Gangliosides, sialylated glycosphingolipids abundant in the nervous system, play crucial roles in neurotransmission, interaction with regulatory proteins, cell-cell recognition, and signaling. Altered gangliosides expression has been correlated with pathological processes, including cancer, inflammatory disorders, and autoimmune diseases. Gangliosides are important endogenous ligands of Siglecs (Sialic acid-binding immunoglobulin-type lectins), I-type lectins mostly expressed by immune cells, that specifically recognize sialylated glycans. Siglec-7, an inhibitory immune receptor on human natural killer cells, represents a potential target for tumor immunotherapy. Notably, the expression of Siglec-7 ligands is high in various cancers, such as pancreatic cancer and melanoma and lead to tumor immune evasion. Siglec-7 binds the disialylated ganglioside GD3, a tumor-associated antigen overexpressed on cancer cells to suppress immune responses. Using a combination of structural biology techniques, including Nuclear Magnetic Resonance (NMR), biophysical, and computational methods, the binding of Siglec-7 to GD3 and Gb3 derivatives is investigated, revealing the importance of ligand conformation in modulating binding energetics and affinity. The greater flexibility of Gb3 derivatives appears to negatively impact binding entropy, leading to lower affinity compared to GD3. A thorough understanding of these interactions could contribute to elucidating molecular mechanisms of cancer immune evasion and facilitate the development of ganglioside-based diagnostic and therapeutic strategies for cancer.
- Klíčová slova
- NMR, gangliosides, siglec‐7, structural biology,
- MeSH
- antigeny diferenciační myelomonocytární * metabolismus chemie MeSH
- gangliosidy * metabolismus chemie MeSH
- lektiny * metabolismus chemie MeSH
- lidé MeSH
- ligandy MeSH
- magnetická rezonanční spektroskopie metody MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- antigeny diferenciační myelomonocytární * MeSH
- gangliosidy * MeSH
- lektiny * MeSH
- ligandy MeSH
- SIGLEC7 protein, human MeSH Prohlížeč
Siglec-7, an immune checkpoint receptor, has emerged as a promising target for cancer immunotherapy due to its involvement in the regulation of immune and inflammatory responses. However, while its participation in immunoediting and immune evasion is well established, understanding its biological context, relevant ligands, and associated signalling pathways remains limited. Understanding these aspects is crucial for the development of effective immunotherapies targeting Siglec-7. In this study, three expression constructs of Siglec-7 were designed, expressed, and characterised, including an analysis of the oligomeric state of its extracellular domain. The N-terminal V-set Ig carbohydrate recognition domain was also produced in an isotopically double-labelled (13C,15N) mammalian cell growth medium. Two stable constructs suitable for biophysical and structural studies were identified. These findings reveal the noncovalent dimerisation of Siglec-7, offering new insights into its possible ligand interactions, signal transduction mechanisms, or receptor/ligand clustering. The dimerisation of Siglec-7 may be essential to achieve multivalent, high-avidity interactions with glycoconjugates, which may result in enhanced or alternative signalling processes within the NK cell immune synapse. In addition, a detailed protocol for generating double-labelled Siglec-7 in HEK293 cells, which may apply to other proteins under similar conditions, was described. These findings contribute to a better understanding of the biophysical and structural properties of Siglec-7 and are key to the design of more precise and effective cancer immunotherapies targeting Siglec-7.
- Klíčová slova
- Dimerisation, HEK293 cells, Isotope labelling, NK cells, NMR spectroscopy, Siglec-7,
- MeSH
- antigeny diferenciační myelomonocytární * chemie metabolismus genetika MeSH
- HEK293 buňky MeSH
- izotopové značení MeSH
- lektiny * chemie metabolismus genetika MeSH
- lidé MeSH
- ligandy MeSH
- multimerizace proteinu * MeSH
- stabilita proteinů MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- antigeny diferenciační myelomonocytární * MeSH
- lektiny * MeSH
- ligandy MeSH
- SIGLEC7 protein, human MeSH Prohlížeč
Galectin-1 (Gal-1) displays unique sensitivity to oxidative inactivation which appears critical in regulating its spatial and temporal activity. The two physicochemical states, i.e. monomer-dimer equilibrium and redox states, are related to Gal-1 varying functionality. In this work, we used chronopotentiometric stripping analysis, intrinsic fluorescence spectroscopy, and mobility shift assay to follow changes in the structure and lectin activity of reduced and oxidized Gal-1 forms. Our results show that monomers and dimers are similarly distributed under mild reduction and oxidation conditions. Gal-1 after its oxidation consists of at least three different monomeric forms while reduced Gal-1 only one. Lectin activity, affinity to N-acetyllactosamine, is relatively similar for low Gal-1 concentrations for both, reduced and oxidized Gal-1. However, at higher Gal-1 concentrations, we observed a ten times higher affinity for reduced than oxidized form. Further, our data indicate that the monoclonal antibodies bind preferentially to Gal-1 dimers and specifically to only some forms of its oxidized form.
- Klíčová slova
- Constant current chronopotentiometric stripping, Galectin-1, Intrinsic fluorescence, Lectin activity of reduced and oxidized form, Mobility shift assay, Oxidative inactivation,
- MeSH
- galektin 1 * chemie metabolismus MeSH
- lektiny * chemie metabolismus MeSH
- lidé MeSH
- multimerizace proteinu * MeSH
- oxidace-redukce MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- galektin 1 * MeSH
- lektiny * MeSH
Pseudomonas aeruginosa is a prevalent opportunistic human pathogen, particularly associated with cystic fibrosis. Among its virulence factors are the LecA and LecB lectins. Both lectins play an important role in the adhesion to the host cells and display cytotoxic activity. In this study, we successfully synthesized hardly hydrolysable carbohydrate ligands targeting these pathogenic lectins, including two bispecific glycans. The interactions between LecA/LecB lectins and synthetic glycans were evaluated using hemagglutination (yeast agglutination) inhibition assays, comparing their efficacy with corresponding monosaccharides. Additionally, the binding affinities of bispecific glycans were assessed using isothermal titration calorimetry (ITC). Structural insight into the lectin-ligand interaction was obtained by determining the crystal structures of LecA/LecB lectins in complex with one of the bispecific ligands using X ray crystallography. This comprehensive investigation into the inhibitory potential of synthetic glycosides against P. aeruginosa lectins sheds light on their potential application in antimicrobial therapy.
- Klíčová slova
- Agglutination, Inhibition, Lectins, Pseudomonas aeruginosa, Synthetic carbohydrates,
- MeSH
- bakteriální adheziny * chemie metabolismus MeSH
- disacharidy * chemie farmakologie MeSH
- krystalografie rentgenová MeSH
- lektiny * chemie antagonisté a inhibitory metabolismus MeSH
- lidé MeSH
- ligandy MeSH
- polysacharidy chemie MeSH
- Pseudomonas aeruginosa * chemie metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- bakteriální adheziny * MeSH
- disacharidy * MeSH
- LecA protein, bacteria MeSH Prohlížeč
- LecB protein, Pseudomonas aeruginosa MeSH Prohlížeč
- lektiny * MeSH
- ligandy MeSH
- polysacharidy MeSH
Mammalian spermatozoa have a surface covered with glycocalyx, consisting of heterogeneous glycoproteins and glycolipids. This complexity arises from diverse monosaccharides, distinct linkages, various isomeric glycans, branching levels, and saccharide sequences. The glycocalyx is synthesized by spermatozoa developing in the testis, and its subsequent alterations during their transit through the epididymis are a critical process for the sperm acquisition of fertilizing ability. In this study, we performed detailed analysis of the glycocalyx on the sperm surface of bull spermatozoa in relation to individual parts of the epididymis using a wide range (24) of lectins with specific carbohydrate binding preferences. Fluorescence analysis of intact sperm isolated from the bull epididymides was complemented by Western blot detection of protein extracts from the sperm plasma membrane fractions. Our experimental results revealed predominant sequential modification of bull sperm glycans with N-acetyllactosamine (LacNAc), followed by subsequent sialylation and fucosylation in a highly specific manner. Additionally, variations in the lectin detection on the sperm surface may indicate the acquisition or release of glycans or glycoproteins. Our study is the first to provide a complex analysis of the bull sperm glycocalyx modification during epididymal maturation.
- Klíčová slova
- cattle, epididymis, glycan, lectin, plasma membrane, sperm surface, spermatozoa,
- MeSH
- epididymis * metabolismus cytologie MeSH
- glykokalyx * metabolismus MeSH
- glykoproteiny metabolismus MeSH
- lektiny * metabolismus MeSH
- polysacharidy metabolismus MeSH
- skot MeSH
- spermie * metabolismus MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- glykoproteiny MeSH
- lektiny * MeSH
- polysacharidy MeSH
Sialic acids are negatively charged carbohydrates that are components of saccharide chains covalently linked to macromolecules. Sialylated glycoproteins are important for most biological processes, including reproduction, where they are associated with spermatogenesis, sperm motility, immune responses, and fertilization. Changes in the glycoprotein profile or sialylation in glycoproteins are likely to affect the quality of ejaculate. The aim of this study was to determine differences in the degree of sialylation between normozoospermic ejaculates and ejaculates with a pathological spermiogram using two lectins, Sambucus nigra (SNA) and Maackia amurensis (MAL II/MAA) recognizing α-2,6 or α-2,3 linkage of Sia to galactosyl residues. Our results show a close relationship between seminal plasma (SP) sialoproteins and the presence of anti-sperm antibodies in the ejaculate, apoptotic spermatozoa, and ejaculate quality. Using mass spectrometry, we identified SP sialoproteins such as, semenogelins, glycodelin, prolactin-inducible protein, lactotransferrin, and clusterin that are associated with spermatozoa and contribute to the modulation of the immune response and sperm apoptosis. Our findings suggest a correlation between the degree of SP glycoprotein sialylation and the existence of possible pathological states of spermatozoa and reproductive organs. Glycoproteins sialylation represents a potential parameter reflecting the overall quality of ejaculate and could potentially be utilised in diagnostics.
- Klíčová slova
- Anti-sperm antibodies, Apoptosis, Ejaculate quality, Glycoprotein, Human,
- MeSH
- analýza spermatu metody MeSH
- apoptóza MeSH
- ejakulace MeSH
- glykodelin metabolismus MeSH
- glykoproteiny metabolismus MeSH
- klusterin metabolismus MeSH
- kyseliny sialové metabolismus MeSH
- laktoferrin metabolismus MeSH
- lektiny metabolismus chemie MeSH
- lidé MeSH
- motilita spermií MeSH
- proteiny semenné plazmy metabolismus MeSH
- sekreční proteiny semenných váčků metabolismus MeSH
- sperma * metabolismus chemie MeSH
- spermie * metabolismus MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- glykodelin MeSH
- glykoproteiny MeSH
- klusterin MeSH
- kyseliny sialové MeSH
- laktoferrin MeSH
- lektiny MeSH
- PAEP protein, human MeSH Prohlížeč
- proteiny semenné plazmy MeSH
- sekreční proteiny semenných váčků MeSH
- seminal vesicle-specific antigen MeSH Prohlížeč
Prostate cancer (PCa) is the second most common cancer. In this paper, the isolation and properties of exosomes as potential novel liquid biopsy markers for early PCa liquid biopsy diagnosis are investigated using two prostate human cell lines, i.e., benign (control) cell line RWPE1 and carcinoma cell line 22Rv1. Exosomes produced by both cell lines are characterised by various methods including nanoparticle-tracking analysis, dynamic light scattering, scanning electron microscopy and atomic force microscopy. In addition, surface plasmon resonance (SPR) is used to study three different receptors on the exosomal surface (CD63, CD81 and prostate-specific membrane antigen-PMSA), implementing monoclonal antibodies and identifying the type of glycans present on the surface of exosomes using lectins (glycan-recognising proteins). Electrochemical analysis is used to understand the interfacial properties of exosomes. The results indicate that cancerous exosomes are smaller, are produced at higher concentrations, and exhibit more nega tive zeta potential than the control exosomes. The SPR experiments confirm that negatively charged α-2,3- and α-2,6-sialic acid-containing glycans are found in greater abundance on carcinoma exosomes, whereas bisecting and branched glycans are more abundant in the control exosomes. The SPR results also show that a sandwich antibody/exosomes/lectins configuration could be constructed for effective glycoprofiling of exosomes as a novel liquid biopsy marker.
- Klíčová slova
- exosomes, microscopy techniques, nanoparticle tracking analysis, prostate cancer, self-assembled monolayer, surface plasmon resonance,
- MeSH
- exozómy * chemie MeSH
- karcinom * metabolismus patologie MeSH
- lektiny analýza metabolismus MeSH
- lidé MeSH
- polysacharidy analýza metabolismus MeSH
- tekutá biopsie MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- lektiny MeSH
- polysacharidy MeSH
Activation of the lectin pathway of the complement system, as demonstrated by elevated levels of mannan-binding lectin proteins (MBL), contributes to vascular pathology in type 1 diabetes (T1D). Vascular complications are greatest in T1D individuals with concomitant insulin resistance (IR), however, whether IR amplifies activiation of the lectin pathway in T1D is unknown. We pooled pretreatment data from two RCTs and performed a cross-sectional analysis on 46 T1D individuals. We employed estimated glucose disposal rate (eGDR), a validated IR surrogate with cut-points of: <5.1, 5.1-8.7, and > 8.7 mg/kg/min to determine IR status, with lower eGDR values conferring higher degrees of IR. Plasma levels of MBL-associated proteases (MASP-1, MASP-2, and MASP-3) and their regulatory protein MAp44 were compared among eGDR classifications. In a subset of 14 individuals, we assessed change in MASPs and MAp44 following improvement in IR. We found that MASP-1, MASP-2, MASP-3, and MAp44 levels increased in a stepwise fashion across eGDR thresholds with elevated MASPs and MAp44 levels conferring greater degrees of IR. In a subset of 14 patients, improvement in IR was associated with significant reductions in MASPs, but not MAp44, levels. In conclusion, IR in T1D amplifies levels of MASP-1/2/3 and their regulator MAp44, and improvement of IR normalizes MASP-1/2/3 levels. Given that elevated levels of these proteins contribute to vascular pathology, amplification of the lectin pathway of the complement system may offer mechanistic insight into the relationship between IR and vascular complications in T1D.
- Klíčová slova
- complement, insulin resistance, mannan-binding lectin-associated serine proteases, type 1 diabetes,
- MeSH
- diabetes mellitus 1. typu * MeSH
- inzulinová rezistence * MeSH
- komplement MeSH
- lektin vázající mannosu * MeSH
- lektiny metabolismus MeSH
- lidé MeSH
- průřezové studie MeSH
- serinové proteasy asociované s proteinem vázajícím mannosu metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- komplement MeSH
- lektin vázající mannosu * MeSH
- lektiny MeSH
- serinové proteasy asociované s proteinem vázajícím mannosu MeSH
Many patients with immunoglobulin A nephropathy (IgAN) progress to kidney failure even with optimal supportive care. An improved understanding of the pathophysiology of IgAN in recent years has led to the investigation of targeted therapies with acceptable tolerability that may address the underlying causes of IgAN or the pathogenesis of kidney injury. The complement system-particularly the lectin and alternative pathways of complement-has emerged as a key mediator of kidney injury in IgAN and a possible target for investigational therapy. This review will focus on the lectin pathway. The examination of kidney biopsies has consistently shown glomerular deposition of mannan-binding lectin (1 of 6 pattern-recognition molecules that activate the lectin pathway) together with IgA1 in up to 50% of patients with IgAN. Glomerular deposition of pattern-recognition molecules for the lectin pathway is associated with more severe glomerular damage and more severe proteinuria and hematuria. Emerging research suggests that the lectin pathway may also contribute to tubulointerstitial fibrosis in IgAN and that collectin-11 is a key mediator of this association. This review summarizes the growing scientific and clinical evidence supporting the role of the lectin pathway in IgAN and examines the possible therapeutic role of lectin pathway inhibition for these patients.
- Klíčová slova
- IgA nephropathy, complement, glomerulonephritis,
- MeSH
- glomerulus patologie MeSH
- IgA nefropatie * patologie MeSH
- imunoglobulin A metabolismus MeSH
- ledviny patologie MeSH
- lektiny metabolismus MeSH
- lidé MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
- Názvy látek
- imunoglobulin A MeSH
- lektiny MeSH
Trisaccharides bind to their interaction partners-lectins relatively weakly, which makes detection of their complexes challenging. In this work, we show that an osmolyte presence improves the distinguishing complexes of lectin Sambucus nigra with trisialyllactoses with various binding affinities. The addition of osmolyte, non-binding sugar mannose significantly improved the precision of binding experiments performed using chronopotentiometric stripping at the electrode surface and fluorescence analysis in solution. Osmolytes minimized nonspecific interactions between binding sugar and lectin. Obtained findings can be utilized in any in vitro methods studying interactions of carbohydrates, respectively their conjugates with proteins. The study of carbohydrate interactions appears important since they play essential roles in a variety of biological processes including carcinogenesis.
- Klíčová slova
- Chronopotentiometric stripping, Impedance C-t curves, Lectin SNA−1 interactions, Osmolytes, Sialylated trisaccharides, Water arrangement,
- MeSH
- bez černý * chemie metabolismus MeSH
- cukry MeSH
- lektiny * metabolismus MeSH
- trisacharidy metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- cukry MeSH
- lektiny * MeSH
- trisacharidy MeSH
