Adult sea cucumbers Apostichopus japonicus were purchased from a fishery. They were maintained in a glass aquarium with artificial seawater (SEA LIFE, Marine Tech) at 13°C. After anesthetization with ice, the body wall, tentacles, Polian vesicle, intestine, and respiratory tree were collected and stored at −80°C until use, if they were not used immediately.

To investigate whether evisceration induces cellular aggregation and wound sealing, we autotransplanted fluorescent-labeled coelomocytes to the animals after evisceration. First, coelomic fluid was obtained using a syringe with 23-gauge needle (Terumo), mixed with an equal volume of Calcein-AM (0.5 mg/ml, Dojindo), and stirred for 10 min. The cells were washed three times with a mixture of 2.4% NaCl, 0.6% KCl, and 0.03 M PBS (3× PBS, pH 7.2) and resuspended in cell-free coelomic fluid (5×10⁶ cell/ml). Next, sterilized distilled water (SDW) (5% of the body weight) was injected into the same individuals to induce evisceration of the digestive tract. After evisceration, 3 ml of the fluorescently labeled coelomocytes was autografted to the coelomic cavity and the animals were kept in an aquarium for 3 h. After anesthetization with ice, the specimen was cut on the ventral side, and a section of the intestinal tissue was observed under a fluorescence microscope (Axioskop2, ZEISS, objective lens ×4) at 25°C. Images were acquired using a digital camera (Axiocam HRc, Zeiss) and the software provided with it (Axiovision, Zeiss), and the contrast was enhanced using the Image J software.

To determine the effects of the intestinal tissue on cellular aggregation in vitro, the freshly obtained intestine was cut into 5 mm square sections and fixed on a glass slide with a drop of 1% agarose solution, which gelled immediately. Fresh coelomic fluid was collected from an animal using a syringe with 23-gauge needle (Terumo). Fifty microliters of the coelomic fluid was dropped onto the intestinal section on a glass slide, covered with a cover slip, and incubated for 30 min. Concomitantly, the same volume of coelomic fluid was dropped onto a glass slide without an intestinal section. The aggregates that were formed were observed under a light microscope (DP26, Olympus), and their areas were measured using the region-of-interest analysis function of the cellSens standard program (version 1.6).

Each tissue, including the body wall, tentacles, intestine, respiratory tree, and Polian vesicle, was homogenized with an equal volume of 3× PBS. After centrifugation at 8,200 × g for 30 min, the supernatant was collected as the tissue extract. Forty microliters of the coelomic fluid and 10 μl of the tissue extract or 3× PBS (as a control) were mixed and placed on a glass slide. Observation and measurement of the area of the aggregates were performed as mentioned above.

Five microliters of the intestinal extract was centrifuged at 8,200 × g for 15 min using an ultrafiltration device (Vivaspin 5k, Sartorius). After centrifugation, the filtrates and concentrates were collected separately. To examine the thermostability of the aggregation-promoting factor, the intestinal extract was heated at 95°C for 5 min. These preparations were mixed with the coelomic fluid at a ratio of 1:9 and cellular aggregation was observed as described above.

First, crude protein was extracted from the intestine according to the procedure described by Ishimoda-Takagi and Ozaki (18) with slight modifications, because we suspected the aggregation-promoting factor to be tropomyosin. The intestine was minced and mixed with an equal volume of SDW and incubated for 30 min. The mixture was filtered through a nylon mesh with a pore size of 100 μm, and the residue was serially washed with 100% ethanol, 50% ethanol, and 97% ethanol. The residue was dried at 20°C and dissolved in 1 M KCl (7 ml/g). The pH of the solution was adjusted to 7 with NaOH and the solution was incubated at 20°C for 12 h. After centrifugation (8,200 × g for 30 min), the supernatant was collected and its pH was adjusted to 4.0 with HCl. After incubation for 1 h and centrifugation (8,200 × g for 30 min), the supernatant was collected and applied to an anion-exchange column of POROS HQ/20 (Applied Biosystems) equilibrated with 50 mM Tris-HCl (pH 8.0) using the BioLogic DuoFlow system (Bio Rad). The bound proteins were eluted with NaCl applied in a gradient from 0 to 0.5 M in 50 mM Tris-HCl. Each eluted fraction was added to the coelomic fluid and the aggregation-promoting activity was examined by microscopic observation. The fraction with the highest activity was subjected to gel-filtration chromatography using a Superdex 200 column (GE Healthcare) equilibrated with 50 mM Tris/HCl containing 150 mM NaCl (pH 7.5). The aggregation-promoting activity of each fraction was examined in the same manner and the fraction with the highest activity was subjected to SDS-PAGE.

To verify our speculation that the aggregation-promoting factor could be a lectin, we examined whether the activity was inhibited by carbohydrates. The intestinal extract was mixed with an equal volume of 0.1 M solution of D (+)-glucose, D (+)-mannose, D (+)-galactose, D (+)-xylose, L (−)-fucose, L (+)-rhamnose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, D (+)-maltose, D (+)-lactose, or D (+)-sucrose solution (in PBS), and incubated overnight. Thereafter, 10 μl of the mixture was added to 40 μl of the coelomic fluid and mounted on a glass slide, covered with a cover slip, and incubated for 30 min in a moist chamber. Observation of the aggregates and measurement of their area were done as mentioned above.

To purify the galactose-binding protein from the intestinal extract, 1 ml of galactose-agarose (Thermo Scientific) and 4 ml of the extract were mixed and gently stirred at 4°C overnight. The non-adsorbed fraction was removed and the galactose-agarose was washed with 3× PBS. The adsorbed protein was eluted with 4 ml of 0.2 M galactose-PBS.

A single 16 kDa band detected on a SDS-polyacrylamide gel after gel-filtration chromatography was subjected to liquid chromatography coupled to time-of-flight high resolution mass spectrometry (LC/Q-TOF/MS; TripleTOF 5600; SCIEX), followed by de novo sequencing. The band on the gel was excised and heated in 100 μl of 50 mM NH₄HCO₃ and 1 μl of 1 M dithiothreitol at 80°C for 10 min for reductive alkylation of the protein. Thereafter, 2 μl of 1 M iodoacetamide was added and incubated at 37°C for 15 min. After removing the liquid, 100 μl of 100% methanol and 100 μl of 10% acetic acid were added to destain the gels. The gels were contracted by shaking in 100% acetonitrile for 5 min. After the gels were dried, 20 μl of trypsin solution (0.5 mg/ml) in 50 mM NH₄HCO₃ was added and incubated overnight at 37°C. The digested proteins were analyzed by LC/Q-TOF/MS with the Analyst TF 1.7 Software (SCIEX). Chromatographic separation was carried out with a Cadenza CD-C18 column (150 × 2 mm, Imtakt, Japan). Elution buffers were 0.5% formic acid in SDW (solvent A) and acetonitrile (solvent B). Raw data were analyzed using the PeakView Software (SCIEX) and DeNovoGUI (Compomics). The single band detected on SDS-polyacrylamide gel after galactose-affinity chromatography was also analyzed in the same manner.

Total RNA was isolated from the intestine and coelomocytes of A. japonicus using ISOGEN (Nippon Gene). The quality and quantity of RNA were assessed with a Nanodrop 2000c spectrophotometer (Thermo Scientific) and 2100 Bioanalyzer using the RNA 6000 Nano kit (Agilent Technologies). A cDNA library was prepared from 1 µg of total RNA, a mixture of equal parts of total RNA from the intestine and CC, using NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England BioLabs), NEBnext Poly (A) mRNA Magnetic Isolation Module (New England BioLabs), and NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1, New England BioLabs) according to the manufacturer’s protocol. The protocol was amended to lower the fragmentation time from 15 min to 10 min to generate larger RNA fragments. The quantity of cDNA library was assessed on a KAPA Library Quantification Kit for Illumina platforms (KapaBiosystems). Sequencing was performed with Miseq (Illumina) using 301 bp paired-end reads with a Miseq reagent kit v3 (600 cycles). The sequencing reads were trimmed on the 3′-end using the statistical software R version 3.4.0 (19) with ShortRead package [version 1.34.0; (20)] and Biostrings package [version 2.44.1; (21)]. Next, trimmed reads were merged using BBmerge (https://sourceforge.net/projects/bbmap/; parameters: verystrict=t). Thereafter, low-quality reads were removed using the FASTX-toolkit (http://hannonlab.cshl.edu/fastx_toolkit/; parameters: -q 30, -p 80). Transcriptome assembly was performed using Trinity version 2.5.1 [ (22); parameters: -SS_lib_type R, min_contig_length 200).

Total RNA was isolated as mentioned above and cDNA was prepared using the SMART™ RACE cDNA amplification kit (Clontech). Sequences similar to those of the peptides obtained by de novo sequencing were searched by BLAST. The hits in the BLAST search were used as queries to search for homologs in the RNA-seq data for the intestine and coelomocytes. Because one sequence that was highly identical to multiple peptide sequences obtained by de novo sequencing was found in the RNA-seq data, cDNA sequencing was performed to confirm the sequence. A pair of primers, Aj-GBP3-1 (5′-TTC GGT CGA CTT TAC AAT ACA CCA ACA G-3′) and Aj-GBP5-1 (5′-TAC ATC GGC CCA TTT ACC ACG CGA ATG C-3′), was designed based on the sequence. PCR was performed using Taq polymerase (Ex Taq, Takara) under the following conditions: 95°C for 3 min; 30 cycles of 95°C for 30 s, 64°C for 30 s, 72°C for 30 s; and 72°C for 7 min. The amplicon was ligated into the pGEM-T Easy vector (Promega) and introduced into Escherichia coli-DH5α competent cells (HIT, RBC Bioscience). DNA sequencing was performed using a Big-Dye Terminator Cycle sequencing kit ver.3.1 (Applied Biosystems) and DNA sequencer (3100-Avant, Applied Biosystems). The 3′-RACE PCR was performed using cDNA extracted from the intestine, Ex Taq, a forward primer, Aj-GBP3-1, and a reverse adapter primer, NUP (5′-AAG CAG TGG TAT CAA CGC AGA GT-3′, Clontech). The conditions for 3′-RACE PCR were the same as described above. A phylogenetic analysis was performed to identify the homologous molecules. Sequences similar to that of the aggregation-promoting factor of A. japonicus were searched using local BLAST of the 807 echinoderm C-type lectin sequences registered in the NCBI database. Of these, 191 sequences had an E-value above 10^-5 and were selected for subsequent analyses. Phylogenetic trees were constructed using the maximum likelihood method with 100 bootstrap replicates based on 191 sequences using MEGA X software.

Total RNA was extracted from the body wall, tentacles, Polian vesicle, intestine, respiratory tree, and coelomocytes, and reverse transcribed as described above. The expression of AjGBCL in these tissues was examined using a pair of specific primers, Aj-GBP3-2 (5′-CCA TAG CAT CGG GCT TGC ATT CGC GTG G-3′) and Aj-GBP5-2 (5′-TAC ACC TGT GGA TCC CAA CTG TTG GTG-3′), and Ex Taq. The PCR conditions were as follows: 95°C for 3 min; 30 cycles of 95°C for 30 s, 64°C for 30 s, and 72°C for 30 s. The expression of actin gene was used as an internal control, using a pair of primers, Actin-F (5′-TCC TTC ACC ACC ACA GCC GA-3′) and Actin-R (5′-ACC GGC GGA CTC CAT TCC AA-3′), which were designed based on the sequence of the A. japonicus actin gene (BAH79732).

Ten milliliters of the coelomic fluid was collected and divided into two 50 ml centrifuge tubes. Five hundred microliters of the intestinal extract was added into one tube, and the same volume of 3× PBS was added to the other as a control. The mixture was allowed to stand for 30 min and passed through a nylon mesh with a pore size of 100 μm to separate the aggregates. After washing three times with 3× PBS, the mesh was turned over and the aggregates were collected and incubated with 200 mM EDTA for 2 h to dissociate the aggregated cells. The dissociated cells were counted using a Burker Turk hemocytometer, and 5.0 × 10⁵ cells were centrifuged onto a glass slide. After staining with May–Grunwald/Giemsa stain, 300 randomly selected cells were classified as described by us in a previous study (15).

The statistical significance of differences in the total area of cellular aggregates, with or without the piece of intestine, was examined using the paired t-test with the PRISM software (Graphpad). In the aggregation experiment using several tissue extracts, the significance of the difference was analyzed by repeated measurement of ANOVA followed by Tukey’s multiple comparison test. Differences in the cellular composition of the aggregates, with or without the intestinal extract, were analyzed by paired t-test. Differences were considered significant at p < 0.05.

To verify the role of cellular aggregation upon tissue damage, the evisceration of A. japonicus was artificially induced, and fluorescently labeled coelomocytes were autografted into the coelom of the animals. As shown in Figure 1 , fluorescence signals were detected on the sectioned surface of the digestive tract at 3 h after this autologous transplantation. This indicates that the aggregates of coelomocytes cover the surface of the digestive tract and seal it after evisceration.

We observed the aggregation of coelomocytes microscopically in the presence of a piece of intestinal tissue. Time-lapse observation revealed that the coelomocytes moved and aggregated more quickly and formed larger aggregates in the presence of the tissue ( Supplementary Video 1 ). Most of these cells moved toward the growing aggregates, but rarely toward the piece of intestine ( Figure 2 ). However, a few aggregates in the vicinity of the tissue eventually got attached to it. After 30 min, the total area of the aggregates formed under this condition was about 4–5-fold larger than that in the control ( Figure 3 ). This suggests that the intestinal tissue contains substances that promote cellular aggregation of the coelomocytes.

Tissue extracts of the body wall, tentacle, intestine, respiratory tree, or Polian vesicle were also examined for their effect on aggregation ( Figure 4 ). The intestinal extract markedly accelerated the cellular aggregation. Other than the intestinal extract, only the respiratory tree extract showed this effect. Interestingly, the extract of the Polian vesicle inhibited the cellular aggregation.

When the intestinal extract was subjected to heat treatment, the total area of the aggregates formed was decreased to nearly the same as that in the control, indicating that the aggregation-promoting factor is heat unstable ( Figures 5A, B, E ). After ultrafiltration of the extract through a 5 kDa membrane, a strong aggregation-promoting effect of the concentrated extract was observed, whereas no effect was observed for the filtrate ( Figures 5C–E ). These results suggested that the factor is a protein.

The protein extracted from the intestinal extract was subjected to anion-exchange chromatography, followed by gel filtration chromatography ( Figures 6A, B ). After anion-exchange chromatography, the aggregation-promoting activity was detected in the fraction indicated in Figure 6A . This fraction was subjected to gel-filtration chromatography, and one of the eluted fractions exhibited the activity ( Figure 6B ). When the fraction was subjected to SDS-PAGE, only a single 16 kDa band was observed ( Figure 6C ).

Assuming that the aggregation-promoting factor is a lectin, we examined the inhibitory effect of sugars on the aggregation-promoting activity of the intestinal extract. The activity was markedly inhibited by galactose but not by other sugars ( Figure 7 ). This suggests that the aggregation-promoting factor is a galactose-binding protein.

The galactose-binding protein was purified from the intestinal extract by affinity chromatography using galactose-agarose. As shown in Figure 8A , we detected a single band of about 16 kDa, similar to that of the aggregation-promoting factor purified by anion-exchange and gel-filtration chromatography, in the fraction eluted with galactose. When the eluted fraction was supplemented to the coelomic fluid, cellular aggregation was accelerated ( Figure 8B ), indicating that the protein was indeed the aggregation-promoting factor.

De novo sequencing of the 16 kDa protein purified by gel-filtration chromatography provided six peptide sequences, and that of the protein obtained by the galactose-affinity chromatography gave three peptide sequences, all of which were also present in the former ( Figure 9A ). Protein sequences were subjected to BLAST search, which revealed that these peptides have similarities to C-type lectins of A. japonicus registered in GenBank (ABC87994 and PIK42223). Sequences similar to these lectins were then searched in the RNA-seq data. We found one sequence with high identity to four peptide sequences obtained by de novo sequencing, which included the three sequences mentioned above ( Figure 9B ). Two sequences that do not match with AjGBCL ( Figure 9A ) did not hit any sequences deposited in databases. We then performed cDNA cloning to determine the sequence, and identified that the cDNA of the aggregation-promoting factor includes a 534 bp open reading frame, encoding a protein with 178 aa residues, and having a 20 aa predicted N-terminal signal peptide ( Figure 9C ). BLAST search of this sequence did not give any identical sequence; however, similarity with C-type lectins of echinoderms was observed. These data indicate that the aggregation-promoting factor is a galactose-binding C-type lectin that has not been reported. Hereafter, the aggregation-promoting factor from A. japonicus will be referred to as “AjGBCL”, a short form for “A. japonicus galactose-binding C-type lectin”. The original contributions presented in the study are publicly available. This data can be found here: (https://www.ncbi.nlm.nih.gov/nuccore/LC552681.1), Accession No. LC552681.

To determine whether homologues of AjGBCL are present in other echinoderm animals, we performed a homology search and phylogenetic analysis. The closest sequence was a C-type lectin of A. japonicus, whose amino acid sequence was 81% similar to that of AjGBCL ( Supplementary Figure 1 ). In other species (Strongylocentrotus purpuratus), a sequence designated as the bone morphogenetic protein-1-like was located in the closest branch; however, the bootstrap value of this node was quite low. This protein showed only 35% homology with AjGBCL. Bootstrap values at some nodes in this tree were quite low, probably due to their moderate resemblance to each other. However, no other sequence with high homology with AjGBCL was found.

Reverse transcription-PCR showed that AjGBCL was expressed in all the examined tissues, namely Polian vesicle, body wall, tentacles, intestine, respiratory tree, and coelomocytes ( Figure 10 ). We also confirmed that all the sequences of the amplicons were identical to that of AjGBCL.

To compare the cellular composition of the aggregates formed with or without the intestinal extract, the aggregates were dissociated and the cells were classified ( Figure 11 ), based on the classification presented in our previous study (15). Amoebocyte was the most abundant cell type without the supplementation of the intestinal extract. However, the proportion of amoebocytes to the total number of cells was lower and those of some other cell types were higher (up to 15%) in the presence of the intestinal extract. In particular, the proportions of the two types of basophilic granulocytes, defined as type K and L in our previous study, were significantly higher than in the case of control (addition of 3× PBS).