Natural killer receptor protein 1 (NKR-P1, a family of proteins), which is a dimeric transmembrane protein predominantly on rat and murine natural killer cells, contains an extracellular motif related to calcium-dependent animal lectins. The domain architecture of this protein and the finding that its cross-linking with antibody results in activation of natural killer cells make it a promising candidate for a receptor function. We have expressed a full-length NKR-P1 protein of the rat in COS cells and prepared soluble extracellular fragments by controlled proteolysis or by expression of truncated cDNA in bacteria. Dimerization of soluble NKR-P1 is predominantly dependent on the presence of an intact juxta-membrane stalk region and independent of N-glycosylation. Binding and inhibition studies using monosaccharides and neoglycoconjugates indicate that NKR-P1 is a lectin with a preference order of GalNAc > GlcNAc >> Fuc >> Gal > Man. At neutral pH, Ca2+ is tightly associated with the protein such that only a proportion can be removed by 10 mM EGTA. However, NKR-P1 can be decalcified completely at pH 10 with a total loss of carbohydrate binding. After recalcification at pH 8, carbohydrate binding is completely restored. Thus, NKR-P1 differs from other calcium-dependent animal lectins investigated so far in its pattern of monosaccharide recognition and in the tightness of Ca2+ binding.

• MeSH
• Antigens, Surface biosynthesis   chemistry   metabolism   MeSH
• Cell Line MeSH
• Killer Cells, Natural immunology   MeSH
• Chlorocebus aethiops MeSH
• Chromatography, Gel MeSH
• Kinetics MeSH
• Cloning, Molecular MeSH
• Rats MeSH
• Kidney MeSH
• NK Cell Lectin-Like Receptor Subfamily B MeSH
• Lectins, C-Type * MeSH
• Lectins metabolism   MeSH
• Macromolecular Substances MeSH
• Membrane Glycoproteins metabolism   MeSH
• Monosaccharides metabolism   MeSH
• Recombinant Proteins biosynthesis   chemistry   metabolism   MeSH
• Substrate Specificity MeSH
• Transfection MeSH
• Calcium pharmacology   MeSH
• Protein Binding MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antigens, Surface MeSH
• NK Cell Lectin-Like Receptor Subfamily B MeSH
• Lectins, C-Type * MeSH
• Lectins MeSH
• Macromolecular Substances MeSH
• Membrane Glycoproteins MeSH
• Monosaccharides MeSH
• Recombinant Proteins MeSH
• Calcium MeSH

Recognition of glycosylation patterns is one of the basic features of innate immunity. Ability of C-type lectin-like receptors such as NKR-P1 to bind saccharide moieties has become recently a controversial issue. In the present study, binding assay with soluble fluorescently labeled recombinant rat NKR-P1A and mouse NKR-P1C proteins revealed apparently no affinity to the various neoglycoproteins. Lack of functional linkage between NKR-P1 and previously described saccharide binder was supported by the fact, that synthetic N-acetyl-D-glucosamine octabranched dendrimer on polyamidoamine scaffold (GN8P) did not change gene expression of NKR-P1 isoforms in C57BL/6 and BALB/c mice divergent in the NK gene complex (both in vitro and in vivo). Surprisingly, N-acetyl-D-glucosamine-coated tetrabranched polyamido-amine dendrimer specifically binds to NKT cells and macrophages but not to NK cells (consistently with changes in cytokine patterns). Despite the fact that GN8P has been tested as an immunomodulator in anti-cancer treatment animal models for many years, surprisingly no changes in cytokine profiles in serum relevant to anti-cancer responses using B16F10 and CT26 harboring mouse strains C57BL/6 and BALB/c are observed. Our results indicate possible indirect involvement of NK cells in GN8P mediated immune responses.

• Keywords
• Anti-tumor immunity, C-type lectin related protein, Carbohydrate dendrimer, Clr, GN4P-A: GlcNAc4-PAMAM-ATTO 565, GN4P-NH(2)-GlcNAc(4)-PAMAM, GN4P: GlcNAc4-PAMAM, GN8P: GlcNAc8-PAMAM, GlcNAc, Gzmb, Macrophages, N-acetyl-d-glucosamine, N-acetyl-d-glucosamine-coated octabranched polyamidoamine dendrimer, N-acetyl-d-glucosamine-coated tetrabranched polyamidoamine dendrimer, N-acetyl-d-glucosamine-coated tetrabranched polyamidoamine dendrimer fluorescently labeled with ATTO 565, N-acetyl-d-glucosamine-coated tetrabranched polyamidoamine dendrimer with free NH(2) group, NK cells, NKG2D, NKR-P1, NKR-P1 receptors, NKT cells, PAMAM dendrimer, PMA, Prf, SBA, SMC, granzyme B, natural killer group 2, member D, natural killer receptor protein 1, perforin, phorbol 12-myristate 13-acetate, polyamidoamine dendrimer, soybean agglutinin, spleen mononuclear cell,
• MeSH
• Acetylglucosamine immunology   metabolism   MeSH
• Killer Cells, Natural immunology   metabolism   MeSH
• Dendrimers metabolism   MeSH
• Neoplasms, Experimental drug therapy   genetics   immunology   MeSH
• Gene Expression drug effects   immunology   MeSH
• Glycoconjugates immunology   metabolism   pharmacology   MeSH
• Interferon-gamma blood   genetics   immunology   MeSH
• Rats MeSH
• Cells, Cultured MeSH
• NK Cell Lectin-Like Receptor Subfamily B genetics   immunology   metabolism   MeSH
• Lectins, C-Type genetics   immunology   metabolism   MeSH
• Macrophages immunology   metabolism   MeSH
• Mice, Inbred BALB C MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• Natural Killer T-Cells immunology   metabolism   MeSH
• Oligosaccharides immunology   metabolism   MeSH
• Polyamines immunology   metabolism   MeSH
• Reverse Transcriptase Polymerase Chain Reaction MeSH
• Protein Isoforms genetics   immunology   metabolism   MeSH
• Flow Cytometry MeSH
• Spleen cytology   immunology   metabolism   MeSH
• Tumor Necrosis Factor-alpha blood   genetics   immunology   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Acetylglucosamine MeSH
• Dendrimers MeSH
• Glycoconjugates MeSH
• Interferon-gamma MeSH
• NK Cell Lectin-Like Receptor Subfamily B MeSH
• Lectins, C-Type MeSH
• Oligosaccharides MeSH
• Poly(amidoamine) MeSH Browser
• Polyamines MeSH
• Protein Isoforms MeSH
• Tumor Necrosis Factor-alpha MeSH

CD69 is a signal transducing molecule of hematopoietic cells. Previous molecular cloning of CD69 has revealed a type II transmembrane orientation and the presence of an extracellular domain related to the Ca(2+)-dependent (C-type) animal lectins. As the predicted amino acid sequence for the lectin-like domain is highly divergent from those of other C-type lectin-like proteins - a feature shared with NKR-P1 of natural killer cells - CD69 and NKR-P1 are among proteins assigned to a separate group, group V. To initiate ligand identification studies, we have prepared soluble forms of CD69 protein by bacterial expression of its extracellular portion. We show that cysteine 68 located in the short membrane-proximal neck region of CD69 which adjoins the C-terminal lectin-like domain is a critical element for dimerization. We have evidence that the soluble dimeric CD69 has a tight association with calcium, a feature shared with NKR-P1, and that it is a carbohydrate-binding protein with N-acetyl-D-glucosamine and N-acetyl-D-galactosamine as the best inhibitors: 4-8 x 10(-5) M giving 50% inhibition of binding to N-acetyl-D-glucosamine neoglycoprotein. Thus, the tight association with calcium and high affinities for carbohydrate binding appear to be features of at least two members of the C-type lectin group V.

• MeSH
• Acetylglucosamine analogs & derivatives   metabolism   MeSH
• Killer Cells, Natural metabolism   MeSH
• Antigens, CD biosynthesis   isolation & purification   metabolism   MeSH
• Antigens, Differentiation, T-Lymphocyte biosynthesis   isolation & purification   metabolism   MeSH
• Electrophoresis, Polyacrylamide Gel MeSH
• Escherichia coli MeSH
• Chromatography, Gel MeSH
• Kinetics MeSH
• Cloning, Molecular MeSH
• Lectins, C-Type MeSH
• Lectins metabolism   MeSH
• Humans MeSH
• Lymphocytes metabolism   MeSH
• Molecular Sequence Data MeSH
• Molecular Weight MeSH
• Monosaccharides pharmacology   MeSH
• Recombinant Proteins biosynthesis   isolation & purification   metabolism   MeSH
• Amino Acid Sequence MeSH
• Serum Albumin, Bovine metabolism   MeSH
• Calcium metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH
• Names of Substances
• Acetylglucosamine MeSH
• Antigens, CD MeSH
• CD69 antigen MeSH Browser
• Antigens, Differentiation, T-Lymphocyte MeSH
• Lectins, C-Type MeSH
• Lectins MeSH
• Monosaccharides MeSH
• N-acetylglucosamine-bovine serum albumin conjugate MeSH Browser
• Recombinant Proteins MeSH
• Serum Albumin, Bovine MeSH
• Calcium MeSH

A recently described bangle lectin (PHL) from the bacterium Photorhabdus asymbiotica was identified as a mainly fucose-binding protein that could play an important role in the host-pathogen interaction and in the modulation of host immune response. Structural studies showed that PHL is a homo-dimer that contains up to seven L-fucose-specific binding sites per monomer. For these reasons, potential ligands of the PHL lectin: α-L-fucopyranosyl-containing mono-, di-, tetra-, hexa- and dodecavalent ligands were tested. Two types of polyvalent structures were investigated - calix[4]arenes and dendrimers. The shared feature of all these structures was a C-glycosidic bond instead of the more common but physiologically unstable O-glycosidic bond. The inhibition potential of the tested structures was assessed using different techniques - hemagglutination, surface plasmon resonance, isothermal titration calorimetry, and cell cross-linking. All the ligands proved to be better than free L-fucose. The most active hexavalent dendrimer exhibited affinity three orders of magnitude higher than that of standard L-fucose. To determine the binding mode of some ligands, crystal complex PHL/fucosides 2 - 4 were prepared and studied using X-ray crystallography. The electron density in complexes proved the presence of the compounds in 6 out of 7 fucose-binding sites.

• MeSH
• Anti-Bacterial Agents chemistry   pharmacology   therapeutic use   MeSH
• Bacterial Infections drug therapy   microbiology   MeSH
• Bacterial Proteins antagonists & inhibitors   chemistry   isolation & purification   metabolism   MeSH
• Dendrimers chemistry   pharmacology   therapeutic use   MeSH
• Erythrocytes MeSH
• Fucose analogs & derivatives   pharmacology   therapeutic use   MeSH
• Hemagglutination drug effects   MeSH
• Host-Pathogen Interactions drug effects   MeSH
• Crystallography, X-Ray MeSH
• Lectins antagonists & inhibitors   chemistry   isolation & purification   metabolism   MeSH
• Humans MeSH
• Ligands MeSH
• Models, Molecular MeSH
• Photorhabdus metabolism   MeSH
• Surface Plasmon Resonance MeSH
• Recombinant Proteins chemistry   isolation & purification   metabolism   MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Anti-Bacterial Agents MeSH
• Bacterial Proteins MeSH
• Dendrimers MeSH
• fucose-binding lectin MeSH Browser
• Fucose MeSH
• Lectins MeSH
• Ligands MeSH
• Recombinant Proteins MeSH

NKR-P1 represent a family of activating receptors in rodent natural killer cells related to C-type animal lectins. We identify here the elements involved in the reactivity of the major receptor of rat, NKR-P1A, with N-linked oligosaccharides of glycoproteins. Plate inhibition assays with isolated, structurally defined N-glycans as inhibitors of binding of NKR-P1A to GlcNAc16-BSA revealed that the removal of both the external sialic acids and the penultimate galactose residues resulted in attaining of significant inhibitory activities. Surprisingly, additional plate inhibition and glycoprotein overlay experiments brought evidence that the core chitobiose, depending on its substitution, can per se support the interaction with NKR-P1A. In a series of linear chitooligomers (n = 2-7), the inhibitory activities reached a maximum for the chitotetraose. The ability of NKR-P1 to recognize both the periphery and the core region of complex type oligosaccharides may define its dual specificity towards carbohydrate components of eukaryotic (e.g., tumor) cell surfaces, but also reflect an evolutionarily conserved reactivity with microbial saccharides important in immune recognition and signaling functions.

• MeSH
• Antigens, Surface metabolism   MeSH
• Killer Cells, Natural metabolism   MeSH
• Disaccharides chemistry   metabolism   MeSH
• Glycosylation MeSH
• Carbohydrate Conformation MeSH
• Rats MeSH
• N-Acetylneuraminic Acid chemistry   metabolism   MeSH
• NK Cell Lectin-Like Receptor Subfamily B MeSH
• Lectins, C-Type * MeSH
• Oligosaccharides metabolism   MeSH
• Ovalbumin metabolism   MeSH
• Ovomucin metabolism   MeSH
• Polysaccharides chemistry   metabolism   MeSH
• Receptors, Immunologic metabolism   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Retracted Publication MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antigens, Surface MeSH
• chitobiose MeSH Browser
• Disaccharides MeSH
• N-Acetylneuraminic Acid MeSH
• NK Cell Lectin-Like Receptor Subfamily B MeSH
• Lectins, C-Type * MeSH
• Oligosaccharides MeSH
• Ovalbumin MeSH
• Ovomucin MeSH
• Polysaccharides MeSH
• Receptors, Immunologic MeSH

Extensive exploitation of titanium dioxide nanoparticles (TiO2NPs) augments rapid release into the marine environment. When in contact with the body fluids of marine invertebrates, TiO2NPs undergo a transformation and adhere various organic molecules that shape a complex protein corona prior to contacting cells and tissues. To elucidate the potential extracellular signals that may be involved in the particle recognition by immune cells of the sea urchin Paracentrotus lividus, we investigated the behavior of TiO2NPs in contact with extracellular proteins in vitro. Our findings indicate that TiO2NPs are able to interact with sea urchin proteins in both cell-free and cell-conditioned media. The two-dimensional proteome analysis of the protein corona bound to TiO2NP revealed that negatively charged proteins bound preferentially to the particles. The main constituents shaping the sea urchin cell-conditioned TiO2NP protein corona were proteins involved in cellular adhesion (Pl-toposome, Pl-galectin-8, Pl-nectin) and cytoskeletal organization (actin and tubulin). Immune cells (phagocytes) aggregated TiO2NPs on the outer cell surface and within well-organized vesicles without eliciting harmful effects on the biological activities of the cells. Cells showed an active metabolism, no oxidative stress or caspase activation. These results provide a new level of understanding of the extracellular proteins involved in the immune-TiO2NP recognition and interaction in vitro, confirming that primary immune cell cultures from P. lividus can be an optional model for swift and efficient immune-toxicological investigations.

• Keywords
• biocorona, echinoderm, extracellular signaling, immune-adhesome, in vitro-ex vivo model, proxy to human,
• MeSH
• Cell Adhesion immunology   MeSH
• Phagocytes immunology   MeSH
• Galectins immunology   MeSH
• Glycoproteins immunology   MeSH
• Sea Urchins immunology   MeSH
• Nanoparticles administration & dosage   MeSH
• Nectins immunology   MeSH
• Paracentrotus immunology   MeSH
• Protein Corona immunology   MeSH
• Proteome immunology   MeSH
• Titanium immunology   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Galectins MeSH
• Glycoproteins MeSH
• Nectins MeSH
• Protein Corona MeSH
• Proteome MeSH
• Titanium MeSH
• titanium dioxide MeSH Browser
• toposome glycoprotein complex MeSH Browser

Interactions between C-type lectin-like NK cell receptors and their protein ligands form one of the key recognition mechanisms of the innate immune system that is involved in the elimination of cells that have been malignantly transformed, virally infected, or stressed by chemotherapy or other factors. We determined an x-ray structure for the extracellular domain of mouse C-type lectin related (Clr) protein g, a ligand for the activation receptor NKR-P1F. Clr-g forms dimers in the crystal structure resembling those of human CD69. This newly reported structure, together with the previously determined structure of mouse receptor NKR-P1A, allowed the modeling and calculations of electrostatic profiles for other closely related receptors and ligands. Despite the high similarity among Clr-g, Clr-b, and human CD69, these molecules have fundamentally different electrostatics, with distinct polarization of Clr-g. The electrostatic profile of NKR-P1F is complementary to that of Clr-g, which suggests a plausible interaction mechanism based on contacts between surface sites of opposite potential.

• MeSH
• Antigens, CD chemistry   immunology   MeSH
• Antigens, Differentiation, T-Lymphocyte chemistry   immunology   MeSH
• Crystallography, X-Ray MeSH
• Lectins, C-Type chemistry   immunology   MeSH
• Humans MeSH
• Ligands MeSH
• Membrane Proteins chemistry   immunology   MeSH
• Mice MeSH
• Receptors, Immunologic chemistry   immunology   MeSH
• Static Electricity MeSH
• Structural Homology, Protein MeSH
• Protein Structure, Tertiary MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antigens, CD MeSH
• CD69 antigen MeSH Browser
• Dcl1 protein, mouse MeSH Browser
• Antigens, Differentiation, T-Lymphocyte MeSH
• Lectins, C-Type MeSH
• Ligands MeSH
• Membrane Proteins MeSH
• Nkrp1f protein, mouse MeSH Browser
• Receptors, Immunologic MeSH

Tandem-repeat C-type lectins (pattern-recognition receptors) with specificity for mannosides are intimately involved in antigen recognition, uptake, routing and presentation in macrophages and dendritic cells. In Langerhans cells, Langerin (CD207), a type-II transmembrane protein with a single C-type carbohydrate recognition domain attached to a heptad repeat in the neck region, which is likely to establish oligomers with an alpha-coiled-coil stalk, has been implicated in endocytosis and the formation of Birbeck granules. The structure of Langerin harbours essential motifs for Ca2+-binding and sugar accommodation. Lectin activity has previously been inferred by diminished antibody binding to cells in the presence of the glycan ligand mannan. In view of the complexity of the C-type lectin/lectin-like network, it is unclear what role Langerin plays for Langerhans cells in binding mannosides. In order to reveal in frozen tissue sections to what extent mannose-binding activity co-localizes with Langerin, we have used a synthetic marker, i.e. a neoglycoprotein carrying mannose maxiclusters, as a histochemical ligand, and computer-assisted fluorescence monitoring in a double-labelling procedure. Mannoside-binding capacity was detected in normal epithelial cells. Double labelling ensured the unambiguous assessment of the binding of the neoglycoprotein in Langerhans cells. Light-microscopically, its localization profile resembled the pattern of immunohistochemical detection of Langerin. This result has implications for suggesting rigorous controls in histochemical analysis of this cell type, because binding of kit reagents, i.e. mannose-rich glycoproteins horseradish peroxidase or avidin, to Langerin (or a spatially closely associated lectin) could yield false-positive signals. To show that recognition of carbohydrate ligands in dendritic cells is not restricted to mannose clusters, we have also documented binding of carrier-immobilized histo-blood group A trisaccharide, a ligand of galectin-3, which was not affected by the presence of a blocking antibody to Langerin. Remarkably, access to the carbohydrate recognition domain of Langerin appeared to be impaired in proliferatively active environments (malignancies, hair follicles), indicating presence of an endogenous ligand with high affinity to saturate the C-type lectin under these conditions.

• MeSH
• Antigens, Surface immunology   metabolism   MeSH
• Antigens, CD MeSH
• Epidermal Cells MeSH
• Epidermis chemistry   metabolism   MeSH
• Carcinoma metabolism   secondary   MeSH
• Langerhans Cells immunology   metabolism   pathology   MeSH
• Lectins, C-Type chemistry   immunology   metabolism   MeSH
• Mannose-Binding Lectins * MeSH
• Humans MeSH
• Mannosides immunology   metabolism   MeSH
• Neoplasms metabolism   pathology   MeSH
• Image Processing, Computer-Assisted MeSH
• Mouth Mucosa cytology   metabolism   MeSH
• Binding Sites, Antibody MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antigens, Surface MeSH
• Antigens, CD MeSH
• CD207 protein, human MeSH Browser
• Lectins, C-Type MeSH
• Mannose-Binding Lectins * MeSH
• Mannosides MeSH

Galectin-3 plays a crucial role in cancerogenesis; its targeting is a prospective pathway in cancer diagnostics and therapy. Multivalent presentation of glycans was shown to strongly increase the affinity of glycoconjugates to galectin-3. Further strengthening of interaction with galectin-3 may be accomplished using artificial glycomimetics with apt aryl substitutions. We established a new, as yet undescribed chemoenzymatic method to produce selective C-3-substituted N,N'-diacetyllactosamine glycomimetics and coupled them to human serum albumin. From a library of enzymes, only β-N-acetylhexosaminidase from Talaromyces flavus was able to efficiently synthesize the C-3-propargylated disaccharide. Various aryl residues were attached to the functionalized N,N'-diacetyllactosamine via click chemistry to assess the impact of the aromatic substitution. In ELISA-type assays with galectin-3, free glycomimetics exhibited up to 43-fold stronger inhibitory potency to Gal-3 than the lactose standard. Coupling to human serum albumin afforded multivalent neo-glycoproteins with up to 4209-fold increased inhibitory potency per glycan compared to the monovalent lactose standard. Surface plasmon resonance brought further information on the kinetics of galectin-3 inhibition. The potential of prepared neo-glycoproteins to target galectin-3 was demonstrated on colorectal adenocarcinoma DLD-1 cells. We investigated the uptake of neo-glycoproteins into cells and observed limited non-specific transport into the cytoplasm. Therefore, neo-glycoproteins primarily act as efficient scavengers of exogenous galectin-3 of cancer cells, inhibiting its interaction with the cell surface, and protecting T-lymphocytes against galectin-3-induced apoptosis. The present neo-glycoproteins combine the advantage of a straightforward synthesis, selectivity, non-toxicity, and high efficiency for targeting exogenous galectin-3, with possible application in the immunomodulatory treatment of galectin-3-overexpressing cancers.

• Keywords
• Cancer, Galectin-3, Glycomimetic, Inhibition, Neo-glycoprotein,
• MeSH
• Biomimetic Materials chemical synthesis   chemistry   pharmacology   MeSH
• Galectins antagonists & inhibitors   genetics   metabolism   MeSH
• Glycoproteins chemistry   metabolism   MeSH
• Kinetics MeSH
• Blood Proteins antagonists & inhibitors   genetics   metabolism   MeSH
• Humans MeSH
• Molecular Structure MeSH
• Dose-Response Relationship, Drug MeSH
• Structure-Activity Relationship MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Galectins MeSH
• Glycoproteins MeSH
• Blood Proteins MeSH
• LGALS3 protein, human MeSH Browser

Signaling by the human C-type lectin-like receptor, natural killer (NK) cell inhibitory receptor NKR-P1, has a critical role in many immune-related diseases and cancer. C-type lectin-like receptors have weak affinities to their ligands; therefore, setting up a comprehensive model of NKR-P1-LLT1 interactions that considers the natural state of the receptor on the cell surface is necessary to understand its functions. Here we report the crystal structures of the NKR-P1 and NKR-P1:LLT1 complexes, which provides evidence that NKR-P1 forms homodimers in an unexpected arrangement to enable LLT1 binding in two modes, bridging two LLT1 molecules. These interaction clusters are suggestive of an inhibitory immune synapse. By observing the formation of these clusters in solution using SEC-SAXS analysis, by dSTORM super-resolution microscopy on the cell surface, and by following their role in receptor signaling with freshly isolated NK cells, we show that only the ligation of both LLT1 binding interfaces leads to effective NKR-P1 inhibitory signaling. In summary, our findings collectively support a model of NKR-P1:LLT1 clustering, which allows the interacting proteins to overcome weak ligand-receptor affinity and to trigger signal transduction upon cellular contact in the immune synapse.

• MeSH
• Antigens, Surface MeSH
• Killer Cells, Natural * MeSH
• X-Ray Diffraction MeSH
• NK Cell Lectin-Like Receptor Subfamily B MeSH
• Lectins, C-Type MeSH
• Humans MeSH
• Ligands MeSH
• Scattering, Small Angle MeSH
• Receptors, Cell Surface * MeSH
• Cluster Analysis MeSH
• Synapses MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antigens, Surface MeSH
• NK Cell Lectin-Like Receptor Subfamily B MeSH
• Lectins, C-Type MeSH
• Ligands MeSH
• Receptors, Cell Surface * MeSH