Human Galectin-3 Research Articles

Scoring System and Diagnosis of Papillary Thyroid Carcinoma Using Human Bone Marrow Endothelium Marker-1, Cytokeratin 19, and Galectin-3.

The increasing incidence of papillary thyroid carcinoma (PTC) and the inadequacy of routine histologic examination in its diagnosis necessitate the application of ancillary studies like immunohistochemistry. This research aimed to investigate the scoring system and diagnosis of PTC with cytokeratin 19 (CK19), human bone marrow endothelium marker-1 (HBME-1), and galectin-3. This experimental laboratory study was performed at Babol University of Medical Sciences, Mazandaran, Iran from April 2017 to March 2019. Neoplastic and nonneoplastic tissue samples of 100 cases with a diagnosis of PTC were selected by convenience sampling. CK19, HBME-1, and galectin-3 immunohistochemistry markers were used on tissue samples. Analysis was performed using the t test and the chi-square test, as well as the receiver operator characteristic (ROC) curve (significance level P < 0.05). The CK19 staining was observed in all 100 (100%) non-neoplastic tissues, but HBME-1 and galectin-3 were positive in 36 (36%) and 14 (14%) of non-neoplastic tissues, respectively. The intensity scores of all the markers and their total had significantly different means in PTC and non-neoplastic tissues (P < 0.001). A significant difference was observed between the total score of each marker and the total score of their combination (P < 0.001). The combination of all 3 markers with an 11.5 0 cut-off for the total score showed the most sensitive (0.99) and specific (1.00) results. Interpreting CK19, HBME-1, and galectin-3 with the aid of the proposed scoring system was fruitful. HBME-1 and galectin-3 can be used individually or in combination for the diagnosis of PTC.

Conserved Protein-Polymer Interactions across Structurally Diverse Polymers Underlie Alterations to Protein Thermal Unfolding.

Protein-polymer conjugates are widely used in many clinical and industrial applications, but lack of experimental data relating protein-polymer interactions to improved protein stability prevents their rational design. Advances in synthetic chemistry have expanded the palette of polymer designs, including development of nonlinear architectures, novel monomer chemical scaffolds, and control of hydrophobicity, but more experimental data are needed to transform advances in chemistry into next generation conjugates. Using an integrative biophysical approach, we investigated the molecular basis for polymer-based thermal stabilization of a human galectin protein, Gal3C, conjugated with polymers of linear and nonlinear architectures, different degrees of polymerization, and varying hydrophobicities. Independently varying the degree of polymerization and polymer architecture enabled delineation of specific polymer properties contributing to improved protein stability. Insights from NMR spectroscopy of the polymer-conjugated Gal3C backbone revealed patterns of protein-polymer interactions shared between linear and nonlinear polymer architectures for thermally stabilized conjugates. Despite large differences in polymer chemical scaffolds, protein-polymer interactions resulting in thermal stabilization appear conserved. We observed a clear relation between polymer length and protein-polymer thermal stability shared among chemically different polymers. Our data indicate a wide range of polymers may be useful for engineering conjugate properties and provide conjugate design criteria.

Cigarette smoke induces epithelial-to-mesenchymal transition, stemness, and metastasis in lung adenocarcinoma cells via upregulated RUNX-2/galectin-3 pathway

AimsAn elevated level of galectin-3, a carbohydrate-binding lectin implicated in tumorigenesis, metastasis, and epithelial-mesenchymal transition (EMT), has been found in cigarette smokers. However, the regulation of its expression and role in the pathogenesis of CS-induced EMT and lung cancer metastasis is unclear. Here, we have investigated the mechanism of CS-induced and galectin-3-mediated EMT in airway epithelial cells (AECs). Main methodsA549 adenocarcinoma cells and primary small airway epithelial cells cultured on an air-liquid interface (ALI) were exposed to cigarette smoke extract (CSE), and MTT, trypan blue, migration, invasion, tumor spheroid and colony formation assays were performed to assess EMT phenotype. Immunoblotting was performed to assess EMT and stemness markers and other regulatory proteins. Key findingsCSE exposure affected cell survival and morphology, migration, invasion, and clonogenicity of AECs, which were concomitant with an increase in the expression of EMT markers, galectin-3, and runt-related transcription factor-2 (RUNX-2), an osteogenic transcription factor and upstream regulator of galectin-3. Chemical inhibition or silencing of RUNX-2 downregulated galectin-3 and modulated EMT marker expression, migration, invasion, and clonogenicity in CSE-exposed AECs. Recombinant human galectin-3 also induced EMT and stemness-associated changes in the AECs, and GB1107, a galectin-3 inhibitor, ameliorated these changes. Further, CSE-induced intracellular ROS enabled an increase in RUNX-2 and galectin-3 expression, which were reversed by n-acetyl-cysteine. SignificanceThese results provide a novel mechanistic insight into CSE-induced EMT via RUNX-2/galectin-3 axis mediated through ROS, which promoted EMT-associated changes, including invasion, migration, and stemness in AECs, which could be implicated in CS-induced lung cancer progression.

Cytosolic galectin-4 enchains bacteria, restricts their motility, and promotes inflammasome activation in intestinal epithelial cells

Galectin-4, a member of the galectin family of animal glycan-binding proteins (GBPs), is specifically expressed in gastrointestinal epithelial cells and is known to be able to bind microbes. However, its function in host-gut microbe interactions remains unknown. Here, we show that intracellular galectin-4 in intestinal epithelial cells (IECs) coats cytosolic Salmonella enterica serovar Worthington and induces the formation of bacterial chains and aggregates. Galectin-4 enchains bacteria during their growth by binding to the O-antigen of lipopolysaccharides. Furthermore, the binding of galectin-4 to bacterial surfaces restricts intracellular bacterial motility. Galectin-4 enhances caspase-1 activation and mature IL-18 production in infected IECs especially when autophagy is inhibited. Finally, orally administered S. enterica serovar Worthington, which is recognized by human galectin-4 but not mouse galectin-4, translocated from the intestines to mesenteric lymph nodes less effectively in human galectin-4-transgenic mice than in littermate controls. Our results suggest that galectin-4 plays an important role in host-gut microbe interactions and prevents the dissemination of pathogens. The results of the study revealed a novel mechanism of host-microbe interactions that involves the direct binding of cytosolic lectins to glycans on intracellular microbes.

Catanionic Vesicles as a Facile Scaffold to Display Natural N-Glycan Ligands for Probing Multivalent Carbohydrate-Lectin Interactions.

Multivalent interactions are a key characteristic of protein-carbohydrate recognition. Phospholipid-based liposomes have been explored as a popular platform for multivalent presentation of glycans, but this platform has been plagued by the instability of typical liposomal formulations in biological media. We report here the exploitation of catanionic vesicles as a stable lipid-based nanoparticle scaffold for displaying large natural N-glycans as multivalent ligands. Hydrophobic insertion of lipidated N-glycans into the catanionic vesicle bilayer was optimized to allow for high-density display of structurally diverse N-glycans on the outer membrane leaflet. In an enzyme-linked competitive lectin-binding assay, the N-glycan-coated vesicles demonstrated a clear clustering glycoside effect, with significantly enhanced affinity for the corresponding lectins including Sambucus nigra agglutinin (SNA), concanavalin A (ConA), and human galectin-3, in comparison with their respective natural N-glycan ligands. Our results showed that relatively low density of high-mannose and sialylated complex type N-glycans gave the maximal clustering effect for binding to ConA and SNA, respectively, while relatively high-density display of the asialylated complex type N-glycan provided maximal clustering effects for binding to human galectin 3. Moreover, we also observed a macromolecular crowding effect on the binding of ConA to high-mannose N-glycans when catanionic vesicles bearing mixed high-mannose and complex-type N-glycans were used. The N-glycan-coated catanionic vesicles are stable and easy to formulate with varied density of ligands, which could serve as a feasible vehicle for drug delivery and as potent inhibitors for intervening protein-carbohydrate interactions implicated in disease.

Human galectin-9 promotes the expansion of HIV reservoirs in vivo in humanized mice.

The human endogenous protein galectin-9 (Gal-9) reactivates latently HIV-infected cells in vitro and ex vivo , which may allow for immune-mediated clearance of these cells. However, Gal-9 also activates several immune cells, which could negatively affect HIV persistence by promoting chronic activation/exhaustion. This potential 'double-edged sword' effect of Gal-9 raises the question of the overall impact of Gal-9 on HIV persistence in vivo . We used the BLT (bone marrow, liver, thymus) humanized mouse model to evaluate the impact of Gal-9 on HIV persistence in vivo during antiretroviral therapy (ART). Two independent cohorts of ART-suppressed HIV-infected BLT mice were treated with either recombinant Gal-9 or phosphate-buffered saline control. Plasma viral loads and levels of tissue-associated HIV DNA and RNA were measured by qPCR. Immunohistochemistry and HIV RNAscope were used to quantify CD4 + T, myeloid, and HIV RNA+ cells in tissues. T cell activation and exhaustion were measured by flow cytometry, and plasma markers of inflammation were measured by multiplex cytokine arrays. Gal-9 did not induce plasma markers of inflammation or T cell markers of activation/exhaustion in vivo . However, the treatment significantly increased levels of tissue-associated HIV DNA and RNA compared to controls ( P = 0.0007 and P = 0.011, respectively, for cohort I and P = 0.002 and P = 0.005, respectively, for cohort II). RNAscope validated the Gal-9 mediated induction of HIV RNA in tissue-associated myeloid cells, but not T cells. Our study highlights the overall adverse effects of Gal-9 on HIV persistence and the potential need to block Gal-9 interactions during ART-suppressed HIV infection.

Blood Group A Enhances SARS-CoV-2 Infection

BACKGROUND: Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) continues to infect millions of individuals worldwide. However, not all individuals are equally susceptible to infection. Many studies have demonstrated that the first polymorphism described in the human population, ABO(H) blood group antigens, are associated with increased risk of SARS-CoV-2 infection. While conflicting data exist, the most common findings suggest that individuals with blood group A exhibit an increased risk of infection when compared to blood group O individuals. However, the mechanism whereby ABO(H) blood group status influences SARS-CoV-2 infection has remained unknown. The receptor binding domain (RBD) of SARS-CoV-2, which facilitates host cell engagement, bears significant similarity to galectins, an ancient family of carbohydrate binding proteins previously shown to recognize ABO(H) blood group antigens. As a result, we hypothesized that SARS-CoV-2 may recognize blood group A directly through its RBD and that this may in part account for increased susceptibility of blood group A individuals for SARS-CoV-2 infection. Methods: The SARS-CoV-2 RBD from the parent strain (Wuhan-Hu-1) and variants (Delta and Omicron), in addition to individual human galectins (1, 2, 3, 4, 7 and 9) were cloned, purified and subjected to glycan binding specificity analysis using a glycan microarray populated with hundreds of distinct glycans, including blood group antigens. This was accomplished by incubating each fluorescently labeled RBD or galectin on the array, followed by detection of bound fluorescence using a microarray scanner (GenePix 4000 B, Molecular devices) and integrated spot intensity analysis using Imagene software (GenePix Pro 7). SARS-CoV-2 with distinct spike proteins (Wuhan-Hu-1, Delta, and Omicron) were used to assess infection using Chinese hamster ovary (CHO) cells engineered to express angiotensin converting enzyme 2 (ACE2) and type 1 blood group A or H (blood group O antigen). A one-way ANOVA with a Tukey's post hoc with a p value <0.05 was considered significant. Results: Sequence analysis of galectins demonstrates that several galectins possess up to 11% sequence identity with the RBDs of SARS-CoV-2 WT (Wuhan-Hu-1) and variants (Delta and Omicron). Each RBD (Wuhan, Delta and Omicron) exhibited glycan binding specificity that overlapped with distinct members of the galectin family, with the highest similarity observed toward the C terminal domain of galectin-4 (Gal-4C). Direct comparison of binding toward ABO(H) antigens demonstrated that each RBD and Gal-4C displayed high specificity for the type 1 blood group A structure uniquely found on respiratory epithelial cells when compared to other blood group antigens (p<0.0001). To specifically examine the impact of blood group A on SARS-CoV-2 infection, CHO cells were engineered to express ACE2 and the type 1 blood group A or H antigen (the antigen expressed in blood group O individuals) normally found on respiratory epithelial cells. Consistent with the increased binding of the RBD to blood group A on the glycan microarray, enhanced binding was observed by each RBD toward blood group A expressing cells (p<0.001). Blood group A cells were also significantly more likely to be infected with Wuhan-Hu-1, Delta and Omicron variants of SARS-CoV-2 when compared to blood group O cells, while pre-incubation of cells with Gal-4C specifically inhibited infection of blood group A expressing cells, while failing to impact infection of blood group O cells (p<0.001). Conclusions: These results demonstrate a direct effect of blood group A on viral infection and suggest that blood group A individuals may exhibit an increased susceptibility to SARS-CoV-2 as a result of direct engagement of the blood group A antigen. However, variations in ACE2 levels, blood group A expression and many other factors likely influence the overall risk of SARS-CoV-2 infection following exposure in a given individual. The present results provide one mechanism whereby blood group A itself may directly influence SARS-CoV-2 infectious risk.

Exploring the In situ pairing of human galectins toward synthetic O-mannosylated core M1 glycopeptides of α-dystroglycan

Dystroglycan (DG), which constitutes a part of the dystrophin–glycoprotein complex, connects the extracellular matrix to the cytoskeleton. The matriglycans presented by the extracellular α-DG serve as a contact point with extracellular matrix proteins (ECM) containing laminin G-like domains, providing cellular stability. However, it remains unknown whether core M1 (GlcNAcβ1-2Man) structures can serve as ligands among the various O-Mannosylated glycans. Therefore, based on the presence of N-acetylLactosamine (LacNAc) in this glycan following the core extension, the binding interactions with adhesion/growth-regulatory galectins were explored. To elucidate this process, the interaction between galectin (Gal)-1, -3, -4 and -9 with α-DG fragment 372TRGAIIQTPTLGPIQPTRV390 core M1-based glycopeptide library were profiled, using glycan microarray and nuclear magnetic resonance studies. The binding of galectins was revealed irrespective of its modular architecture, adding galectins to the list of possible binding partners of α-DG core M1 glycoconjugates by cis-binding (via peptide- and carbohydrate-protein interactions), which can be abrogated by α2,3-sialylation of the LacNAc units. The LacNAc-terminated α-DG glycopeptide interact simultaneously with both the S- and F-faces of Gal-1, thereby inducing oligomerization. Furthermore, Gal-1 can trans-bridge α-DG core M1 structures and laminins, which proposed a possible mechanism by which Gal-1 ameliorates muscular dystrophies; however, this proposal warrants further investigation.

Synthesis, binding affinity, and inhibitory capacity of cyclodextrin-based multivalent glycan ligands for human galectin-3

Human galectin 3 (Gal-3) has been implicated to play important roles in different biological recognition processes such as tumor growth and cancer metastasis. High-affinity Gal-3 ligands are desirable for functional studies and as inhibitors for potential therapeutic development. We report here a facile synthesis of β-cyclodextrin (CD)-based Tn and TF antigen-containing multivalent ligands via a click reaction. Binding studies indicated that the synthetic multivalent glycan ligands demonstrated a clear clustering effect in binding to human Gal-3, with up to 153-fold enhanced relative affinity in comparison with the monomeric glycan ligand. The GalNAc (Tn antigen) containing heptavalent ligand showed the highest affinity for human Gal-3 among the synthetic ligands tested, with an EC50 of 1.4 μM in binding to human Gal-3. A cell-based assay revealed that the synthetic CD-based multivalent ligands could efficiently inhibit Gal-3 binding to human airway epithelial cells, with an inhibitory capacity consistent with their binding affinity measured by SPR. The synthetic cyclodextrin-based ligands described in this study should be valuable for functional studies of human Gal-3 and potentially for therapeutic applications.

Atomic-Scale View of Protein-PEG Interactions that Redirect the Thermal Unfolding Pathway of PEGylated Human Galectin-3.

PEGylation is a promising approach to address the central challenge of applying biologics, i.e., lack of protein stability in the demanding environment of the human body. Wider application is hindered by lack of atomic level understanding of protein-PEG interactions, preventing design of conjugates with predicted properties. We deployed an integrative structural and biophysical approach to address this critical challenge with the PEGylated carbohydrate recognition domain of human galectin-3 (Gal3C), a lectin essential for cell adhesion and potential biologic. PEGylation dramatically increased Gal3C thermal stability, forming a stable intermediate and redirecting its unfolding pathway. Structural details revealed by NMR pointed to a potential role of PEG localization facilitated by charged residues. Replacing these residues subtly altered the protein-PEG interface and thermal unfolding behavior, providing insight into rationally designing conjugates while preserving PEGylation benefits.

Multicomponent reaction derived small di- and tri-carbohydrate-based glycomimetics as tools for probing lectin specificity.

Lectins, carbohydrate-binding proteins, play important functions in all forms of life from bacteria and viruses to plants, animals, and humans, participating in cell-cell communication and pathogen binding. In an attempt to modify lectin functions, artificial lectin ligands were made usually as big dendrimeric or cluster multivalent glycomimetic structures. Here we synthesized a novel set of glycomimetic ligands through protection/deprotection multicomponent reactions (MCR) approach. Multivalent di-and tri-carbohydrate glycomimetics containing D-fructose, D-galactose, and D-allose moieties were prepared in 63-96% yield. MCR glycomimetics demonstrated different binding abilities for plant lectins Con A and UEA I, and human galectin-3. Information gained about the influence of molecule structure, multivalency and optical purity on the lectin binding ability can be used in lectin detection and sensitivity measurements to further facilitate understanding of carbohydrate recognition process.

Identification of Monosaccharide Derivatives as Potent, Selective, and Orally Bioavailable Inhibitors of Human and Mouse Galectin-3.

Galectin-3 (Gal-3), a member of the β-galactoside-binding protein family, is implicated in a wide variety of human diseases. Identification of Gal-3 inhibitors with the right combination of potency (against both human and mouse Gal-3) and pharmacokinetic properties to fully evaluate the potential of Gal-3 for therapeutic intervention has been a major challenge due to the characteristics of its binding pocket: high hydrophilicity and key structural differences between human Gal-3 and the mouse ortholog. We report the discovery of a novel series of monosaccharide-based, highly potent, and orally bioavailable inhibitors of human and mouse Gal-3. The novel monosaccharide derivatives proved to be selective for Gal-3, the only member of the chimeric type of galectins, over Gal-1 and Gal-9, representative of the prototype and tandem-repeat type of galectins, respectively. The proposed binding mode for the newly identified ligands was confirmed by an X-ray cocrystal structure of a representative analogue bound to Gal-3 protein.

Exploring the Molecular Interactions of Symmetrical and Unsymmetrical Selenoglycosides with Human Galectin-1 and Galectin-3.

Galectins (Gals) are small cytosolic proteins that bind β-galactoside residues via their evolutionarily conserved carbohydrate recognition domain. Their dysregulation has been shown to be associated with many diseases. Consequently, targeting galectins for clinical applications has become increasingly relevant to develop tailored inhibitors selectively for one galectin. Accordingly, binding studies providing the molecular details of the interaction between galectin and inhibitor may be useful for the rational design of potent and selective antagonists. Gal-1 and Gal-3 are among the best-studied galectins, mainly for their roles in cancer progression; therefore, the molecular details of their interaction with inhibitors are demanded. This work gains more value by focusing on the interaction between Gal-1 and Gal-3 with the selenylated analogue of the Gal inhibitor thiodigalactose, characterized by a selenoglycoside bond (SeDG), and with unsymmetrical diglycosyl selenides (unsym(Se). Gal-1 and Gal-3 were produced heterologously and biophysically characterized. Interaction studies were performed by ITC, NMR spectroscopy, and MD simulation, and thermodynamic values were discussed and integrated with spectroscopic and computational results. The 3D complexes involving SeDG when interacting with Gal-1 and Gal-3 were depicted. Overall, the collected results will help identify hot spots for the design of new, better performing, and more specific Gal inhibitors.

CORRELATION OF SERUM GALECTIN-3 LEVELS WITH EJECTION FRACTIONS IN HEALTHY SUBJECTS AND HEART FAILURE PATIENTS

Background: High Galectin-3 levels are indicative of severity of heart diseases and are associated with increased risk of major adverse cardiovascular events including heart failure. The objective of this study was to determine correlation between ejection fraction and serum Galectin-3 levels recorded by simple and quick test. Methods: This cross-sectional analytical study was conducted at the Armed Forces Institute of Cardiology (AFIC) Rawalpindi. Thirty (30) healthy adults and 60 heart failure patients were selected through randomised sampling. Diagnosed cases were classified using modified NYHA classification. Group 1 included healthy adults, Group 2 included 30 mild heart failure subjects of NYHA class I and II, Group 3 included 30 severe heart failure subjects of NYHA class III and IV. Ejection fraction was recorded at AFIC with trans-thoracic echocardiography in supine and left lateral positions. Blood samples of the participants were used for estimation of Galectin-3 by ELISA using human serum Galectin-3 ELISA kit Catalogue No. E1951Hu (Bio Assay technology). Observations were recorded on proforma, Pearson’s correlation test and ANOVA were performed using SPSS-24. Results: The LVEF was 70.7±2.78% in Group-1, 59.3±3.4% in Group-2, and 38.96±12.27% in Group-3. The LVEF showed a substantial negative correlation with serum Galectin-3 levels (r= -0.630, p&lt;0.001). Conclusion: There is a significant negative correlation between Serum Galectin-3 and extent of functional changes of left ventricle recorded by Ejection fraction on echocardiography. Pak J Physiol 2022;18(2):28–30

CLINICAL VALUE OF GALECTIN-3 IN DIFFERENTIATING PAPILLARY THYROID CANCER FROM NODULAR GOITER

"Introduction: Thyroid cancer in one of the most common cancers worldwide, with an increasing incidence over the last decades. Serum Galectin-3 (Gal-3) is used as biomarker in different types of cancer (gastric, hepatic), but its utility in papillary thyroid cancer differential diagnostic in still inconclusive. Objectives: The present study aims to evaluate the diagnostic value of preoperative serum Galectin-3 levels in differentiating between papillary thyroid cancer and nodular goiter. Methods: 277 of serum samples from patients with thyroid lesions were divided into two subgroups: nodular goiter (NG; n = 121) and papillary thyroid carcinoma (PTC; n = 136). Serum Gal-3 levels were measured before surgery for the entire group, and for 62 patients (33 NG, 29 PTC) it was measured a month post-surgery. Gal-3 was measured by ELISA, using Human Galectin-3 Quantikinine ELISA kit (R&amp;D Systems) and statistical analysis were performed using MedCalc Software. All paticipants signed the written informed consent and the study was approved by the Ethics Commitee of the C.I. Parhon National Institute of Endocrinology, Bucharest. Results: Circulating Gal-3 values show that this biomarker has a higher value in PTC patients compared with NG patients (7.17 ng/ml vs. 8.285 ng/ml, p = 0.0038), before thyroidectomy. There were not observed significant statistical correlations between serum Gal-3 values and histological subtype, focality, invasivity or dominant nodule dimension. Conclusion: Our results regarding Gal-3 values show that this biomarker has a higher value in PTC patients compared with NG patients, but its clinical value in preoperative diagnosis and evaluation of thyroid nodules needs further investigations. Keywords: thyroid cancer, papillary thyroid carcinoma, galectin-3"

Galectin-1 and -3 in high amounts inhibit angiogenic properties of human retinal microvascular endothelial cells in vitro.

PurposeGalectin-1 and -3 are β-galactoside binding lectins with varying effects on angiogenesis and apoptosis. Since in retinal pigment epithelial cells high amounts of human recombinant galectin (hr-GAL)1 and 3 inhibit cell adhesion, migration and proliferation, we investigated if hr-GAL1 and 3 have homologous effects on human retinal microvascular endothelial cells (HRMEC) in vitro.MethodsTo investigate the effect of galectin-1 and -3 on HRMEC, proliferation, apoptosis and viability were analyzed after incubation with 30, 60 and 120 μg/ml hr-GAL1 or 3 by BrdU-ELISA, histone-DNA complex ELISA, live/dead staining and the WST-1 assay, respectively. Further on, a cell adhesion as well as tube formation assay were performed on galectin-treated HRMEC. Migration was investigated by the scratch migration assay and time-lapse microscopy. In addition, immunohistochemical staining on HRMEC for β-catenin, galectin-1 and -3 were performed and β-catenin expression was investigated by western blot analysis.ResultsIncubation with hr-GAL1 or 3 lead to a decrease in proliferation, migration, adhesion and tube formation of HRMEC compared to the untreated controls. No toxic effects of hr-GAL1 and 3 on HRMEC were detected. Intriguingly, after treatment of HRMEC with hr-GAL1 or 3, an activation of the proangiogenic Wnt/β-catenin signaling pathway was observed. However, incubation of HRMEC with hr-GAL1 or 3 drew intracellular galectin-1 and -3 out of the cells, respectively.ConclusionExogenously added hr-GAL1 or 3 inhibit angiogenic properties of HRMEC in vitro, an effect that might be mediated via a loss of intracellular endogenous galectins.

The S1 Subunit of the SARS-CoV-2 Spike Protein Activates Human Monocytes to Produce Cytokines Linked to COVID-19: Relevance to Galectin-3.

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), rapidly evolved into a pandemic –the likes of which has not been experienced in 100 years. While novel vaccines show great efficacy, and therapeutics continue to be developed, the persistence of disease, with the concomitant threat of emergent variants, continues to impose massive health and socioeconomic issues worldwide. Studies show that in susceptible individuals, SARS-CoV-2 infection can rapidly progress toward lung injury and acute respiratory distress syndrome (ARDS), with evidence for an underlying dysregulated innate immune response or cytokine release syndrome (CRS). The mechanisms responsible for this CRS remain poorly understood, yet hyper-inflammatory features were also evident with predecessor viruses within the β-coronaviridae family, namely SARS-CoV-1 and the Middle East Respiratory Syndrome (MERS)-CoV. It is further known that the spike protein (S) of SARS-CoV-2 (as first reported for other β-coronaviruses) possesses a so-called galectin-fold within the N-terminal domain of the S1 subunit (S1-NTD). This fold (or pocket) shows structural homology nearly identical to that of human galectin-3 (Gal-3). In this respect, we have recently shown that Gal-3, when associated with epithelial cells or anchored to a solid phase matrix, facilitates the activation of innate immune cells, including basophils, DC, and monocytes. A synthesis of these findings prompted us to test whether segments of the SARS-CoV-2 spike protein might also activate innate immune cells in a manner similar to that observed in our Gal-3 studies. Indeed, by immobilizing S components onto microtiter wells, we show that only the S1 subunit (with the NTD) activates human monocytes to produce a near identical pattern of cytokines as those reported in COVID-19-related CRS. In contrast, both the S1-CTD/RBD, which binds ACE2, and the S2 subunit (stalk), failed to mediate the same effect. Overall, these findings provide evidence that the SARS-CoV-2 spike protein can activate monocytes for cytokines central to COVID-19, thus providing insight into the innate immune mechanisms underlying the CRS and the potential for therapeutic interventions.

An Evaluation of the Mechanisms of Galacto-Oligosaccharide (GOS)-Induced IgE Cross-Linking on Basophils in GOS Allergy.

The prebiotics, galacto-oligosaccharides (GOS), are small carbohydrate molecules with 1–7 galactose units linked to glucose and have been shown to trigger IgE-mediated anaphylaxis in some cases following ingestion. It is still an unresolved question of how GOS cross-links IgE on basophils. In this study, we examined whether human galectins, a class of lectins that bind specifically to β-galactoside carbohydrates, are involved in GOS-induced basophil activation. Basophil activation test to GOS and control allergen, Blomia tropicalis (Blo t) extract were performed in the presence or absence of four sugar-based galectin inhibitors (lactose, thiodigalactoside [TDG], TD139, and GB1107) and one peptide-based inhibitor, G3-C12. Results showed that TD139, GB1107, and G3-C12 did not display a specific inhibitory effect on GOS-induced basophil activation as compared to control allergen. An inhibitory effect of lactose and TDG on GOS-induced basophil activation was observed and varied between subjects with up to 100% inhibition at low doses of GOS. The results of competitive ELISA suggest that the inhibitory effects of high dose lactose and TDG on the basophil activation is likely due to the cross-reactivity of GOS-specific IgE to lactose and TDG. Basophil activation is performed using purified basophils suggested that cell surface receptors on other blood cells were not required to induce basophil activation. In conclusion, our results suggest that GOS, a low molecular weight sugar, is able to cross-link IgE independently.

Investigation of the Molecular Details of the Interactions of Selenoglycosides and Human Galectin-3.

Human galectin-3 (hGal-3) is involved in a variety of biological processes and is implicated in wide range of diseases. As a result, targeting hGal-3 for clinical applications has become an intense area of research. As a step towards the development of novel hGal-3 inhibitors, we describe a study of the binding of two Se-containing hGal-3 inhibitors, specifically that of di(β-D-galactopyranosyl)selenide (SeDG), in which two galactose rings are linked by one Se atom and a di(β-D-galactopyranosyl)diselenide (DSeDG) analogue with a diseleno bond between the two sugar units. The binding affinities of these derivatives to hGal-3 were determined by 15N-1H HSQC NMR spectroscopy and fluorescence anisotropy titrations in solution, indicating a slight decrease in the strength of interaction for SeDG compared to thiodigalactoside (TDG), a well-known inhibitor of hGal-3, while DSeDG displayed a much weaker interaction strength. NMR and FA measurements showed that both seleno derivatives bind to the canonical S face site of hGal-3 and stack against the conserved W181 residue also confirmed by X-ray crystallography, revealing canonical properties of the interaction. The interaction with DSeDG revealed two distinct binding modes in the crystal structure which are in fast exchange on the NMR time scale in solution, explaining a weaker interaction with hGal-3 than SeDG. Using molecular dynamics simulations, we have found that energetic contributions to the binding enthalpies mainly differ in the electrostatic interactions and in polar solvation terms and are responsible for weaker binding of DSeDG compared to SeDG. Selenium-containing carbohydrate inhibitors of hGal-3 showing canonical binding modes offer the potential of becoming novel hydrolytically stable scaffolds for a new class of hGal-3 inhibitors.

77Se-Enriched Selenoglycoside Enables Significant Enhancement in NMR Spectroscopic Monitoring of Glycan-Protein Interactions.

Detailed investigation of ligand–protein interactions is essential for better understanding of biological processes at the molecular level. Among these binding interactions, the recognition of glycans by lectins is of particular importance in several diseases, such as cancer; therefore, inhibition of glycan-lectin/galectin interactions represents a promising perspective towards developing therapeutics controlling cancer development. The recent introduction of 77Se NMR spectroscopy for monitoring the binding of a selenoglycoside to galectins prompted interest to optimize the sensitivity by increasing the 77Se content from the natural 7.63% abundance to 99%. Here, we report a convenient synthesis of 77Se-enriched selenodigalactoside (SeDG), which is a potent ligand of the medically relevant human galectin-3 protein, and proof of the expected sensitivity gain in 2D 1H, 77Se correlation NMR experiments. Our work opens perspectives for adding isotopically enriched selenoglycans for rapid monitoring of lectin-binding of selenated as well as non-selenated ligands and for ligand screening in competition experiments.