Siglecs, sialic-acid-binding immunoglobulin-like lectins, are key immune cell receptors that recognize sialic acid residues on cell surfaces. Pathogens and tumor cells exploit Siglecs to evade immune responses and modulate immunity, contributing significantly to infectious disease and cancer pathogenesis. Siglec-7, primarily expressed on natural killer (NK) cells, functions as an inhibitory receptor, tightly regulating the immune activity. This study investigates the interaction between Siglec-7 and the capsular polysaccharide (CPS) of Neisseria meningitidis serogroup Y (Men-Y), a bacterium whose sialylated CPS is critical for virulence. We demonstrate that Men-Y CPS binds to inhibitory Siglec-7, potentially dampening immune recognition. We employed a multifaceted approach, combining biochemical and biophysical techniques to dissect this interaction. Enzyme-linked immunosorbent assays (ELISAs) and fluorescence titrations quantified the binding specificity and affinity. Ligand- and protein-based nuclear magnetic resonance (NMR) spectroscopy, coupled with computational modeling, provides detailed molecular insights. We highlight the critical influence of the Men-Y CPS conformation and sialic acid presentation on Siglec-7 binding. The specific arrangement of α-2,6-linked sialic acids on the CPS is crucial for Siglec-7 binding, demonstrating the importance of the CPS 3D structure. Preliminary immunological assays using stimulated U937 cells (a promonocytic cell line) further support the immunomodulatory role of Siglec-7 mediated by Men-Y CPS. These results offer valuable insights into the development of targeted therapeutic strategies against bacterial infections.

• Klíčová slova
• Neisseria meningitidis serogroup Y CPS, Siglec-7, binding studies, immune evasion, sialic acid,
• Publikační typ
• časopisecké články MeSH

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

INTRODUCTION: The gene encoding myelin-associated glycoprotein (MAG) has been implicated in autosomal-recessive spastic paraplegia type 75. To date, only four families with biallelic missense variants in MAG have been reported. The genotypic and phenotypic spectrum of MAG-associated disease awaits further elucidation. METHODS: Four unrelated patients with complex neurologic conditions underwent whole-exome sequencing within research or diagnostic settings. Following determination of the underlying genetic defects, in-depth phenotyping and literature review were performed. RESULTS: In all case subjects, we detected ultra-rare homozygous or compound heterozygous variants in MAG. The observed nonsense (c.693C > A [p.Tyr231*], c.980G > A [p.Trp327*], c.1126C > T [p.Gln376*], and 1522C > T [p.Arg508*]) and frameshift (c.517_521dupAGCTG [p.Trp174*]) alleles were predicted to result in premature termination of protein translation. Affected patients presented with variable combinations of psychomotor delay, ataxia, eye movement abnormalities, spasticity, dystonia, and neuropathic symptoms. Cerebellar signs, nystagmus, and pyramidal tract dysfunction emerged as unifying features in the majority of MAG-mutated individuals identified to date. CONCLUSIONS: Our study is the first to describe biallelic null variants in MAG, confirming that loss of myelin-associated glycoprotein causes severe infancy-onset disease with central and peripheral nervous system involvement.

• Klíčová slova
• Ataxia, Dystonia, Loss-of-function variants, MAG, Spastic paraplegia,
• MeSH
• atrofie optického nervu genetika   MeSH
• cerebelární ataxie genetika   MeSH
• dítě MeSH
• dospělí MeSH
• dystonické poruchy genetika   MeSH
• dystonie genetika   MeSH
• genotyp MeSH
• glykoprotein asociovaný s myelinem genetika   MeSH
• lidé MeSH
• mentální retardace genetika   MeSH
• mutace genetika   MeSH
• předškolní dítě MeSH
• rodokmen MeSH
• spastická paraplegie dědičná genetika   MeSH
• spinocerebelární ataxie genetika   MeSH
• svalová spasticita genetika   MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• glykoprotein asociovaný s myelinem MeSH
• MAG protein, human MeSH Prohlížeč

BACKGROUND: Tick-borne encephalitis (TBE) is the main tick-borne viral infection in Eurasia. Its manifestations range from inapparent infections and fevers with complete recovery to debilitating or fatal encephalitis. The basis of this heterogeneity is largely unknown, but part of this variation is likely due to host genetic. We have previously found that BALB/c mice exhibit intermediate susceptibility to the infection of TBE virus (TBEV), STS mice are highly resistant, whereas the recombinant congenic strain CcS-11, carrying 12.5% of the STS genome on the background of the BALB/c genome is even more susceptible than BALB/c. Importantly, mouse orthologs of human TBE controlling genes Oas1b, Cd209, Tlr3, Ccr5, Ifnl3 and Il10, are in CcS-11 localized on segments derived from the strain BALB/c, so they are identical in BALB/c and CcS-11. As they cannot be responsible for the phenotypic difference of the two strains, we searched for the responsible STS-derived gene-locus. Of course the STS-derived genes in CcS-11 may operate through regulating or epigenetically modifying these non-polymorphic genes of BALB/c origin. METHODS: To determine the location of the STS genes responsible for susceptibility of CcS-11, we analyzed survival of TBEV-infected F2 hybrids between BALB/c and CcS-11. CcS-11 carries STS-derived segments on eight chromosomes. These were genotyped in the F2 hybrid mice and their linkage with survival was tested by binary trait interval mapping. We have sequenced genomes of BALB/c and STS using next generation sequencing and performed bioinformatics analysis of the chromosomal segment exhibiting linkage with TBEV survival. RESULTS: Linkage analysis revealed a novel suggestive survival-controlling locus on chromosome 7 linked to marker D7Nds5 (44.2 Mb). Analysis of this locus for polymorphisms between BALB/c and STS that change RNA stability and genes' functions led to detection of 9 potential candidate genes: Cd33, Klk1b22, Siglece, Klk1b16, Fut2, Grwd1, Abcc6, Otog, and Mkrn3. One of them, Cd33, carried a nonsense mutation in the STS strain. CONCLUSIONS: The robust genetic system of recombinant congenic strains of mice enabled detection of a novel suggestive locus on chromosome 7. This locus contains 9 candidate genes, which will be focus of future studies not only in mice but also in humans.

• Klíčová slova
• Candidate gene, Chromosome 7, Mouse model, Survival, Susceptibility locus, Tick-borne encephalitis virus (TBEV),
• MeSH
• ABC transportéry genetika   MeSH
• genotyp MeSH
• lidé MeSH
• lidské chromozomy, pár 7 genetika   MeSH
• mapování chromozomů * MeSH
• myši MeSH
• transportní proteiny genetika   MeSH
• virové nemoci mortalita   MeSH
• viry klíšťové encefalitidy patogenita   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• ABC transportéry MeSH
• transportní proteiny MeSH