Lectin Binding Research Articles

Identification of immune subsets with distinct lectin binding signatures using multi-parameter flow cytometry: correlations with disease activity in systemic lupus erythematosus.

Cell surface glycosylation can influence protein-protein interactions with particular relevance to changes in core fucosylation and terminal sialylation. Glycans are ligands for immune regulatory lectin families like galectins (Gals) or sialic acid immunoglobulin-like lectins (Siglecs). This study delves into the glycan alterations within immune subsets of systemic lupus erythematosus (SLE). Evaluation of binding affinities of Galectin-1, Galectin-3, Siglec-1, Aleuria aurantia lectin (AAL, recognizing core fucosylation), and Sambucus nigra agglutinin (SNA, specific for α-2,6-sialylation) was conducted on various immune subsets in peripheral blood mononuclear cells (PBMCs) from control and SLE subjects. Lectin binding was measured by multi-parameter flow cytometry in 18 manually gated subsets of T-cells, NK-cells, NKT-cells, B-cells, and monocytes in unstimulated resting state and also after 3-day activation. Stimulated pre-gated populations were subsequently clustered by FlowSOM algorithm based on lectin binding and activation markers, CD25 or HLA-DR. Elevated AAL, SNA and CD25+/CD25- SNA binding ratio in certain stimulated SLE T-cell subsets correlated with SLE Disease Activity Index 2000 (SLEDAI-2K) scores. The significantly increased frequencies of activated AALlow Siglec-1low NK metaclusters in SLE also correlated with SLEDAI-2K indices. In SLE, activated double negative NKTs displayed significantly lower core fucosylation and CD25+/CD25- Siglec-1 binding ratio, negatively correlating with disease activity. The significantly enhanced AAL binding in resting SLE plasmablasts positively correlated with SLEDAI-2K scores. Alterations in the glycosylation of immune cells in SLE correlate with disease severity, which might represent potential implications in the pathogenesis of SLE.

Sperm Incubation in Biggers-Whitten-Whittingham Medium Induces Capacitation-Related Changes in the Lizard Sceloporus torquatus.

Sperm capacitation involves biochemical and physiological changes that enable sperm to fertilize the oocyte. It can be induced in vitro under controlled conditions that simulate the environment of the oviduct. While extensively studied in mammals, its approach in lizards remains absent. Understanding the mechanisms that ensure reproduction is essential for advancing the implementation of assisted reproductive technologies in this group. We aimed to perform a sperm analysis to determine if capacitation-related changes were induced after incubation with capacitating media. Fifteen males of Sceloporus torquatus were collected during the early stage of the reproductive season. The sperm were isolated from the seminal plasma and then diluted up to a volume of 150 μL using BWW medium to incubate with 5% CO2 at 30 °C for a maximum duration of 3 h. A fraction was retrieved hourly for ongoing sperm assessment. The sperm analysis included assessments of its motility, viability, the capacitation status using the chlortetracycline (CTC) assay, and the acrosome integrity with the lectin binding assay to detect changes during incubation. We found that total motility was maintained up to 2 h post incubation, after which it decreased. However, sperm viability remained constant. From that moment on, we observed a transition to a deeper and less symmetrical flagellar bending in many spermatozoa. The CTC assay indicated a reduction in the percentage of sperm showing the full (F) pattern and an increase in those exhibiting the capacitated (B) and reactive (RA) patterns, accompanied by an elevation in the percentage of damaged acrosomes as revealed by the lectin binding assay. In mammals, these changes are often associated with sperm capacitation. Our observations support the notion that this process may also occur in saurian. While sperm analysis is a valuable method for assessing certain functional changes, additional approaches are required to validate this process.

Carbohydrate-Functionalized Anthracene Carboximides as Multivalent Ligands and Bio-Imaging Agents.

Anthracene carboximides (ACIs) conjugated with gluco-, galacto- and mannopyranosides are synthesized, by glycosylation of N-hydroxyethylanthracene carboximide acceptor with glycosyl donors. Glycoconjugation of anthracene carboximide increases the aq. solubility by more than 3-fold. The glycoconjugates display red-shifted absorption and emission, as compared to anthracene. Large Stokes shift (λabs/λem=445/525 nm) and high fluorescence quantum yields (Φ) of 0.86 and 0.5 occur in THF and water, respectively. The ACI-glycosides undergo facile photodimerization in aqueous solutions, leading to the formation of the head-to-tail dimer, as a mixture of syn and anti-isomers. Solution phase and solid-state characterizations by dynamic light scattering (DLS), microscopic imaging by atomic force (AFM) and transmission electron (TEM) microscopies reveal self-assembled vesicle structures of ACI glycosides. These self-assembled structures act as multivalent glycoclusters for ligand-specific lectin binding, as evidenced by the binding of Man-ACI to Con A, by fluorescence and turbidity assays. The conjugates do not show cellular cytotoxicity (IC50) till concentrations of 50 μM with HeLa and HepG2 cell lines and are cell-permeable, showing strong fluorescence inside the cells. These properties enable the glycoconjugates to be used in cell imaging. The non-selective cellular uptake of the glycoconjugates suggests a passive diffusion through the membrane.

Lectin binding to pectoral fin of neonate little skates reared under ambient and projected-end-of-century temperature regimes.

The glycosylation of macromolecules can vary both among tissue structural components and by adverse conditions, potentially providing an alternative marker of stress in organisms. Lectins are proteins that bind carbohydrate moieties and lectin histochemistry is a common method to visualize microstructures in biological specimens and diagnose pathophysiological states in human tissues known to alter glycan profiles. However, this technique is not commonly used to assess broad-spectrum changes in cellular glycosylation in response to environmental stressors. In addition, the binding of various lectins has not been studied in elasmobranchs (sharks, skates, and rays). We surveyed the binding tissue structure specificity of 14 plant-derived lectins, using both immunoblotting and immunofluorescence, in the pectoral fins of neonate little skates (Leucoraja erinacea). Skates were reared under present-day or elevated (+5°C above ambient) temperature regimes and evaluated for lectin binding as an indicator of changing cellular glycosylation and tissue structure. Lectin labeling was highly tissue and microstructure specific. Dot blots revealed no significant changes in lectin binding between temperature regimes. In addition, lectins only detected in the elevated temperature treatment were Canavalia ensiformis lectin (Concanavalin A) in spindle cells of muscle and Ricinus communis agglutinin in muscle capillaries. These results provide a reference for lectin labeling in elasmobranch tissue that may aid future investigations.

Mannose Binding Lectin and C3 Serum Levels in Coronary Artery Disease: A Cross-Sectional Study

ABSTRACT Background The process of tissue injury in coronary artery disease (CAD) has been associated with activation of the complement system, partly due to the action of mannose-binding lectin (MBL) and C3, which are expressed in atherosclerotic lesions. Objective The aim of this study was to evaluate the serum levels of MBL and C3 in patients with CAD and to compare them with healthy controls. Additionally, we aim to assess the correlation between MBL and C3 levels and cardiometabolic parameters. Methods MBL and C3 serum concentration were determined by ELISA and immunoturbidimetry, respectively, in up to 119 patients undergoing coronary angiography for CAD evaluation, comprising 48 individuals diagnosed with acute myocardial infarction (MI) and 71 without MI. A total of 93 paired healthy controls were included in the study. Results Individuals with CAD had MBL serum concentration higher than controls (p = .002), regardless of the presence of MI (p = .006). In addition, high concentration of MBL (>2000 ng/mL) was more frequent in patients with CAD (p = .007; OR = 2.6; 95% CI = 1.3–5.1). C3 levels were not significantly associated with any of the patient groups but were positively correlated with cardiometabolic parameters such as body mass index (BMI) and triglycerides levels. Conclusions Higher concentrations of MBL were found to be associated with CAD, whereas C3 levels were found to be associated with cardiovascular risk factors.

Neisseria gonorrhoeae scavenges host sialic acid for Siglec-mediated, complement-independent suppression of neutrophil activation.

Neisseria gonorrhoeae, the bacterium that causes gonorrhea, is an urgent global health concern due to increasing infection rates, widespread antibiotic resistance, and its ability to thwart protective immune responses. The mechanisms by which Gc subverts protective immune responses remain poorly characterized. One way N. gonorrhoeae evades human immunity is by adding sialic acid that is scavenged from the host onto its lipooligosaccharide, using the sialyltransferase Lst. Here, we found that sialylation enhances N. gonorrhoeae survival from neutrophil assault and inhibits neutrophil activation, independently of the complement system. Our results implicate bacterial binding of sialic acid-binding lectins (Siglecs) on the neutrophil surface, which dampens neutrophil antimicrobial responses. This work identifies a new role for sialylation in protecting N. gonorrhoeae from cellular innate immunity, which can be targeted to enhance the human immune response in gonorrhea.

Infection with different Neospora caninum strains causes differences in the glycosylation pattern in the uteri and placentae of Neospora caninum-infected heifers

Neospora caninum is an obligate intracellular parasite that causes abortion in ruminants. Different strains produce differences in the severity of disease outcomes. These differences may cause physiological or pathological changes in cells, modifying the intercellular interactions and intracellular transport pathways that could be evidenced by identifying the terminal sugars. This study aimed to characterize the oligosaccharide pattern in the bovine placenta and uterus after infection with tachyzoites of three different strains of N. caninum (Nc-1, Nc-6 Argentina and Nc Spain-7) during early gestation. Fourteen heifers were inoculated intravenously on day 70 of gestation with 2 × 108 N. caninum tachyzoites and samples of placentae and uteri were analysed by histology and lectin histochemistry. In the infected groups, severe placentitis was associated with changes in lectin binding in the vascular endothelium by Lens culinaris agglutinin (LCA), Pisum sativum agglutinin (PSA) and Ricinus communis I (RCA-I) lectins, in the epithelial cells of the endometrial glands by RCA-I, Dolichos biflorus agglutinin (DBA), succinylated wheat germ agglutinin, peanut agglutinin (PNA), concanavalin-A (CON-A), LCA, PSA and Phaseolus vulgaris erythroagglutinin (PHA-e), and in the trophoblast layer by PNA, CON-A, LCA, PSA, PHA-e, soybean agglutinin, RCA-I, DBA and Bandieraea simplicifolia agglutinin (BSA-I). The results suggest that N. caninum causes changes in the glycosylation pattern in the maternofetal interface tissues and might cause abortions in early gestation due to changes in the cellular structure of the placenta.

Deciphering the Chemical Language of the Immunomodulatory Properties of Veillonella parvula Lipopolysaccharide

AbstractVeillonella parvula, prototypical member of the oral and gut microbiota, is at times commensal yet also potentially pathogenic. The definition of the molecular basis tailoring this contrasting behavior is key for broadening our understanding of the microbiota‐driven pathogenic and/or tolerogenic mechanisms that take place within our body. In this study, we focused on the chemistry of the main constituent of the outer membrane of V. parvula, the lipopolysaccharide (LPS). LPS molecules indeed elicit pro‐inflammatory and immunomodulatory responses depending on their chemical structures. Herein we report the structural elucidation of the LPS from two strains of V. parvula and show important and unprecedented differences in both the lipid and carbohydrate moieties, including the identification of a novel galactofuranose and mannitol‐containing O‐antigen repeating unit for one of the two strains. Furthermore, by harnessing computational studies, in vitro human cell models, as well as lectin binding solid‐phase assays, we discovered that the two chemically diverse LPS immunologically behave differently and have attempted to identify the molecular determinant(s) governing this phenomenon. Whereas pro‐inflammatory potential has been evidenced for the lipid A moiety, by contrast a plausible “immune modulating” action has been proposed for the peculiar O‐antigen portion.

Deciphering the Chemical Language of the Immunomodulatory Properties of Veillonella parvula Lipopolysaccharide.

Veillonella parvula, prototypical member of the oral and gut microbiota, is at times commensal yet also potentially pathogenic. The definition of the molecular basis tailoring this contrasting behavior is key for broadening our understanding of the microbiota-driven pathogenic and/or tolerogenic mechanisms that take place within our body. In this study, we focused on the chemistry of the main constituent of the outer membrane of V. parvula, the lipopolysaccharide (LPS). LPS molecules indeed elicit pro-inflammatory and immunomodulatory responses depending on their chemical structures. Herein we report the structural elucidation of the LPS from two strains of V. parvula and show important and unprecedented differences in both the lipid and carbohydrate moieties, including the identification of a novel galactofuranose and mannitol-containing O-antigen repeating unit for one of the two strains. Furthermore, by harnessing computational studies, in vitro human cell models, as well as lectin binding solid-phase assays, we discovered that the two chemically diverse LPS immunologically behave differently and have attempted to identify the molecular determinant(s) governing this phenomenon. Whereas pro-inflammatory potential has been evidenced for the lipid A moiety, by contrast a plausible "immune modulating" action has been proposed for the peculiar O-antigen portion.

Human ST3Gal II and ST6GalNAc IV genes increase human serum-mediated cytotoxicity to xenogeneic cells.

Carbohydrate-antigens widely existed on glycoproteins and glycosphingolipids of all mammalian cells play a crucial role in self-defense and immunity. Xeno-reactive antibodies included in natural human sera play a protecting role in an acute phase-rejection of xenotransplantation. In this study, we investigated the effect of an alteration of glycosylation-pattern, caused by human sialyltransferases such as hST3Gal II or hST6GalNAc IV, on human serum mediated cytotoxicity in pig kidney PK15 cells. From LDH cytotoxicity assay, cytotoxicity to human serum was significantly increased in hST3Gal II and hST6GalNAc IV-transfected PK15 cells, as compared to the control. In the hST6Gal I-carrying cells, the cytotoxicity to human serum was rather decreased. Moreover, flow cytometry analysis revealed that an alteration of pig glycosylation-pattern by hST3Gal II or hST6GalNAc IV influences on a binding of human IgM or IgG, respectively, in pig kidney cells, regardless of Gal antigen alteration. Finally, we found that hST6GalNAc IV contributed to increase of terminal disialylated tetrasaccharide structure, disialyl T antigen, as evidenced by increase of the MAL II lectin binding capacity in the hST6GalNAc IV-transfected PK15 cells, compared with control. Therefore, our results suggest that carbohydrate antigens, such as disialyl T antigen, newly synthesized by the ST3Gal II- and ST6GalNAc IV are potentially believed to be new xeno-reactive elements.

A171 A MISSENSE MUTATION IN FCGBP, A STRUCTURAL COMPONENT OF MUC2 MUCUS, ALTERS THE GLYCOMICS PROFILE AND FUNCTION OF COLONIC MUCUS

Abstract Background MUC2 mucin produced by colonic goblet cells, form a mucus bilayer that provides a physical barrier between potential pathogens in the lumen and the underlying epithelial cells. Mucus is thus the first line of innate host defense in the gastrointestinal (GI) tract. Many GI diseases including inflammatory bowel disease and colon cancer affect the glycosylation of mucus. Goblet cells produce a variety of proteins that are associated with the mucus layer. Of these proteins, FCGBP is of significant interest due to its structural similarities to MUC2 mucin with unknown functions. In this study, we investigated if a missense mutation in FCGBP altered the glycosylation of goblet cell MUC2 and affected its barrier functions. Aims Aims: 1) To determine mechanistically how FCGBP impeded the structural integrity of the mucus layer 2) To quantify MUC2 glycoprotein modifications in the altered mucus layer Methods To investigate whether FCGBP impaired mucus barrier functions, two cell types were investigated: wildtype LS174T (WT) MUC2 mucus-producing goblet cells and LS174T cells with a missense mutation in FCGBP (FCGBP-Mut). To determine if FCGBP-Mut led to loss in barrier function in the mucus layer, the penetration of 0.2, 1, and 2 μm fluorescent beads (to mimic bacteria) through the mucus layer were quantified. To determine if the differences in penetrability were caused by differences in MUC2 glycosylation in the goblet cell lines, sensitive glycomic analyses were performed by high-performance liquid chromatography-mass spectrometry (HPLC-MS) and capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). Both cells and purified MUC2 mucin granules were analyzed and confirmed by lectin binding assays. To enumerate differences in the glycomics profiles, RT-PCR was performed on over 30 human glycosyltransferases. Results FCGBP-Mut cells exhibited an altered glycomics profile with a significant increase in sialylated/fucosylated glycans as quantified by HPLC-MS and an increase in sialyl- and fructosyltransferase mRNA expression. In contrast to WT goblet cells, FCGBP-Mut cells exhibited significant loss in MUC2 mucus barrier function as quantified by fluorescent beads penetration through the mucus layer temporally. The altered glycosylation in FCGBP-Mut cells triggered increased metabolic stress and ROS production that enhanced susceptibility to Salmonella enterica binding, invasion and cell death. Conclusions This study demonstrates that a single missense mutation in FCGBP altered the penetrability of the mucus layer associated with an increase in sialylated/fucosylated glycans, a signature hallmark of numerous colonic diseases. These findings underscore the importance of FCGBP in providing structural integrity of the mucus layer and homeostatic maintenance of goblet cell glycosylation profiles. Funding Agencies CIHR

FRET-Sensing of Multivalent Protein Binding at the Interface of Biomimetic Microparticles Functionalized with Fluorescent Glycolipids.

Cell adhesion is a central process in cellular communication and regulation. Adhesion sites are triggered by specific ligand-receptor interactions inducing the clustering of both partners at the contact point. Investigating cell adhesion using microscopy techniques requires targeted fluorescent particles with a signal sensitive to the clustering of receptors and ligands at the interface. Herein, we report on simple cell or bacterial mimics, based on liquid microparticles made of lipiodol functionalized with custom-designed fluorescent lipids. These lipids are targeted toward lectins or biotin membrane receptors, and the resulting particles can be specifically identified and internalized by cells, as demonstrated by their phagocytosis in primary murine bone marrow-derived macrophages. We also evidence the possibility to sense the binding of a multivalent lectin, concanavalin A, in solution by monitoring the energy transfer between two matching fluorescent lipids on the surface of the particles. We anticipate that these liquid particle-based sensors, which are able to report via Förster resonance energy transfer (FRET) on the movement of ligands on their interface upon protein binding, will provide a useful tool to study receptor binding and cooperation during adhesion processes such as phagocytosis.

Influence of maternal rats' hypothyroidism on morpho-functional peculiarities and glycome of progeny skin.

We studied the influence of maternal hypothyroidism on progeny skin morphogenesis by means of histological, histochemical and lectin-histochemical methods. Hypothyroid conditions in rats were achieved by daily food supplementation with antithyroid drug Mercazolil. The experiment was conducted on 10 control and 10 hypothyroid rats, which delivered 70 and 46 offsprings, respectively. We discovered that maternal hypothyroidism induces the accumulation of mast cells (MCs) in the skin of progeny on the 1st, 10th and 20th postnatal days, with decrease of these cell's count returning to control level on 40th postnatal day. These results indicate that offsprings developing under conditions of maternal hypothyroidism are a risk group for changes in immune status and the occurrence of allergic reactions. The stratum corneum of epidermis, its lipid barrier as well as pilosebaceous units, in both control and experimental group animals, at the early stages of postnatal ontogenesis are enriched with carbohydrate determinants of αDMan, βDGal, βDGal(1-3)DGalNAc, αLFuc, αDGalNAc, αDGlcNAc, Neu5Ac. Galanthus nivalis agglutinin (GNA) is a selective histochemical marker of MCs, while Lactarius torminosus fungus agglutinin (LTFA) is a selective label of Langerhans cells. Maternal hypothyroidism resulted in reduction of lectin binding with the structural components of progeny skin and its derivatives. We speculate that alterations in glycoconjugate processing and degradation sequences have an impact on the cell signaling, formation of adhesive contacts, cellular proliferation and differentiation. The lectin set we used clearly demonstrated specific labeling of cellular subpopulations, monitoring glycoconjugates processing and degradation under physiological and pathological conditions in all skin components.

P139 A potential protective rule of Mannose Binding lectin (MBL) against SARS-COV2 infection in IBD patients

Abstract Background Since the beginning of the COVID-19 pandemic, IBD patients have appeared to be less susceptible to a severe form of the disease, despite undergoing immunosuppressive treatments. Mannose binding lectin (MBL) is a humoral fluid-phase pattern recognition molecule (PRM) crucial for activating the lectin complement pathway. It is capable of binding to the spike protein and blocking SARS-CoV-2 infection. Low MBL expression has been linked to poor outcomes in both SARS and SARS- CoV-2 infections. In IBD patients, the role of MBL is controversial; low concentrations seem to predispose individuals to the development of IBD, while high concentrations have been observed in severe forms of Crohn's disease. Methods Since April 2020, 104 IBD patients have been enrolled, with 20% treated with mesalazine, 50% with Anti-TNFa, 20% with Vedolizumab, and 20% receiving no treatment. Additionally, 45 healthy controls (HC) from North Italy were included. Serum samples and colon biopsies from inflamed and non- inflamed mucosa were collected. Serum samples underwent an ELISA test to quantify circulating MBL concentration, and MBL gene expression in biopsies was analyzed using qPCR. Results Both Crohn's disease and Ulcerative Colitis patients exhibited significantly higher circulating MBL levels than HC (411.6 ± 45.5 ng/ml vs. 160.9 ± 35.3 ng/ml, p < 0.05). Patients treated with Anti-TNFa showed significantly higher circulating MBL levels compared to those not undergoing therapy (530.1 ± 76.5 ng/ml vs. 225.4 ± 61.6 ng/ml, p < 0.05) and patients receiving Vedolizumab (530.1 ± 76.5 ng/ml vs. 155.3 ± 32.1 ng/ml, p < 0.05). MBL intestinal gene expression is higher in IBD patients than HC (5.55 ± 0.9 vs. 2.36 ± 0.6; p=0.591) and tended to be elevated at similar levels in inflamed and non-inflamed mucosa (5.14 ± 1.4 vs. 5.79 ± 1.1; p=0.337), suggesting a prevalent genetic determination of MBL concentration regardless of the inflammatory background. Conclusion MBL appears to play a key role in SARS-CoV-2 infection. We demonstrate that IBD patients have, on average, higher circulating MBL levels than HC independently of their inflammatory status. The highest MBL levels were observed in patients treated with Anti-TNFa. These findings contribute to understanding the reported low mortality and hospitalization for COVID-19 among IBD patients treated with Anti-TNFa.

Mannose-specific plant and microbial lectins as antiviral agents: A review.

Lectins are non-immunological carbohydrate-binding proteins classified on the basis oftheir structure, origin, and sugar specificity. The binding specificity of such proteins with the surface glycan moiety determines their activity and clinical applications. Thus, lectins hold great potential as diagnostic and drug discovery agents and as novel biopharmaceutical products. In recent years, significant advancements have been made in understanding plant and microbial lectins as therapeutic agents against various viral diseases. Among them, mannose-specific lectins have being proven aspromising antiviral agents against a variety of viruses, such as HIV,Influenza, Herpes, Ebola, Hepatitis, Severe Acute Respiratory Syndrome Coronavirus-1 (SARS-CoV-1), Middle Eastern Respiratory Syndrome Coronavirus (MERS-CoV) and most recent Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). The binding of mannose-binding lectins (MBLs) from plants and microbes to high-mannose containing N-glycans (which may be simple or complex) of glycoproteins found on the surface of viruses has been found to be highly specific and mainly responsible for their antiviral activity. MBLs target various steps in the viral life cycle, including viral attachment, entry and replication. The present review discusses the brief classification and structure of lectins along with antiviral activity of various mannose-specific lectins from plants and microbial sources and their diagnostic and therapeutic applications against viral diseases.

Punica granatum sarcotesta lectin (PgTeL) inhibits Pseudomonas aeruginosa replication, viability, aggregation, and biofilms

Pseudomonas aeruginosa is included in the ESKAPEE group, which includes pathogens responsible for nosocomial infections. PgTeL is a lectin from pomegranate sarcotesta active against human pathogenic microrganisms. This study evaluated the antibacterial effects of PgTeL on three strains of P. aeruginosa, one non-resistant strain (ATCC 27853) and two multidrug-resistant (MDR) strains (UFPEDA 261 and UFPEDA 262). Minimal inhibitory (MIC) and bactericidal (MBC) concentrations were determined. The anti-aggregating and antibiofilm effects of PgTeL were also evaluated as well as its synergistic effects with antibiotics. Fluorescent nanoprobes (PgTeL conjugated with quantum dots; QDs-PgTeL) were used to investigate differences in the lectin binding to the cell surface of the strains as well as possible involvement of the carbohydrate-recognizing domain (CRD) in the binding to planktonic and biofilm cells. PgTeL showed an MIC of 12.5–25.0 μg/mL, an MBC of 50.0–100.0 μg/mL and an anti-aggregating effect. Biofilm formation was inhibited by more than 50 % at PgTeL concentrations of 25.0, 3.12 and 0.78 μg/mL for ATCC 27853, UFPEDA 261, and UFPEDA 262, respectively. The lectin (0.78–3.12 μg/mL) also promoted eradication of preformed biofilms. The QDs-PgTeL conjugates revealed a higher binding efficiency of PgTeL to UFPEDA 261 (74.2 %), followed by UFPEDA262 (54.5 %) and ATCC 27853 (44.8 %). The PgTeL CRD is involved in the interaction of this lectin with planktonic or biofilm cells, as the nanoprobes had their effectiveness decreased to 8.2–21.6 % when previously incubated with fetuin. Finally, the combination of PgTeL and ceftazidime showed synergistic effects against the MDR strains (ΣFIC of 0.5 and 0.25, respectively). In conclusion, PgTeL impaired replication, viability, aggregation, and biofilm formation of P. aeruginosa and differences in the effects for the three strains may be due to distinct glycidic composition on their cell surfaces and, consequently, differential binding efficiency of PgTeL.

Synthesis of the 3'-O-sulfated TF antigen with a TEG-N3 linker for glycodendrimersomes preparation to study lectin binding.

The synthesis of gram quantities of the TF antigen (β-ᴅ-Gal-(1→3)-α-ᴅ-GalNAc) and its 3'-sulfated analogue with a TEG-N3 spacer attached is described. The synthesis of the TF antigen comprises seven steps, from a known N-Troc-protected galactosamine donor, with an overall yield of 31%. Both the spacer (85%) and the galactose moiety (79%) were introduced using thioglycoside donors in NIS/AgOTf-promoted glycosylation reactions. The 3'-sulfate was finally introduced through tin activation in benzene/DMF followed by treatment with a sulfur trioxide-trimethylamine complex in a 66% yield.

A novel biomimetic probe for galectin‐3 recognition: Chemical synthesis and structural characterization of a β‐galactose branched sodium hyaluronate

AbstractSodium hyaluronate (HA), a derivative of hyaluronan, is a natural and biocompatible polysaccharide that interacts with cluster of differentiation‐44 receptor to promote fine‐tuning of inflammation, fibrosis, and tissue remodeling. HA has a smaller molecular weight than hyaluronan and is overall more stable being less prone to oxidation. In this study, we report a novel lactose‐functionalized sodium hyaluronate, named HYLACH®. Functionalization with multiple β‐galactose residues facilitates its interaction with galectin‐3, a β‐galactose binding lectin implicated in various pathological processes including inflammation, host defense, and fibrosis, especially critical in idiopathic pulmonary fibrosis (IPF). Our strategy was to modify HA, to varying extents, at carboxyl sites with 1‐amino‐1‐deoxy‐lactitol, in the presence of 4‐(4,6‐dimethoxy‐1,3,5‐triazin‐2‐yl)‐4‐methyl morpholinium chloride in aqueous media. We characterized the chemical structure, molecular weight, and degree of substitution of HYLACH® using NMR spectroscopy and size exclusion chromatography. We further determined several key parameters including its stability toward enzymatic degradation and the binding affinity and conformational changes of galectin‐3 interaction with HYLACH®. Collectively, the generation of a novel functionalized HA with an ability to bind and suppress galectin‐3 function, in combination with safety and biocompatibility, offers the opportunity to test this compound in therapeutic trials of devastating fibrotic diseases such as IPF.

Inhibition of Pro-Fibrotic Molecules Expression in Idiopathic Pulmonary Fibrosis-Derived Lung Fibroblasts by Lactose-Modified Hyaluronic Acid Compounds.

Idiopathic pulmonary fibrosis (IPF) is a chronic inflammatory and fibrotic pathological condition with undefined effective therapies and a poor prognosis, partly due to the lack of specific and effective therapies. Galectin 3 (Gal-3), a pro-fibrotic ß-galactoside binding lectin, was upregulated in the early stages of the pathology, suggesting that it may be considered a marker of active fibrosis. In the present in vitro study, we use Hylach®, a lactose-modified hyaluronic acid able to bind Gal-3, to prevent the activation of lung myofibroblast and the consequent excessive ECM protein cell expression. Primary human pulmonary fibroblasts obtained from normal and IPF subjects activated with TGF-β were used, and changes in cell viability, fibrotic components, and pro-inflammatory mediator expression at both gene and protein levels were analyzed. Hylach compounds with a lactosylation degree of about 10% and 30% (Hylach1 and Hylach 2), administrated to TGF-β-stimulated lung fibroblast cultures, significantly downregulated α-smooth muscle actin (α-SMA) gene expression and decreased collagen type I, collagen type III, elastin, fibronectin gene and protein expression to near baseline values. This anti-fibrotic activity is accompanied by a strong anti-inflammatory effect and by a downregulation of the gene expression of Smad2 for both Hylachs in comparison to the native HA. In conclusion, the Gal-3 binding molecules Hylachs attenuated inflammation and TGF-β-induced over-expression of α-SMA and ECM protein expression by primary human lung fibroblasts, providing a new direction for the treatment of pulmonary fibrotic diseases.

Amphiphilic Low-Molecular-Weight Gelators Bearing β-S-N-Acetylglucosamine Linked to a Tartaric Acid Scaffold: Synthesis, Self-Assembly and Wheat Germ Agglutinin Binding.

The self-assembly of carbohydrate-based amphiphiles can lead to colloidal soft materials such as supramolecular gels featuring highly desirable characteristics like biodegradability and biocompatibility. The report herein presents the synthesis, characterization and supramolecular self-assembly, physical gelation and wheat lectin binding of two structurally related amphiphilic compounds having β-S-N-acetylglucosamine residues linked to a 2,3-diacyl-N,N'-dipropargylated-l-tartaric diamide. A 1-thio-β-N-acetyl-d-glucosamine precursor attached to a conveniently functionalized linker with an azido group was synthesized by means of a one-pot procedure followed by deprotection. A click reaction successfully led to the two amphiphiles, which differed in length of the fatty acid attached to the tartaric acid scaffold. Although both compounds are poorly soluble in water and organic solvents, the difference in terms of hydrophilic moieties provided them with distinct supramolecular gelation properties. While the presence of an octadecyl chain produced a hydrogelator, the dodecadecyl homologue would only form weak gels in DMSO. SEM and rheology experiments confirmed the characteristic fibrillar morphology and viscoelastic properties, in agreement with the presence of physical gels. Both amphiphiles were able to interact reversibly with wheat germ agglutinin (WGA), a lectin that specifically recognizes GlcNAc residues, indicating a potential use in the food industry, as a gluten sensitivity manager, as well as in health-related industries, for example, for drug delivery systems.