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 Cells MeSH
• Rats MeSH
• NK Cell Lectin-Like Receptor Subfamily B chemistry   genetics   metabolism   MeSH
• Humans MeSH
• Protein Multimerization MeSH
• Calcitonin Receptor-Like Protein chemistry   genetics   metabolism   MeSH
• Protein Domains MeSH
• Protein Engineering methods   MeSH
• Recombinant Proteins chemistry   genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't 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
• 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

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
• NK Cell Lectin-Like Receptor Subfamily B chemistry   metabolism   MeSH
• Models, Molecular * MeSH
• Protein Multimerization * MeSH
• Mice, Inbred BALB C MeSH
• Mice MeSH
• Proteomics * MeSH
• Protein Structure, Secondary MeSH
• Structure-Activity Relationship MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't 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
• Inclusion Bodies MeSH
• Disulfides chemistry   MeSH
• Escherichia coli metabolism   MeSH
• Mass Spectrometry MeSH
• NK Cell Lectin-Like Receptor Subfamily B biosynthesis   chemistry   genetics   MeSH
• Magnetic Resonance Spectroscopy MeSH
• Molecular Sequence Data MeSH
• Mice MeSH
• Protein Refolding MeSH
• Recombinant Proteins biosynthesis   MeSH
• Amino Acid Sequence MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

• MeSH
• Killer Cells, Natural MeSH
• Lectins MeSH
• Ligands MeSH
• Peptides MeSH
• Carbohydrates MeSH
• Protein Binding MeSH
• Publication type
• Review MeSH

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

• Publication type
• Published Erratum 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
• Glycosylation MeSH
• Protein Structure, Quaternary MeSH
• NK Cell Lectin-Like Receptor Subfamily B chemistry   genetics   metabolism   MeSH
• Lectins, C-Type chemistry   genetics   metabolism   MeSH
• Humans MeSH
• Protein Multimerization * MeSH
• Receptors, Cell Surface chemistry   genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't 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
• 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

The C-type lectin-like receptors include the Nkrp1 protein family that regulates the activity of natural killer (NK) cells. Rat Nkrp1a was reported to bind monosaccharide moieties in a Ca2+-dependent manner in preference order of GalNac > GlcNAc > Fuc > Gal > Man. These findings established for rat Nkrp1a have been extrapolated to all additional Nkrp1 receptors and have been supported by numerous studies over the past two decades. However, since 1996 there has been controversy and another article showed lack of interactions with saccharides in 1999. Nevertheless, several high affinity saccharide ligands were synthesized in order to utilize their potential in antitumor therapy. Subsequently, protein ligands were introduced as specific binders for Nkrp1 proteins and three dimensional models of receptor/protein ligand interaction were derived from crystallographic data. Finally, for at least some members of the NK cell C-type lectin-like proteins, the "sweet story" was impaired by two reports in recent years. It has been shown that the rat Nkrp1a and CD69 do not bind saccharide ligands such as GlcNAc, GalNAc, chitotetraose and saccharide derivatives (GlcNAc-PAMAM) do not directly and specifically influence cytotoxic activity of NK cells as it was previously described.

• MeSH
• Killer Cells, Natural * chemistry   immunology   metabolism   MeSH
• Antigens, CD * chemistry   immunology   metabolism   MeSH
• Antigens, Differentiation, T-Lymphocyte * chemistry   immunology   metabolism   MeSH
• Rats MeSH
• NK Cell Lectin-Like Receptor Subfamily B * chemistry   immunology   metabolism   MeSH
• Lectins, C-Type * chemistry   immunology   metabolism   MeSH
• Humans MeSH
• Oligosaccharides * chemistry   immunology   metabolism   MeSH
• Protein Structure, Tertiary MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't 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.

• MeSH
• Bioreactors MeSH
• Killer Cells, Natural metabolism   MeSH
• Th17 Cells metabolism   MeSH
• Escherichia coli genetics   MeSH
• HEK293 Cells MeSH
• NK Cell Lectin-Like Receptor Subfamily B biosynthesis   genetics   isolation & purification   MeSH
• Lectins, C-Type metabolism   MeSH
• Humans MeSH
• Ligands MeSH
• Receptors, Cell Surface metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH