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.

• Keywords
• CD161, HEK293, LLT1, NK cells, NKR-P1, klrb1,
• 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
• Names of Substances
• CLEC2D protein, human MeSH Browser
• KLRB1 protein, human MeSH Browser
• NK Cell Lectin-Like Receptor Subfamily B MeSH
• Lectins, C-Type MeSH
• Ligands MeSH
• Receptors, Cell Surface 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.

• Keywords
• C-type lectin-like ligand, LLT1,
• 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
• Names of Substances
• CLEC2D protein, human MeSH Browser
• KLRB1 protein, human MeSH Browser
• NK Cell Lectin-Like Receptor Subfamily B MeSH
• Lectins, C-Type MeSH
• Receptors, Cell Surface 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
• Names of Substances
• Antigens, CD * MeSH
• CD69 antigen MeSH Browser
• Antigens, Differentiation, T-Lymphocyte * MeSH
• KLRB1 protein, human MeSH Browser
• NK Cell Lectin-Like Receptor Subfamily B * MeSH
• Lectins, C-Type * MeSH
• Oligosaccharides * 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
• Antigens, Surface metabolism   MeSH
• Killer Cells, Natural immunology   MeSH
• Antigens, CD metabolism   MeSH
• Phosphorylation MeSH
• Phosphotyrosine metabolism   MeSH
• NK Cell Lectin-Like Receptor Subfamily B MeSH
• NK Cell Lectin-Like Receptor Subfamily D MeSH
• Lectins, C-Type * MeSH
• Humans MeSH
• Macromolecular Substances MeSH
• Membrane Glycoproteins metabolism   MeSH
• Molecular Weight MeSH
• Precipitin Tests MeSH
• Receptors, Immunologic metabolism   MeSH
• Signal Transduction MeSH
• src Homology Domains MeSH
• Protein-Tyrosine Kinases metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Names of Substances
• Antigens, Surface MeSH
• Antigens, CD MeSH
• Phosphotyrosine MeSH
• KLRB1 protein, human MeSH Browser
• KLRD1 protein, human MeSH Browser
• NK Cell Lectin-Like Receptor Subfamily B MeSH
• NK Cell Lectin-Like Receptor Subfamily D MeSH
• Lectins, C-Type * MeSH
• Macromolecular Substances MeSH
• Membrane Glycoproteins MeSH
• Receptors, Immunologic MeSH
• Protein-Tyrosine Kinases MeSH

NK cells represent a potential tool for adoptive immunotherapy against tumors. Membrane-bound Hsp70 acts as a tumor-specific marker enhancing NK cell activity. Using flow cytometry the effect of in vitro stimulation with IL-2 or IL-15 alone or in combination with Hsp70-derived 14-mer peptide (TKD) on cell surface expression of NK activatory receptors (CD16, NKG2D, NKG2C, NKp46, NKp44, NKp30, KIR2DL4, DNAM-1, and LAMP1) and NK inhibitory receptors (NKG2A, KIR2DL2/L3, LIR1/ILT-2, and NKR-P1A) in healthy individuals was studied. Results were expressed as the percentage of receptor expressing cells and the amount of receptor expressed by CD3(-)CD56(+) cellular population. CD94, NKG2D, NKp44, NKp30, KIR2DL4, DNAM-1, LAMP1, NKG2A, and NKR-P1A were upregulated after the stimulation with IL-2 or IL-15 alone or in combination with TKD. KIR2DL2/L3 was upregulated only by IL-15 and IL-15/TKD. Concurrently, an increase in a number of NK cells positive for CD94, NKp44, NKp30, KIR2DL4, and LAMP1 was observed. IL-15 and IL-15/TKD caused also cell number rise positive for KIR2DL2/L3 and NKR-P1A. Cell number positive for NKG2C and NKG2A was increased only by IL-2 and IL-2/TKD. The diverse effect of IL-2 or IL-15 w or w/o TKD on cell surface expression was observed in CD16, NKp46, and LIR1/ILT-2.

• MeSH
• Killer Cells, Natural drug effects   metabolism   MeSH
• Antigens, Differentiation, T-Lymphocyte metabolism   MeSH
• Interleukin-15 pharmacology   MeSH
• Interleukin-2 pharmacology   MeSH
• Cells, Cultured MeSH
• NK Cell Lectin-Like Receptor Subfamily B metabolism   MeSH
• NK Cell Lectin-Like Receptor Subfamily C metabolism   MeSH
• NK Cell Lectin-Like Receptor Subfamily K metabolism   MeSH
• Leukocytes, Mononuclear drug effects   metabolism   MeSH
• Humans MeSH
• Lysosomal Membrane Proteins metabolism   MeSH
• Peptides chemistry   pharmacology   MeSH
• HSP70 Heat-Shock Proteins chemistry   MeSH
• Natural Cytotoxicity Triggering Receptor 1 metabolism   MeSH
• Natural Cytotoxicity Triggering Receptor 2 metabolism   MeSH
• Natural Cytotoxicity Triggering Receptor 3 metabolism   MeSH
• Receptors, IgG metabolism   MeSH
• Receptors, KIR2DL2 MeSH
• Receptors, KIR2DL4 metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Antigens, Differentiation, T-Lymphocyte MeSH
• Interleukin-15 MeSH
• Interleukin-2 MeSH
• KIR2DL2 protein, human MeSH Browser
• KIR2DL4 protein, human MeSH Browser
• KLRB1 protein, human MeSH Browser
• KLRC2 protein, human MeSH Browser
• KLRK1 protein, human MeSH Browser
• LAMP1 protein, human MeSH Browser
• NK Cell Lectin-Like Receptor Subfamily B MeSH
• NK Cell Lectin-Like Receptor Subfamily C MeSH
• NK Cell Lectin-Like Receptor Subfamily K MeSH
• Lysosomal Membrane Proteins MeSH
• NCR1 protein, human MeSH Browser
• NCR2 protein, human MeSH Browser
• NCR3 protein, human MeSH Browser
• Peptides MeSH
• HSP70 Heat-Shock Proteins MeSH
• Natural Cytotoxicity Triggering Receptor 1 MeSH
• Natural Cytotoxicity Triggering Receptor 2 MeSH
• Natural Cytotoxicity Triggering Receptor 3 MeSH
• Receptors, IgG MeSH
• Receptors, KIR2DL2 MeSH
• Receptors, KIR2DL4 MeSH
• T Lineage-Specific Activation Antigen 1 MeSH