Yuki Fujii

Functional divergence among lectins can arise even in the absence of variation in carbohydrate recognition domains (CRDs), yet the underlying mechanisms remain poorly understood. Here, we investigated the structure–function relationships of hRTLs, a family of six prototype galectins from the sponge Chondrilla australiensis. Recombinant hRTLs lacking the predicted 23-amino-acid non-canonical signal peptide were expressed in Escherichia coli and analyzed for glycan-binding specificity using glycan arrays. Four members (hRTL-1, 2, 5, and 6) exhibited glycan-binding activity, whereas hRTL-3 and 4 showed markedly weaker activity. hRTL-1 and 2 displayed similar binding profiles, including recognition of TF-related glycans, while hRTL-5 preferentially bound type-1 LacNAc and hRTL-6 recognized blood group-related glycans. Structural analyses revealed distinct oligomeric states, with dimeric and tetrameric species supported by native mass spectrometry and size-exclusion chromatography, while additional higher-molecular-weight (HMW) species were observed by non-heated SDS–PAGE. Far-UV CD spectroscopy further showed that representative active and low-activity hRTLs retained β-sheet-rich secondary structures. Notably, thermal stability assays showed that multimer-forming hRTLs retained hemagglutination activity after prolonged incubation at 60 °C, whereas non-multimerizing members exhibited reduced stability. These findings show that distinct oligomeric organizations are associated with functional diversification among structurally conserved galectin CRDs and suggest that modulation of multimerization may have contributed to the evolution of early metazoan lectins.

The structure of a lectin, OXYL, derived from the feather star, Anneissia japonica (phylum Echinodermata), was analyzed from biochemistry, informatics, and 3D structural prediction. OXYL’s primary structure resembled the globular domain of complement C1q in vertebrates. Structural simulations identified OXYL as part of the C1q/TNF superfamily, characterized by a jellyroll β-sandwich fold. The subunits can form monomer to nonamer (9-mer), with trimers being the most stable. However, gel permeation chromatography indicated tetramers as the significant form. In hemagglutination inhibition tests, OXYL bound type-2 N-acetyllactosamine (LacNAc: Galβ1-4GlcNAc) and inhibited agglutination but did not bind lactose (Lac: Galβ1-4Glc). Glycan microarray analysis with 20 immobilized glycans confirmed OXYL’s specificity for type-2 LacNAc and its sialic acid α2-3 linkage. The structural simulation showed a suitable molecular docking between type-2 LacNAc and the lectin rather than lactose. OXYL bound to the LacNAc glycan on the microarray was also elucidated to capture human IgG. This finding suggests that the invertebrate C1q/TNF family lectin will mimic vertebrate C1qs, despite lacking evolutionally antibody systems, possess the potential to interact with antibodies.

Background/Objectives: Nucleoside precursors and derivatives play pivotal roles in the development of antimicrobial and antiviral therapeutics. The 2022 global outbreak of monkeypox (Mpox) across more than 100 nonendemic countries underscores the urgent need for novel antiviral agents. This study aimed to synthesize and evaluate a series of 5′-O-(palmitoyl) derivatives (compounds 2–6), incorporating various aliphatic and aromatic acyl groups, for their potential antimicrobial activities. Methods: The structures of the synthesized derivatives were confirmed through physicochemical, elemental, and spectroscopic techniques. In vitro antibacterial efficacy was assessed, including minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) determinations for the most active compounds (4 and 5). The antifungal activity was evaluated based on mycelial growth inhibition. Density functional theory (DFT) calculations were employed to investigate the electronic and structural properties, including the global reactivity, frontier molecular orbital (FMO), natural bond orbital (NBO), and molecular electrostatic potential (MEP). Molecular docking studies were conducted against the monkeypox virus and the Marburg virus. The top-performing compounds (3, 5, and 6) were further evaluated via 200 ns molecular dynamics (MD) simulations. ADMET predictions were performed to assess drug-likeness and pharmacokinetic properties. Results: Compounds 4 and 5 demonstrated remarkable antibacterial activity compared with the precursor molecule, while most derivatives inhibited fungal mycelial growth by up to 79%. Structure-activity relationship (SAR) analysis highlighted the enhanced antibacterial/antifungal efficacy with CH₃(CH₂)₁₀CO– and CH₃(CH₂)₁₂CO–acyl chains. In silico docking revealed that compounds 3, 5, and 6 had higher binding affinities than the other derivatives. MD simulations confirmed the stability of the protein-ligand complexes. ADMET analyses revealed favorable drug-like profiles for all the lead compounds. Conclusions: The synthesized compounds 3, 5, and 6 exhibit promising antimicrobial and antiviral activities. Supported by both in vitro assays and comprehensive in silico analyses, these derivatives have emerged as potential candidates for the development of novel therapeutics against bacterial, fungal, and viral infections, including monkeypox and Marburg viruses.

We here report the purification of a novel member of the galectin family, the β-galactoside-binding lectin hRTL, from the marine sponge Chondrilla australiensis. The hRTL lectin is a tetrameric proto-type galectin with a subunit molecular weight of 15.5 kDa, consisting of 141 amino acids and sharing 92% primary sequence identity with the galectin CCL from the congeneric species C. caribensis. Transcriptome analysis allowed for the identification of additional sequences belonging to the same family, bringing the total number of hRTLs to six. Unlike most other galectins, hRTLs display a 23 amino acid-long signal peptide that, according to Erdman degradation, is post-translationally cleaved, leaving an N-terminal end devoid of acetylated modifications, unlike most other galectins. Moreover, two hRTLs display an internal insertion, which determines the presence of an unusual loop region that may have important functional implications. The characterization of the glycan-binding properties of hRTL revealed that it had high affinity towards TF-antigen, sialyl TF, and type-1 N-acetyl lactosamine with a Galβ1-3 structure. When administered to DLD-1 cells, a colorectal carcinoma cell line expressing mucin-associated TF-antigen, hRTL could induce glycan-dependent cytotoxicity.

We here report the purification of a novel member of the galectin family, the β-Galactoside-binding lectin hRTL, from the marine sponge Chondrilla australiensis. The hRTL lectin is a tetrameric proto-type galectin with a subunit molecular weight of 15.5 kDa, consisting of 141 amino acids and sharing 92% primary sequence identity with the galectin CCL from the congeneric species C. caribensis. Transcriptome analysis allowed the identification of additional sequences belonging to the same family, bringing the total number of hRTLs to six. Unlike most other galectins, hRTLs display a 23 amino acids long signal peptide that, according to Erdman degradation, is post-translationally cleaved, leaving a N-terminal end devoid of acetylated modifications, unlike most other galectins. Moreover, two hRTLs display an internal insertion, which determines the presence of an unusual loop region that may have important functional implications. The characterization of the glycan binding properties of hRTL revealed that it had high affinity to-wards TF-antigen, sialyl TF, and type-1 N-acetyl lactosamine with a Galβ1-3 structure. When administered to DLD-1 cells, a colorectal carcinoma cell line expressing mucin-associated TF-antigen, hRTL could induce glycan-dependent cell death by activating MAP kinases, thereby inhibiting cancer cell proliferation.

SeviL, a galactoside-binding lectin previously isolated from the mussel Mytilisepta virgata, was demonstrated to trigger apoptosis in HeLa ovarian cancer cells. Here, we show that this lectin can promote the polarization of macrophage cell lines toward an M1 functional phenotype at low concentrations. The administration of SeviL to monocyte and basophil cell lines reduced their growth in a dose-dependent manner. However, low lectin concentrations induced proliferation in the RAW264.7 macrophage cell line, which was supported by the significant up-regulation of TOM22, a component of the mitochondrial outer membrane. Furthermore, the morphology of lectin-treated macrophage cells markedly changed, shifting from a spherical to an elongated shape. The ability of SeviL to induce the polarization of RAW264.7 cells to M1 macrophages at low concentrations is supported by the secretion of proinflammatory cytokines and chemokines, as well as by the enhancement in the expression of IL-6- and TNF-α-encoding mRNAs, both of which encode inflammatory molecular markers. Moreover, we also observed a number of accessory molecular alterations, such as the activation of MAP kinases and the JAK/STAT pathway and the phosphorylation of platelet-derived growth factor receptor-α, which altogether support the functional reprogramming of RAW264.7 following SeviL treatment. These results indicate that this mussel β-trefoil lectin has a concentration-dependent multifunctional role in regulating cell proliferation, phenotype, and death in macrophages, suggesting its possible involvement in regulating hemocyte activity in vivo.

In this review, chemical and biological assays performed in the pharmaceutical industry to determine the potency and bioactivity of antibiotics are discussed. Though commonly employed chemical methods can measure the potency of antibiotics, inefficiency in estimating the bioactivity is one of their major limitations. Due to their sensitivity and cost-effectiveness, common microbiological assays can serve as alternative methods. Several factors like doses of antibiotics, homogeneity of agar medium, inoculum concentration, the chemical composition of agar media, size and solubility of samples or drug molecules, pH, relative humidity and exposure time can influence microbiological assays. Based on specific requirements and experimental targets, agar diffusion assays are designed focusing on their costs, errors, accuracy and simplicity. To avoid the misuse and overuse of antibiotics that lead to drug-resistance, parameters like zone of inhibition, minimum inhibitory concentration, minimum bactericidal concentration, mutation prevention concentration and critical concentration are also discussed in this study. Finally, microbiological and high-performance liquid chromatography methods were specifically compared for their sensitivity, accuracy and assessment of biological activity with minimal cost. Due to their advantages and disadvantages, parallel use of both bioassays and chemical methods are suggested to precisely determine the potency of antibiotics.

A Cucurbita phloem exudate lectin (CPL) from summer squash (Cucurbita pepo) fruits was isolated and its sugar-binding properties and biological activities were studied. The lectin was purified by affinity chromatography and the hemagglutination assay method was used to determine its pH, heat stability, metal-dependency and sugar specificity. Antimicrobial and anticancer activities were also studied by disc diffusion assays and in vivo and in vitro methods. The molecular weight of CPL was 30 ± 1 KDa and it was stable at different pH (5.0 to 9.0) and temperatures (30 to 60 °C). CPL recovered its hemagglutination activity in the presence of Ca²⁺. 4-nitrophenyl-α-D-glucopyranoside, lactose, rhamnose and N-acetyl-D-glucosamine strongly inhibited the activity. With an LC₅₀ value of 265 µg/mL, CPL was moderately toxic and exhibited bacteriostatic, bactericidal and antibiofilm activities against different pathogenic bacteria. It also exhibited marked antifungal activity against Aspergillus niger and agglutinated A. flavus spores. In vivo antiproliferative activity against Ehrlich ascites carcinoma (EAC) cells in Swiss albino mice was observed when CPL exerted 36.44% and 66.66% growth inhibition at doses of 3.0 mg/kg/day and 6.0 mg/kg/day, respectively. A 12-day treatment by CPL could reverse their RBC and WBC counts as well as restore the hemoglobin percentage to normal levels. The MTT assay of CPL performed against human breast (MCF-7) and lung (A-549) cancer cell lines showed 29.53% and 18.30% of inhibitory activity at concentrations of 128 and 256 µg/mL, respectively.