Receptors belonging to NKR-P1 family and their specific Clr ligands form an alternative missing self recognition system critical in immunity against tumors and viruses, elimination of tumor cells subjected to genotoxic stress, activation of T cell dependent immune response, and hypertension. The three-dimensional structure of the extracellular domain of the mouse natural killer (NK) cell receptor mNKR-P1Aex has been determined by X-ray diffraction. The core of the C-type lectin domain (CTLD) is homologous to the other CTLD receptors whereas one quarter of the domain forms an extended loop interacting tightly with a neighboring loop in the crystal. This domain swapping mechanism results in a compact interaction interface. A second dimerization interface resembles the known arrangement of other CTLD NK receptors. A functional dimeric form of the receptor is suggested, with the loop, evolutionarily conserved within this family, proposed to participate in interactions with ligands.

• MeSH
• buňky NK metabolismus   MeSH
• difrakce rentgenového záření MeSH
• lektinové receptory NK-buněk - podrodina B chemie   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• myši MeSH
• Ramanova spektroskopie MeSH
• sekundární struktura proteinů MeSH
• sekvence aminokyselin MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• lektinové receptory NK-buněk - podrodina B MeSH

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.

• Klíčová slova
• CD161, HEK293, LLT1, NK cells, NKR-P1, klrb1,
• 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
• Názvy látek
• CLEC2D protein, human MeSH Prohlížeč
• KLRB1 protein, human MeSH Prohlížeč
• lektinové receptory NK-buněk - podrodina B MeSH
• lektiny typu C MeSH
• ligandy MeSH
• receptory buněčného povrchu MeSH

Human natural killer (NK) cells express on their surface several members of the C-type lectin family such as NKR-P1, CD94, and NKG2 that are probably involved in recognition of target cells and delivery of signals modulating NK cell cytotoxicity. To elucidate the mechanisms involved in signaling via these receptors, we solubilized in vitro cultured human NK cells by a mild detergent, Brij-58, immunoprecipitated molecular complexes containing the NKR-P1 or CD94 molecules, respectively, by specific monoclonal antibodies, and performed in vitro kinase assays on the immunoprecipitates. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, autoradiography, and phospho-amino acid analysis revealed the presence of in vitro tyrosine phosphorylated proteins that were subsequently identified by re-precipitation (and/or by western blotting) as the respective C-type lectin molecules and Src family kinases Lck, Lyn, and Fyn. The NKR-P1 and the CD94-containing complexes were independent of each other and both very large, as judged by Sepharose 4B gel chromatography. Crosslinking of NKR-P1 on the cell surface induced transient in vivo tyrosine phosphorylation of cellular protein substrates. These results indicate involvement of the associated Src-family kinases in signaling via the NKR-P1 and CD94 receptors.

• MeSH
• antigeny povrchové metabolismus   MeSH
• buňky NK imunologie   MeSH
• CD antigeny metabolismus   MeSH
• fosforylace MeSH
• fosfotyrosin metabolismus   MeSH
• lektinové receptory NK-buněk - podrodina B MeSH
• lektinové receptory NK-buněk - podrodina D MeSH
• lektiny typu C * MeSH
• lidé MeSH
• makromolekulární látky MeSH
• membránové glykoproteiny metabolismus   MeSH
• molekulová hmotnost MeSH
• precipitinové testy MeSH
• receptory imunologické metabolismus   MeSH
• signální transdukce MeSH
• src homologní domény MeSH
• tyrosinkinasy metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Názvy látek
• antigeny povrchové MeSH
• CD antigeny MeSH
• fosfotyrosin MeSH
• KLRB1 protein, human MeSH Prohlížeč
• KLRD1 protein, human MeSH Prohlížeč
• lektinové receptory NK-buněk - podrodina B MeSH
• lektinové receptory NK-buněk - podrodina D MeSH
• lektiny typu C * MeSH
• makromolekulární látky MeSH
• membránové glykoproteiny MeSH
• receptory imunologické MeSH
• tyrosinkinasy 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
• Názvy látek
• antigeny povrchové MeSH
• lektinové receptory NK-buněk - podrodina B MeSH
• lektiny typu C MeSH
• ligandy MeSH
• receptory buněčného povrchu * MeSH

Natural killer (NK) and T cells express a superfamily of proteins with structural features of C-type lectins. Recombinantly prepared soluble form of rat NKR-P1 (CD161) recognized carbohydrate GalNac and GlcNac moieties. Ganglioside GM2 and heparin related-IS oligosaccharides representing the high affinity ligands for this receptor, increased the sensitivity of targets for killing by the rat effectors isolated from blood and spleen in vitro. Based on these results, we investigated in vivo the possible therapeutic effect of GM2 and IS carried by liposomes during induced rat colorectal carcinogenesis. The reduction of cancer incidence versus the controls (50% vs 88.88%), approached the 5-fluorouracil treatment (41.66%).

• MeSH
• antigeny povrchové * účinky léků   MeSH
• azoxymethan MeSH
• Escherichia coli MeSH
• fluoruracil terapeutické užití   MeSH
• G(M2) gangliosid terapeutické užití   MeSH
• karcinogeny MeSH
• kolorektální nádory chemicky indukované   farmakoterapie   MeSH
• krysa rodu Rattus MeSH
• lektinové receptory NK-buněk - podrodina B MeSH
• lektiny typu C * MeSH
• liposomy MeSH
• nosiče léků MeSH
• oligosacharidy terapeutické užití   MeSH
• potkani Sprague-Dawley MeSH
• protinádorové antimetabolity terapeutické užití   MeSH
• screeningové testy protinádorových léčiv MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny povrchové * MeSH
• azoxymethan MeSH
• fluoruracil MeSH
• G(M2) gangliosid MeSH
• karcinogeny MeSH
• lektinové receptory NK-buněk - podrodina B MeSH
• lektiny typu C * MeSH
• liposomy MeSH
• nosiče léků MeSH
• oligosacharidy MeSH
• protinádorové antimetabolity 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.

• Klíčová slova
• C-type lectin-like ligand, LLT1,
• 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
• Názvy látek
• CLEC2D protein, human MeSH Prohlížeč
• KLRB1 protein, human MeSH Prohlížeč
• lektinové receptory NK-buněk - podrodina B MeSH
• lektiny typu C MeSH
• receptory buněčného povrchu MeSH

NKR-P1 protein is an important activating receptor at the surface of the rat natural killer cells. GlcNAc and chitooligomers were identified as strong activation ligands in vitro and in vivo. Their clustering brings about increase of their affinity to the NKR-P1 by 3-6 orders. Here we describe novel methodology for preparation of neoglycoproteins based on BSA carrying the chitooligomers (n = 2-5). Further on we developed novel methodology of the coupling of glycosylamines via aromatic-SCN activated linker both to protein or synthetic cores. Inhibition studies of chitooligomer glycoconjugates with the NKR-P1 receptor show that our neoglycoproteins are very strong ligands with high binding affinity (-log IC(50) = 13-15). In analogy with our previous observations with GlcNAc clustered on protein or PAMAM backbones the synthetic chitooligomer clusters should provide considerably better ligands in the in vivo antitumor treatment.

• MeSH
• aminy chemie   MeSH
• antigeny povrchové metabolismus   MeSH
• buňky NK metabolismus   MeSH
• chitin chemie   MeSH
• chromatografie metody   MeSH
• glykoproteiny chemická syntéza   metabolismus   MeSH
• imunoblotting MeSH
• inhibiční koncentrace 50 MeSH
• isothiokyanatany chemie   MeSH
• krysa rodu Rattus MeSH
• kyselina N-acetylneuraminová metabolismus   MeSH
• lektinové receptory NK-buněk - podrodina B MeSH
• lektiny typu C metabolismus   MeSH
• ligandy MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• sérový albumin hovězí chemie   MeSH
• skot MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   MeSH
• thioglykosidy chemická syntéza   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aminy MeSH
• antigeny povrchové MeSH
• chitin MeSH
• glykoproteiny MeSH
• isothiokyanatany MeSH
• kyselina N-acetylneuraminová MeSH
• lektinové receptory NK-buněk - podrodina B MeSH
• lektiny typu C MeSH
• ligandy MeSH
• sérový albumin hovězí MeSH
• thioglykosidy MeSH

Aminosugars have a good affinity for the NKR-P1A protein, the major activating receptor at the surface of rat natural killer cells. We have systematically investigated the structural requirements of the recombinant soluble dimeric form of the receptor for its optimal carbohydrate ligands. While N-acetylD-mannosamine was the best neutral monosaccharide ligand, its participation in the context of an extended oligosaccharide sequence was equally important. The IC(50) value for the GalNAcbeta1 --> ManNAc disaccharide was nearly 10(-10) M with a further possible increase depending on the type of the glycosidic linkage and the aglycon nature. From the point of view of its availability, stability, and affinity for the receptor and a potential in vivo use, these studies are pivotal for the design of an oligosaccharide or glycomimetics suitable for further clustering into the multivalent glycodendrimers.

• MeSH
• antigeny povrchové chemie   účinky léků   genetika   metabolismus   MeSH
• buňky NK účinky léků   metabolismus   MeSH
• konformace sacharidů MeSH
• krysa rodu Rattus MeSH
• lektinové receptory NK-buněk - podrodina B MeSH
• lektiny typu C * MeSH
• oligosacharidy chemie   farmakologie   MeSH
• rekombinantní proteiny chemie   účinky léků   metabolismus   MeSH
• sacharidy chemie   MeSH
• sbalování proteinů MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• odvolaná publikace MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny povrchové MeSH
• lektinové receptory NK-buněk - podrodina B MeSH
• lektiny typu C * MeSH
• oligosacharidy MeSH
• rekombinantní proteiny MeSH
• sacharidy MeSH

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
• antigeny povrchové biosyntéza   chemie   metabolismus   MeSH
• buněčné linie MeSH
• buňky NK imunologie   MeSH
• Cercopithecus aethiops MeSH
• gelová chromatografie MeSH
• kinetika MeSH
• klonování DNA MeSH
• krysa rodu Rattus MeSH
• ledviny MeSH
• lektinové receptory NK-buněk - podrodina B MeSH
• lektiny typu C * MeSH
• lektiny metabolismus   MeSH
• makromolekulární látky MeSH
• membránové glykoproteiny metabolismus   MeSH
• monosacharidy metabolismus   MeSH
• rekombinantní proteiny biosyntéza   chemie   metabolismus   MeSH
• substrátová specifita MeSH
• transfekce MeSH
• vápník farmakologie   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny povrchové MeSH
• lektinové receptory NK-buněk - podrodina B MeSH
• lektiny typu C * MeSH
• lektiny MeSH
• makromolekulární látky MeSH
• membránové glykoproteiny MeSH
• monosacharidy MeSH
• rekombinantní proteiny MeSH
• vápník MeSH

Receptor proteins at the cell surface regulate the ability of natural killer cells to recognize and kill a variety of aberrant target cells. The structural features determining the function of natural killer receptor proteins 1 (NKR-P1s) are largely unknown. In the present work, refined homology models are generated for the C-type lectin-like extracellular domains of rat NKR-P1A and NKR-P1B, mouse NKR-P1A, NKR-P1C, NKR-P1F, and NKR-P1G, and human NKR-P1 receptors. Experimental data on secondary structure, tertiary interactions, and thermal transitions are acquired for four of the proteins using Raman and infrared spectroscopy. The experimental and modeling results are in agreement with respect to the overall structures of the NKR-P1 receptor domains, while suggesting functionally significant local differences among species and isoforms. Two sequence regions that are conserved in all analyzed NKR-P1 receptors do not correspond to conserved structural elements as might be expected, but are represented by loop regions, one of which is arranged differently in the constructed models. This region displays high flexibility but is anchored by conserved sequences, suggesting that its position relative to the rest of the domain might be variable. This loop may contribute to ligand-binding specificity via a coupled conformational transition.

• MeSH
• aminokyselinové motivy MeSH
• fylogeneze MeSH
• konzervovaná sekvence * MeSH
• krysa rodu Rattus MeSH
• lektinové receptory NK-buněk - podrodina B chemie   klasifikace   MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• Ramanova spektroskopie MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• simulace molekulární dynamiky MeSH
• spektroskopie infračervená s Fourierovou transformací MeSH
• strukturní homologie proteinů MeSH
• terciární struktura proteinů MeSH
• termodynamika MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• lektinové receptory NK-buněk - podrodina B MeSH