High-level expression of soluble form of mouse natural killer cell receptor NKR-P1C(B6) in Escherichia coli

Mouse NKR-P1C(B6) receptor corresponding to NK1.1 alloantigen is one of the most widespread surface markers of mouse NK and NKT cells in C57BL/6 mice detected by monoclonal antibody PK136. Although functional studies revealed the ability of this receptor to activate both natural killing and production of cytokines upon antibody crosslinking, the ligand for NKR-P1C(B6) remains unknown. In order to initiate ligand identification, structural studies, and epitope mapping experiments, we developed a simple and efficient expression and purification protocol allowing to produce large amounts of pure soluble monomeric mouse NKR-P1C(B6). Our protein encompassed approximately half of the stalk region and the entire C-terminal globular ligand binding domain. The identity of protein that was devoid of N-terminal initiation methionine and had all three expected disulfides closed was confirmed using high resolution ion cyclotron resonance mass spectrometry. Protein produced into inclusion bodies in Escherichia coli was efficiently refolded into a unique three dimensional structure as confirmed by NMR using (1)H-(15)N-HSQC spectra of uniformly labeled protein. The exceptional purity of the protein should allow its crystallization and detailed structural investigations, and is a prerequisite for its use as a probe in ligand identification and antibody epitope mapping experiments.     

The novel inhibitory NKR-P1C receptor and Ly49s3 identify two complementary, functionally distinct NK cell subsets in rats

The proximal region of the NK gene complex encodes the NKR-P1 family of killer cell lectin-like receptors which in mice bind members of the genetically linked C-type lectin-related family, while the distal region encodes Ly49 receptors for polymorphic MHC class I molecules. Although certain members of the NKR-P1 family are expressed by all NK cells, we have identified a novel inhibitory rat NKR-P1 molecule termed NKR-P1C that is selectively expressed by a Ly49-negative NK subset with unique functional characteristics. NKR-P1C(+) NK cells efficiently lyse certain tumor target cells, secrete cytokines upon stimulation, and functionally recognize a nonpolymorphic ligand on Con A-activated lymphoblasts. However, they specifically fail to kill MHC-mismatched lymphoblast target cells. The NKR-P1C(+) NK cell subset also appears earlier during development and shows a tissue distribution distinct from its complementary Ly49s3(+) subset, which expresses a wide range of Ly49 receptors. These data suggest the existence of two major, functionally distinct populations of rat NK cells possessing very different killer cell lectin-like receptor repertoires.     

Genomic structure and strain-specific expression of the natural killer cell receptor NKR-P1.

NK cells are able to lyse a variety of virally infected and neoplastic cells in an MHC-unrestricted manner. The cell-surface protein NKR-P1 is thought to play a key role in this process. NKR-P1, initially identified in rat IL-2 activated NK cells, is encoded in the mouse by at least three similar, but not identical, genes. We previously reported the isolation and characterization of three different NKR-P1 cDNA, termed cDNA 2, 34, and 40, from IL-2 activated mouse NK cells. This report describes the structure of the gene encoding NKR-P1 cDNA 2, the smallest of these three cDNA. Gene 2 is composed of six exons spanning approximately 14 kb of genomic DNA. The first exon encodes the N-terminal intracellular domain, and exons 4, 5, and 6 contain the sequences coding for the CRD. This organization is similar to that of other genes that encode C-type animal lectins. The expression of the NKR-P1 genes in A-LAK cells from 13 mouse strains was examined by Northern blot analysis. NKR-P1 expression appears to coincide with that of the NK1.1 Ag. This observation further supports the hypothesis that the NK1.1 Ag is encoded by one of the NKR-P1 genes. Nucleotide sequence analysis of the promoter region of the three NKR-P1 genes in BALB/c and C57BL/6 mice suggests that differences in the level of expression probably do not result from alterations in the upstream regions of these genes, but may be caused by the expression of strain-specific transacting factors.     

Molecular cloning of the NK1.1 antigen, a member of the NKR-P1 family of natural killer cell activation molecules.

In mice, the NK1.1 alloantigen is expressed on all NK cells and it initiates transmembrane signals that activate cytotoxicity. NK1.1 has been mapped to a chromosomal region that encodes two families of receptor-like molecules, the NKR-P1 family and the Ly-49 family. Both of these gene families encode type II integral membrane proteins whose extracellular domains are homologous with known C-type lectins. We have isolated and expressed a member of the NKR-P1 gene family (mNKR-P1.9) and have demonstrated both by immunofluorescence and by immunoprecipitation that this cDNA encodes NK1.1. These findings, which demonstrate the receptor-like structure of NK1.1, will facilitate studies regarding the role of NK1.1 in natural killing and regarding the identification of possible NK1.1 ligands.     

Toward an optimal oligosaccharide ligand for rat natural killer cell activation receptor NKR-P1

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.     

Functional requirements for signaling through the stimulatory and inhibitory mouse NKR-P1 (CD161) NK cell receptors

The NK cell receptor protein 1 (NKR-P1) (CD161) molecules represent a family of type II transmembrane C-type lectin-like receptors expressed predominantly by NK cells. Despite sharing a common NK1.1 epitope, the mouse NKR-P1B and NKR-P1C receptors possess opposing functions in NK cell signaling. Engagement of NKR-P1C stimulates cytotoxicity of target cells, Ca2+ flux, phosphatidylinositol turnover, kinase activity, and cytokine production. In contrast, NKR-P1B engagement inhibits NK cell cytotoxicity. Nonetheless, it remains unclear how different signaling outcomes are mediated at the molecular level. Here, we demonstrate that both NKR-P1B and NKR-P1C associate with the tyrosine kinase, p56(lck). The interaction is mediated through the di-cysteine CxCP motif in the cytoplasmic domains of NKR-P1B/C. Disrupting this motif leads to abrogation of both stimulatory and inhibitory NKR-P1 signals. In addition, mutation of the consensus ITIM (LxYxxL) in NKR-P1B abolishes both its Src homology 2-containing protein tyrosine phosphatase-1 recruitment and inhibitory function. Strikingly, engagement of NKR-P1C on NK cells obtained from Lck-deficient mice failed to induce NK cytotoxicity. These results reveal a role for Lck in the initiation of NKR-P1 signals, and demonstrate a requirement for the ITIM in NKR-P1-mediated inhibition.     

Ras activation induces expression of raet1 family NK receptor ligands

NK cells play a crucial role in innate immunity against tumors. In many human tumors, Ras is chronically active, and tumor cells frequently express ligands for the activating NK cell receptor NKG2D. In this study, we report that Ras activation upregulates the expression of Raet1 protein family members Rae1α and Rae1β in mouse and ULBP1-3 in human cells. In addition, Ras also induced MHC class I chain-related protein expression in some human cell lines. Overexpression of the constitutively active H-RasV12 mutant was sufficient to induce NKG2D ligand expression. H-RasV12-induced NKG2D ligand upregulation depended on Raf, MAPK/MEK, and PI3K, but not ATM or ATR, two PI3K-like kinases previously shown to induce NKG2D ligand expression. Analysis of the 5' untranslated regions of Raet1 family members suggested the presence of features known to impair translation initiation. Overexpression of the rate-limiting translation initiation factor eIF4E induced Rae1 and ULBP1 expression in a Ras- and PI3K-dependent manner. Upregulation of NKG2D ligands by H-RasV12 increased sensitivity of cells to NK cell-mediated cytotoxicity. In summary, our data suggest that chronic Ras activation is linked to innate immune responses, which may contribute to immune surveillance of H-Ras transformed cells.     

Synthesis of chitooligomer-based glycoconjugates and their binding to the rat natural killer cell activation receptor NKR-P1

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 carying 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 IC₅₀ = 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.