86 ISJ 18: 86-97, 2021 ISSN 1824-307X RESEARCH REPORT A C-type lectin (AjSJL-1) containing DPN/WVD motifs in Apostichopus japonicus recognizes multiple microbes Q Zhao1,4,5, H Wang1,4,5, W Wang1,4,5, J Li1,4,5, Y Liu1,4,5, Z Xue1,4,5, Z Liu1,4,5, L Wang1,3,4,5, L Song1,2,3,4,5* 1Liaoning Key Laboratory of Marine Animal Immunology, Dalian Ocean University, Dalian 116023, China 2Southern Laboratory of Ocean Science and Engineering (Guangdong, Zhuhai), Zhuhai, 519000, China 3Laboratory of Marine Fisheries Science and Food Production Process, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266235, China 4Liaoning Key Laboratory of Marine Animal Immunology and Disease Control, Dalian Ocean University, Dalian 116023, China 5Dalian Key Laboratory of Aquatic Animal Disease Prevention and Control, Dalian Ocean University, Dalian 116023, China This is an open access article published under the CC BY license Accepted May 14, 2021 Abstract C-type lectins (CTLs) are a superfamily of Ca2+ dependent carbohydrate-recognition proteins with diversified functions ranging from embryonic development to immune defense. In the present study, a CTL containing only one CRD domain with new motifs Asp-Pro-Asn (DPN) and Trp-Val-Asp (WVD) in its Ca2+ binding site 2 (designated as AjSJL-1) was identified from sea cucumber Apostichopus japonicus. The deduced amino acid sequence of AjSJL-1 was homologous to CTLs from other animals with the identities ranging from 19 % to 28.4 %. The mRNA transcripts of AjSJL-1 were detected in all the examined tissues with varied abundance. The expression level of AjSJL-1 mRNA in coelomocyte was up-regulated significantly at 12 h after Vibrio splendidus challenge. The recombinant protein of AjSJL-1 (rAjSJL-1) displayed significant binding activity to lipopolysaccharide, peptidoglycan, mannose and D-galactose in a Ca2+-dependent manner. Moreover, rAjSJL-1 exhibited strong binding capability to V. splendidus but week to Staphylococcus aureus, Bifidobacterium breve, Pichia pastoris and Yarrowia lipolytica in the presence of Ca2+. These results collectively suggested that AjSJL-1 with new DPN/WVD motifs served as a pattern recognition receptor in sea cucumber with the capability to bind broad-spectrum microbes and initiate the immune response against invaders. Key Words: C-type lectin; Apostichopus japonicus; DPN/WVD motifs; binding capability; innate immunity Introduction The C-type lectin (CTL) is a superfamily of Ca2+ dependent carbohydrate-recognition proteins with carbohydrate recognition domains (CRDs) (Wang et al., 2011). Most C-type lectins contain one CRD, but some have two or more CRDs (East and Isacke, 2002). Studies in invertebrates have implied that the clustering of multiple CRDs in one molecule endow C-type lectin with broader spectrum and higher affinity of binding pathogen-associated molecular patterns (PAMPs) (Wang et al., 2011). For instance, CfLec-3 from Chlamys farreri with three CRDs can bind more PAMPs than CfLec-1 and CflLec-2 with single CRD (Zhang et al., 2009). In addition, the key _________________________________________ Corresponding author: Linsheng Song Dalian Ocean University Dalian 116023, China E-mail: lshsong@dlou.edu.cn motifs in CRD of CTLs also affect the recognition range of carbohydrates. In vertebrate CTLs, there are four Ca2+-binding sites in CRD, among which the Ca2+-binding site 2 is known to be involved in the carbohydrate binding (Zelensky and Gready, 2005). In the Ca2+-binding site 2, the first motif is always Glu-Pro-Asn (EPN) or Gln-Pro-Asp (QPD), which displays specific affinity to mannose or galactose, respectively. The second motif is always Trp-Asn-Asp (WND), which can improve the binding affinity and specificity to carbohydrate (Drickamer, 1992; Zelensky and Gready, 2005). In invertebrate CTLs, there are many novel motifs such as Glu-Pro-Asp (EPD), Gln-Pro-Gly (QPG), Gln-Pro-Ser (QPS), Tyr-Pro-Gly (YPG), Tyr- Pro-Thr (YPT) for the first motif, and Trp-Ile-Asp (WID), Trp-Ser-Asp (WSD), Trp-His-Asp (WHD), Phe-Ser-Asp (FSD) and Leu-Ser-Asp (LSD) for the 87 Table 1 Designations and nucleotide sequences of the primers were used in this study Primer Sequence(5'-3') AjSJL-1-p1 ATGGCTCTGTCTCTGGTCACTG AjSJL-1-p2 TTCAATACATGGAAGTTTACAAATGA AjSJL-1-M1 GGATCCATGGCTCTGTCTCTGGTCACTG AjSJL-1-M2 GAATTCTTCAATACATGGAAGTTTACAAATGA Cytb-p1 CGTAGTTCAGTTTCTCCTT Cytb-p2 AAGGGAAAAGGAAGTGAAAG T7 TAATACGACTCACTATAGGG T7-terminator TGCTAGTTATTGCTCAGCGG M13-47 CGCCAGGGTTTTCCCAGTCACGAC M13-48 GAGCGGATAACAATTTCACACAGG RT-SJL-1-p1 TGCTTCAGTCCATTCAACCGA RT-SJL-1-p2 GGTCATTTCCGCACCAGTTCT second motif (Wang et al., 2011). The existence of diversified motifs in invertebrate CTLs suggests that there are flexible binding sites to recognize and bind various carbohydrates, which endow them with multiple immune functions (Huang et al., 2015). For example, a CTL with EPD/WFD motifs (CfLec-2) from scallop C. farreri exhibited the PAMP binding activity to LPS, PGN, mannan, and zymosan (Yang et al., 2010). EPD motif containing lectin AiCTL-7 in scallop Argopecten irradians was able to bind LPS, PGN, mannan, yeast glucan and poly I:C in vitro, and involved in agglutination of fungi and bacteria (Wang et al., 2011). FcLec3 with EPS motif identified from Chinese white shrimp Fenneropenaeus chinensis recognized muramic acid and peptidoglycan, and functioned in the recognition of bacterial and viral pathogens (Wang et al., 2009). PtCLec1 with YPD motif from swimming crab Portunus trituberculatus bound many PAMPs, including lipopolysaccharides (LPS), peptidoglycan (PGN) and glucan (GLU), and numerous microorganisms (Su et al., 2020). The information about the CTLs with novel motifs as well as their functions in the immune response of invertebrates is helpful for the comprehensive understanding of the diversity and complexity of CTL family (Wang et al., 2007; Li et al., 2015b). Sea cucumber Apostichopus japonicus is a special group of marine invertebrates, which is commercially important aquaculture species in Asian countries. Recently, the frequently outbreaks of diseases have caused severe economic losses and threatens the healthy development of sea cucumber framing industry. As an invertebrate, sea cucumber lacks adaptive immunity and solely relies on innate immunity to defend against various invaders. CTL plays an important role in recognizing and binding to microorganisms as PRR in invertebrates to initiate immune responses (Drickamer and Taylor, 1993). The knowledge about CTL family members in A. japonicus would provide new insights into the immune defense mechanisms of sea cucumber (Wang et al., 2018). In the present study, a previously identified CTL from A. japonicus (Ono et al., 2018) was further investigated with the objectives to (1) characterize new motifs in CRD, (2) examine its mRNA expression after Vibrio splendidus challenge, and (3) determine its activity to bind various PAMPs and microbes. Materials and Methods Sea cucumber and microbes Sea cucumbers (Apostichopus japonicus) approximately 100 ± 25 g in weight were collected from a local commercial farm (Dalian, China) and maintained in aerated seawater at 18 ± 2 °C for 7 days before experiments. Lipopolysaccharide (LPS from Escherichia coli 055:B5, Sigma-Aldrich, USA), D-galactose (Sigma- Aldrich, USA), peptidoglycan (PGN from Staphylococcus aureus, Sigma-Aldrich, USA) and mannose (Man from Saccharomyces cerevisiae, Sigma-Aldrich, USA) were dissolved in TBS-Ca2+ buffer (50 mM Tris-HCl, 50 mM NaCl, 20 mM CaCl2, pH 7.4) or TBS buffer (50 mM Tris-HCl, 50 mM NaCl, pH 7.4) at the final concentration of 1 M, respectively. These solutions were stored at -20 °C for following experiment. Gram-negative bacteria Vibrio splendidus and Vibrio anguillarum (laboratory preservation), Gram- positive bacteria Streptococcus aureus (laboratory preservation), Bifidobacterium breve (Cellbio, Shanghai), and Fungi Pichia pastoris and Yarrowia lipolytica (laboratory preservation) were cultured in 2216E medium (Solarbio, Beijing) at 28 °C for 24 h, LB medium at 37 °C for 24 h, BS medium (Hopebio, Qingdao) at 37 °C for 24 h, YPD medium (Solarbio, Beijing) at 28 °C for 24 h, respectively. 88 Tissue collection and immune challenge Six unstimulated sea cucumbers were sacrificed, and the tissues including body wall, muscle, gonad, gut, tube-foot and respiratory tree were collected to investigate the mRNA distribution of AjSJL-1. Coelomic fluid was also collected and immediately centrifuged at 800 × g, 4 °C for 10 min to harvest the coelomocytes. All these tissues and coelomocyte samples were stored at -80 °C after addition of 1 mL Trizol reagent (Thermo Fisher Scientific, China) for subsequent RNA extraction (Ren et al., 2014; Yan et al., 2018). A total of 72 sea cucumbers were randomly divided into two groups for soaking stimulation. Thirty-six individuals in control group were soaked in 100 L sterile sea water. Another 36 sea cucumbers in the stimulation group were soaked in sterile sea water adding with V. splendidus (1 × 109 CFU/mL, dissolved in seawater) (Wang et al., 2018). Then six sea cucumbers were randomly collected from two groups at 0, 3, 6, 12, 24 and 48 h post-challenge (hpc) for coelomocytes collection as previously described (Jiang et al., 2014). RNA extraction and cDNA synthesis Total RNA was extracted from the different tissues of sea cucumber A. japonicus by using the Easy Pure RNA Kit (TransGen, Beijing, China). The sample was lysed in Trizol reagent and total RNA was extracted in cold chloroform and pelleted by centrifugation. Then the RNA was purified and washed twice in 70 % ethanol for following steps. According to Prime Script™ First Strand cDNA Synthesis Kit (TaKaRa, Dalian, China), the first- strand of cDNA was synthesized at 37 °C for 15 min, and terminated at 85 °C for 5 s. The cDNA mix was stored at 80 °C for subsequent experiments. Cloning of AjSJL-1 gene Based on the published sequence (GenBank accession No. LC214944.1), two primers AjSJL-1-p1 and AjSJL-1-p2 (Table 1) were designed to clone the open reading frame (ORF) of AjSJL-1 (Ono et al., 2018). The PCR products were gel-purified, cloned into the pMD19-T simple vector (TransGen, Beijing, China). The recombinant plasmid was transformed into Trans-5α chemically competent cell (TransGen, Beijing, China), and three positive clones were sequenced by Sangon Biotech (Shanghai). Sequence analysis of AjSJL-1 The sequence of AjSJL-1 was analyzed using the BLAST algorithm at the National Center for Biotechnology Information. The amino acid sequence of AjSJL-1 and its theoretical molecular weight as well as the isoelectric point were predicated with an online translation tool (http://web.expasy.org/translate). The functional domains were revealed by the simple modular architecture research tool (SMART) version 4.0 (http://smart.embl-heidelberg.de/). The presumed tertiary structure of the CRD in AjSJL-1 was established using the SWISS-MODEL prediction algorithm (http://swissmodel.expasy.org/) and displayed by PyMOL according to previous reports (Arnold et al., 2006; Kiefer et al., 2009). Multiple sequences alignment was created by the Clustal X and performed by online tools (http://www.bio- soft.net/sms/). A phylogenetic tree was constructed based on the deduced amino acid sequences of AjSJL-1 and other C-type lectins by the neighbor- joining (NJ) algorithm using the MEGA 4.1 software (Kumar et al., 2004). To derive confidence value for the phylogeny analysis, bootstrap trials were replicated 1000 times. Quantitative Real-time PCR analysis of AjSJL-1 mRNA expression pattern The expression patterns of AjSJL-1 in different tissues (coelomocyte, gut, muscle, gonad, tube-foot, body wall, and respiratory tree) were analyzed by using the ABI Quantstudio Sequence Detection System (Applied Biosystems, USA). The fragment of cytochrome b (cytb) gene (GenBank accession No. 7802877) amplified with primers Cytb-p1and Cytb-p2 (Table 1) from A. japonicus was served as the internal control (Sun et al., 2010). The 2- △△Ct method was used to analyze the relative expression levels of AjSJL-1 in different tissues with the calibrator of coelomocyte (Livak and Schmittgen, 2001). Preparation of recombinant AjSJL-1 and polyclonal antibody The ORF fragment encoding AjSJL-1 was amplified with specific primers AjSJL-1-M1 and AjSJL-1-M2 (Table 1). The PCR products with BamH I and EcoR I sites were cloned into pET-22b expression vector with His-tag (TransGen, Beijing, China). The recombinant plasmid (pET-22b-AjSJL-1) was transformed into Transetta (DE3) chemically competent cell (TransGen, Beijing, China). After PCR screening with T7 primer (Table 1), the positive clones were confirmed by nucleotide sequencing. The positive transformants were incubated in LB medium (containing 50 mg/mL ampicillin) at 37 °C with shaking at 220 rpm. When the culture medium reached OD600 of 0.6-0.8, isopropyl β-D-1- thiogalactopyranoside (IPTG, Sigma, USA) was added at the final concentration of 1 mM, and cultured at 16 °C, 100 rpm for additional 12 h. The recombinant protein (designated rAjSJL-1) was purified through the Ni-Sepharose FF column, refolded, verified and quantified, and then stored at - 80 °C as previous report (Jiang et al., 2014). The rAjSJL-1 was used to prepare polyclonal antibody according to the previous report (Wei et al., 2015). Briefly, 500 μL of rAjSJL-1 (0.64 mg/mL) protein was emulsified with 500 μL complete Freund's adjuvant (Sigma, USA) to immunize 6-8 weeks old mice (bought from Dalian Medical University) by subcutaneous implantation. The second and third inoculations were performed on the 14th and 30th day with incomplete Freund's adjuvant (Sigma, USA). The anti-rAjSJL-1 serum was harvested on the 40th day and obliquely placed at 4 °C overnight. The immune serum (containing anti- rAjSJL-1 antibody) was collected after centrifugation at 4 °C, 3000 g for 30min, and stored at -80 °C before use. The specificity of polyclonal antibody against rAjSJL-1 was verified by western blotting assay. The rAjSJL-1 proteins were subjected to 12% SDS-PAGE, and the proteins in the gel were transferred to a 0.22 μm pore nitrocellulose membrane for western blotting http://smart.embl-heidelberg.de/ http://swissmodel.expasy.org/ http://www.bio-soft.net/sms/ http://www.bio-soft.net/sms/ 89 analysis. The anti-rAjSJL-1 antibody (diluted at 1:1000) was used as the primary antibody, and the HRP-linked goat-anti-mouse IgG (BBI, USA) (diluted at 1:1000) was used as the secondary antibody. The immune-blotted protein bands were visualized by ECL chemiluminescent substrate reagent kit (Thermo Fisher Scientific, China) and developed by Herosbio gel-imaging system (GE Healthcare, USA). Non-immunized mouse serum was used as negative control. PAMPs binding assay The PAMPs-binding ability of rAjSJL-1 was examined by enzyme-linked immune sorbent assay (ELISA) according to previous report with some modification (Loizou et al., 1985; Devi et al., 2010; Yang et al., 2016; Liu et al., 2021). Briefly, 100 μL solutions of LPS, PGN, Man and D-galactose dissolved in TBS or TBS-Ca2+ buffer at the final concentration of 0.1 mg/mL were used to coat 96-well plate, and the wells were blocked with 3% BSA (Sangon Biotech, China) in TBST (TBS solution containing 0.05 % Tween-20, pH 7.4) at 37 °C for 1 h. After three times of washing with TBST, 100 μL rAjSJL-1 with gradient dilution was added, and the reaction was incubated at 37 °C for 1 h. The same concentration tag protein (rTrx-His) expressed by the pET-32a null vector was added to the wells as negative group at the same time. Meanwhile, the wells filled with 200 μL TBS were used as blank. The plate was firstly incubated with 100 μL of anti-AjSJL- 1 serum (1:1000 dilution in 3% BSA) at 37 °C for 1 h, followed by four times of washing with TBST, and then incubated with 100 μL of goat-anti-mouse Ig- alkaline peroxidase conjugate (Sangon Biotech, China) second antibody (1:4000 dilution in 3% BSA). After the last washing, 100 μL of tetramethylbenzidine (TMB) single component substrate solution (Solarbio, Beijing) was added and incubated at 37 °C for 15 min. The reaction was stopped by adding 50 μL of 2 M HCl per well, and the absorbance was measured at 450 nm (TECAN, Switzerland). The light absorption values of rAjSJL-1 group, rTrx-His group and TBS group were employed as sample group (P), negative group (N) and blank group (B), respectively. Samples with P (sample) – B (blank)/N (negative) - B (blank) > 2.1 were considered as positive. Three replications were performed for each sample, and the data were presented as mean ± SD (N = 3). Microbial binding assay Microbial-binding activity of rAjSJL-1 was measured according to previous report with some Fig. 1 Sequence features of AjSJL-1. A: Nucleotide and amino acid sequences of AjSJL-1. The motif putative for ligand binding specificity is boxed in blue and the cysteine residues in the CRD are marked with yellow boxes. B: The functional domains predicted by SMART program. Light red represents CRD, and red represents signal peptide. C: The spatial structure of CRD in AjSJL-1 predicted by SWISS-MODEL program. Random coil, β-stands and α- helices are marked as pink, purple, and blue, respectively. There are four cysteines marked as yellow (C35, C52, C118, and C138) involved in forming disulfide bridges 90 Fig. 2 The multiple sequence alignment and phylogenetic tree of AjSJL-1 with other CTLs. Multiple sequence alignment (A) and Phylogenetic tree (B) of 13 CTL from 12 organisms were analyzed. CTLs from various species include A. japonicus (LC214944.1; JN133520), P. crispus (KFQ60316.1), C. canorus (KFO79987.1), H. sapiens (NP_001243649.1), D. rerio (NP_001315005.1), S. purpuratus (XP_030844747.1), C. gigas (EKC23119.1), Drosophila melanogaster (ADV37094.1), Argopecten irradians (AEE36500.1), Penaeus chinensis (ACJ06431.1), Eriocheir sinensis (AFF59979.1), Penaeus vannamei (ADW08726.1) modification (Yu et al., 2007). Gram-negative bacteria (V. splendidus and V. anguillarum), Gram- positive bacteria (S. aureus and B. breve) and fungi (P. pastoris and Y. lipolytica) were employed to detect the microbial-binding ability of rAjSJL-1. The microbes were suspended in TBS or TBS-Ca2+ buffer, and incubated with rAjSJL-1 (300 μL, 0.64 mg/mL) under slight rotation at 4 °C overnight. After rinsed by TBS for five times, the bound proteins were dissociated from the microorganisms by loading buffer and analyzed by SDS-PAGE and western blot. rTrx (300 μL, 0.5 mg/mL) protein was employed as negative control. The anti-His monoclonal antibody (Sangon Biotech, China) was used for western blot to analysis the bacteria binding activity of rTrx. Statistical analysis All data were given as means ± SD and subjected to one-way analysis of variance (one-way ANOVA) followed by a multiple comparison (LSD). Differences were considered significant at p < 0.05 (Livak and Schmittgen, 2001). 91 Results Molecular characters and predicted spatial structure of AjSJL-1 The ORF sequence of AjSJL-1 was of 492 bp, which encoded a polypeptide of 163 amino acids with molecular mass of 17.74 kDa and theoretical pI of 4.48 (Fig. 1A). There were a signal peptide and only one CRD domain predicted in AjSJL-1 by SMART analysis (Fig. 1B). Two new motifs, DPN and WVD, were identified in CRD domain of AjSJL-1 for carbohydrate binding specificity (Fig. 1A). The potential tertiary structure of CRD in AjSJL- 1 was established by SWISS-MODEL prediction algorithm based on the template 1egi.1.B. The CRD adopted a typical long-form double-loop structure consisting of two parts, a lower part that was constituted of two α-helices and two β-strands, and an upper part that was composed of three β-strands. Moreover, four conserved cysteines (C35, C52, C118, and C138) were predicated to form two disulfide bridges at the bases of the loops. The Cys35 and Cys138 linked the whole domain loop, while Cys52 and Cys118 linked the long loop region (Fig. 1C). In addition, another two cysteine residues (Cys157 and Cys161) were identified at the N-terminus of the CRD, indicating a long-form CRD in AjSJL-1 (Fig. 1A). Homology comparison of AjSJL-1 BLAST homology analysis revealed that AjSJL- 1 shared 19% to 28.4 % identities with CTLs from A. japonicus (JN133520) and other animals, such as Pelecanus crispus (KFQ60316.1), Cuculus canorus (KFO79987.1), Homo sapiens (NP_001243649.1), Danio rerio (NP_001315005.1), and Strongylocentrotus purpuratus (XP_030844747.1) (Fig. 2A). A neighbor-joining tree was constructed based on multiple sequence alignment of AjSJL-1 and other ten CTLs from different species. There were generally three groups in the phylogenetic tree. AjSJL-1 and the CTLs from A. japonicus (AjCTL) and S. purpuratus (SpCTL) were first clustered into the echinoderm group, which formed a sister group to the CTLs from vertebrates. The CTLs from insects, molluscs and crustaceans were well separated and clustered together into the other invertebrate group (Fig. 2B). The distribution of AjSJL-1 mRNA in tissues AjSJL-1 mRNA transcripts were found to be distributed in all the examined tissues of sea cucumber A. japonicus, with the highest level in respiratory tree and the lowest level in coelomocytes. The expression levels were significantly higher in respiratory tree (27.26-fold, p < 0.01), body wall (10.38-fold, p < 0.01) and tube-foot (4.70-fold, p < 0.05) than that in coelomocytes (Fig. 3A). Temporal expression of AjSJL-1 in coelomocytes after the V. splendidus stimulation The mRNA expression level of AjSJL-1 in coelomocytes was monitored post the stimulation with V. splendidus. It was significantly up-regulated at 6 h (5.01-fold of that in control group, p < 0.05), reached the highest level at 12 h (19.27-fold of that in control group, p < 0.01), down-regulated sharply at 24 h, and increased again at 48 h (9.22-fold of that in control group, p < 0.01) after stimulation (Fig. 3B). The recombination protein of AjSJL-1 and the specificity of its polyclonal antibody. The recombinant plasmid (pET-22b-AjSJL-1) was transformed into Transetta (DE3). After IPTG induction with shake (100 rpm) at 16 °C for 12 h, the whole cell lysate was analyzed by 12 % SDS–PAGE. A distinct band was revealed with a molecular mass of 26 kDa, which was consistent with the predicted molecular mass of AjSJL-1 plus His-Tag (Fig. 4A). Fig. 3 The temporal and spatial expression patterns of AjSJL-1 mRNA. A: AjSJL-1 mRNA expression in different tissues of sea cucumber A. japonicus. Vertical bars represent the mean ± S.D (N =3). The different letters indicate significant differences comparing with other groups (p < 0.05, ANOVA). B: The mRNA expression levels of AjSJL- 1 in coelomocyte of A. japonicus after V. splendidus challenge. The cytb gene was used as an internal control to calibrate the cDNA template for all the samples. Each value is shown as mean ± S.D. (N =3). The different letters indicate significant differences comparing with other groups (p < 0.05, ANOVA) 92 Fig. 4 The recombinant protein of AjSJL-1 and the specificity for its polyclonal antibodies. A: SDS-PAGE analyzes of rAjSJL-1. Lane M: standard protein molecular weight marker; Lane 1: blank control for rAjSJL-1 (without plasmid pET-22b-AjSJL-1); Lane 2: IPTG-induced expression of rAjSJL-1; Lane 3: purified rAjSJL-1. B: Western blot analyzes of the specificity of polyclonal antibody. Lane M: standard protein molecular weight marker; Lane 4: western-bolt based on the sample of purified rAjSJL-1 The purified rAjSJL-1 was used to prepare antibody, and the specificity of the antibody against rAjSJL-1 was assayed by western blotting. A clear reaction band with 26 kDa was revealed (Fig. 4B). No visible reaction band was found in the negative control (data not shown). Carbohydrate binding capacity of AjSJL-1 The binding assay was performed to examine the PAMPs binding spectrum of AjSJL-1. The PAMP binding capacity was recorded as P/N at 405 nm, and the samples with P/N > 2.1 were considered as positive. In the presence of Ca2+, P/N values of rAjSJL-1 toward LPS, PGN, D-galactose and Man were all higher than 2.1 (Fig. 5A). rAjSJL-1 exhibited relatively higher affinity to PGN and Man, with the minimum concentration of 0.005 mg/mL. Its affinity to D-galactose and LPS was lower with the minimum concentration of 0.025 mg/mL. The values of P/N increased correspondingly with the increasing of rAjSJL-1 concentration, which suggested that the binding activities of rAjSJL-1 towards PGN, Man, D-galactose and LPS were of dose-dependence. In the absence of Ca2+, the P/N values of rAjSJL-1 toward four PAMPs were all less than 2.1 (Fig. 5B). As control, the P/N values of rTrx for all the examined PAMPs were less than 2.1 (data not shown). Microbial binding activity of rAjSJL-1 The microbe binding assay was carried out to determine the ability of rAjSJL-1 to bind Gram- negative bacteria, Gram-positive bacteria and fungi. In the presence of Ca2+, a strong band was detected for V. splendidus, and shallow bands were detected for S. aureus, B. breve, P. pastoris and Y. lipolytica. However, when microbes were incubated with rAjSJL-1 in the absence of Ca2+, there were no visible bands detected in all the lanes (Fig. 6A). The result suggested that rAjSJL-1 displayed no obviously binding ability towards the tested microbes without Ca2+. As a control, no binding affinity was found in the group of rTrx-His (Fig. 6B). Discussion CTL is a key type of PRR to play significant roles in pathogen recognition through binding to the terminal sugars of glycoprotein in a Ca2+ dependent manner. In the past decades, an increasing number of CTLs have been identified in various species (Mcnulty et al., 2012; Zhang et al., 2012; Huang et al., 2014; Zhang et al., 2017), which function in immune response either as cytomembrane receptors in immunocytes or as soluble proteins existing in tissue fluids (Wang et al., 2012; Huang et al., 2013a; Hoving et al., 2014). 93 Fig. 5 The interaction between rAjSJL-1 and the PAMPs revealed by ELISA analysis. A: The interaction between rAjSJL-1 and the PAMPs with TBS-Ca2+ buffer. B: The interaction between rAjSJL-1 and the PAMPs with TBS buffer. Each value is shown as mean ± S.D. (N =3) The CTLs harbor at least one CRD, and every CRD has four Ca2+-binding sites, in which Ca2+- binding site 2 is considered as the key site to determine the binding spectrum of CTL (Drickamer, 1999; Zelensky and Gready, 2005; Wang et al., 2011). In Ca2+-binding site 2, there are two conserved motifs determining the CRD binding ability, and the first one is always EPN or QPD in vertebrates (Weis et al., 1998; Zelensky and Gready, 2005). It is found that the motifs in the invertebrate CTLs are more diversified than the vertebrate ones. AjSJL-1 was previously identified as a CTL in sea cucumber containing only one CRD with 163 amino acid residues (Ono et al., 2018). Six conserved cysteine residues (Cys35, Cys52, Cys118, Cys138, Cys157 and Cys161) were found in the Ca2+ binding site 2 of AjSJL- 1, among which Cys157 and Cys161 allowed the CRD to adopt a typical long-form double-loop structure (Drickamer, 1999). In the present study, a novel DPN/WVD motif was identified in the Ca2+ binding site 2 of CRD from AjSJL-1, which was different from the previously reported motifs of AJCTL (EPN/WTD) (Han et al., 2012), AjCTL-1 (EPN/WND) and AjCTL- 2 (EPN/WND). In the phylogenetic tree, AjSJL-1 was closer to CTL-19s from vertebrate. These results indicated that there was functional differentiation among AjSJL-1 and other CTLs from A. japonicus. DPN was only found in Gal/GalNAc lectin from Entamoeba histolytica, which was able to recognize galactose/N-acetylgalac-tosamine (Weber et al., 2006; Yadav et al., 2016). It is implied that AjSJL-1 might display similar recognition ability to Gal/GalNAc lectin from E. histolytica. All these results indicated that AjSJL-1 was a member of the CTL with novel DPN/WVD motifs in sea cucumber A. japonicus. The distribution of AjSJL-1 mRNA and the alternation of its expression level in coelomocyte after bacterial challenge were examined to understand its involvement in the immune response of sea cucumber. AjSJL-1 mRNA transcripts were found to be widely expressed in water pipe system of sea cucumber, with the highest expression level in respiratory trees (Ono et al., 2018). AjCTL-1 was reported to be highly expressed in gonad and functioned as a PRR in A. japonicus to recognize microbes (Wei et al., 2015). AjCTL-2 was highly expressed in coelomocyte, indicating that it might be an important molecule in the coelomocyte mediated immune defense (Wang et al., 2018). The different expression profiles of those CTLs in tissues indicated that they might execute different functions in the innate immunity of A. japonicus. Respiratory tree is considered as a unique tissue for breathing in A. japonica. The higher expressions of AjSJL-1 in respiratory tree suggested that AjSJL-1 might play an important role in mucosal immunity of sea cucumber. Coelomocytes are the main component of the immune system and play important roles in sea cucumber to resist pathogen invasion (Wang et al., 2015). When the sea cucumbers were challenged by V. splendidus, the expression level of AjSJL-1 mRNA in coelomocyte increased significantly (p < 0.01) at 12 h, suggesting it was induced by immune stimulation similar to the CTLs in other invertebrates (Wang et al., 2012; Drummond and Brown, 2013; Huang et al., 2013a; Huang et al., 2013b; Li et al., 2015a; Yang et al., 2015; Zhou and Sun, 2015). All these results indicated that AjSJL-1 was involved in the immune response against microbial infection. The protein-carbohydrate interaction mediated by CTLs is benefited from their CRDs, and the conserved motifs at the Ca2+-binding site 2 of CRDs determine their binding ability (Kong et al., 2011; Huang et al., 2013b; Yang et al., 2015). In the present 94 Fig. 6 The microbe binding activity of rAjSJL-1 and rTrx revealed by western blotting. A: The microbe binding activity of rAjSJL-1 with TBS-Ca2+ or TBS buffer. B: The microbe binding activity of rTrx. Lane M: pre-stained marker (kDa); Lane 1: V. splendidus; Lane 2: V. anguillarum; Lane 3: S. aureus; Lane 4: B. breve; Lane 5: P. pastoris; Lane 6: Y. lipolytica study, AjSJL-1 was found to bind all the tested PAMPs including LPS, PGN, mannose and D- galactose in a Ca2+ dependent manner. It is noteworthy that a new DPN/WVD motif was found in the CRD of AjSJL-1. Different from the previously identified Gal/GalNAc lectin from E. histolytica with DPN motif to recognize galactose/N-acetylgalac- tosamine (Weber et al., 2006; Yadav et al., 2016), the new DPN/WVD motif gifted AjSJL-1 with the broad- spectrum binding activity. It is a common feature for all the identified vertebrate and invertebrate CRDs that the second amino acid in the motifs of Ca2+ binding site 2 is always “Pro” (Mitra and Das, 2001; Wei et al., 2012; De Melo et al., 2014; Drickamer and Taylor, 2015; Zhou and Sun, 2015). Structurally, the side chain of “Pro” is essential to keep the stability and bind metal ion (Zelensky and Gready, 2005; Yang et al., 2015). Moreover, the first and third amino acid in the motifs of Ca2+-binding site 2 affects the binding spectrum of CRD (Mullin et al., 1997; Zelensky and Gready, 2005; Wang et al., 2012). In the present study, the DPN motif endowed AjSJL-1 with binding activity to various PAMPs, which was different from the specific binding capability of EPN and QPD in vertebrate CTL. According to previous report that the third amino acid “Asn” in the motif of Ca2+-binding site 2 determined the binding specificity of CTL towards mannose (Yang et al., 2015), the first amino acid “Asp” in the motif of AjSJL-1 might be the key determinant for its binding activity to D-galactose (Yadav et al., 2016). In addition, compared with AjCTL-2, AjSJL-1 displayed stronger binding ability to various PAMPs, which was suspected to be determined by the WVD motif. The second motif has been reported to affect the affinity with carbohydrates (Zelensky and Gready, 2005; Yang et al., 2015). AjSJL-1 lost its binding ability to all PAMPs in the absence of Ca2+. These results collectively suggested that AjSJL-1 was a Ca2+-dependent CTL with novel DPN/WVD motifs responsible for the recognition of diverse PAMPs in immune response of sea cucumber. The predominant microbe recognition and binding capability of CTLs make them indispensable in the first defense line against pathogen infection (Drummond and Brown, 2013). In the present study, 95 AjSJL-1 was found to bind Gram-negative bacteria, Gram-positive bacteria and fungi in the presence of Ca2+, which was consistent with its broad binding spectrum to PAMPs. The broad microbe binding spectrum manifested its indispensable roles in innate immunity (Yu et al., 2007), which favored that AjSJL- 1 was involved in immune response against various pathogens as a PRR. It was previously found that some CTLs with a single CRD identified from invertebrates could bind to limited spectrum of microorganisms. For instance, AiCTL-7 only aggregated P. pastoris (Kong et al., 2011), and CgCLec-2 was able to bind LPS, PGN, mannan and zymosan rather than β-glucan (Yang et al., 2010). Considering that there was a novel DPN/WVD motif in the Ca2+-binding site 2 of AjSJL-1, it was speculated that this new DPN/WVD motifs might endow AjSJL-1 with a broad recognition spectrum of microorganisms. In conclusion, AjSJL-1 was characterized as a new member of CTL family with novel DPN/WVD motifs in sea cucumber A. japonicus. AjSJL-1 mRNA was highly expressed in the respiratory tree, and its expression level in coelomocyte increased significantly at 12 h after V. splendidus stimulation. It recognized and bound various PAMPs and microbes with broad spectrum. Acknowledgement We are grateful to all the laboratory members for their technical advice and helpful discussions. 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