Carbohydrate Recognition Domain Research Articles

Design, synthesis, biological evaluation and docking study of some new aryl and heteroaryl thiomannosides as FimH antagonists

FimH is a mannose-recognizing lectin that is expressed by Escherichia coli guiding its ability to adhere and infect cells. It is involved in pathogenesis of urinary tract infections and Chron’s disease. Several X-ray structure-guided ligand design studies were extensively utilized in the discovery and optimization of small molecule aryl mannoside FimH antagonists. These antagonists retain key specific interactions of the mannose scaffolds with the FimH carbohydrate recognition domains. Thiomannosides are attractive and stable scaffolds, and this work reports the synthesis of some of their new aryl and heteroaryl derivatives as FimH antagonists. FimH-competitive binding assays as well as biofilm inhibition of the new compounds (24–32) were determined in comparison with the reference n-heptyl α-d-mannopyranoside (HM). The affinity among these compounds was found to be governed by the structure of the aryl and heteroarylf aglycones. Two compounds 31 and 32 revealed higher activity than HM. Molecular docking and total hydrophobic to topological polar surface area ratio calculations attributed to explain the obtained biological results. Finally, the SAR study suggested that introducing an aryl or heteroaryl aglycone of sufficient hydrophobicity and of proper orientation within the tyrosine binding site considerably enhance binding affinity. The potent and synthetically feasible FimH antagonists described herein hold potential as leads for the development of sensors for detection of E. coli and treatment of its diseases.

Biophysical and Structural Characterization of the Interaction between Human Galectin-3 and the Lipopolysaccharide from Pseudomonas aeruginosa.

Given the significant involvement of galectins in the development of numerous diseases, the aim of the following work is to further study the interaction between galectin-3 (Gal3) and the LPS from Pseudomonas aeruginosa. This manuscript focused on the study of the interaction of the carbohydrate recognition domain of Gal3 with the LPS from Pseudomonas aeruginosa by means of different complementary methodologies, such as circular dichroism; spectrofluorimetry; dynamic and static light scattering and evaluation of the impact of Gal3 on the redox potential membranes of Escherichia coli and P. aeruginosa cells, as well as ITC and NMR studies. This thorough investigation reinforces the hypothesis of an interaction between Gal3 and LPS, unraveling the structural details and providing valuable insights into the formation of these intricate molecular complexes. Taken together, these achievements could potentially prompt the design of therapeutic drugs useful for the development of agonists and/or antagonists for LPS receptors such as galectins as adjunctive therapy for P. aeruginosa.

Crystal structure of the complex of CLEC12A and an antibody that interferes with binding of diverse ligands.

C-type lectin receptors (CLRs) are a family of pattern recognition receptors, which detect a broad spectrum of ligands via small carbohydrate-recognition domains (CRDs). CLEC12A is an inhibitory CLR that recognizes crystalline structures such as monosodium urate crystals. CLEC12A also recognizes mycolic acid, a major component of mycobacterial cell walls, and suppresses host immune responses. Although CLEC12A could be a therapeutic target for mycobacterial infection, structural information on CLEC12A was not available. We report here the crystal structures of human CLEC12A (hCLEC12A) in ligand-free form and in complex with 50C1, its inhibitory antibody. 50C1 recognizes human-specific residues on the top face of hCLEC12A CRD. A comprehensive alanine scan demonstrated that the ligand-binding sites of mycolic acid and monosodium urate crystals may overlap with each other, suggesting that CLEC12A utilizes a common interface to recognize different types of ligands. Our results provide atomic insights into the blocking and ligand-recognition mechanisms of CLEC12A and leads to the design of CLR-specific inhibitors.

Multivalent, calcium-independent binding of surfactant protein A and D to sulfated glycosaminoglycans of the alveolar epithelial glycocalyx.

Lung surfactant collectins, surfactant protein A (SP-A) and D (SP-D), are oligomeric C-type lectins involved in lung immunity. Through their carbohydrate recognition domain, they recognize carbohydrates at pathogen surfaces and initiate lung innate immune response. Here, we propose that they may also be able to bind to other carbohydrates present in typical cell surfaces, such as the alveolar epithelial glycocalyx. To test this hypothesis, we analyzed and quantified the binding affinity of SP-A and SP-D to different sugars and glycosaminoglycans (GAGs) by microscale thermophoresis (MST). In addition, by changing the calcium concentration, we aimed to characterize any consequences on the binding behavior. Our results show that both oligomeric proteins bind with high affinity (in nanomolar range) to GAGs, such as hyaluronan (HA), heparan sulfate (HS) and chondroitin sulfate (CS). Binding to HS and CS was calcium-independent, as it was not affected by changing calcium concentration in the buffer. Quantification of GAGs in bronchoalveolar lavage (BAL) fluid from animals deficient in either SP-A or SP-D showed changes in GAG composition, and electron micrographs showed differences in alveolar glycocalyx ultrastructure in vivo. Taken together, SP-A and SP-D bind to model sulfated glycosaminoglycans of the alveolar epithelial glycocalyx in a multivalent and calcium-independent way. These findings provide a potential mechanism for SP-A and SP-D as an integral part of the alveolar epithelial glycocalyx binding and interconnecting free GAGs, proteoglycans, and other glycans in glycoproteins, which may influence glycocalyx composition and structure.NEW & NOTEWORTHY SP-A and SP-D function has been related to innate immunity of the lung based on their binding to sugar residues at pathogen surfaces. However, their function in the healthy alveolus was considered as limited to interaction with surfactant lipids. Here, we demonstrated that these proteins bind to glycosaminoglycans present at typical cell surfaces like the alveolar epithelial glycocalyx. We propose a model where these proteins play an important role in interconnecting alveolar epithelial glycocalyx components.

Galectin-3 – A novel ligand of complement protein C1q

In the present study we report a novel interaction of human C1q, a primary activator of the Complement system, with human Galectin-3 (Gal-3). We investigated the potential recognition between C1q and Gal-3 on a solid hydrophobic surface by ELISA, by fluorescence spectroscopy, molecular docking and molecular dynamics (MD). The data showed that C1q and Gal-3 had a pronounced affinity for protein-protein interaction and supramolecular binding, locating the binding sites within the globular domains of C1q (gC1q) and on the backside of the carbohydrate recognition domain (CRD) of Gal-3. Fluorescence spectroscopy gave quantitative assessment of the recognition with KD value of 0.04 μM. MD analysis showed that when the active AAs of the two proteins interacted, electrostatic attraction, aided by a large number of hydrogen bonds, was dominant for the stabilization of the complex. When the contact of C1q and Gal-3 was not limited to active residues, the complex between them was stabilized mainly by Van der Waals interactions and smaller in number but stronger hydrogen bonds. This is the first report analyzing the interaction of Gal-3 with C1q, which could open the way to new applications of this protein-protein complex.

Mannose Ligands for Mannose Receptor Targeting.

The mannose receptor (MR, CD 206) is an endocytic receptor primarily expressed by macrophages and dendritic cells, which plays a critical role in both endocytosis and antigen processing and presentation. MR carbohydrate recognition domains (CRDs) exhibit a high binding affinity for branched and linear oligosaccharides. Furthermore, multivalent mannose presentation on the various templates like peptides, proteins, polymers, micelles, and dendrimers was proven to be a valuable approach for the selective and efficient delivery of various therapeutically active agents to MR. This review provides a detailed account of the most relevant and recent aspects of the synthesis and application of mannosylated bioactive formulations for MR-mediated delivery in treatments of cancer and other infectious diseases. It further highlights recent findings related to the necessary structural features of the mannose-containing ligands for successful binding to the MR.

Induced CTL-S15 gene expression by Bacillus thuringiensis declines susceptibility in Spodoptera exigua

It has been reported that C-type lectins (CTLs), which are pattern recognition receptors of the insect innate immunity response, may compete with Cry toxin for the receptor alkaline phosphatase to decrease its toxicity in insects. However, to date, which CTLs affect larval susceptibility to Bt in Spodoptera exigua is not clear. In this study, 33 CTL genes were identified from S. exigua. Based on the number of carbohydrate-recognition domains (CRDs) and the domain architectures, they were classified into three groups: (1) nineteen CTL-S (single-CRD), (2) eight immulectin (dual-CRD) and (3) six CTL-X (CRD with other domains). RT-qPCR analysis revealed that expression levels of SeCTL-S15, IML-4 and CTL-X6 were upregulated after challenge with Bt and Cry1Ab. Tissue and developmental stage expression analysis showed that only SeCTL-S15 was mainly expressed in the midgut and larva, respectively. Knockdown of SeCTL-S15 significantly increased Bt susceptibility, as indicated by reduced survival and larval weight. These results suggest that CTL-S15 might play a vital role in the low susceptibility of larvae to Bt in S. exigua. Our results provide new insights into CTL function in insects.

Unprecedented selectivity for homologous lectin targets: differential targeting of the viral receptors L-SIGN and DC-SIGN.

DC-SIGN (CD209) and L-SIGN (CD209L) are two C-type lectin receptors (CLRs) that facilitate SARS-CoV-2 infections as viral co-receptors. SARS-CoV-2 manipulates both DC-SIGN and L-SIGN for enhanced infection, leading to interest in developing receptor antagonists. Despite their structural similarity (82% sequence identity), they function differently. DC-SIGN, found in dendritic cells, shapes the immune response by recognizing pathogen-associated carbohydrate patterns. In contrast, L-SIGN, expressed in airway epithelial endothelial cells, is not directly involved in immunity. COVID-19's primary threat is the hyperactivation of the immune system, potentially reinforced if DC-SIGN engages with exogenous ligands. Therefore, L-SIGN, co-localized with ACE2-expressing cells in the respiratory tract, is a more suitable target for anti-adhesion therapy. However, designing a selective ligand for L-SIGN is challenging due to the high sequence identity of the Carbohydrate Recognition Domains (CRDs) of the two lectins. We here present Man84, a mannose ring modified with a methylene guanidine triazole at position 2. It binds L-SIGN with a K D of 12.7μM ± 1 μM (ITC) and is the first known L-SIGN selective ligand, showing 50-fold selectivity over DC-SIGN (SPR). The X-ray structure of the L-SIGN CRD/Man84 complex reveals the guanidinium group's role in achieving steric and electrostatic complementarity with L-SIGN. This allows us to trace the source of selectivity to a single amino acid difference between the two CRDs. NMR analysis confirms the binding mode in solution, highlighting Man84's conformational selection upon complex formation. Dimeric versions of Man84 achieve additional selectivity and avidity in the low nanomolar range. These compounds selectively inhibit L-SIGN dependent trans-infection by SARS-CoV-2 and Ebola virus. Man84 and its dimeric constructs display the best affinity and avidity reported to date for low-valency glycomimetics targeting CLRs. They are promising tools for competing with SARS-CoV-2 anchoring in the respiratory tract and have potential for other medical applications.

Galactomannan inhibits Trichinella spiralis invasion of intestinal epithelium cells and enhances antibody-dependent cellular cytotoxicity related killing of larvae by driving macrophage polarization.

Previous studies have shown that recombinant Trichinella spiralis galectin (rTsgal) is characterized by a carbohydrate recognition domain sequence motif binding to beta-galactoside, and that rTsgal promotes larval invasion of intestinal epithelial cells. Galactomannan is an immunostimulatory polysaccharide composed of a mannan backbone with galactose residues. The aim of this study was to investigate whether galactomannan inhibits larval intrusion of intestinal epithelial cells and enhances antibody-dependent cellular cytotoxicity (ADCC), killing newborn larvae by polarizing macrophages to the M1 phenotype. The results showed that galactomannan specially binds to rTsgal, and abrogated rTsgal facilitation of larval invasion of intestinal epithelial cells. The results of qPCR, Western blotting, and flow cytometry showed that galactomannan and rTsgal activated macrophage M1 polarization, as demonstrated by high expression of iNOS (M1 marker) and M1 related genes (IL-1β, IL-6, and TNF-α), and increased CD86+ macrophages. Galactomannan and rTsgal also increased NO production. The killing ability of macrophage-mediated ADCC on larvae was also significantly enhanced in galactomannan- and rTsgal-treated macrophages. The results demonstrated that Tsgal may be considered a potential vaccine target molecule against T. spiralis invasion, and galactomannan may be a novel adjuvant therapeutic agent and potential vaccine adjuvant against T. spiralis infection.

Largemouth bass galectin, MsGal-9: Mediating various functions as a pattern recognition receptor and a potential damage-associated molecular pattern

Galectins are lectins that bind to β-galactose and are widely expressed in immune system tissues, playing pivotal roles in innate immunity through their conserved carbohydrate-recognition domains (CRDs). In this present investigation, a tandem-repeat galectin was discovered in the largemouth bass, Micropterus salmoides (designated as MsGal-9). The open reading frame of MsGal-9 encodes two CRDs, each containing two consensus motifs that are essential for ligand binding. MsGal-9 is expressed in various tissues of the largemouth bass, with particularly high expression levels in the liver and spleen. The full-length form of MsGal-9, as well as the N-terminal (MsGal-9-N) and C-terminal (MsGal-9-C) CRDs, were individually recombined. Their ability for nonself recognition was studied. The three recombinant proteins were able to bind to glucan (GLU), peptidoglycan (PGN), and lipopolysaccharide (LPS), with MsGal-9 displaying the highest binding activity. Furthermore, rMsGal-9-N exhibited higher binding activity towards GLU in comparison to rMsGal-9-C. Further investigations revealed that the full-length rMsGal-9 could significantly bind to Gram-positive bacteria, Gram-negative bacteria, and fungi, while rMsGal-9-C specifically bound to Escherichia coli. However, rMsGal-9-N did not exhibit significant binding activity towards any microbes. These findings indicate that MsGal-9 requires both CRDs to cooperate in order to fulfill its nonself recognition function. All three recombinant proteins demonstrated agglutination activity towards various microbes, with MsGal-9 and MsGal-9-N displaying a similar broad binding spectrum, while MsGal-9-C agglutinated three types of bacteria. Moreover, both MsGal-9 and MsGal-9-N were capable of coagulating largemouth bass red blood cells, whereas MsGal-9-C lacked this ability. However, MsGal-9-C played a significant role in enhancing the encapsulation of leukocytes in comparison to MsGal-9-N. All three proteins acted as potential damage-associated molecular patterns (DAMPs), inducing apoptosis in leukocytes.

New l-Rhamnose-Binding Lectin from the Bivalve Glycymeris yessoensis: Purification, Partial Structural Characterization and Antibacterial Activity.

In this study, a new l-rhamnose-binding lectin (GYL-R) from the hemolymph of bivalve Glycymeris yessoensis was purified using affinity and ion-exchange chromatography and functionally characterized. Lectin antimicrobial activity was examined in different ways. The lectin was inhibited by saccharides possessing the same configuration of hydroxyl groups at C-2 and C-4, such as l-rhamnose, d-galactose, lactose, l-arabinose and raffinose. Using the glycan microarray approach, natural carbohydrate ligands were established for GYL-R as l-Rha and glycans containing the α-Gal residue in the terminal position. The GYL-R molecular mass determined by MALDI-TOF mass spectrometry was 30,415 Da. The hemagglutination activity of the lectin was not affected by metal ions. The lectin was stable up to 75 °C and between pH 4.0 and 12.0. The amino acid sequence of the five GYL-R segments was obtained with nano-ESI MS/MS and contained both YGR and DPC-peptide motifs which are conserved in most of the l-rhamnose-binding lectin carbohydrate recognition domains. Circular dichroism confirmed that GYL is a α/β-protein with a predominance of the random coil. Furthermore, GYL-R was able to bind and suppress the growth of the Gram-negative bacteria E. coli by recognizing lipopolysaccharides. Together, these results suggest that GYL-R is a new member of the RBL family which participates in the self-defense mechanism against bacteria and pathogens with a distinct carbohydrate-binding specificity.

A prototype galectin-1 (also known as galecin-2) from large yellow croaker (Larimichthys crocea): Molecular and function study

Galectin-1 (also known as galecin-2), one member of galectins family, has multiple functions as a pattern recognition receptor (PRR) in innate immune defense system. In the present study, LcGal-1, a prototype galectin, was identified and function investigated in large yellow croaker (Larimichthys crocea). LcGal-1 consists of one carbohydrate recognition domain (CRD), which contains two carbohydrate binding motifs HFNPR and WG-E-R. LcGal-1 had a ubiquitous tissues profile with the highest and lowest expression in spleen and muscle, respectively. Moreover, it was in cytoplasm and nucleus of head-kidney cells in large yellow croaker. RT-qRCR showed that P. plecoglossicida induced LcGal-1 up-regulated expression in liver and gills, and the results were validated by immunohistochemistry analysis. Additionally, the recombinant LcGal-1 (rLcGal-1) showed agglutinate activity on erythrocytes, and the histidine (His) in the HFNPR motif was a key locus to the activity. The agglutination effect of rLcGal-1 on erythrocytes could be inhibited by LPS, α-lactase and d-galactose. The rLcGal-1 was able to bind and agglutinate Gram+ and Gram-bacteria, and damage bacterial membrane as confirmed by PI staining and SEM observation. Transcriptome analysis showed that the overexpressed LcGal-1 in HEK 293T cells could induce 176 DGEs, including 172 boosting genes and 4 falling genes. Collectively, LcGal-1 was a key immune gene involved in the recognition, conjunction, and elimination of pathogens in L. crocea, as well as multiple physiological and pathological regulatory processes.

Molecular characterization and functional study of a galectin-9 from a teleost fish, Boleophthalmus pectinirostris

Galectin-9, a tandem-repeat galectin, plays an important role in the regulation of innate immune response against various microbial infections. Here, galectin-9 from mudskipper (Boleophthalmus pectinirostris) was identified and named as BpGal-9. Putative BpGal-9 contains two conserved carbohydrate recognition domains (CRDs), one CRD within N-terminal (N-CRD) and the other one within C-terminal (C-CRD). Multi-alignment analysis indicated that BpGal-9 shared the highest amino acid sequence identity of 64.3 % with that of Southern platyfish (Xiphophorus maculatus). Phylogenetic analysis showed that BpGal-9 grouped tightly with other teleosts galectin-9 and was most closely related to that of Southern platyfish. BpGal-9 transcripts were more abundant in the intestine, and its expression upregulated significantly in the intestine, kidney, spleen, gills, and skin after Edwardsiella tarda infection. Meanwhile, BpGal-9 expression significantly increased in hemocytes and serum of mudskipper infected by E. tarda. The recombinant BpGal-9 (rBpGal-9) and rBpGal-9C-CRD could agglutinate all tested bacteria, whereas rBpGal-9N-CRD could only agglutinate three kinds of bacteria. When targeting the same bacteria, rBpGal-9 showed stronger agglutinating activities than rBpGal-9C-CRD or rBpGal-9N-CRD. In addition, the induction effect of three recombinant proteins on the mRNA expression of anti-inflammatory cytokines (BpIL-10 and BpTGF-β) was better than that on the pro-inflammatory cytokines (BpIL-1β and BpTNF-α). Our result suggested that the N-CRD and C-CRD of galectin-9 contribute differently to its multiple functions in innate immunity in teleosts.

Identification and characterization of an L-type lectin from obscure puffer Takifugu obscurus in response to bacterial infection

L-type lectins (LTLs) contain a carbohydrate recognition domain homologous to leguminous lectins, and have functions in selective protein trafficking, sorting and targeting in the secretory pathway of animals. In this study, a novel LTL, designated as ToERGIC-53, was cloned and identified from obscure puffer Takifugu obscurus. The open reading frame of ToERGIC-53 contained 1554 nucleotides encoding 517 amino acid residues. The deduced ToERGIC-53 protein consisted of a signal peptide, a leguminous lectin domain (LTLD), a coiled-coil region, and a transmembrane region. Quantitative real-time PCR showed that ToERGIC-53 was expressed in all examined tissues, with the highest expression level in the liver. The expression of ToERGIC-53 was significantly upregulated after infection with Vibrio harveyi and Staphylococcus aureus. Recombinant ToERGIC-53-LTLD (rToERGIC-53-LTLD) protein could not only agglutinate and bind to one Gram-positive bacterium (S. aureus) and three Gram-negative bacteria (V. harveyi, V. parahaemolyticus and Aeromonas hydrophila), but also bind to glycoconjugates on the surface of bacteria such as lipopolysaccharide, peptidoglycan, mannose and galactose. In addition, rToERGIC-53-LTLD inhibited the growth of bacteria in vitro. All these results suggested that ToERGIC-53 might be a pattern recognition receptor involved in antibacterial immune response of T. obscurus.

Sialyl LewisX glycomimetics bearing an extended anionic chain targeting E- and P- selectin binding sites

Neutrophil binding to vascular P- and E-selectin is the rate-limiting step in the recruitment of immune cells to sites of inflammation. Many diseases, including sickle cell anemia, post-myocardial infarction reperfusion injury, and acute respiratory distress syndrome are characterized by dysregulated inflammation. We have recently reported sialyl Lewisx analogues as potent antagonists of P- and E-selectin and demonstrated their in vivo immunosuppressive activity. A key component of these molecules is a tartrate diester that serves as an acyclic tether to orient the fucoside and the galactoside moiety in the required gauche conformation for optimal binding. The next stage of our study involved attaching an extended carbon chain onto one of the esters. This chain could be utilized to tether other pharmacophores, lipids, and contrast agents in the context of enhancing pharmacological applications through the sialyl Lewisx / receptor-mediated mechanism. Herein, we report our preliminary studies to generate a small library of tartrate based sialyl Lewisx analogues bearing extended carbon chains. Anionic charged chemical entities are attached to take advantage of proximal charged amino acids in the carbohydrate recognition domain of the selectin receptors. Starting with a common azido intermediate, synthesized using copper-catalyzed Huisgen 1,3-dipolar cycloadditions, these molecules demonstrate E- and P-selectin binding properties.

Ginseng-derived type I rhamnogalacturonan polysaccharide binds to galectin-8 and antagonizes its function

BackgroundPanax ginseng Meyer polysaccharides exhibit various biological functions, like antagonizing galectin-3-mediated cell adhesion and migration. Galectin-8 (Gal-8), with its linker-joined N- and C-terminal carbohydrate recognition domains (CRDs), is also crucial to these biological processes, and thus plays a role in various pathological disorders. Yet the effect of ginseng-derived polysaccharides in modulating Gal-8 function has remained unclear. MethodsP. ginseng-derived pectin was chromatographically isolated and enzymatically digested to obtain a series of polysaccharides. Biolayer Interferometry (BLI) quantified their binding affinity to Gal-8, and their inhibitory effects on Gal-8 was assessed by hemagglutination, cell migration and T-cell apoptosis. ResultsOur ginseng-derived pectin polysaccharides consist mostly of rhamnogalacturonan-I (RG-I) and homogalacturonan (HG). BLI shows that Gal-8 binding rests primarily in RG-I and its β-1,4-galactan side chains, with sub-micromolar KD values. Both N- and C-terminal Gal-8 CRDs bind RG-I, with binding correlated with Gal-8-mediated function. ConclusionP. ginseng RG-I pectin β-1,4-galactan side chains are crucial to binding Gal-8 and antagonizing its function. This study enhances our understanding of galectin-sugar interactions, information that may be used in the development of pharmaceutical agents targeting Gal-8.

Isomerization of proline-46 in the N-terminal tail of galectin-3 enhances T cell apoptosis via the ROS-ERK pathway

Galectin-3 (Gal-3) is unique in the galectin family, due to the presence of a long N-terminal tail (NT) arising from its conserved carbohydrate recognition domain (CRD). Although functional significance of the NT has remained elusive, our previous studies demonstrated the importance of NT prolines to Gal-3 function. Here, we show that during the time Gal-3 stands in solution for three or more days, Gal-3 NT undergoes a slow, intra-molecular, time-dependent conformational/dynamical change associated with proline cis-trans isomerization. From initial dissolution of Gal-3 in buffer to three days in solution, Gal-3-mediated T cell apoptosis is enhanced from 23 % to 37 %. Western blotting and flow cytometry show that the enhancement occurs via the ROS-ERK pathway, and not by the PKC-ERK pathway. To assess which proline(s) is (are) responsible for this effect, we individually mutated all 14 NT prolines within the first 68 residues to alanines, and assessed their effect on ROS production. Our study shows that isomerization of P46 alone is responsible for the upregulation of ROS and T cell apoptosis. NMR studies show that this unique effect is mediated by a change in dynamic interactions between the NT and CRD F-face, which in turn leads to this change in Gal-3 function.

Molecular cloning, expression, and functional analysis of a putative lectin from the pearl oyster (Pinctada fucata, Gould 1850)

Marine lectins are a group of proteins that possess specific carbohydrate recognition and binding domains. They exhibit various activities, including antimicrobial, antitumor, antiviral, and immunomodulatory effects. In this study, a novel galectin-binding lectin gene named PFL-96 (GenBank: OQ561753.1) was cloned from Pinctada fucata. The PFL-96 gene has an open reading frame of 324 base pairs (bp) and encodes a protein comprising 107 amino acids. The protein has a molecular weight of 11.95 kDa and an isoelectric point of 9.27. It contains an N-terminal signal peptide and a galactose-binding lectin domain. The sequence identity to lectin proteins from fish, echinoderms, coelenterates, and shellfish ranges from 31.90 to 40.00 %. In the phylogenetic analysis, it was found that the PFL-96 protein is closely related to the lectin from Pteria penguin. The PFL-96 recombinant protein exhibited coagulation activity on 2 % rabbit red blood cells at a concentration of ≥8 μg/mL. Additionally, it showed significant hemolytic activity at a concentration of ≥32 μg/mL. The PFL-96 recombinant protein exhibited significant antibacterial activity against Bacillus subtilis, Staphylococcus aureus, Candida albicans, and Vibrio alginolyticus, with minimum inhibitory concentrations (MIC) of 4, 8, 16, and 16 μg/mL, respectively. The minimum bactericidal concentrations (MBC) were determined to be 8, 16, 32, and 32 μg/mL, respectively. Furthermore, the PFL-96 recombinant protein exhibited inhibitory effects on the proliferation of Hela tumor cells, HepG2 tumor cells, and C666-1 tumor cells, with IC50 values of 7.962, 8.007, and 9.502 μg/mL, respectively. These findings suggest that the recombinant protein PFL-96 exhibits significant bioactivity in vitro, contributing to a better understanding of the active compounds found in P. fucata. The present study establishes a fundamental basis for further investigation into the mechanism of action and structural optimization of the recombinant protein PFL-96. The aim is to develop potential candidates for antibacterial and anti-tumor agents.

Molecular characterization and functional analysis of galectin-1 from silver pomfret (Pampus argenteus)

Galectins, as members of lectin families, exhibit a high affinity for β-galactosides and play diverse roles in biological processes. They function as pattern recognition receptors (PRRs) with important roles in immune defense. In this study, galectin-1, designated as SpGal-1, was identified and characterized from silver pomfret (Pampus argenteus). The SpGal-1 comprises an open reading frame (ORF) spanning 396 base pairs (bp) and encodes a deduced amino acid (aa) sequence containing a single carbohydrate recognition domain (CRD). Sublocalization analysis revealed that SpGal-1 was mainly expressed in the cytoplasm. The mRNA transcripts of SpGal-1 were ubiquitously detected in various tissues, with a higher expression level in the intestine. In addition, when exposed to Photobacterium damselae subsp. damselae (PDD) infection, both the liver and head kidney exhibited significantly increased SpGal-1 mRNA expression. The recombinant protein of SpGal-1 (named as rSpGal-1) demonstrated hemagglutination against red blood cells (RBCs) from Larimichthys crocea and P. argenteus in a Ca2+ or β-Mercaptoethanol (β-ME)-independent manner. Notably, rSpGal-1 could bind with various pathogen-associated molecular patterns (PAMPs) including D-galactose, D-mannose, lipopolysaccharide (LPS), and peptidoglycan (PGN), with highest affinity to PGN. Moreover, rSpGal-1 effectively interacted with an array of bacterial types encompassing Gram-positive bacteria (Staphylococcus aureus and Nocardia seriolae) and Gram-negative bacteria (PDD and Escherichia coli, among others), with the most robust binding affinity towards PDD. Collectively, these findings highlight that SpGal-1 is a crucial PRR with involvement in the host immune defense of silver pomfret.

Enzymatically-derived oligo-carrageenans interact with α-Gal antibodies and Galectin-3

Carrageenans are linear sulfated galactans synthesized in the Gigartinales, Rhodophyceae species with a varied range of biological properties that are of value to the pharmaceutical and cosmetic sectors. It is unknown how the fine structure of carrageenans dictates their capacity to affect molecular and cellular responses important to wound healing, or the ability to mitigate oxidative, hemostatic and inflammatory processes. Here we use specific endo-carrageenases, from the marine bacterium Zobellia galactanivorans, to produce enzymatically defined neo-series oligosaccharides from carrageenans with 3,6-anhydro-D-galactose on the non-reducing end. Further enzymatic modification of the oligosaccharides was done by treating with the 3,6-anhydro-D-galactosidases from the same bacterium which hydrolyze non-reducing end 3,6-anhydro-D-galactose moieties from neo-carrageenan oligosaccharides. Using the enzymatically produced oligosaccharides, we demonstrate binding to natural human serum antibodies and a monoclonal anti-αGal Ab (m86). The significant interactions with the Galα(1,3)Gal reactive antibodies produced by humans makes them potential potent inducers of complement-dependent reactions and attractive for therapeutic applications. We also demonstrate modulation of the galectin selectivity for the Gal-3 Carbohydrate Recognition Domain (CRD) relative to Gal-1 which has implications to targeting specific biological pathways regulated by the galectins.