incubator (Eppendorf, Germany) overnight at 4 C, Ò Quantitative RT-PCR was performed by SYBR Premix Ex TaqÔ the hemocytes were incubated at room temperature for 3 h with (TaKaRa, Japan) with EsDscam gene specific primer pairs designed rabbit anti-Dscam antibody in PBS (1% BSA and 0.2% Triton-X100), in conserved domain Ig9 (Table S1, Fig. S1), producing 179 bp and then reacted with FITC-conjugated goat anti-rabbit IgG anti- amplicon. PCR were carried out in instrument CFX96 (Bio-Rad, body (Huabio, China) at room temperature for 1 h. All antibodies USA) as follows: 30 cycles at 94 C for 30 s, 58 C for 30 s, and 72 C were diluted 1:200 in PBS containing 0.1% BSA. The nuclei were for 1 min. Internal control PCR reactions for b-actin were performed counterstained with 40,6-diamidino-2-phenylindole (Huabio, in a separate tube, as described above with the exception of an China) for 30 min, and then was added to the slide and hemocytes alternative gene-specific primer pair (Table S1), which was were observed by fluorescence microscopy (Leica, Germany). designed based upon a cloned E. sinensis b-actin cDNA fragment to produce a 276 bp amplicon. All qRT-PCR experiment were 2.10. Statistical analysis completed in triplicate using independently extracted RNA. Both for the tissue transcription assay and the immune challenge tran- Statistical analysis was performed using SPSS software (Ver11.0). scription assay, the PCR template were obtained as Section 2.1. The The data are represented as the mean standard error (S.E.). Sta- DD EsDscam relative expression levels were calculated by the 2 Ct tistical significance was determined by one-way ANOVA and post comparative CT method [25]. hoc Duncan multiple range tests. Significance was set at P < 0.05. Besides, in the tissue transcription assay, qRT-PCR products of b- actin from each tissue were size separated on an ethidium bromide 3. Results stained 1.5% agarose gel, visualized under ultraviolet light, and images were captured with a Gel Doc 2000 System (Tannon, China). 3.1. Characterization of EsDscam

2.7. EsDscam membrane-bound and secreted isoforms detection The obtained E. sinensis Dscam cDNA were designed as EsDscam (GenBank accession numbers JX679085). Under the analysis of To identify the membrane-bound and secreted isoforms in the deduced amino acid sequence by the SMART program, EsDscam EsDscam TM variable regions, we designed the specific forward was detected to have typical domain architecture compared with primer in FNIII 1 domain (Table S1, Fig. S1) combined with Oligo-dT other Dscam orthologs, including one signal-peptide, 10 immuno- (Takara, Japan), used cDNA templates (sampled from more than 3 globulin (Ig) domains, 6 fibronectin type III (FNIII) domains, one individuals each) from testis, health hemocytes and hemocytes transmembrane (TM) domain and one cytoplasmic tail (Fig. 1A). post LPS (2 h), PG (6 h) and Glu (12 h) challenged (peak expressed The full-length sequence of EsDscam was 6324 bp, containing a time interval) respectively, subsequently carried out RT-PCR and 5946-bp ORF that encoded a 1981-amino-acid protein (Fig. 1B), a sequencing according to Section 2.4. 201-bp 50 UTR (Fig. 1C), and a 177-bp 30 UTR (Fig. 1D). Besides, ClustalW2 alignment results demonstrated that EsDs- 2.8. Western blotting assay cam contains several short motifs in cytoplasmic tail which are highly conserved between crustaceans and insects, including SH3- Base on the highly similarity of antigen sequences alignment binding motif (Red), endocytosis/phagocytosis motif (Yellow), SH2- with fruit fly Dscam as Section 2.5, we purchased the anti-Dscam binding motif (Blue), Polyproline motif (Green), Postsynaptic den- rabbit polyclonal antibody (#ab43847,Abcam Ltd, Hong Kong). To sity, Disc large, and Zo-I protein (PDZ) domains motif (Purple) and test the antibody specificity against EsDscam protein, a western ITIM (immune tyrosineebased inhibition motif) motif (included in blot was performed using E. sinensis total protein, testis, cell-free box) [15e18] (Fig. S2). hemolymph and hemocyte fractions. The tissues were extracted according to Section 2.1, specially, hemocytes were collected by 3.2. EsDscam hypervariable regions centrifugation, washed for three times by TBS (10 mM TriseHCl, pH 7.5, 150 mM NaCl) and homogenized in cell lysis buffer (Beyotime, In order to identify the hypervariable regions of EsDscam, several China), and 1 mM phenylmethanesulfonyl fluoride (PMSF). The pairs of specific primers were designed (Table S1, Fig. S1). Approx- homogenate was centrifuged at 3000 g for 5 min and the super- imately 50 clones of the amplicons from testis and hemocytes were natant collected for total protein quantification using a Bio-Rad two-way sequenced, and aligned using ClustalW in deduced amino protein assay kit (Bio-Rad, USA). The total protein from the cell- acids format. Alternatively spliced mRNA location and relevant X.-K. Jin et al. / Fish & Shellfish Immunology 35 (2013) 900e909 903

Fig. 1. Characterization of E. sinensis Dscam. A) Schematic diagram demonstrating the domain architecture of the EsDscam protein. B) Deduced EsDscam amino acid sequence. Signal peptide was single underlined; the Immunoglobulin (Ig) domains (1e10) were shaded with light gray; the Fibronectin type III (FNIII) domains (1e6) were shaded with dark gray; the transmembrane (TM) domain was included in box; the cytoplasmic tail was double underlined. C) 50 UTR of EsDscam full-length mRNA. A start codon (ATG) is bold and underlined. D) 30 UTR of EsDscam full-length mRNA. A stop codon (TGA) and the polyadenylation signal (AATAA) were both bold and underlined respectively. segment counts of the EsDscam were discovered in the N-terminal E. sinensis, especially in immune organs such as hepatopancreas, halves of Ig2 domain (25) and Ig3 domain (30), the entire Ig7 domain gills and hemocytes; in nerve organs such as thoracic ganglia and (18) and the transmembrane domain (2), potentially generate not brain; in digestive organs stomach and intestine (Fig. 4). less than 27,000 unique isoforms (Fig. 2AeE). Besides, in each of the detected hypervariable region, several 3.5. Temporal expression of EsDscam post PAMPs immune conserved amino acids motifs (listed solely in single letter), challenged comparatively conserved amino acids motifs (listed in several let- ters) and variable amino acids motifs (listed in no letters) showed in Based on the results of Real-time qRT-PCR measurements, Fig. S3. EsDscam expression in hemocytes were differentially inducible post Glu, LPS and PG challenged. EsDscam expression was signifi- 3.3. Bioinformatic analysis of EsDscam cantly higher than the blank control after 2, 6 and 12 h post Glu injection (P < 0.05), peaking up to 4 times above the blank control Base on the comparison with the other reported Dscams from after 12 h (Fig. 5A). EsDcam expression was immediately up- arthropods, the EsDscam extracellular domains were highly regulated after 2 h than the blank control post LPS injection conserved, and most relative with decapod Pacifastacus leniusculus (P < 0.05) (Fig. 5B). EsDscam expression levels peaking up after 6 h (Fig. 3A). Maximum-Likelihood-phylogenetic tree was produced post PG injection, but significantly decreased in 24 h (Fig. 5C). based on phylogenetic analysis of EsDscam conserved sequence Control reactions, in which were induced with ESS (Eriochir sinensis from Ig8 to FNIII4 with representative invertebrate and vertebrate saline), yielded no significant variation in expression levels (white orthologs. The tree contained two distinct clades distinguishing bars in Fig. 5AeC). invertebrate Dscams (mostly arthropods) from vertebrate DSCAMs. EsDscam together with other two decapods P. leniusculus and 3.6. EsDscam membrane-bound and secreted isoforms L. vannamei shared in the same subclade of invertebrate, but in distinct subclades with insects (Fig. 3B). In order to identify the EsDscam membrane-bound and secreted isoforms, we performed a PCR reaction using specific primer 3.4. Tissue distribution of EsDscam designed in the FNIII 1 domain pairing with Oligo-dT, and cDNA template from health hemocytes and testis, or hemocytes post As determined by Real-time quantitative RT-PCR, EsDscam PAMPs challenged (EsDscam peaking expressed time interval, Glu expression were widely observed in all the detected tissues of 12 h, LPS 2 h and PG 6 h). Based on the results of agarose gel 904 X.-K. Jin et al. / Fish & Shellfish Immunology 35 (2013) 900e909

Fig. 2. Hypervariable regions of EsDscam protein. A) Schematic diagram demonstrating the locations of detected alternatively spliced exons of EsDscam. The hypervariable regions are located in the N-terminal halves of Ig2 domain (red) and Ig3 domain (yellow), complete Ig7 domain (blue) and TM domain (green) respectively. The numbers of alternative exons detected are presented under the relevant domains. BeE) Multiple amino acid sequence alignment of detected EsDscam hypervariable regions of N-terminal halves of Ig2 domain (B) and Ig3 domain (C), entire Ig7 domain (D) and TM domain (E) by ClustalW. Identical (*) and similar (. or :) residues are indicated. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) electrophoresis (Fig. 6A), EsDscam had only one kind of 3000 bp was found on the surface of hemocytes (Fig. 6D-cec00), nuclei amplicon from testis; one major kind of 3000 bp amplicon from (blue) was found in localized areas of DAPI-stained hemocytes health hemocytes; both 3000 bp and 1600 bp amplicon from he- (Fig. 6D-beb00). Control staining experiments performed using: a) mocytes post PAMPs injection. primary antibody alone (data not show), and b) FITC-conjugated As Fig. 6B showed, E. sisnesis Dscam has a high antigen sequence goat anti-rabbit IgG secondary antibodies alone did not yield any similarity (70%) with D. melanogaster Dscam. Based on this result, positive fluorescent signal in the hemocytes, hence, ensure the we purchased the commercial anti-Dscam polyclonal antibody. specificity of cross-reactivity between Dscam-antibodies and he- The specificity against the EsDscam protein (amino acid residues mocyte EsDscam proteins (Fig. S4). 1142e1242) was tested by western blot using protein lysates from E. sinensis whole protein, cell-free hemolymph and hemocyte ex- 4. Discussion tracts. The antibody reacted specifically to a band around 220 kD from crab whole proteins, and to a band around 160 kDa both from In the event of pathogens intruding, their conserved pathogen- hemocytes and from hemolymph (Fig. 6C). These results ensure the associated molecular patterns (PAMPs), such as lipopolysaccharides availability of commercial anti-Dscam polyclonal antibody for (LPS), peptidoglycans (PG) and b-1, 3-glucans (Glu), i.e., are essential subcellular location assay. and unique components of virtually all microorganisms, but absent in higher organisms [26] could be discriminated by a wide range of 3.7. EsDscam subcellular location pattern recognition receptors (PRRs) that are highly conserved in evolution, then encountered a variety of defense mechanisms [27]. To identify EsDscam in hemocytes, we performed an immuno- Whereas, invertebrate pattern-recognition were gradually localization assay. EsDscam membrane-bound isoforms (green) unveiled to have previously unrecognized feature of alternative X.-K. Jin et al. / Fish & Shellfish Immunology 35 (2013) 900e909 905

Fig. 3. Comparison of EsDscam with other orthologs. A) Conservation of “high diversity” Dscam receptors in arthopods. Dscam extracellular domains of Pacifastacus leniusculus, Daphnia magna, Drosophila melanogaster, Anopheles gambiae and Tribolium castaneum were aligned using ClustalW. Conservation is given in % amino acid identity to the corre- sponding domains in Eriocheir sinensis. For Ig2 and Ig3, values are given for the C-terminal halves (encoded by constant exons) only. B) Maximum Likelihood phylogenetic tree of DSCAMs (blue branch) and Dscams (red branch) based on the conserved sequence from Ig8 to FNIII4. Numbers at branch nodes represented the node’s percent bootstrap support based on 3000 replications. GenBank accession numbers of chosen amino acid sequences were shown in Table S2. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

splicing, like somatic rearrangement of vertebrate immune receptors peptide, 10 immunoglobulin (Ig) domains, 6 fibronectin type III [12e17]. These molecular processes both aim at diversified the domains (FNIII), one transmembrene domain (TM) and one cyto- repertoire of immune receptors in order to sufficiently recognize a plasmic tail (Fig. 1). The Dscams extensively exist in many species plethora of various intruding pathogenetic objects [9]. including vertebrates and invertebrates, nevertheless, alter- In the present study, EsDscam has the identical domain natively splicing of Dscam was solely found in some arthropods in component as all the other Dscam orthologs, including one signal- current knowledge, such as insecta and crustacea [28]. The 906 X.-K. Jin et al. / Fish & Shellfish Immunology 35 (2013) 900e909

Fig. 4. Tissue distribution of EsDscam transcript. A) The EsDscam transcript was examined in various tissues by Real-time quantitative RT-PCR. The b-actin gene from E. sinensis was used as an internal control. B) The b-actin PCR products in corre- sponding tissues were detected on 1.0% agarose gel.

hypervariable regions of EsDscam are located in the N-terminal halves of Ig2 domain and Ig3 domain, the entire Ig7 domain and the transmembrane domain respectively, with capability to generate more than 27,000 unique isoforms (Fig. 2). Dscam gene family is comparatively conserved especially in the Ig and FNIII domains, except for the alternative splicing regions [15]. Additionally, there are many highly conserved functional motifs intensively arranged in the cytoplasmic domain of Dscams. The cytoplasmic domain is only associated with transmembrane isoform, in which these motifs play important roles in immunological signaling [15]. In the daphnia, the endocytosis/phagocytosis motif (YXXL), and the ITAM, an immunoreceptor tyrosineebased activation motif (YXXL/ V-6 to 17 X-YXXL/V) [29] are both involved in down-stream protein tyrosine kinase (PTK)-mediated immunoreceptor signaling after ligand binding, and regulated the expression of surface membrane receptors [15]. At the very end of the carboxyl terminal of cyto- plasmic tail, there are the postsynaptic density, disc large, and zo-I protein (PDZ) domains motif and immune tyrosine-based inhibi- tion motif (ITIM) (I/S/V/LXYXXV/L). The PDZ domains could provide Dscam with different functions via different ligands or localization in the cellular membrane [30]. ITIM could be phosphorylated in tyrosine site by Src kinases, subsequently interact with the extra- cellular part of the receptor, which then allows it to recruit phos- photyrosine phosphatase that in turn decreases the activity of the cell [29]. In our findings, the EsDscam also contains several short motifs in cytoplasmic tail which are highly conserved among ar- thropods, including SH3-binding motif, endocytosis/phagocytosis motif, Polyproline motif, PDZ domains motif. Interestingly, the ITIM motif (I/S/V/LXYXXV/L) is absent in EsDscam, which was also not found in crayfish PlDscam, indicating the decapoda Dscams exhibited only positive transmembrane signaling feature (Fig. S2). Recently, several alternative splicing regulatory factors were discovered in L. vannamei, such as heterogeneous nuclear ribonu- cleoprotein hrp36 and serine/arginine (SR)-rich protein B52 [31,32]. However, the specific function of these motifs discussed above and alternative splicing regulatory mechanism of Dscam in Fig. 5. EsDscam transcription was induced post different PAMPs challenge. EsDscam E. sinensis are still require further research. Phylogenetic clustering expression was significantly induced compared with the control (white bars) as fol- results of Dscams clearly distinguish invertebrate clade from lows: A) 2 h, 6 h and 12 h post Glu challenge; B) 2 h post LPS challenge and C) 6 h post vertebrate clade. All decapoda Dscams share the same subclade of PG challenge. Statistical significance is indicated with an asterisk (P < 0.05). invertebrate Dscams, yet in distinct subclade with insects (Fig. 3B). These observations indicated that EsDscam is likely to serve the X.-K. Jin et al. / Fish & Shellfish Immunology 35 (2013) 900e909 907

Fig. 6. EsDscam cytoplasmic tail isoforms. A)Two major sizes (3000 bp and 1600 bp) of amplicons detected on 1.0% agarose gel indicated EsDscam mRNA isoforms with (3000 bp) or without (1600 bp) cytoplasmic tail. B) Schematic diagram demonstrating the sequence position of anti-Dscam binding site, and the antigen sequences’ identities between fly Dscam and crab Dscam. C) Western blotting of anti-Dscam antibody against protein extracts from different crab tissues as follows: 1, total protein; 2, testis; 3, hemolymph; 4e6, hemocytes. D) EsDscam immuno-localization assay in hemocytes. EsDscam membrane-bound isoforms (green) was found on the surface of hemocytes (cec00), nuclei (blue) was found in DAPI- stained localized areas (beb00). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 908 X.-K. Jin et al. / Fish & Shellfish Immunology 35 (2013) 900e909 same function in the process of invertebrate internal defense as the in the characteristics and function of Dscams in the innate immune other arthropod Dscams [18]. response of crustacean. The Dscam were initially identified as an axon guidance receptor in animal nerve system [20,21]. Subsequent research extensively 4.1. Concluding remarks detected Dscam existence in immune system such as fat body cells and hemocytes of insects and crustaceans [13e18]. Information In the present study, we report the cloning, sequence analysis, extracted from EsDscam tissue-dependent mRNA expression tissue-specific distribution, immune responsiveness and localiza- showed that these transcripts were widely distributed in all tion of a novel Dscam in crustacean Eriocheir sinensis. The conser- examined tissues, especially in immune organs such as hepato- vation of key motifs and sequence types between taxa support their pancreas, gills and hemocytes, in nerve organs thoracic ganglia and common functionality respectively. Future research, with respect to brain as well. Interestingly, EsDscam was found highly transcribed the alternative splicing regulatory mechanisms within the innate in crabs’ digestive organ, such as intestine, which might aroused by immune system of E. sinensis Dscam, may elucidate the exact the defenses of alimentary canal to protect themselves from immunological actions, providing a vehicle for further invertebrate invading pathogens [3,33]. In this study, we observed E. sinensis immunology research. acute infection symptoms (lose mobility) in the period of 24 h post different PAMPs challenged, and also found the counts of drawn out hemocytes severely decreased, finally resulted in individual Acknowledgments motality compared with control respectively [34,35]. Meanwhile, the responsiveness of EsDscam transcription after different PAMPs This research was supported by grants from the Innovation stimulation in hemocytes were differentially induced, which were Program of Shanghai Municipal Education Commission (13zz031), possibly due to the distinct molecular structure of Glu, LPS and PG. the National Science and Technology Support Program of China Overall, the elicitor-and time-dependent expression profile of (2012BAD26B04), and the National Research Foundation for the EsDscam indicated its capacity of possessing some degree of Doctoral Program of Higher Education of China (20110076110016). specificity, which was consistent with the other reported arthropod Dscams [14,17,18]. Appendix A. Supplementary data Hemocytes constitute vitally important cells of the invertebrate immune system [36]. Most proteins are secreted into hemolymph, Supplementary data related to this article can be found at http:// the crustacean equivalent of blood serum, comprise an important dx.doi.org/10.1016/j.fsi.2013.06.029. component of the humoral immune defense [37]. In contrast, the other proteins associated with the hemocytes membrane are References involved in a series of cellular defense strategies [38]. By the 30 RACE and western blotting assay, the membrane-bound and [1] Litman GW, Cooper MD. Why study the evolution of immunity? Nature secreted Dscam isoforms in E. sinensis were detected, and that Immunology 2007;8:547e8. extensively transcribed isoforms post different PAMPs challenge [2] Herrin BR, Cooper MD. Alternative adaptive immunity in jawless vertebrates. The Journal of Immunology 2010;185:1367e74. were also shown, combined results possibly explained the up- [3] Rowley AF, Powell A. Invertebrate immune systems-specific, quasi-specific, or regulated expression profile of EsDscam. In the other studies, the nonspecific? The Journal of Immunology 2007;179:7209e14. fruit fly Dscams with or without a cytoplasmic domain had been [4] Little TJ, O’Connor B, Colegrave N, Watt K, Read AF. Maternal transfer of strain- fi specific immunity in an invertebrate. Current Biology 2003;13:489e92. found to exhibit isoform-speci c homophilic binding, participated [5] Sadd BM, Schmid-Hempel P. Insect immunity shows specificity in protection in axon guidance and immune recognition [13,39]. In the mosquito, upon secondary pathogen exposure. Current Biology 2006;16:1206e10. Dscam gene had been proved to produce both membrane-bound [6] Pham LN, Dionne MS, Shirasu-Hiza M, Schneider DS. A specific primed im- and soluble isoforms, and potentially serve as a mechanism of mune response in Drosophila is dependent on phagocytes. PLoS Pathogens 2007;3:e26. opsonizing pathogens with secreted Dscam splice isoforms and [7] Roth O, Sadd BM, Schmid-Hempel P, Kurtz J. Strain-specific priming of resis- present them to cell surface expressing transmembrane isoforms tance in the red flour beetle, Tribolium castaneum. Proceedings of the Royal e with the same exon representations [14,19]. Furthermore, we also Society B: Biological Sciences 2009;276:145 51. [8] Pope EC, Powell A, Roberts EC, Shields RJ, Wardle R, Rowley AF. Enhanced locate EsDscam in the membrane of crab hemocytes via immuno- cellular immunity in shrimp Litopenaeus vannamei after ‘vaccination’. PLoS localization assay, which is consistent with evenly distributed One 2011;6:e20960. Dscams on the cell surface of insects’ hemocytes [13,14]. In crus- [9] Kurtz J, Armitage SAO. Alternative adaptive immunity in invertebrates. Trends e fi in Immunology 2006;27:493 6. tacean, the rst Dscam was isolated and characterized from prawn [10] Cooper MD, Alder MN. The evolution of adaptive immune systems. 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Science 2005;309:1874e8. insects and crustaceans from intruding pathogens, and enhanced [14] Dong Y, Taylor HE, Dimopoulos G. AgDscam, a hypervariable immunoglobulin phagocytic response are the critical effectors of the priming domain-containing receptor of the Anopheles gambiae innate immune system. PLoS Biology 2006;4:e229. response upon second bacteria exposure [6,8]. In crayfish [15] Brites D, McTaggart S, Morris K, Anderson J, Thomas K, Colson I, et al. The P. leniusculus, various Dscam isoforms with specific binding motifs Dscam homologue of the crustacean daphnia is diversified by alternative e were induced post immune challenge, then participating in bac- splicing like in insects. Molecular Biology and Evolution 2008;25:1429 39. [16] Chou P-H, Chang H-S, Chen IT, Lin H-Y, Chen Y-M, Yang H-L, et al. The terial clearance and phagocytosis in hemocytes [18]. In summary, putative invertebrate adaptive immune protein Litopenaeus vannamei the collective results indicate that EsDscam is a kind of constitutive Dscam (LvDscam) is the first reported Dscam to lack a transmembrane and inducible acute-phase PRRs involved in defense response domain and cytoplasmic tail. Developmental & Comparative Immunology 2009;33:1258e67. against PAMPs challenge with previously unrecognized alternative [17] Chou P-H, Chang H-S, Chen IT, Lee C-W, Hung H-Y, Han-Ching Wang KC. splicing character. Hence, the current study provided new insights Penaeus monodon Dscam (PmDscam) has a highly diverse cytoplasmic tail and X.-K. Jin et al. / Fish & Shellfish Immunology 35 (2013) 900e909 909

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