Adult C. gigas, 10–15 cm in length and 150–200 g in weight, were collected from a farm in Qingdao, Shandong Province, China, and acclimated in aerated seawater at 18°C for two weeks prior to use. BALB/C mice were purchased from Qingdao institute for the control of drug products. All the experiments were conducted according to the regulations of local and central government. All animal-involving experiments of this study were approved by the Ethics Committee of Institute of Oceanology, Chinese Academy of Sciences (23).

The shelled fresh C. gigas were crushed and homogenized with a Dounce tissue grinders (Sigma, USA), and 500 g of the wet mass was suspended and extracted with 1,000 ml TBS (50 mM Tris–HCl, pH 7.4, 150 mM NaCl) at 4°C with continuously agitation over night. The extract was centrifuged at 12,000 g for 30 min, and the crude proteins in the supernatant were precipitated with 80% (w/v) ammonium sulfate at 4°C over night. The protein precipitate was collected by centrifugation at 12,000 g for 1 h, followed by extensively dialysis against TBS for three times at 4°C. The supernatant was collected after centrifugation at 15,000 g for 1 h and filtered through a 0.45 µm membrane.

Carbohydrate coupled Sepharose 6B matrix was prepared according to our previous study (24). In brief, epoxy-activated Sepharose 6B matrix (GE Healthcare, Sweden) was washed extensively with distilled water, and then mixed with four kinds of carbohydrates, including d-lactose, N-acetyl-d-glucosamine (d-GlcNAc), d-mannose, and l-fucose (Sigma-Aldrich, Buchs, Switzerland), at a final concentration of 200 µM in distilled water (pH 13.0). The mixtures were incubated at 30°C with gentle shaking for 16 h, and 1 M ethanolamine (pH 8.0) was used to block the remaining active groups after TBS washing. The carbohydrate coupled Sepharose 6B matrix was then washed extensively with TBS, and packed into Teflon columns with 100 mm long and 10 mm inner diameter, respectively.

The d-lactose, d-GlcNAc, d-mannose, and l-fucose coupled Sepharose 6B affinity columns were preequilibrated with TBS, and the crude proteins from C. gigas were passed through the carbohydrate coupled Sepharose 6B affinity columns at a flow rate of 1 ml/min on an AKTA avant chromatography system (GE Healthcare, USA), respectively. After extensive TBS washing, the carbohydrate binding proteins were eluted with 200 mM d-lactose, d-GlcNAc, d-mannose, and l-fucose in TBS (pH 7.4) corresponding to each carbohydrate coupled Sepharose 6B column. The eluates were then dialyzed against TBS to remove carbohydrates, and concentrated by a centrifugal filter with 3 kDa cutoff (Millipore, USA).

For the peptide mass fingerprinting (PMF) analysis, SDS-PAGE was performed to separate the eluate proteins, and the protein bands were visualized by Coomassie Brilliant Blue R-250 (Sigma, USA) staining. The protein band from the d-mannose eluate was excised and tryptically digested, and the peptides were desalted with a ZipTip^TM (Millipore, USA) according to the standard protocol (25). MALDI-TOF/TOF-mass spectrometry was performed on an UltrafleXtrem (Bruker Daltonics, Germany). MS spectra were interpreted with the MASCOT software (Matrix Sciences, UK) (26).

The coding sequence of CgDM9CP-1 was identified by searching the PMF against C. gigas genome database. The homology searches of amino acid sequence of CgDM9CP-1 were conducted with BLAST at the National Center for Biotechnology Information (NCBI).¹ The protein domain was predicted with the simple modular architecture research tool (SMART)² and conserved domain database (CDD) search service.³ Multiple alignment was performed with the Clustal X⁴ and the alignment show software Jalview.⁵ Conserved amino acid residues among DM9CPs were identified based on the sequence alignment and presented using WebLogo V3.⁶

Phylogenetic tree was constructed as previously reported (27). Briefly, the amino acid sequences of DM9CPs were searched against NCBI and the Joint Genome Institute.⁷ Multiple sequence alignments were generated using Clustal W⁸ with default parameters. The alignment was imported into the phylogenetic analysis program MEGA,⁹ and a maximum likelihood tree was generated. A circular phylogenetic tree was then constructed using the interactive tree of life server.¹⁰

Total RNA was isolated from the hemocytes of C. gigas using Trizol reagent (Invitrogen, USA) according to the manufactor’s instruction. RQ1 RNase-free DNase (Promega, USA) was used to digest genomic DNA, and Moloney murine leukemia virus reverse transcriptase (Promega, USA) was used to synthesize cDNA from 1 µg of total RNA. The CgDM9CP-1 coding sequence was amplified using ExTaq polymerase (TaKaRa, Japan) and primers listed in Table 1 with the temperature profile as following: 5 min denaturation step at 95°C and completed by a 10 min extension step at 72°C, with 30 s at 94°C, 20 s at 50°C, and 30 s at 72°C for 30 cycles. The amplified product was purified and cloned into pET-30a vector (Novagen, USA) according to the manufacturer’s instruction. The coding sequence of CgDM9CP-1 was confirmed by sequencing, and the plasmid was then transformed into E. coli Transetta (DE3) cells.

The mutagenic primers were designed using the PrimerX tool¹¹ (Table 1). CgDM9CP-1 mutants, including D22A, K43A and H52A, were constructed by PCR amplification. The coding sequence of mutants were cloned and inserted into the pET28a (+) vector (Novagen, USA), which encoded 6× His-tag at the N-terminal of mutants. All the mutants were confirmed by DNA sequencing, and the plasmid was then transformed into E. coli Transetta (DE3) cells.

The E. coli Transetta (DE3) cells expressing wild-type recombinant CgDM9CP-1 (rCgDM9CP-1) with His-tag were incubated in LB medium at 37°C with shaking at 220 rpm for 3 h. Induction of recombinant proteins was performed with 0.5 mM isopropyl β-d-thiogalactopyranoside (IPTG) at mid-exponential phase (OD 600 nm of 0.4–0.6). The bacteria were then grown at 18°C with shaking at 200 rpm over night. The bacteria was harvested and lysed, and the supernatant was pooled, loaded on to a d-mannose-coupled Sepharose 6B column. rCgDM9CP-1 was eluted using 200 mM d-mannose after extensive washing with TBS. The His-tagged recombinant mutants (D22A, K43A, and H52A) were expressed under the same culture condition with 0.1 mM IPTG induction. The His-tagged recombinant proteins were purified by Ni-NTA affinity chromatography. All the purified proteins were dialyzed over night against TBS at 4°C to remove free ligands.

To determine the carbohydrate-binding specificity of rCgDM9CP-1, glycan array screening was performed by the consortium for functional glycomics (Core H)¹² according to the standard protocol (28). The glycan array (version 5.1) was printed with 610 different natural and synthetic glycans. In the glycan array screening, His-tagged wild-type rCgDM9CP-1 was incubated with slides, and the bound rCgDM9CP-1 was determined by fluorescence labeled anti-His tag antibodies. The fluorescence intensity was detected using a ScanArray 5000 confocal scanner (PerkinElmer, USA). ImaGene image analysis software (BioDiscovery, USA) was used to analyze the image. The relative binding for each glycan was expressed as mean relative fluorescence unit (RFU) of four from the six replicates, with the highest and lowest RFU removed.

Isothermal titration calorimetry experiments on the interaction of rCgDM9CP-1 and its mutants with carbohydrates were performed at 25°C with a VP-ITC isothermal titration calorimeter (Microcal, USA). The freshly purified wild-type rCgDM9CP-1 was dialyzed overnight in PBS (pH 7.4) at 4°C and the protein concentration in the microcalorimeter cell (1.4478 ml volume) was adjusted to 0.05 mM. Carbohydrate solutions, including d-mannose, l-mannose, d-glucose, d-galactose, d-lactose, l-fucose, and d-GlcNAc at a final concentration of 5 mM in PBS were placed in the syringe. After the first injection with 4 µl, 27 injections of 10 µl were conducted with a stirring rate at 300 rpm. The dilution heats of the carbohydrates were measured by injecting different carbohydrate solutions into buffers alone and were subtracted from the experimental curves prior to data analysis. The determination of interactions between mutants and d-mannose were performed by the procedure described above, except that the d-mannose concentration in the syringe was adjusted to 2.5 mM in PBS. The experimental data was fitted to a theoretical titration curve using Microcal ORIGIN software supplied with the instrument, and the standard molar enthalpy change for the binding, ΔbHm0, and the dissociation constant, K_d, were derived. The standard molar free energy change, ΔbGm0, and the standard molar entropy change, ΔbSm0, for the binding reaction were calculated by using the following thermodynamic equations: (1)ΔbGm0=RTlnKd, (2)ΔbSm0=(ΔbHm0−ΔbGm0)/T.

The purified proteins were concentrated to 10 mg/ml by a centrifugal filter with 3 kDa cutoff (Millipore, MA, USA) at 4°C. The protein precipitate was removed by centrifugation at 12,000 g for 30 min, and the supernatant was further filtered through a 0.45 µm filter (Millipore, MA, USA). The protein sample was loaded onto a Superdex 200 10/300 GL gel-filtration column (GE Healthcare, Sweden) equilibrated with TBS at a flow rate of 0.5 ml/min on an AKTA avant chromatography system. The eluates corresponding to the peak areas were collected and used for crystallizations. Standard crystallization screening was carried out using a CrystalMation robot system (Rigaku, USA). Crystallization screens were purchased from Sigma (Sigma, USA), Jena Bioscience (Jena Bioscience, Germany), and Qiagen (Qiagen, Germany). Three hundred nanoliters of protein solution were mixed with crystallization buffer in a ratio of 1:1, sealed and kept at 18°C. Crystallization was done by vapor diffusion in sitting drops. A 300 nl aliquot of wild-type protein rCgDM9CP-1 (dataset name of DM9CPm, 20 mg/ml) was mixed with 300 nl of a reservoir solution composed of 20% (v/v) Poly (ethylene glycol) 2000 monomethyl ether and 100 mM Tris–HCl buffer (pH 7.0). The crystals appeared within 2 days. Crystals of DM9CPm with d-mannose cofactor were obtained within 3 days when adding a 300 nl aliquot of DM9CPm (20 mg/ml) in TBS with 10 mM d-mannose to 300 nl of a reservoir solution composed of 20% (v/v) poly (ethylene glycol) 6000 and 100 mM MES buffer (pH 6.0). For crystallization of D22A mutant (dataset name of DM9CPd), a 300 nl aliquot of DM9CPd (5 mg/ml) in TBS was mixed with 300 nl of a reservoir solution composed of 30% (v/v) poly (ethylene glycol) 3350 and 100 mM HEPES buffer (pH 7.0), and 0.12 M magnisium chloride hexahydrate. The crystal grew within 2 weeks. A 300 nl aliquot of K43A mutant (dataset name of DM9CPk, 5 mg/ml) was mixed with 300 nl of a reservoir solution composed of 25% poly(ethylene glycol) 3350 and 0.1 M Bis-Tris (pH 6.0), in presence of 0.2 M lithium sulfate monohydrate; crystals were obtained after 10 days.

Native data were collected at beamline X10SA at the Swiss Light Source (SLS). Long wavelength data for native single-wavelength anomalous diffraction (SAD) phasing were collected at beamline X06DA at SLS. For collection, the crystal was reoriented twice during data collection using the PRIGO mutiaxis goniometer (29). According to the single crystal native SAD phasing strategy described before (30), 3 × 360° long wavelength data were collected at 6 keV, employing phi-fine slicing (31) at three different crystal orientations (chi angles of 0, 10, and 20). Both data sets of mutants D22A and K43A, and native binding with cofactor d-mannose were collected at Beamline X10SA, indexed and processed with the program XDS (32) to a resolution of 1.3, 1.6, and 1.1 Å, respectively.

The structure was solved using the SHELXC/D/E pipeline with hkl2map as graphical user interface. Four sites were readily identified with SHELXD with a resolution cutoff of 2.6 Å resulting in CCall and CCweak of 46.0 and 28.5, respectively. Density modification in SHELXE was carried out for 60 cycles using the high resolution native data up to 1.1 Å resolution with a solvent content of 0.45. The initial model was auto built using BUCCANEER and completed with 144 of 144 residues built. The other structures were determined employing the Molecular Replacement Method using the native model DM9CP (PDB ID: 5MH0) as an ensemble in the program PHASER (33).

The structures were refined initially using REFMAC5 (34) and PHENIX REFINE (35) for the final stages. Necessary model improvements as well as search for solvent molecules were performed using COOT (36) and “update water” in PHENIX REFINE. Anisotropic thermal displacement factors were refined at 1.3 Å or better resolution, otherwise using the TLS model. Glycerol was tentatively built in the model to allow for the corresponding electron density as well as d-mannose in the density of DM9CPm.

Vibrio splendidus was grown in 2216E media at 28°C, 220 rpm for 12 h. E. coli, S. aureus, and Bacillus subtilis were grown in LB media at 37°C, 220 rpm for 8 h. Pichia pastoris and Yarrowia lipolytica were grown in YPD media at 30°C, 220 rpm for 24 h. All microbes were grown to mid-log phase, harvested by centrifugation at 6,000 g for 15 min, and washed three times with PBS.

Microbes were collected, fixed with 4% paraformaldehyde (PFA) as previously reported (37), and mixed with 1 mg/ml FITC (Sigma, USA) in 0.1 M NaHCO₃ (pH 9.0) buffer with continuous gentle stirring at room temperature overnight. The FITC-labeled microbes were washed with PBS for three times to eliminate free FITC molecules.

Microbes including E. coli, V. splendidus, B. subtilis, S. aureus, P. pastoris, and Y. lipolytica (10⁸ cells/ml) were incubated with 3% BSA in PBS for 1 h to block the non-specific binding sites. After three times PBS washing, microbes were incubated with or without rCgDM9CP-1 (control group) at a final concentration of 0.5 mg/ml for 30 min at room temperature. The cells were then washed three times with PBS and incubated with FITC labeled anti-His tag antibody for 1 h at room temperature. After extensive washing, the samples were examined by flow cytometry to detect the microbial binding activity, and analyzed by the fluorescence microscopy to determine the microbial agglutination activity.

The antibiotic assay was performed as described previously (38). Briefly, microbes (10⁸ cells/ml) were incubated with 3% BSA in PBS for 1 h, followed by incubation with rCgDM9CP-1 at a final concentration of 0.5 mg/ml at room temperature for 2 h. The cells were then washed three times with PBS and incubated with propidium iodide (5 µg/ml) at room temperature for 10 min. After extensive washing, the samples were examined by flow cytometry.

Pathogen-associated molecular pattern binding activity was determined by modified enzyme-linked immunosorbent assay (ELISA). Briefly, lipopolysaccharide (LPS, from E. coli O55:B5, Sigma-Aldrich), peptidylglycan (PGN, from B. subtilis, Sigma-Aldrich), mannan (from Saccharomyces cerevisiae, Sigma-Aldrich), and β-1,3-glucan (from Euglena gracilis, Sigma-Aldrich) were coated on wells of a 96-well microtiter plate (20 μg/well) at 37°C for 1 h. After PBST (PBS, pH7.4, 0.1% Tween-20) washing, the wells were blocked with 3% BSA in PBS at room temperature for 1 h. His-tagged rCgDM9CP-1 was added to each well by incubation at 4°C overnight followed by three times PBST washing. For the carbohydrate inhibition assay, His-tagged rCgDM9CP-1 was preincubated with 200 mM d-glucose, d-GlcNAc, l-fucose, d-lactose, and d-mannose (Sigma-Aldrich, Buchs, Switzerland) for 30 min, respectively, then added to each well of PAMPs coated 96-well microtiter plate and incubation at 4°C overnight followed by three times PBST washing. Horseradish peroxidase (HRP)-labeled anti-His tag monoclonal antibody was added to each well and incubated at room temperature for 1 h. Tetramethylbenzidine substrates (Pierce, Rockford, IL, USA) was added to each well and incubated for 20 min after five times PBST washing, followed by addition of 1 M H₂SO₄ to terminate the reaction. The value of each well was recorded at 450 nm by microplate reader (Biotek, USA). The dissociation constant (K_d) was calculated using GraphPad Prism 5 with nonlinear regression curve fit and a one-site binding model analysis. As A = A_max [L]/(K_d + [L]), where A is the absorbance at 450 nm and [L] is the concentration of the rCgDM9CP-1.

BALB/C mice were immunized subcutaneously with 50 µg recombinant rCgDM9CP-1 in complete Freund’s adjuvant, and boosted three times in incomplete Freund’s adjuvant at 2-weeks intervals. At 1 week after the final immunization, the blood samples were collected and serum was separated by centrifugation at 2,000 g, 4°C for 15 min. The serum titers of the polyclonal antibodies against rCgDM9CP-1 were determined by ELISA. The serum was then buffered by PBS and loaded onto a 2 ml protein A column (GE Healthcare, Sweden). After PBS washing, immunoglobulin G (IgG) was eluted with 100 mM glycine-HCl (pH 2.8). The eluate was rapidly neutralized with 1 M Tris–HCl (pH 8.5), and dialyzed extensively against PBS over night. The eluted IgG was concentrated and the binding specificity toward rCgDM9CP-1 was determined by western blotting.

Hemolymph samples from 10 adult C. gigas were prepared as described previously (39). After centrifugation at 800 g, 4°C for 10 min, supernatant was collected, and hemocytes were pelleted. Hemolymph was centrifuged at 12,000 g, 4°C for 10 min to remove cell debris. Hemocytes were washed with PBS (pH 7.2) for three times at 800 g, 4°C for 10 min, and lysed in RIPA buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate and 0.1% SDS) on ice for 15 min. The cell lysate was collected after centrifugation at 12,000 g, 4°C for 10 min. Hepatopancreas, gill, mantle, and adductor muscle from 10 C. gigas were collected and homogenized in RIPA buffer using Dounce tissue grinders (Sigma, USA). The supernatant was collected by centrifugation at 12,000 g, 4°C for 20 min. The protein concentration was determined by BCA Protein Assay Kit (Pierce, USA). The samples with same amount of proteins (30 µg) were separated by SDS-PAGE. The proteins were transferred from the gel to the polyvinylidene difluoride membranes (Millipore, USA), and the membrane was soaked with 5% BSA in TBST. The membrane was then incubated with antibodies against rCgDM9CP-1 and β-tubulin for 1 h, respectively, followed by HRP labeled secondary antibodies incubation for 1 h. The immune-reactive protein bands were visualized by using an enhanced chemiluminescence kit (Pierce, USA).

Hemolymph was extracted from the posterior adductor muscle sinus using a 2 ml syringe equipped with a 23 G sterile needle, and immediately mixed with pre-chilled anticoagulant citrate dextrose solution A (ACD-A, 0.1 mol/l trisodium citrate, 0.11 mol/l dextrose, and 71 mmol/l citric acid monohydrate) at a volume ratio of 7:1. The hemocytes were harvested by centrifugation at 1,000 g at 4°C for 10 min, washed twice, and suspended in modified Leibovitz L15 medium (supplemented with 0.54 g/l KCl, 0.60 g/l CaCl₂, 1.00 g/l MgSO₄, 3.90 g/l MgCl₂, and 20.20 g/l NaCl). After incubation with 3% BSA for 1 h, the hemocytes were incubated with polyclonal IgG against rCgDM9CP-1 for 1 h, while the control hemocytes were incubated with isotype IgG under the same condition. After extensive washing, the hemocytes were incubated with FITC labeled goat anti mouse IgG for 1 h. The fluorescence intensity and the mean fluorescence intensity (10,000 cells) of hemocytes were determined on a FACSAria II flow cytometer (BD Biosciences, USA).

Hemocytes were prepared as described above, and plated on glass-bottom culture dishes and incubated at 18°C for 3 h. For the CgDM9CP-1 distribution analysis, 4% PFA was added to fix cells at 4°C for 15 min followed by three times PBS washing, and 0.1% Triton X-100 was added for permialization for 10 min. After PBS washing, 3% BSA in PBS was added to block the nonspecific binding sites for 1 h. Polyclonal IgG against rCgDM9CP-1 were incubated with hemocytes for 1 h, and Alexa Fluor 488 labeled goat antimouse IgG was incubated with hemocytes for 1 h after extensive PBS washing. Hemocytes were further incubated with Dil for 30 min to stain cytoplasmic membrane, and incubated with DAPI for 5 min to stain cell nucleus. For the phagocytosis assay, hemocytes were precultured with FITC labeled microbes for 1 h, and then fixed and permealized as described above. Polyclonal IgG against rCgDM9CP-1 were incubated with hemocytes for 1 h. After PBS washing, Alexa Fluor 594 labeled goat anti-mouse IgG was incubated with hemocytes for 1 h, followed by DAPI staining for 5 min. The hemocytes were monitored and the fluorescent images were taken using Carl Zeiss LSM 710 confocal microscope (Carl Zeiss, Germany).

The two-sample Student’s t-test was used for comparisons between groups. Statistical analysis was performed with GraphPad Prism 5 software. Results are shown as means ± SEM, and statistical significance was defined as P < 0.05.

Carbohydrate affinity chromatography was employed to isolate glycan binding proteins from crude protein extract of C. gigas. A single protein was highly enriched by d-mannose affinity chromatography, whereas d-lactose, N-acetyl-d-glucosamine (d-GlcNAc), and l-fucose affinity chromatography yielded far less (Figures S1A,B in Supplementary Material). The d-mannose binding protein was analyzed by MALDI-TOF/TOF-MS, and the amino acid sequence was identified by searching ten tryptic peptides against the genome of C. gigas (Figures S1C,D and Table S1 in Supplementary Material). The d-mannose binding protein was identical to the recently reported protein (22). Sequence alignment revealed that CgDM9CP-1 was not homologous to any other known lectins or carbohydrate binding proteins. Protein domain analysis using NCBI’s CDD and SMART showed that CgDM9CP-1 was only composed of two DM9 domains (Figure S2A in Supplementary Material), and they shared 33% sequence identity (Figure S2B in Supplementary Material). The carbohydrate binding specificity of rCgDM9CP-1 was determined by high-throughput glycan microarray printed with 610 different glycans (version 5.1, the consortium for functional glycomics). His-tagged rCgDM9CP-1 was found to bind non-reducing mannosyl glycans with the highest binding specificity. These glycans can be either branched high-mannose oligosaccharides or mannosylated bi- or triantennary hybrid oligosaccharides (Figure 1A; Table S2 in Supplementary Material). The former, for example, glycan #2, #5, #6, and #8, are uniformly composed of mannose residues, while the latter contain at least one branch antenna modified with non-reducing mannose termini, for instance, glycan #1, #3, #4, and #7. rCgDM9CP-1 also exhibited strong binding activity toward glycan #9 [6S(3S)Galβ1-4(6S)GlcNAc], which did not contain mannose residues, suggesting that there might exist another carbohydrate binding mechanism different from that of mannosylated glycans. The carbohydrate binding capacity of rCgDM9CP-1 was further revealed by ITC. The calorimetric data of d-mannose binding was fitted to one binding site model, and the dissociation constant (K_d) was determined to be 122.6 ± 19.7 µM (Figure 1B, left panel and Table 2). Compared with the relatively high binding affinity to d-mannose, rCgDM9CP-1 showed almost no binding affinity to other carbohydrates, including d-glucose, d-galactose, d-lactose, l-fucose, and d-GlcNAc (Figure S3 in Supplementary Material). Moreover, it did not exhibit any binding activity toward the l-isomer of mannose (Figure 1B, right panel).

Recombinant CgDM9CP-1 was prepared for crystallization by d-mannose-Sepharose 6B affinity chromatography followed by gel filtration chromatography (Figure 2A; Figure S4A in Supplementary Material). The crystal structure of rCgDM9CP-1 was determined at 1.24 Å resolution using the single crystal native SAD phasing strategy (PDB: 5MH0, Figures S5A,B in Supplementary Material). The diffraction data collection and structure refinement were summarized in Table 3. The crystal structure of rCgDM9CP-1 in complex with d-mannose was solved to reveal the potential residues involved in the d-mannose binding (5MH1, Figure S5C in Supplementary Material), which exhibited similar features to that of native CgDM9CP-1 reported by Unno et al. (22). Moreover, the side chain from Gly128 and four water molecules were also found to participate in the formation of hydrogen bond net work between d-mannose and rCgDM9CP-1 (Figure 2E).

In order to confirm the determinants of interaction between rCgDM9CP-1 and d-mannose, the residues of Asp22, Lys43, and His52 were individually mutated to alanine, and the His-tagged mutants were purified by Ni-NTA affinity chromatography followed by gel filtration chromatography (Figure 2A; Figures S4B–D in Supplementary Material). The mutations D22A and K43A completely abolished the d-mannose binding activities (Figures 2B,C), while the mutation of H52A exhibited a limited effect on the d-mannose binding activity (Figure 2D). The crystal structures of D22A (PDB: 5MH2) and K43A (PDB: 5MH3) were superimposed with that of rCgDM9CP-1 in complex with d-mannose, respectively. Compared with the wild-type protein, both the two mutants exhibited much smaller nonpolar side chains, which significantly impaired the hydrogen bond formation between d-mannose and rCgDM9CP-1 (Figures 2F,G).

The flow cytometric analysis revealed that rCgDM9CP-1 could bind to a number of microbes, including Gram-negative bacteria V. splendidus and E. coli, gram-positive bacteria S. aureus and B. subtilis, and fungi P. pastoris and Y. lipolytica (Figure 3A). Immunofluorescence microscopy further demonstrated the strong microbial binding and agglutination activities of rCgDM9CP-1 toward these microbes (Figure 3B). The binding activities of rCgDM9CP-1 toward different PAMPs, including LPS, PGN, mannan, and β-1,3-glucan, were determined by ELISA. rCgDM9CP-1 could directly bind LPS, PGN, mannan and β-1, 3-glucan in a concentration dependent manner with a saturable process from 0 to 10 µM (Figures 3C–F). The apparent K_d of rCgDM9CP-1 toward LPS, PGN, mannan and β-1, 3-glucan, calculated from the saturation curve, were 1.2 × 10⁻⁶, 2.1 × 10⁻⁷, 4.5 × 10⁻⁸, and 1.6 × 10⁻⁷ M, respectively. The binding activities of rCgDM9CP-1 toward LPS, PGN, mannan, and β-1,3-glucan significantly decreased after it was pre-saturated with 200 mM d-mannose (Figure 4A). Correspondingly, the PAMP binding activities of rCgDM9CP-1 significantly changed after the key amino acid residues were mutated. Mutants D22A and K43A showed much lower binding activity toward the four PAMPs even at high concentrations (5 µM), while H52A exerted PAMP binding activity in a concentration dependent manner from 0.05 to 5 µM, which was similar to that of wild-type rCgDM9CP-1 (Figures 4B–E).

The distribution of CgDM9CP-1 in different tissues was examined by Western blotting. CgDM9CP-1 was highly expressed in hepatopancreas, mantle and hemocytes, lower expressed in gill, while it was hard to be detected in muscle and hemolymph (Figure 5A; Figure S6 in Supplementary Material). Flow cytometric analysis revealed that CgDM9CP-1 was distributed on the outer membrane of hemocytes with high abundance (Figure 5B). Confocal analysis further confirmed that CgDM9CP-1 was mainly distributed on the hemocyte membrane, while less in the cytoplasm under non-challenged condition (Figure 5C). When the hemocytes were incubated with microbes, including E. coli, V. splendidus, S. aureus, and Y. lipolytica, to induce phagocytosis, CgDM9CP-1 could internalize from the cell membrane into the cytoplasm, and was found to colocalize with or surround the engulfed microbes (Figures 5D–G). Moreover, CgDM9CP-1 was found to exhibited antibiotic activity toward Y. lipolytica, while its antibiotic activity toward Gram-negative and Gram-positive bacteria was weak (Figure S7 in Supplementary Material).

To date, up to 477 DM9CPs have been annotated in the released genomes of a wide range of organisms from the Procaryotae, Fungi, Protista and Animalia Kingdoms (Figure S8A and Table S3 in Supplementary Material). DM9CPs are found to be of multi-copy in animal species, especially in invertebrates, with approximately ten similar genes in each Drosophila species (Figure 6A; Figure S8B in Supplementary Material). In C. gigas, seven DM9CPs were annotated, and they all contained two DM9 domains and shared high similarity with each other (Figures S2C,D in Supplementary Material). In order to further analyze the potential biological function of DM9CP family members, CgDM9CP-1 was aligned with another 476 DM9CPs using WebLogo (Figure 6B). The primary structure alignment illustrated that the amino acid sequence of DM9 domains were highly conserved throughout evolution, such as Asp22 and Lys43, which were found to be essential for the ligand binding in CgDM9CP-1.