Lectin Binding Research Articles

Targeting the Leloir Pathway with Galactose-Based Antimetabolites in Glioblastoma.

Glioblastoma (GBM) uses Glut3 and/or Glut14 and the Leloir pathway to catabolize D-Galactose (Gal). UDP-4-deoxy-4-fluorogalactose (UDP-4DFG) is a potent inhibitor of the two key enzymes, UDP-galactose-4-epimerase (GALE) and UDP-Glucose 6-dehydrogenase (UGDH), involved in Gal metabolism and in glycan synthesis. The Gal antimetabolite 4-deoxy-4-fluorogalactose (4DFG) is a good substrate for Glut3/Glut14 and acts as a potent glioma chemotherapeutic. Primary GBM cell cultures were used to examine toxicity and alterations in glycan composition via lectin binding in fixed cells and by Western blots. Toxicity/efficacy in vivo data was performed in mouse flank and intracranial models. The effect of 4DFG on D-glucose (Glc) metabolism in GBM cells was assessed by using 13C NMR-based tracer studies. 4DFG is moderately potent against GBM cells (IC50: 125-300 µM). GBM glycosylation is disrupted by 4DFG. Survival analysis in an intracranial mouse model showed that treatment with 4DFG (6 × 25 mg/kg of 4DFG, intravenously) improved outcomes by three-fold (p < 0.01). Metabolic flux analysis revealed that both glycolytic and mitochondrial metabolic fluxes of [U-13C]Glc were significantly decreased in the presence of 4DFG in GBM cells. A functional Gal-scavenging pathway in GBM allows Gal-based antimetabolites to act as chemotherapeutics. 4DFG is metabolized by GBM in vitro and in vivo, is lethal to GBM tumors, and is well tolerated in mice.

Selective Glycan Presentation in Liquid‐Ordered or ‐Disordered Membrane Phases and its Effect on Lectin Binding

Glycan‐protein interactions play a key role in various biological processes from fertilization to infections. Many of these interactions take place at the glycocalyx – a heavily glycosylated layer at the cell surface. Despite its significance, studying the glycocalyx remains challenging due to its complex, dynamic, and heterogeneous nature. This study introduces a glycocalyx model allowing for the first time to control spatial organization and heterogeneity of the glycan moieties. Glycan‐mimetics with lipid‐moieties that partition into either liquid‐ordered (Lo, lipid rafts) or liquid‐disordered (Ld) phases of giant unilamellar vesicles (GUVs), which serve as simplified cell membrane models micking lipid rafts, are developed. This phase‐specific allocation allows controlled placement of glycan motifs in distinct membrane environments, creating heteromultivalent systems that replicate the natural glycocalyx's complexity. We show that phase localization of glycan mimetics significantly influences recruitment of protein receptors to the membrane. Glycan‐conjugates in the ordered phase demonstrate enhanced lectin binding, supporting the idea that raft‐like domains facilitate stronger receptor interactions. This study provides a platform for systematically investigating spatial and dynamic presentation of glycans in biological systems and presents the first experimental evidence that glycan accumulation in lipid rafts enhances receptor binding affinity, offering deeper insights into the glycocalyx's functional mechanisms.

Selective Glycan Presentation in Liquid-Ordered or -Disordered Membrane Phases and its Effect on Lectin Binding.

Glycan-protein interactions play a key role in various biological processes from fertilization to infections. Many of these interactions take place at the glycocalyx - a heavily glycosylated layer at the cell surface. Despite its significance, studying the glycocalyx remains challenging due to its complex, dynamic, and heterogeneous nature. This study introduces a glycocalyx model allowing for the first time to control spatial organization and heterogeneity of the glycan moieties. Glycan-mimetics with lipid-moieties that partition into either liquid-ordered (Lo, lipid rafts) or liquid-disordered (Ld) phases of giant unilamellar vesicles (GUVs), which serve as simplified cell membrane models micking lipid rafts, are developed. This phase-specific allocation allows controlled placement of glycan motifs in distinct membrane environments, creating heteromultivalent systems that replicate the natural glycocalyx's complexity. We show that phase localization of glycan mimetics significantly influences recruitment of protein receptors to the membrane. Glycan-conjugates in the ordered phase demonstrate enhanced lectin binding, supporting the idea that raft-like domains facilitate stronger receptor interactions. This study provides a platform for systematically investigating spatial and dynamic presentation of glycans in biological systems and presents the first experimental evidence that glycan accumulation in lipid rafts enhances receptor binding affinity, offering deeper insights into the glycocalyx's functional mechanisms.

Sugar-binding Profiles of the Mesothelial Glycocalyx in Frozen Tissues of Mice Revealed by Lectin Staining

The mesothelium is a non-adhesive protective surface that lines the serosal cavities and organs within the body. The glycocalyx is a complex structure that coats the outer layer of the mesothelium. However, due to the limitations of conventional fixation techniques, studies on glycans are limited. In this study, lectin staining of frozen tissues was performed to investigate the diversity of glycans in the glycocalyx of mesothelial cells in mice. Datura stramonium lectin (DSL), which recognizes lactosamine and binds to Galectin-3 and -1, was broadly bound to the mesothelial cells of the visceral and parietal peritoneum but not to the pancreas, liver, intestine, or heart. Furthermore, human mesothelial cells in the omentum and parietal peritoneum were positive for DSL. Erythrina cristagalli lectin binding was specific to mesothelial cells in the parietal peritoneum, that is, the pleura, diaphragm, and peritoneum. Intriguingly, surface sialylation, the key element in reducing peritoneal dissemination and implantation, and promoting ascites formation by ovarian carcinoma cells, was much higher in the parietal peritoneum than in the omentum. These findings revealed slight differences in the glycans of mesothelial cells of different organs, which may be related to clinical diseases. These results also suggest that there may be differences in the functions of parietal and visceral mesothelial cells.

Accelerated wound healing and reduced scar formation induced by D-mannose: a possible role of mannose binding lectin

Wounds are most commonly caused by accidents, surgery and burns, and can be internal or external. Naturally, the wound healing process can take a long time and lead to scarring. In this study we present a technique to shorten wound healing time and prevent or mitigate scarring using D-mannose that is applied directly on the wound. The results showed that the healing time is almost halved compared to treatment with povidone-iodine solution which is an antiseptic widely used in surgery. D-Mannose is neither an antisepsis nor an antibiotic. We propose a posssible mechanism by which D-mannose binds to D-mannose binding lectin and immediately activates the innate immune system that ultimately phagocytizes pathogens and clears the wound of degraded cells and materials, which reduces inflammation and implicitly wound healing time. D-Mannose also intervenes in the coagulation process by binding to fibrinogen, generating a finer and denser fibrin, which visibly reduces collagen scars. Our findings show that applying D-mannose directly on the wound as a powder shortens wound healing time and visibly attenuates scarring. Apart from the unaesthetic appearance, these scars can also cause a certain tissue dysfunction, regardless of the affected organ.Graphical abstract

Sub-10 μm Soft Interlayers Integrating Patterned Multivalent Biomolecular Binding Environments.

Designing surfaces that enable controlled presentation of multivalent ligand clusters (e.g., for rapid screening of biomolecular binding constants or design of artificial extracellular matrices) is a cross-cutting challenge in materials and interfacial chemistry. Existing approaches frequently rely on complex building blocks or scaffolds and are often specific to individual substrate chemistries. Thus, an interlayer chemistry that enabled efficient nanometer-scale patterning on a transferrable layer and subsequent integration with other classes of materials could substantially broaden the scope of surfaces available for sensors and wearable electronics. Recently, we have shown that it is possible to assemble nanometer-resolution chemical patterns on substrates including graphite, use diacetylene polymerization to lock the molecular pattern together, and then covalently transfer the pattern to amorphous materials (e.g., polydimethylsiloxane, PDMS), which would not natively enable high degrees of control over ligand presentation. Here, we develop a low-viscosity PDMS formulation that generates very thin films (<10 μm) with dense cross-linking, enabling high-efficiency surface functionalization with polydiacetylene arrays displaying carbohydrates and other functional groups (up to 10-fold greater than other soft materials we have used previously) on very thin films that can be integrated with other materials (e.g., glass and soft materials) to enable a highly controlled multivalent ligand display. We use swelling and other characterization methods to relate surface functionalization efficiency to the average distance between cross-links in the PDMS, developing design principles that can be used to create even thinner transfer layers. In the context of this work, we apply this approach using precision glycopolymers presenting structured arrays of N-acetyl glucosamine ligands for lectin binding assays. More broadly, this interlayer approach lays groundwork for designing surface layers for the presentation of ligand clusters on soft materials for applications including wearable electronics and artificial extracellular matrix.

Reduction of N-Acetylglucosaminyltransferase-I Activity Promotes Neuroblastoma Invasiveness and EGF-Stimulated Proliferation In Vitro

Aberrant N-glycosylation has been associated with progression of the pediatric cancer neuroblastoma (NB) but remains understudied. Here we investigated oligomannose N-glycans in NB by genetic editing of MGAT1 in a human NB cell line, BE(2)-C, called BE(2)-C(MGAT1−/−). Lectin binding studies confirmed that BE(2)-C(MGAT1−/−) had decreased complex and increased oligomannose N-glycans. The relevance of 2D and 3D cell cultures was demonstrated for cell morphology, cell proliferation, and cell invasion, thereby highlighting the necessity for 3D cell culture in investigating cancerous properties. Western blotting revealed that oligomannosylated EGFR had increased autophosphorylation. Proliferation was decreased in BE(2)-C(MGAT1−/−) using 2D and 3D cultures, but both cell lines had similar proliferation rates using 3D cultures without serum. Upon EGF treatment, BE(2)-C(MGAT1−/−), but not BE(2)-C, showed increased proliferation, and furthermore, the mutant proliferated much faster than BE(2)-C under 3D conditions. Cell spheroid invasiveness was greatly increased in BE(2)-C(MGAT1−/−) compared with BE(2)-C. Moreover, invasiveness was reduced in both cell lines with either EGF or RhoA activator treatment, regardless of the N-glycan population. Thus, this study further extends our earlier findings that oligomannose N-glycans enhance NB cell invasiveness, and that EGF stimulation of oligomannosylated EGFR greatly enhances cell proliferation rates, underlining the role of oligomannose N-glycans in the promotion of NB.

Phylogenetic variations in a novel family of hyperstable apple snail egg proteins: insights into structural stability and functional trends.

The relationship between protein stability and functional evolution is little explored in proteins purified from natural sources. Here, we investigated a novel family of egg proteins (Perivitellin-1, PV1) from Pomacea snails. Their remarkable stability and clade-related functions in most derived clades (Canaliculata and Bridgesii) make them excellent candidates for exploring this issue. To that aim, we studied PV1 (PpaPV1) from the most basal lineage, Flagellata. PpaPV1 displays unparalleled structural and kinetic stability, surpassing PV1s from derived clades, ranking among the most hyperstable proteins documented in nature. Its spectral features contribute to a pale egg coloration, exhibiting a milder glycan binding lectin activity with a narrower specificity than PV1s from the closely related Bridgesii clade. These findings provide evidence for substantial structural and functional changes throughout the genus' PV1 evolution. We observed that structural and kinetic stability decreased in a clade-related fashion and was associated with large variations in defensive traits. For instance, pale PpaPV1 lectin turns potent in the Bridgesii clade, adversely affecting gut morphology, while giving rise to brightly colored PV1s providing eggs with a conspicuous, probably warning signal in the Canaliculata clade. This work provides a comprehensive comparative analysis of PV1s from various apple snail species within a phylogenetic framework, offering insights into the interplay among their structural features, stability profiles and functional roles. More broadly, our work provides one of the first examples from natural evolution showing the crucial link among protein structure, stability and evolution of new functions.

The galectin-3 inhibitor selvigaltin reduces liver inflammation and fibrosis in a high fat diet rabbit model of metabolic-associated steatohepatitis.

Galectin-3 is a pro-fibrotic β-galactoside binding lectin highly expressed in fibrotic liver and implicated in hepatic fibrosis. Selvigaltin (previously known as GB1211) is a novel orally active galectin-3 small molecule inhibitor that has high affinity for galectin-3 (human KD = 25nM; rabbit KD = 12nM) and high oral bioavailability in rabbits and man. In this study the efficacy of selvigaltin was investigated in a high fat diet (HFD) rabbit model of metabolic-associated steatohepatitis (MASH). Male New Zealand White rabbits were individually caged under standard conditions in a temperature and humidity-controlled room on a 12h light/darkness cycle. After 1week of regular diet (RD), rabbits were randomly assigned for 8 or 12weeks to different groups: RD/vehicle, RD/selvigaltin, HFD (8weeks), HFD/vehicle and HFD/selvigaltin (0.3, 1.0, 5.0 or 30mg/kg selvigaltin with vehicle/selvigaltin p.o. dosed therapeutically q.d. 5days per week from week 9 or 12). Liver inflammation, steatosis, ballooning, and fibrosis was measured via blood metabolic markers, histomorphological evaluation [Oil Red O, Giemsa, Masson's trichome, picrosirius red (PSR) and second harmonic generation (SHG)], and mRNA and protein expression. Steatosis, inflammation, ballooning, and fibrosis were all increased from RD to HFD/vehicle groups. Selvigaltin demonstrated target engagement by significantly decreasing galectin-3 levels in the liver as measured via immunohistochemistry and mRNA analysis. Selvigaltin dose-dependently reduced biomarkers of liver function (AST, ALT, bilirubin), inflammation (cells foci), and fibrosis (PSR, SHG), as well as decreasing the mRNA and protein expression of several key inflammation and fibrosis biomarkers (e.g., IL6, TGFβ3, SNAI2, collagen). Doses of 1.0 or 5.0mg/kg demonstrated consistent efficacy across most biological endpoints supporting the current clinical doses of selvigaltin being investigated in liver disease. Selvigaltin significantly reduced hepatic inflammation and fibrosis in an HFD rabbit model of MASH following therapeutic dosing for 4 weeks in a dose-dependent manner. These data support the human selvigaltin dose of 100mg b.i.d. that has been shown to reduce key liver biomarkers during a clinical study in liver cirrhosis.

Synthesis and Thermodynamic Evaluation of Sialyl-Tn MUC1 Glycopeptides Binding to Macrophage Galactose-Type Lectin.

Interactions between the tumor-associated carbohydrate antigens of Mucin 1 (MUC1) and the carbohydrate-binding proteins, lectins, often lead to the creation of a pro-tumor microenvironment favoring tumor initiation, progression, metastasis, and immune evasion. Macrophage galactose binding lectin (MGL) is a C-type lectin receptor found on antigen-presenting cells that facilitates the uptake of carbohydrate antigens for antigen presentation, modulating the immune response homeostasis, autoimmunity, and cancer. Considering the crucial role of tumor-associated forms of MUC1 and MGL in tumor immunology, a thorough understanding of their binding interaction is essential for it to be exploited for cancer vaccine strategies. The synthesis of MUC1 glycopeptide models carrying a single or multiple Tn and/or sialyl-Tn antigen(s) is described. A novel approach for the sialyl-Tn threonine building block suitable for the solid phase peptide synthesis was developed. The thermodynamic profile of the binding interaction between the human MGL and MUC1 glycopeptide models was analyzed using isothermal titration calorimetry. The measured dissociation constants for the sialyl-Tn-bearing peptide epitopes were consistently lower compared to the Tn antigen and ranged from 10 μM for mono- to 1 μM for triglycosylated MUC1 peptide, respectively. All studied interactions, regardless of the glycan's site of attachment or density, exhibited enthalpy-driven thermodynamics.

Impact of 5' Near Gene Variants of Mannose Binding Lectin (MBL2) on Breast Cancer Risk.

The immune system plays a bifaceted role in tumour development through modulation of inflammation. MBL binds to damage-associated molecular patterns and induces inflammation through the activation of complement pathway. Dysregulated inflammation plays a major role in breast cancer pathogenesis, thereby suggesting its contribution towards breast cancer risk. Literature asserts single-nucleotide polymorphisms (SNPs) modulating serum MBL levels. Therefore, studying MBL2 SNPs in breast cancer might provide valuable insight in the disease pathogenesis. The present case-control association study aimed to elucidate the association between MBL2 5' near gene SNPs and breast cancer risk. Breast cancer patients were recruited from Government Medical College, G.N.D. Hospital, Amritsar. The age- and gender-matched genetically unrelated healthy individuals, from adjoining regions, with no history of malignancy up to three generations were recruited as controls. The SNPs of MBL2 from the 5' near gene region with putative functional significance were selected based upon the in silico analysis and literature review. The genotypic, allelic and haplotype frequencies for the studied variants were assessed and compared in the study participants by ARMS-PCR and PCR-RFLP. No difference in allelic, genotypic and haplotype frequencies was reported for rs7096206, rs7084554 and rs11003125 in both the participant groups. rs7084554 (CC) was found to confer risk towards hormone receptor-positive breast cancer. An intermediate LD was observed between rs7084554 and rs11003125. The study reports association between MBL2 variant (rs7084554) and hormone receptor-positive breast cancer risk. Further research in this direction might validate the findings.

Impaired myoblast differentiation and muscle IGF-1 receptor signaling pathway activation after N-glycosylation inhibition.

The role of N-glycosylation in the myogenic process remains poorly understood. Here, we evaluated the impact of N-glycosylation inhibition by Tunicamycin (TUN) or by phosphomannomutase 2 (PMM2) gene knockdown, which encodes an enzyme essential for catalyzing an early step of the N-glycosylation pathway, on C2C12 myoblast differentiation. The effect of chronic treatment with TUN on tibialis anterior (TA) and extensor digitorum longus (EDL) muscles of WT and MLC/mIgf-1 transgenic mice, which overexpress muscle Igf-1Ea mRNA isoform, was also investigated. TUN-treated and PMM2 knockdown C2C12 cells showed reduced ConA, PHA-L, and AAL lectin binding and increased ER-stress-related gene expression (Chop and Hspa5 mRNAs and s/uXbp1 ratio) compared to controls. Myogenic markers (MyoD, myogenin, and Mrf4 mRNAs and MF20 protein) and myotube formation were reduced in both TUN-treated and PMM2 knockdown C2C12 cells. Body and TA weight of WT and MLC/mIgf-1 mice were not modified by TUN treatment, while lectin binding slightly decreased in the TA muscle of WT (ConA and AAL) and MLC/mIgf-1 (ConA) mice. The ER-stress-related gene expression did not change in the TA muscle of WT and MLC/mIgf-1 mice after TUN treatment. TUN treatment decreased myogenin mRNA and increased atrogen-1 mRNA, particularly in the TA muscle of WT mice. Finally, the IGF-1 production and IGF1R signaling pathways activation were reduced due to N-glycosylation inhibition in TA and EDL muscles. Decreased IGF1R expression was found in TUN-treated C2C12 myoblasts which was associated with lower IGF-1-induced IGF1R, AKT, and ERK1/2 phosphorylation compared to CTR cells. Chronic TUN-challenge models can help to elucidate the molecular mechanisms through which diseases associated with aberrant N-glycosylation, such as Congenital Disorders of Glycosylation (CDG), affect muscle and other tissue functions.

Exploring galectin interactions with human milk oligosaccharides and blood group antigens identifies BGA6 as a functional galectin-4 ligand

Galectins (Gals), a family of multifunctional glycan-binding proteins, have been traditionally defined as β-galactoside binding lectins. However, certain members of this family have shown selective affinity towards specific glycan structures including human milk oligosaccharides (HMOs) and blood group antigens. In this work, we explored the affinity of human galectins (particularly Gal-1, -3, -4, -7 and -12) towards a panel of oligosaccharides including HMOs and blood group antigens using a complementary approach based on both experimental and computational techniques. While prototype Gal-1 and Gal-7 exhibited differential affinity for type I vs. type II Lac/LacNAc residues and recognized fucosylated neutral glycans, chimera-type Gal-3 showed high binding affinity towards poly-LacNAc structures including LNnH and LNnO. Notably, the tandem-repeat human Gal-12 showed preferential recognition of 3-fucosylated glycans, a unique feature among members of the galectin family. Finally, Gal-4 presented a distinctive glycan-binding activity characterized by preferential recognition of specific blood group antigens, also validated by saturation transfer difference nuclear magnetic resonance (STD-NMR) experiments. Particularly, we identified oligosaccharide blood group A type 6 (BGA6) as a biologically relevant Gal-4 ligand, which specifically inhibited IL-6 secretion induced by this lectin on human peripheral blood mononuclear cells. These findings highlight unique determinants underlying specific recognition of HMOs and blood group antigens by human galectins, emphasizing the biological relevance of Gal-4-BGA6 interactions, with critical implications in the development and regulation of inflammatory responses.

Chorioamnionitis: clinical, anamnestic and molecular-genetic parallels

Aim: to determine clinical, anamnestic and molecular-genetic parallels in emergence of clinical chorioamnionitis (CA) and severe forms of intrauterine infections (IUI) in high-risk pregnant women.Materials and Methods. A single-center prospective cohort comparative case-control study was conducted by examining 58 pregnant female patients aged 18 to 42 years with a verified CA diagnosis during pregnancy and childbirth at different gestation stages (main group), and 35 age-matched pregnant women with uncomplicated pregnancy and no significant extragenital pathology, aggravated factors of obstetric and gynecological history and risk factors for developing CA (control group), observed and performed a delivery in Yudin City Clinical Hospital. All women underwent clinical, anamnestic, laboratory, instrumental and molecular-genetic examitation. We studied the polymorphism of genes FCGR2A (Fc fragment of immunoglobulin G receptor IIa), IFN-γ (interferon gamma), IL-10 (interleukin-10), IL-6 (interleukin-6) and MBL2 (mannose binding lectin 2) to determine their role in assessing a risk of maternal and neonatal infection.Results. Among the patients with developed clinical CA vs. control subjects, more of them had a history of abortion and miscarriages (17.24 %), comorbid with chronic arterial hypertension (13.79 %), previous surgical interventions (27.59 %), as well as chronic inflammatory diseases (chronic tonsillitis, bronchitis, pyelonephritis, sinusitis; 27.59 % vs. 17.14 %). In addition to risk factors directly related to the infectious and inflammatory unfavorable background, they also had a significantly higher rate of obstetric complications: moderate preeclampsia – 6 (10.34 %) cases, threat of miscarriage or premature birth – 14 (24.14 %) cases vs. 1 (2.86 %) case in control group (p = 0.007), polyhydramnions – 4 (6.9 %) cases, placental insufficiency – 6 (10.34 %) cases. The frequency of premature rupture of membranes was 31.03 % in women with CA. Questionable cardiotocography (CTG) type was found in 24 (41.38 %) women with CA vs. 4 (11.4 3%) women without CA (p = 0.003), the pathological CTG type was observed only in women with CA. In the group with clinical CA and neonatal IUI, the combination of genotypes AG rs1801274 FCGR2A, ТT rs2430561 (IFN-γ)+874, GC rs1800795 (IL-6)-174 occurs in 80.65 % (25/31), whereas in women without severe neonatal IUI – in 37.04 % (10/27) (odds ratio (OR) =7.08; 95 % confidence interval (CI) = 2.166–23.166). In addition, the combination of alleles TT rs2430561 (IFN-γ)+874, GC+CC rs1800795 (IL-6)-174, AA rs1800450 MBL2 codon 54 was detected in 60.86 % (62/102) vs. 47.52 % (370/778) in main and control group (OR = 11.667; 95 % CI = 2.842–47.886), respectively.Conclusion. The study data evidence about importance of identifying genes for developing CA and neonatal septic complications to optimize and personalize management of high-risk patients (premature birth, infections during pregnancy, premature rupture of membranes).

Synthesis of mannose conjugated biodegradable polyester-based nanocarriers and their binding study with Concanavalin A

In this paper, we aim to create fully biodegradable glyconanoparticles formed via self-assembly of glycopolymers, comprising of biodegradable aliphatic polyester conjugated with mannose moiety. To accomplish the goal, a series of random copolymers comprised of poly(propargyl glycolide-co-lactide) were synthesized. Alkyne moiety of the poly (propargyl glycolide) component was then clicked with mannose ethyl azide to produce amphiphilic glycopolymers in good yield (60–75 %). The glycopolymers were then self-assembled to form glyconanoparticles of 15–23 nm size in dry state and 78–88 nm size in hydrated state (hydrodynamic diameter). The copolymers were characterized by NMR and FTIR, whereas the nanoparticles were thoroughly characterized by DLS, FESEM, and HR-TEM and explored for their lectin binding efficiency. Isothermal calorimetry (ITC) experiments suggest a stronger binding efficiency of glyconanoparticles towards mannose-specific lectin such as Concanavalin A, as compared to its corresponding glycopolymers (∼2 fold) and monomeric mannose unit (∼7-fold) as well. Moreover, curcumin was selected as the model drug to be encapsulated (∼76 % encapsulation efficiency) and released (74 % in 24 h) from the glyconanoparticles. Apart from these, excellent haemocompatibility, cell viability, and cellular uptake of the nanoparticles (more than 80 % cells showed uptake of the nanoparticles), further supported their potential as drug carriers having sugar as a targeting moiety.

Candida and Long Covid: Mannan Not from Heaven

The pandemic has supercharged growing awareness of the gut microbiome as a critical determinant of human health. “Long haulers” share microbiomes similar to those seen in myalgic encephalomyelitis/chronic fatigue syndrome and fibromyalgia, all frequently associated with Candida overgrowth (CO). Candida can synthesize its own IDO, altering tryptophan metabolism (ATM). Zonulin, a circulating protein that increases intestinal and endothelial permeability, has emerged as a central player. Candida hyphal walls express proteins analogous to gliadin/gluten, e.g., celiac disease (CeD), and mannans, e.g., Crohn’s disease (CrD), that may trigger antigliadin and anti-Gq coupled GPCR auto-antibodies linked to their lectin binding domain respectively. Hyphal mannan may induce auto-antibodies to AT1Rs, α1-ARs, mAChRs, and β2-ARs, prominent in LC, and regulate T cell receptors (TCRs) and regulatory B cell function, compromised in not only LC (vitiligo, psoriasis, alopecia) but also SLE, RA, and many other autoimmune diseases. All are Gq coupled GPCRs. The spike protein S on SARS CoV2 can attach to both the ACE2 receptor (required for tryptophan absorption) and Toll-like receptor4 (TLR4) bearing endothelial cells and enterocytes. Spike protein S is persistent in most with LC and, as a ligand for TLR4, can also activate zonulin. S can also activate the NLRP3 inflammasome, as can candidalysin. This inflammasome is directly connected to dementia, cancer, autoimmunity and obesity. Candidalysin causes hypercitrullination, instrumental in creating ACPAs (anti-citrullinated peptide antibodies) linked to LC, MCAS (mast cell activation syndrome), HSD (hypermobility spectrum disorder), and APS (antiphospholipid syndrome). A hypothetical pathophysiologic model is proposed implicating pre-existing CO, aggravated by Covid-19, in not only the genesis of LC but also that of autoimmune disease, dementia, cancer, many chronic diseases, and aging.

Evaluation of non-clinical safety of Wsmol, a chitin binding lectin of Moringa oleifera seeds in adult zebrafish (Danio rerio)

Previous studies have shown several biotechnological and lectin biomedical activities provided from Moringa oleifera seeds. The Wsmol (water soluble chitin binding lectin) has affinity for n-acetyl-D-glucosamine-glycans present in various cell receptors and it is able to realize many biological activities. This study was conducted to evaluate the nonclinical safety of Wsmol in adult Zebrafish (Danio rerio). The action of Wsmol (1.0 mg / mL; 20 μL; ip) and Vehicle (0.9 % NaCl, 20 μL, ip) on the locomotor activity of adult zebrafish (ZFa; n = 6 / group) was evaluated in the open field test. A naïve group was included. After 24 h of treatments, mortality rate was analyzed and the concentration able to kill 50 % (LC50) of the zebrafish was calculated. Wsmol, active in the physiological environment of Zebrafish did not modify the locomotor activity nor did it prove to be toxic to the animals, suggesting that its lectin activity, although can promote pharmacological activities when recognizing glycans of several ion channels and membrane protein receptors, can not be associated with toxicity or locomotor damage.

Novel genetically glycoengineered human dendritic cell model reveals regulatory roles of α2,6-linked sialic acids in DC activation of CD4+ T cells and response to TNFα.

Dendritic cells (DCs) are central for the initiation and regulation of appropriate immune responses. While several studies suggest important regulatory roles of sialoglycans in DC biology, our understanding is still inadequate primarily due to a lack of appropriate models. Previous approaches based on enzymatic- or metabolic-glycoengineering and primary cell isolation from genetically modified mice have limitations related to specificity, stability, and species differences. This study addresses these challenges by introducing a workflow to genetically glycoengineer the human DC precursor cell line MUTZ-3, described to differentiate and maturate into fully functional dendritic cells, using CRISPR-Cas9, thereby providing and validating the first isogenic cell model for investigating glycan alteration on human DC differentiation, maturation, and activity. By knocking out (KO) the ST6GAL1 gene, we generated isogenic cells devoid of ST6GAL1-mediated α(2,6)-linked sialylation, allowing for a comprehensive investigation into its impact on DC function. Glycan profiling using lectin binding assay and functional studies revealed that ST6GAL1 KO increased the expression of important antigen presenting and co-stimulatory surface receptors and a specifically increased activation of allogenic human CD4 + T cells. Additionally, ST6GAL1 KO induces significant changes in surface marker expression and cytokine response to TNFα-induced maturation, and it affects migration and the endocytic capacity. These results indicate that genetic glycoengineering of the isogenic MUTZ-3 cellular model offers a valuable tool to study how specific glycan structures influence human DC biology, contributing to our understanding of glycoimmunology.

Boltzmann Model Predicts Glycan Structures from Lectin Binding.

Glycans are complex oligosaccharides that are involved in many diseases and biological processes. Unfortunately, current methods for determining glycan composition and structure (glycan sequencing) are laborious and require a high level of expertise. Here, we assess the feasibility of sequencing glycans based on their lectin binding fingerprints. By training a Boltzmann model on lectin binding data, we predict the approximate structures of 88 ± 7% of N-glycans and 87 ± 13% of O-glycans in our test set. We show that our model generalizes well to the pharmaceutically relevant case of Chinese hamster ovary (CHO) cell glycans. We also analyze the motif specificity of a wide array of lectins and identify the most and least predictive lectins and glycan features. These results could help streamline glycoprotein research and be of use to anyone using lectins for glycobiology.

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.