Molecular cloning of NKp46: a novel member of the immunoglobulin superfamily involved in triggering of natural cytotoxicity

Abstract

NKp46 has been shown to represent a novel, natural killer (NK) cell-specific surface molecule, involved in human NK cell activation. In this study, we further analyzed the role of NKp46 in natural cytotoxicity against different tumor target cells. We provide direct evidence that NKp46 represents a major activating receptor involved in the recognition and lysis of both human and murine tumor cells. Although NKp46 may cooperate with other activating receptors (including the recently identified NKp44 molecule) in the induction of NK-mediated lysis of human tumor cells, it may represent the only human NK receptor involved in recognition of murine target cells. Molecular cloning of the cDNA encoding the NKp46 molecule revealed a novel member of the immunoglobulin (Ig) superfamily, characterized by two C2-type Ig-like domains in the extracellular portion. The transmembrane region contains the positively charged amino acid Arg, which is possibly involved in stabilizing the association with CD3zeta chain. The cytoplasmic portion, spanning 30 amino acids, does not contain immunoreceptor tyrosine-based activating motifs. Analysis of a panel of human/hamster somatic cell hybrids revealed segregation of the NKp46 gene on human chromosome 19. Assessment of the NKp46 mRNA expression in different tissues and cell types unambiguously confirmed the strict NK cell specificity of the NKp46 molecule. Remarkably, in line with the ability of NKp46 to recognize ligand(s) on murine target cells, the cDNA encoding NKp46 was found to be homologous to a cDNA expressed in murine spleen. In conclusion, this study reports the first characterization of the molecular structure of a NK-specific receptor involved in the mechanism of NK cell activation during natural cytotoxicity.

Figure 1

Inhibition of natural cytotoxicity by mAb-mediated masking of NKp46 molecules. Seven representative NK cell clones, derived from two donors, were analyzed for cytotoxic activity in a ⁵¹Cr-release assay against the following FcγR-negative target cell lines: BW1502 (murine thymoma), M14 (human melanoma), and IGROV (human ovarian carcinoma), in either the absence (□) or presence of BAB281 (anti-NKp46, ▪), or in Z231 (anti-NKp44, ◪) mAb. Both mAbs are IgG1. The E/T ratio was 5:1 for human target cells and 8:1 for murine target cells. Each bar represents the mean of triplicate experiments.

Figure 2

(A) Cell surface expression of NKp46 protein in COS-7 transfected cells. COS-7 cells, transfected with clone 2F cDNA (left) or vector alone (right), were stained with anti-NKp46 mAb, followed by PE-conjugated goat anti–mouse IgG1 and were analyzed by flow cytometry. White profiles represent cells incubated with second reagent alone (i.e., negative controls). (B) Nucleotide and predicted amino acid sequences of NKp46. The beginning of translation is marked by an arrow. The putative signal peptide is indicated in lowercase letters, the minimal predicted transmembrane region is underlined, and the charged amino acid Arg is circled and shaded in gray. Cysteines involved in the Ig-like fold are circled and putative N- and O-glycosylation sites are boxed. DNA and protein sequence analysis were performed using GeneWorks, MacVector suites (Oxford Molecular Group Inc., Oxford, UK), NetOGlyc 2.0 (http: //www.cbs.dtu.dk/services/NetOGlyc/), and PSORT (http://psort. nibb.ac.jp/) Prediction Servers. These sequence data are available from EMBL/GenBank/DDBJ under accession number AJ001383.

Figure 2

(A) Cell surface expression of NKp46 protein in COS-7 transfected cells. COS-7 cells, transfected with clone 2F cDNA (left) or vector alone (right), were stained with anti-NKp46 mAb, followed by PE-conjugated goat anti–mouse IgG1 and were analyzed by flow cytometry. White profiles represent cells incubated with second reagent alone (i.e., negative controls). (B) Nucleotide and predicted amino acid sequences of NKp46. The beginning of translation is marked by an arrow. The putative signal peptide is indicated in lowercase letters, the minimal predicted transmembrane region is underlined, and the charged amino acid Arg is circled and shaded in gray. Cysteines involved in the Ig-like fold are circled and putative N- and O-glycosylation sites are boxed. DNA and protein sequence analysis were performed using GeneWorks, MacVector suites (Oxford Molecular Group Inc., Oxford, UK), NetOGlyc 2.0 (http: //www.cbs.dtu.dk/services/NetOGlyc/), and PSORT (http://psort. nibb.ac.jp/) Prediction Servers. These sequence data are available from EMBL/GenBank/DDBJ under accession number AJ001383.

Figure 3

Biochemical analysis of NKp46 glycoprotein. (a) A polyclonal NK cell population (A) and COS-7 cells, untransfected (B) or transfected with NKp46 cDNA (C), were surface-labeled with ¹²⁵I and immunoprecipitated with BAB281 (anti-NKp46) mAb. Samples were analyzed in an 11% SDS-PAGE under reducing conditions. (b) NKp46 molecules, purified from a ¹²⁵I surface-labeled NK cell population, were treated with various enzymes as indicated. Samples were run in a 9% SDS-PAGE under reducing conditions.

Figure 3

Biochemical analysis of NKp46 glycoprotein. (a) A polyclonal NK cell population (A) and COS-7 cells, untransfected (B) or transfected with NKp46 cDNA (C), were surface-labeled with ¹²⁵I and immunoprecipitated with BAB281 (anti-NKp46) mAb. Samples were analyzed in an 11% SDS-PAGE under reducing conditions. (b) NKp46 molecules, purified from a ¹²⁵I surface-labeled NK cell population, were treated with various enzymes as indicated. Samples were run in a 9% SDS-PAGE under reducing conditions.

Figure 4

Alignment of amino acid sequences corresponding to the extracellular regions of NKp46 and ILT3 proteins. The putative signal peptides were deleted from both sequences. Consensus sequence is indicated on top, dashes were introduced to maximize homologies, and amino acids identical to the consensus are indicated by dots.

Figure 5

Northern blot analysis of NKp46 transcript expression. Total RNA was isolated from cells of different origins as follows: polyclonal NK cell populations (NK-LM and NK-FG); NK cell clones (KK41 and TB34); a polyclonal CD3⁺ T cell population (PHA blasts); a T lymphoma cell line (Jurkat); Burkitt's lymphoma B cell lines (Raji and Daudi); an EBV-transformed B cell line (LCL721.221); a histiocytic lymphoma cell line (U937); and an acute promyelocytic leukemia cell line (HL60). 10 μg of each RNA preparation (2 μg of RNA from NK cell clones KK41 and TB34) were hybridized with the 1.3-kb NKp46 probe. The positions of 28S and 18S ribosomal RNA subunits are indicated on the left.

Figure 6

Chromosomal localization of NKp46 gene. Genomic DNA derived from a panel of hamster/human somatic cell hybrids, or from human, hamster, or mouse tissues, and digested with EcoRI, was hybridized with the 1.3-kb NKp46 probe. The hybrid cell lines containing chromosome 19 are indicated on top. The positions of the 23.1-, 9.4-, and 6.6-kb fragments of the λ HindIII-digested molecular weight marker are indicated on the right side of each autoradiograph.