Multiple interactions at the natural killer workshop
Abstract
The cell surface receptors and their ligands that control natural killer (NK) cell adhesion, homing, cytokine secretion and target cell recognition are beginning to yield their secrets, as ‘NKologists’ adopt the same ‘multiple interaction strategy’ as the cells themselves. This report from the recent NK workshop∗ updates progress on these receptors and on the origin, regulation and physiology o f the cells.
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A signal peptide peptidase (SPP) reporter activity assay based on the cleavage of type II membrane protein substrates provides further evidence for an inverted orientation of the SPP active site relative to presenilin
2004, Journal of Biological ChemistrySignal peptide peptidase (SPP) is an intramembrane-cleaving protease identified by its cleavage of several type II membrane signal peptides after signal peptidase cleavage. Here we describe a novel, quantitative, cell-based SPP reporter assay. This assay utilizes a substrate consisting of the NH2 terminus of the ATF6 transcription factor fused to a transmembrane domain susceptible to SPP cleavage in vitro. In cells, cleavage of the substrate releases ATF6 from the membrane. This cleavage can be monitored by detection of an epitope that is unmasked in the cleaved substrate or by luciferase activity induced by the cleaved ATF6 substrate binding to and activating an ATF6 luciferase reporter construct. Using this assay we show that (i) SPP is the first aspartyl intramembrane-cleaving protease whose activity increases proportionally to its overexpression and (ii) selectivity of various SPP and γ-secretase inhibitors can be rapidly evaluated. Because this assay was designed based on data suggesting that SPP has an orientation distinct from presenilin and cleaves type II membrane proteins, we determined whether the segment of SPP located between the two presumptive catalytic aspartates was in the lumen or cytoplasm. Using site-directed mutagenesis to insert an N-linked glycosylation site we show that a portion of this region is present in the lumen. These data provide strong evidence that although the SPP and presenilin active sites have some similarities, their presumptive catalytic domains are inverted. This assay should prove useful for additional functional studies of SPP as well as evaluation of SPP and γ-secretase inhibitors.
Signal Peptide Peptidase Forms a Homodimer That Is Labeled by an Active Site-directed γ-Secretase Inhibitor
2004, Journal of Biological ChemistryPresenilin (PS) is the presumptive catalytic component of the intramembrane aspartyl protease γ-secretase complex. Recently a family of presenilin homologs was identified. One member of this family, signal peptide peptidase (SPP), has been shown to be a protease, which supports the hypothesis that PS and presenilin homologs are related intramembrane-cleaving aspartyl proteases. SPP has been reported as a glycoprotein of ∼45 kDa. Our initial characterization of SPP isolated from human brain and cell lines demonstrated that SPP is primarily present as an SDS-stable ∼95-kDa protein on Western blots. Upon heating or treatment of this ∼95-kDa SPP band with acid, a ∼45-kDa band could be resolved. Co-purification of two different epitope-tagged forms of SPP from a stably transfected cell line expressing both tagged versions demonstrated that the ∼95-kDa band is a homodimer of SPP. Pulse-chase metabolic labeling studies demonstrated that the SPP homodimer assembles rapidly and is metabolically stable. In a glycerol velocity gradient, SPP sedimented from ∼100–200 kDa. Significantly the SPP homodimer was specifically labeled by an active site-directed photoaffinity probe (III-63) for PS, indicating that the active sites of SPP and PS/γ-secretase are similar and providing strong evidence that the homodimer is functionally active. Collectively these data suggest that SPP exists in vivo as a functional dimer.
Nonspecific cytotoxic cell receptor protein-1: A novel (predicted) type III membrane receptor on the teleost equivalent of natural killer cells recognizes conventional antigen
1998, Cellular ImmunologyThe phylogenetic model for “conventional” antigen recognition by NK cells may be a protein (NCCRP-1) recently identified from catfish nonspecific cytotoxic cells (NCC). NCCRP-1 may be a Type III membrane protein. The antigen binding domain was identified by competition experiments using synthetic peptides. Within this domain, a 38-mer peptide (aa 104–140) inhibited NCC killing of IM-9, HL-60, NC-37, U937, and MOLT-4 target cells. Biotinylated 38-mer also bound to IM-9 target cells. A mab which inhibited conjugate formation between NCC and target cells also bound to the 38-mer. Nonbiotinylated 38-mer inhibited mab 5C6 binding to immobilized homologous biotinylated peptide in cold competition ELISA experiments. Peptide 104–140 was truncated into two peptides. Amino acid 104–119 bound to (68%) and inhibited lyis of IM-9 target cells, whereas aa 120–140 had no activity. A predicted structure–function algorithm suggested an N-terminal domain containing BOX-1 motifs for cytokine activation; a C-terminal domain containing abundant phosphorylation sites (i.e., Y, S, and T amino acids); and an extracellular antigen binding domain..
Anti-candidial activity of natural killer (NK) and lymphokine activated killer (LAK) lymphocytes in vitro
1996, ImmunobiologyThe natural cytotoxic effects of peripheral blood lymphocytes (PBL) on Candida stellatoidea and several other Candida species were examined by a colony forming inhibition (CFI) assay. Peripheral blood mononuclear cells (PBMC), were incubated with C. stellatoidea yeast cells. After the incubation period the colony-forming ability of the yeast was significantly reduced. In similar experiments, six different Candida species (C. albicans, C. krusei, C. stellatoidea, C. tropicalis, C. pseudotropicalis, C. guillermondii) were used as target cells. There was no statistically significant difference in the anticandidial activities of PBL against the Candida species used. It was demonstrated that a fraction of lymphocytes, natural killer cells (NK), had the major natural anti-candidial activity by using anti-Leu M1 (CD 15) and anti-Leu 11b (CD 16) monoclonal antibodies (mAbs) plus complement (C′). It was observed that inhibition of colony-forming ability of C. stellatoidea was significantly (78–96 %) reduced when anti-Leu 11 b plus C’ were used. In addition, the colony formation inhibition capacity of NK cells was increased by recombinant human interleukin-2 (rhIL-2) while anti-interferon-7 (IFN-7) had no effect. Besides the fact that NK cells are among those responsible for natural immunity against Candida species, this colony-forming inhibition assay performed with C. stellatoidea yeast cells as target and monocyte-depleted PBMC as effector cells, is a simple method to assess NK cell activity.
'Harpoon' model for cell-cell adhesion and recognition of target cells by the natural killer cells
1996, Journal of Theoretical BiologyThe mechanism of recognition by natural killer (NK) cells is still unknown. A dynamic model is formulated describing recognition or NK-sensitive target cells (TCs) by NK cells of NK-like cells. This model does not assume the presence of the specific NK-receptor(s) on the membrane of NK cells and corresponding specific ligands on the NK-sensitive TCs. We suggest: (1) the expression of various kinds of “non-NK receptors” and corresponding ligands (counter-receptors) on the plasma membrane of the same NK cell and, possibly, of TCs (e.g. LFA-1 and ICAM-1-ICAM3, CD2 and LFA-3; receptors for TNF and corresponding ligand etc.); (2) the presence of multiple disorders in the organization of “extracellular matrix-surface membrane-submembrane cytoskeleton” assembly of the NK-sensitive TCs; (3) non-specific primary linking of NK cell with TCs, which induces a transfer of vesicles or membrane fragments from the NK surface to the target cell surface (and perhaps vice versa). These processes may also permit the transfer of many types of receptor and counter-receptor molecules from the surface of one conjugated cell to another by vesicles or membrane fragments. After transferral through the intercellular cleft, the free receptors and counter-receptors will be localized on both cell surfaces at the contact region between conjugated cells. By this model the NK cell can “harpoon” the TC and enhance the binding forces between cells up to the critical level and then switch on killing mechanisms for the TC. By means of this “harpoon” model of cell recognition, it seems possible to explain the nature of the wide polymorphism of TCs which are sensitive to the effect of NK and NK-like cells. A mathematical model of the NK cell cytotoxic reaction is described. The model describes many nonlinear peculiarities of the cytotoxic process and predicts some new phenomena. We suggest new approaches of manipulation of cell membranes which can transform NK-resistant target cells in NK sensitive cells and vice versa.
AK-5 tumor-induced expression of interleukin-12: Role of IL-12 in NK-mediated AK-5 regression
1995, Cellular ImmunologySpontaneous regression of AK-5, a histiocytic tumor, is mediated by CD3-, CD8+ NK cells through ADCC. The onset of AK-5 regression is associated with the induction of humoral immune response and the augmentation of effector function. The mechanism of tumor cell death involves both necrosis and apoptosis. Interleukin-12, a 75-kDa heterodimeric cytokine, has multiple effects on T and NK cells. We have investigated the role of IL-12 in the NK cell-mediated AK-5 tumor regression process. Subcutaneous transplantation of AK-5 tumor induced the expression of IL-12 (p35 and p40) message by Day 6-8 in the splenocytes of syngenic rats. Similarly, analysis of serum samples from tumor-bearing animals showed the presence of circulating IL-12 around the same time. Interaction of immune cells with antibody-tagged AK-5 cells in vitro also triggered the expression of IL-12 message and protein by 3 hr. The circulating IL-12 in the sera of tumor-rejecting animals, as well as rIL-12, stimulated NK cell proliferation, expression of CD16 and CD25, and the activation of NK cell function. These observations suggest that the ability of the AK-5 tumor to induce the endogenous production of IL-12 may be responsible for keeping the NK cells constantly in an activated state, thus demonstrating an efficient mechanism for the complete regression of the tumor.