Working at the border between innate and adaptive immunity, natural killer (NK) cells play a key role in the immune system by protecting healthy cells and by eliminating malignantly transformed, stressed or virally infected cells. NK cell recognition of a target cell is mediated by a receptor "zipper" consisting of various activating and inhibitory receptors, including C-type lectin-like receptors. Among this major group of receptors, two of the largest rodent receptor families are the NKR-P1 and the Clr receptor families. Although these families have been shown to encode receptor-ligand pairs involved in MHC-independent self-nonself discrimination and are a target for immune evasion by tumour cells and viruses, structural mechanisms of their mutual recognition remain less well characterized. Therefore, we developed a non-viral eukaryotic expression system based on transient transfection of suspension-adapted human embryonic kidney 293 cells to produce soluble native disulphide dimers of NK cell C-type lectin-like receptor ectodomains. The expression system was optimized using green fluorescent protein and secreted alkaline phosphatase, easily quantifiable markers of recombinant protein production. We describe an application of this approach to the recombinant protein production and characterization of native rat NKR-P1B and Clr-11 proteins suitable for further structural and functional studies.

• MeSH
• HEK293 buňky MeSH
• krysa rodu rattus MeSH
• lektinové receptory NK-buněk - podrodina B chemie   genetika   metabolismus   MeSH
• lidé MeSH
• multimerizace proteinu MeSH
• protein podobný kalcitoninovému receptoru chemie   genetika   metabolismus   MeSH
• proteinové domény MeSH
• proteinové inženýrství metody   MeSH
• rekombinantní proteiny chemie   genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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.

• MeSH
• acetylglukosamin imunologie   metabolismus   MeSH
• buňky NK imunologie   metabolismus   MeSH
• dendrimery metabolismus   MeSH
• experimentální nádory farmakoterapie   genetika   imunologie   MeSH
• exprese genu účinky léků   imunologie   MeSH
• glykokonjugáty imunologie   metabolismus   farmakologie   MeSH
• interferon gama krev   genetika   imunologie   MeSH
• krysa rodu rattus MeSH
• kultivované buňky MeSH
• lektinové receptory NK-buněk - podrodina B genetika   imunologie   metabolismus   MeSH
• lektiny typu C genetika   imunologie   metabolismus   MeSH
• makrofágy imunologie   metabolismus   MeSH
• myši inbrední BALB C MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• NKT buňky imunologie   metabolismus   MeSH
• oligosacharidy imunologie   metabolismus   MeSH
• polyaminy imunologie   metabolismus   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• protein - isoformy genetika   imunologie   metabolismus   MeSH
• průtoková cytometrie MeSH
• slezina cytologie   imunologie   metabolismus   MeSH
• TNF-alfa krev   genetika   imunologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The cytotoxicity of mouse natural killer (NK) cells in response to pathological changes in target cells is regulated via the Nkrp1b receptor. Here, we characterized the Nkrp1b structure and structural features (stalk, loop, and oligomerization state) that affect its interactions. To study the Nkrp1b protein structure and the functional importance of its stalk, two Nkrp1b protein variants differing by the presence of the stalk were prepared. These variants were studied using a combination of structural mass spectrometry approaches with computational modeling to derive structural models. In addition, information about biological activity and localization in mammalian cells was acquired using scanning microscopy techniques and western blotting. Based on these methods, we obtained the structure of Nkrp1b ectodomain in its monomeric and dimeric conformations, identified the dimerization interface, and determined disulfide connections within the molecule. We found that Nkrp1b occurs as a mixture of monomers and homodimers, both in vitro and in vivo. SIGNIFICANCE: Despite the long-standing assumption that Nkrp1 proteins are homodimers connected by disulfide bonds in the stalk region, our data showed that both Nkrp1b protein variants form monomers and homodimers irrespective of the presence of the stalk. We demonstrated that the stalk is not crucial for protein dimerization or ligand binding and that Nkrp1b interacts with its natural ligands only in its monomeric conformation; therefore, dimers may have another regulatory function. Using a unique combination of computational, biochemical, and biological methods, we revealed the structural conformation and behavior of Nkrp1b in its native state. In addition, it is a first report utilizing the intermolecular chemical cross-linking of light- and heavy-labeled protein chains together with ion mobility-mass spectrometry to design the structural models of protein homodimers.

• MeSH
• lektinové receptory NK-buněk - podrodina B chemie   metabolismus   MeSH
• molekulární modely * MeSH
• multimerizace proteinu * MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• proteomika * MeSH
• sekundární struktura proteinů MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• buňky NK MeSH
• lektiny MeSH
• ligandy MeSH
• peptidy MeSH
• sacharidy MeSH
• vazba proteinů MeSH
• Publikační typ
• přehledy MeSH

Human LLT1 is a C-type lectin-like ligand of NKR-P1 (CD161, gene KLRB1), a C-type lectin-like receptor of natural killer cells. Using X-ray diffraction, the first experimental structures of human LLT1 were determined. Four structures of LLT1 under various conditions were determined: monomeric, dimeric deglycosylated after the first N-acetylglucosamine unit in two forms and hexameric with homogeneous GlcNAc2Man5 glycosylation. The dimeric form follows the classical dimerization mode of human CD69. The monomeric form keeps the same fold with the exception of the position of an outer part of the long loop region. The hexamer of glycosylated LLT1 consists of three classical dimers. The hexameric packing may indicate a possible mode of interaction of C-type lectin-like proteins in the glycosylated form.

• MeSH
• glykosylace MeSH
• kvarterní struktura proteinů MeSH
• lektinové receptory NK-buněk - podrodina B chemie   genetika   metabolismus   MeSH
• lektiny typu C chemie   genetika   metabolismus   MeSH
• lidé MeSH
• multimerizace proteinu * MeSH
• receptory buněčného povrchu chemie   genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

As a part of the innate immunity, NK (Natural Killer) cells provide an early immune response to different stimuli, e.g. viral infections and tumor growths. However, their functions are more complex; they play an important role in reproduction, alloimmunity, autoimmunity and allergic diseases. NK cell activities require an intricate system of regulation that is ensured by many different receptors on a cell surface which integrate signals from interacting cells and soluble factors. One way to understand NK cell biology is through the structure of NK receptors, which can reveal ligand binding conditions. We present a modified protocol for recombinant expression in Escherichia coli and in vitro refolding of the ligand-binding domain of the inhibitory Nkrp1b (SJL/J) protein. Nkrp1b identity and folding was confirmed using mass spectrometry (accurate mass of the intact protein and evaluation of disulfide bonds) and one-dimensional nuclear magnetic resonance spectroscopy. The intention is to provide the basis for conducting structural studies of the inhibitory Nkrp1b protein, since only the activating Nkrp1a receptor structure is known.

• MeSH
• buněčná inkluze MeSH
• disulfidy chemie   MeSH
• Escherichia coli metabolismus   MeSH
• hmotnostní spektrometrie MeSH
• lektinové receptory NK-buněk - podrodina B biosyntéza   chemie   genetika   MeSH
• magnetická rezonanční spektroskopie MeSH
• molekulární sekvence - údaje MeSH
• myši MeSH
• refolding proteinů MeSH
• rekombinantní proteiny biosyntéza   MeSH
• sekvence aminokyselin MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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
• antigeny povrchové MeSH
• buňky NK * MeSH
• difrakce rentgenového záření MeSH
• lektinové receptory NK-buněk - podrodina B MeSH
• lektiny typu C MeSH
• lidé MeSH
• ligandy MeSH
• maloúhlový rozptyl MeSH
• receptory buněčného povrchu * MeSH
• shluková analýza MeSH
• synapse MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Diplomová práce, která se zaměřila na možnou vazbu mezi Hsp65 a z něj odvozených peptidů na protein CD69 a na úlohu chaperoninu Hsp60.

• MeSH
• bakteriální proteiny MeSH
• chaperoniny MeSH
• chemické techniky analytické MeSH
• leukocyty MeSH
• ligandy MeSH
• proteiny teplotního šoku MeSH
• vazba proteinů MeSH
• Publikační typ
• vysokoškolské kvalifikační práce MeSH

• Konspekt
• Biochemie. Molekulární biologie. Biofyzika
• NLK Obory
• biochemie
• alergologie a imunologie

Human natural killer receptor protein 1 (NKR-P1, CD161, gene klrb1) is a C-type lectin-like receptor of natural killer (NK) cells responsible for recognition of its cognate protein ligand lectin-like transcript 1 (LLT1). NKR-P1 is the single human orthologue of the prototypical rodent NKR-P1 receptors. Naturally, human NKR-P1 is expressed on the surface of NK cells, where it serves as inhibitory receptor; and on T and NKT cells functioning as co-stimulatory receptor promoting secretion of IFNγ. Most notably, it is expressed on Th17 and Tc17 lymphocytes where presumably promotes targeting into LLT1 expressing immunologically privileged niches. We tested effect of different protein tags (SUMO, TRX, GST, MsyB) on expression of soluble NKR-P1 in E. coli. Then we optimized the expression construct of soluble NKR-P1 by preparing a library of expression constructs in pOPING vector containing the extracellular lectin-like domain with different length of the putative N-terminal stalk region and tested its expression in Sf9 and HEK293 cells. Finally, a high-level expression of soluble NKR-P1 was achieved by stable expression in suspension-adapted HEK293S GnTI- cells utilizing pOPINGTTneo expression vector. Purified soluble NKR-P1 is homogeneous, deglycosylatable, crystallizable and monomeric in solution, as shown by size-exclusion chromatography, multi-angle light scattering and analytical ultracentrifugation.

• MeSH
• bioreaktory MeSH
• buňky NK metabolismus   MeSH
• buňky Th17 metabolismus   MeSH
• Escherichia coli genetika   MeSH
• HEK293 buňky MeSH
• lektinové receptory NK-buněk - podrodina B biosyntéza   genetika   izolace a purifikace   MeSH
• lektiny typu C metabolismus   MeSH
• lidé MeSH
• ligandy MeSH
• receptory buněčného povrchu metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

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
• CD antigeny chemie   imunologie   MeSH
• diferenciační antigeny T-lymfocytů chemie   imunologie   MeSH
• krystalografie rentgenová MeSH
• lektiny typu C chemie   imunologie   MeSH
• lidé MeSH
• ligandy MeSH
• membránové proteiny chemie   imunologie   MeSH
• myši MeSH
• receptory imunologické chemie   imunologie   MeSH
• statická elektřina MeSH
• strukturní homologie proteinů MeSH
• terciární struktura proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH