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Identification and molecular characterization of NKp30, a novel triggering receptor involved in natural cytotoxicity mediated by human natural killer cells.

Pende D, Parolini S, Pessino A, Sivori S, Augugliaro R, Morelli L, Marcenaro E, Accame L, Malaspina A, Biassoni R, Bottino C, Moretta L, Moretta A - J. Exp. Med. (1999)

Bottom Line: This novel receptor is associated with CD3zeta chains that become tyrosine phosphorylated upon sodium pervanadate treatment of NK cells.Molecular cloning of NKp30 cDNA revealed a member of the immunoglobulin superfamily, characterized by a single V-type domain and a charged residue in the transmembrane portion.Moreover, we show that NKp30 is encoded by the previously identified 1C7 gene, for which the function and the cellular distribution of the putative product were not identified in previous studies.

View Article: PubMed Central - PubMed

Affiliation: Istituto Nazionale per la Ricerca sul Cancro, 16132 Genova, Italy.

ABSTRACT
Two major receptors involved in human natural cytotoxicity, NKp46 and NKp44, have recently been identified. However, experimental evidence suggested the existence of additional such receptor(s). In this study, by the generation of monoclonal antibodies (mAbs), we identified NKp30, a novel 30-kD triggering receptor selectively expressed by all resting and activated human natural killer (NK) cells. Although mAb-mediated cross-linking of NKp30 induces strong NK cell activation, mAb-mediated masking inhibits the NK cytotoxicity against normal or tumor target cells. NKp30 cooperates with NKp46 and/or NKp44 in the induction of NK-mediated cytotoxicity against the majority of target cells, whereas it represents the major triggering receptor in the killing of certain tumors. This novel receptor is associated with CD3zeta chains that become tyrosine phosphorylated upon sodium pervanadate treatment of NK cells. Molecular cloning of NKp30 cDNA revealed a member of the immunoglobulin superfamily, characterized by a single V-type domain and a charged residue in the transmembrane portion. Moreover, we show that NKp30 is encoded by the previously identified 1C7 gene, for which the function and the cellular distribution of the putative product were not identified in previous studies.

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Cytofluorimetric analysis of the NKp30 molecules expressed in COS-7 cell transfectants; amino acid sequence and hydrophobicity plot of NKp30. (A) COS-7 cells transfected with clone 5C cDNA construct were stained with anti-NKp30 (A76, AZ20, and Z25) or with anti-NKp46 (BAB281) mAbs followed by PE-conjugated goat anti–mouse IgG1, and analyzed by flow cytometry. White profiles represent cells incubated with the second reagent alone (i.e., negative controls). (B) The putative signal peptide is indicated in lowercase letters, and the transmembrane region is underlined. Cysteines involved in the Ig-like fold are circled, and putative N-glycosylation sites are boxed. A Kyte-Doolittle hydrophobicity plot is shown at the bottom. DNA and protein sequence analyses were performed using GeneWorks, MacVector suites, NetOGlyc version 2.0 (available at http://www.cbs.dtu.dk/services/NetOGlyc), and PSORT Prediction Servers (available at http://psort.nibb.ac.jp:8800/). The sequence data are available from EMBL/GenBank/DDBJ under accession no. AJ223153.
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Figure 7: Cytofluorimetric analysis of the NKp30 molecules expressed in COS-7 cell transfectants; amino acid sequence and hydrophobicity plot of NKp30. (A) COS-7 cells transfected with clone 5C cDNA construct were stained with anti-NKp30 (A76, AZ20, and Z25) or with anti-NKp46 (BAB281) mAbs followed by PE-conjugated goat anti–mouse IgG1, and analyzed by flow cytometry. White profiles represent cells incubated with the second reagent alone (i.e., negative controls). (B) The putative signal peptide is indicated in lowercase letters, and the transmembrane region is underlined. Cysteines involved in the Ig-like fold are circled, and putative N-glycosylation sites are boxed. A Kyte-Doolittle hydrophobicity plot is shown at the bottom. DNA and protein sequence analyses were performed using GeneWorks, MacVector suites, NetOGlyc version 2.0 (available at http://www.cbs.dtu.dk/services/NetOGlyc), and PSORT Prediction Servers (available at http://psort.nibb.ac.jp:8800/). The sequence data are available from EMBL/GenBank/DDBJ under accession no. AJ223153.

Mentions: In an attempt to identify the cDNA encoding the NKp30 molecule, a cDNA expression library was generated from the mRNA of human polyclonal NK cells 24. COS-7 cells transfected with different cDNA library pools were stained with A76 mAb by an immunocytochemical detection method. A 674-bp cDNA (NK-A1 clone 5C; sequence data available from EMBL/GenBank/DDBJ under accession no. AJ223153) was isolated that contained a single open reading frame (ORF) of 573 bp. Transfection of COS-7 cells with clone 5C cDNA construct resulted in the surface expression of a molecule that was recognized by all the various anti-NKp30 mAbs (Fig. 7 A), but not by anti-NKp46 mAbs as assessed by cytofluorimetric analysis. As shown in Fig. 7 B, clone 5C ORF encoded a putative 190 amino acid polypeptide belonging to the Ig-SF, characterized by a signal peptide of 18 amino acids and by an extracellular region of 120 amino acids forming an Ig-like domain of the V type. The extracellular portion contains two potential N-linked glycosylation sites and no consensus sequences for O-linked glycosylation. A region rich in hydrophobic amino acids, potentially involved in protein–protein interactions, is connecting the Ig V-like domain with the transmembrane region. The 19 amino acid transmembrane region contains the positively charged amino acid, Arg, and the 33 amino acid cytoplasmic portion lacks typical immunoreceptor tyrosine-based activating motif (ITAM) consensus sequences. The presence of a charged amino acid in the transmembrane domain is a feature common to other triggering receptors expressed on NK cells 242534353637. These charged residues are usually thought to be involved in the association with ITAM-containing signaling polypeptides.


Identification and molecular characterization of NKp30, a novel triggering receptor involved in natural cytotoxicity mediated by human natural killer cells.

Pende D, Parolini S, Pessino A, Sivori S, Augugliaro R, Morelli L, Marcenaro E, Accame L, Malaspina A, Biassoni R, Bottino C, Moretta L, Moretta A - J. Exp. Med. (1999)

Cytofluorimetric analysis of the NKp30 molecules expressed in COS-7 cell transfectants; amino acid sequence and hydrophobicity plot of NKp30. (A) COS-7 cells transfected with clone 5C cDNA construct were stained with anti-NKp30 (A76, AZ20, and Z25) or with anti-NKp46 (BAB281) mAbs followed by PE-conjugated goat anti–mouse IgG1, and analyzed by flow cytometry. White profiles represent cells incubated with the second reagent alone (i.e., negative controls). (B) The putative signal peptide is indicated in lowercase letters, and the transmembrane region is underlined. Cysteines involved in the Ig-like fold are circled, and putative N-glycosylation sites are boxed. A Kyte-Doolittle hydrophobicity plot is shown at the bottom. DNA and protein sequence analyses were performed using GeneWorks, MacVector suites, NetOGlyc version 2.0 (available at http://www.cbs.dtu.dk/services/NetOGlyc), and PSORT Prediction Servers (available at http://psort.nibb.ac.jp:8800/). The sequence data are available from EMBL/GenBank/DDBJ under accession no. AJ223153.
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Related In: Results  -  Collection

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Figure 7: Cytofluorimetric analysis of the NKp30 molecules expressed in COS-7 cell transfectants; amino acid sequence and hydrophobicity plot of NKp30. (A) COS-7 cells transfected with clone 5C cDNA construct were stained with anti-NKp30 (A76, AZ20, and Z25) or with anti-NKp46 (BAB281) mAbs followed by PE-conjugated goat anti–mouse IgG1, and analyzed by flow cytometry. White profiles represent cells incubated with the second reagent alone (i.e., negative controls). (B) The putative signal peptide is indicated in lowercase letters, and the transmembrane region is underlined. Cysteines involved in the Ig-like fold are circled, and putative N-glycosylation sites are boxed. A Kyte-Doolittle hydrophobicity plot is shown at the bottom. DNA and protein sequence analyses were performed using GeneWorks, MacVector suites, NetOGlyc version 2.0 (available at http://www.cbs.dtu.dk/services/NetOGlyc), and PSORT Prediction Servers (available at http://psort.nibb.ac.jp:8800/). The sequence data are available from EMBL/GenBank/DDBJ under accession no. AJ223153.
Mentions: In an attempt to identify the cDNA encoding the NKp30 molecule, a cDNA expression library was generated from the mRNA of human polyclonal NK cells 24. COS-7 cells transfected with different cDNA library pools were stained with A76 mAb by an immunocytochemical detection method. A 674-bp cDNA (NK-A1 clone 5C; sequence data available from EMBL/GenBank/DDBJ under accession no. AJ223153) was isolated that contained a single open reading frame (ORF) of 573 bp. Transfection of COS-7 cells with clone 5C cDNA construct resulted in the surface expression of a molecule that was recognized by all the various anti-NKp30 mAbs (Fig. 7 A), but not by anti-NKp46 mAbs as assessed by cytofluorimetric analysis. As shown in Fig. 7 B, clone 5C ORF encoded a putative 190 amino acid polypeptide belonging to the Ig-SF, characterized by a signal peptide of 18 amino acids and by an extracellular region of 120 amino acids forming an Ig-like domain of the V type. The extracellular portion contains two potential N-linked glycosylation sites and no consensus sequences for O-linked glycosylation. A region rich in hydrophobic amino acids, potentially involved in protein–protein interactions, is connecting the Ig V-like domain with the transmembrane region. The 19 amino acid transmembrane region contains the positively charged amino acid, Arg, and the 33 amino acid cytoplasmic portion lacks typical immunoreceptor tyrosine-based activating motif (ITAM) consensus sequences. The presence of a charged amino acid in the transmembrane domain is a feature common to other triggering receptors expressed on NK cells 242534353637. These charged residues are usually thought to be involved in the association with ITAM-containing signaling polypeptides.

Bottom Line: This novel receptor is associated with CD3zeta chains that become tyrosine phosphorylated upon sodium pervanadate treatment of NK cells.Molecular cloning of NKp30 cDNA revealed a member of the immunoglobulin superfamily, characterized by a single V-type domain and a charged residue in the transmembrane portion.Moreover, we show that NKp30 is encoded by the previously identified 1C7 gene, for which the function and the cellular distribution of the putative product were not identified in previous studies.

View Article: PubMed Central - PubMed

Affiliation: Istituto Nazionale per la Ricerca sul Cancro, 16132 Genova, Italy.

ABSTRACT
Two major receptors involved in human natural cytotoxicity, NKp46 and NKp44, have recently been identified. However, experimental evidence suggested the existence of additional such receptor(s). In this study, by the generation of monoclonal antibodies (mAbs), we identified NKp30, a novel 30-kD triggering receptor selectively expressed by all resting and activated human natural killer (NK) cells. Although mAb-mediated cross-linking of NKp30 induces strong NK cell activation, mAb-mediated masking inhibits the NK cytotoxicity against normal or tumor target cells. NKp30 cooperates with NKp46 and/or NKp44 in the induction of NK-mediated cytotoxicity against the majority of target cells, whereas it represents the major triggering receptor in the killing of certain tumors. This novel receptor is associated with CD3zeta chains that become tyrosine phosphorylated upon sodium pervanadate treatment of NK cells. Molecular cloning of NKp30 cDNA revealed a member of the immunoglobulin superfamily, characterized by a single V-type domain and a charged residue in the transmembrane portion. Moreover, we show that NKp30 is encoded by the previously identified 1C7 gene, for which the function and the cellular distribution of the putative product were not identified in previous studies.

Show MeSH
Related in: MedlinePlus