2.1 Silkworm Rearing

B. mori silkworms (F₁ Baekok-Jam hybrid) were provided by the Department of Agricultural Biology, National Institute of Agricultural Science, Republic of Korea, and were reared on fresh mulberry leaves in accordance with the silkworm breeding standards outlined by the Rural Development Administration (Kim and Jin 2017; Lee et al. 2018; Park et al. 2024).

2.2 Cloning and Sequence Analysis of BmCTL4

To clone the BmCTL4 cDNA, total RNA was extracted from the entire body of B. mori larvae using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Complementary DNA (cDNA) was synthesized from 1 μg of total RNA using the AccuPower RT-PCR PreMix (BIONEER, Daejeon, Republic of Korea) and 10 pmol of each primer (Table S1) at 42°C for 60 min. PCR amplification was then conducted under the following cycling conditions: initial denaturation at 95°C for 5 min; 33 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 1 min; followed by a final extension at 72°C for 5 min. The PCR products were then ligated into the pGEM-T vector (Promega, Madison, WI, USA), and the fidelity of the nucleotide sequences was verified using a 3730XL DNA Analyzer (Applied Biosystems, Foster City, CA, USA).

For the analysis of the BmCTL4 amino acid sequence, the SignalP 5.0 program (http://www.cbs.dtu.dk/services/SignalP), MOTIF Search (https://www.genome.jp/tools/motif), known sugar-binding sites (Kolatkar and Weis 1996; Huang et al. 2013), and multiple sequence alignment using MacVector (Version 6.5, Oxford Molecular Ltd., Oxford, UK) were used. Phylogenetic analysis was conducted using MEGA 11.0 with the maximum likelihood method based on the Kimura 2-parameter model and 1000 bootstrap replicates. Lectin amino acid sequences from insects were aligned using ClustalW with default parameters prior to phylogenetic tree construction.

2.3 Microbial Injection and Reverse Transcription-Quantitative PCR (RT-qPCR)

For tissue-specific expression analysis, fat body, hemocytes, and epidermis were collected from 3-day-old fifth-instar larvae. To examine BmCTL4 expression induction by microorganisms, 3-day-old fifth-instar larvae were injected with 1.0 × 10⁵ Escherichia coli in 50 μL per individual, while larvae injected with 50 μL of PBS served as the control (Park et al. 2024). Fat bodies were collected at 3, 6, 12, and 24 h post-injection. Total RNA was isolated using RNAiso Plus (TaKaRa Bio, Shiga, Japan) and quantified with a NanoDrop One spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was synthesized using the AccuPower RT PreMix (BIONEER, Daejeon, Korea) with 2 μg of total RNA for RT-qPCR. BmCTL4 expression levels were quantified using TB Green Premix Ex Taq (TaKaRa Bio) in a 25 μL reaction mixture containing 0.2 nmol/L of each primer (listed in Table S1) and 80 ng of cDNA. RT-qPCR was conducted using a CronoSTAR 96 real-time PCR system (Clontech, now TaKaRa Bio USA, San Jose, CA, USA). The thermal cycling conditions were 95°C for 5 min, followed by 40 cycles of 95°C for 15 s and 60°C for 30 s. BmCTL4 expression was normalized to the internal control gene actin A4 (Lee et al. 2018; Park et al. 2024). Relative gene expression was calculated using the 2^−ΔΔCT method (Livak and Schmittgen 2001) on three biological replicates for each treatment.

2.4 Purification of Recombinant BmCTL4 and Antibody Production

Recombinant BmCTL4 protein was produced using a baculovirus expression system with Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Sf9 insect cells (Je et al. 2001). Primers (Table S1) used to amplify BmCTL4 cDNA from fat body RNA carried restriction sites for cloning into the pBacPAK8 vector and a 6×His tag for purification. Sf9 cells were co-transfected with AcMNPV DNA and pBacPAK8-BmCTL4 using Lipofectin (Gibco BRL, Gaithersburg, MD, USA). The recombinant virus was propagated in TC100 medium at 27°C. The protein was purified with the MagneHis system (Promega, Madison, WI, USA) and concentrated using Vivaspin 500 (Sartorius Stedim Biotech, Göttingen, Germany). Its molecular weight was estimated using a standard calibration curve (Dunker and Rueckert 1969; Shapiro et al. 1967; Shapiro et al. 1969; Weber and Osborn 1969).

Anti-BmCTL4 antibodies were produced as described in previous studies (Lee et al. 2015; Zou et al. 2015; Kim and Jin 2017; Lee et al. 2018; Park et al. 2024). BALB/c mice (Samtako Bio Korea Co., Osan, Republic of Korea) were injected with ~5 μg of recombinant BmCTL4 protein once a week for 4 weeks. Freund's complete adjuvant was included in the initial dose, Freund's incomplete adjuvant was used in the subsequent two doses (Sigma-Aldrich, St. Louis, MI, USA), and the final dose consisted solely of protein. Blood was collected 3 days after the last injection. Antibodies were obtained by centrifugation (9600 ×g, 10 min, 4°C) and stored at −70°C for western blot analysis.

2.5 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Western Blot Analysis

Proteins from fat bodies were extracted for western blot analysis using BioMasher II tubes (NIPPI, Tokyo, Japan), followed by centrifugation to obtain the supernatant. Protein concentrations were determined using a protein assay reagent (Bio-Rad, Hercules, CA, USA).

Following protocols reported in previous studies (Lee et al. 2015; Zou et al. 2015; Kim and Jin 2017; Lee et al. 2018; Park et al. 2024), protein samples (~10 μg) were separated on a 12% SDS-PAGE. The separated proteins were transferred and analyzed through western blotting using anti-BmCTL4 or anti-His antibodies (BETHYL Laboratories, Montgomery, TX, USA), and detected with an enhanced chemiluminescence (ECL) system (Amersham Biosciences, Piscataway, NJ, USA), following the manufacturer's instructions. A horseradish peroxidase-conjugated anti-mouse IgG (1:5000 dilution, v/v; Enzo Life Sciences, Farmingdale, NY, USA) was used as the secondary antibody. Band intensities were quantified using an Alpha Imager 1220 v5.5 (Alpha Innotech Co., San Leandro, CA, USA).

2.6 Binding Properties of Recombinant BmCTL4

The binding properties of recombinant BmCTL4 as a PRR were evaluated using a binding assay as described by Kim and Jin (2017) and Park et al. (2024). Microbial cell wall components—LPS, mannan, N-acetyl-D-glucosamine, and peptidoglycan (PGN) (all from Sigma-Aldrich)—were coated onto a 96-well plate. The plate was subsequently blocked with 0.1 mg/mL bovine serum albumin (BSA) solution in Tris buffer (50 mmol/L Tris–HCl, 50 mmol/L NaCl, pH 8.0). After washing four times with Tris buffer, the plate was incubated with 0, 20, 40, 60, 80, or 100 nmol/L of recombinant BmCTL4, with or without 5 mmol/L CaCl₂, for 3 h at room temperature. After washing with Tris buffer, the plate was then incubated with polyclonal anti-His antibodies (1:1000, v/v) for 1 h at room temperature. Subsequently, it was washed again and incubated with HRP-conjugated goat anti-mouse IgG (1:5000, v/v; Enzo Life Sciences) for an additional hour. After a final wash, 100 μL of TMB substrate was added to each well and was allowed to incubate for 10 min. The reaction was terminated with 100 μL of 2 mol/L H₂SO₄, and absorbance was measured at 450 nm using an Infinite F50 microplate reader (Tecan, Grödig, Austria).

Next, to verify the binding of recombinant BmCTL4 to the surface of living microorganisms, cultures of E. coli, B. thuringiensis, and B. bassiana were prepared for the microbial binding assay following a protocol of a previous study (Park et al. 2024). Upon reaching an optical density of 0.4 at 600 nm (OD₆₀₀), 4 mL of each culture was harvested, washed twice with PBS, and resuspended in 40 μL of PBS containing 1 μg of recombinant BmCTL4. The mixtures were incubated at room temperature for 10 min and then centrifuged at 1500 ×g for 5 min to collect the microbial pellet. After a PBS wash, the pellet was resuspended in 40 μL of PBS. To assess binding, both pellet and supernatant fractions were subjected to western blot analysis using anti-His antibodies (BETHYL Laboratories; 1:10,000, v/v), following previously described methods. Microbial immunofluorescence staining (Park et al. 2024) was subsequently carried out by fixing the microorganism pellets onto slides with cold methanol (−20°C) for 2 min. Microorganisms were subsequently incubated with anti-BmCTL4 antibodies, followed by fluorescein-conjugated goat anti-mouse secondary antibodies (Santa Cruz Biotech. Inc., Santa Cruz, CA, USA). The samples were visualized using a laser scanning confocal microscope (Carl Zeiss LSM 510, Zeiss, Jena, Germany).

2.7 Statistics

Statistical analysis was carried out using SPSS Statistics 22.0 (IBM, Armonk, NY, USA). Each treatment comprised three biological replicates and is presented as the mean ± standard deviation. Comparisons between two groups were performed using the Wilcoxon rank sum test, followed by an independent unpaired two-tailed Student's t-test. Statistical significance was defined as *p < 0.05 and **p < 0.001.

3.1 Structural Feature of BmCTL4

B. mori CTL4 (BmCTL4) cDNA encodes a protein of 221 amino acids, including a 19-amino-acid signal peptide and a single carbohydrate recognition domain (CRD) of 172 amino acids. The predicted molecular weight and isoelectric point of the mature BmCTL4 were 23.1 kDa and 8.2, respectively. The amino acid sequence of the cloned BmCTL4 contains eight conserved cysteine residues and a Gln-Pro-Asp (QPD) motif, which is associated with potential galactose-binding specificity (Figure S1). The phylogenetic tree was divided into two groups: one containing lepidopteran and coleopteran CTLs, and the other including dipteran, hymenopteran, and hemipteran CTLs. BmCTL4 showed the highest homology with Antheraea pernyi CTL4 (Figure 1).

3.2 Tissue Expression Profiling and Induction Analysis

The tissue-specific expression of BmCTL4 in hemocytes, fat body, and epidermis of 3-day-old fifth-instar larvae was assessed with RT-qPCR. BmCTL4 expression was predominantly observed in the fat body and epidermis, whereas its expression in hemocytes was negligible (Figure 2A). To investigate whether BmCTL4 functions as a PRR responsive to E. coli, its expression in the fat body was further evaluated at multiple time points following E. coli injection. BmCTL4 expression was gradually upregulated starting at 3 h, with higher expression levels observed at 12 and 24 h following E. coli injection (Figure 2B). Next, western blot analysis was performed to confirm BmCTL4 upregulation at the protein level. Consistent with the RT-qPCR results, BmCTL4 protein expression was induced in the fat body, exhibiting a time-dependent increase in band intensity (Figure 2C).

3.3 BmCTL4 Binding Affinity to Carbohydrates and Microorganisms

To investigate the functional role of BmCTL4, a baculovirus expression system was employed for the production of the recombinant BmCTL4 protein. The 24.5-kDa recombinant protein was purified from total cellular lysates and used to immunize mice for the generation of polyclonal antibodies (Figure 3A). Binding assays demonstrated that the recombinant BmCTL4 interacted with LPS (Figure 3B), mannan (Figure 3C), N-acetyl-D-glucosamine (Figure 3D), and PGN (Figure 3E) in a dose- and a Ca²⁺-dependent manner.

To further assess its binding target recognition capabilities, we tested whether recombinant BmCTL4 could bind to the surfaces of live microorganisms, including B. thuringiensis, E. coli, and B. bassiana. Western blot-based microbial binding assays confirmed its affinity and binding to all three organisms (Figure 4A). Furthermore, BmCTL4 was localized to the cell walls of bacteria, fungi, and yeast, as evidenced by immunofluorescence staining (Figure 4B). Collectively, these findings indicate that BmCTL4 is a CTL capable of recognizing both Gram-positive and Gram-negative bacteria, as well as fungi.