Biochemistry 1993, 32, 65 15-6522
6515
Identification and Partial Characterization of an EctoATPase Expressed by Human Natural Killer Cellst Kenneth E. Dombrowski,’JJ James M. Trevillyan,gsII J. Catherine Cone,l.# Yiling L U , and ~ Catherine A. Phillipst*~Jl Department of Veterans Affairs Medical Center, Amarillo, Texas 79106, Department of Internal Medicine and Department of Biochemistry and Molecular Biology, Texas Tech University Health Sciences Center, Amarillo, Texas 79106, and Department of Biology, Texas A&M University, College Station, Texas 77843 Received December 23, 1992; Revised Manuscript Received April 15, 1993
ABSTRACT: An extracellular membrane-associated ectoATPase has been identified on the human natural killer cell line NK3.3. The enzyme is distinct from other classes of ATPases, kinases, and phosphatases. NK3.3 ectoATPase demonstrated a K, for A T P of 41 FM and a V,,, of 0.2 Mmollmin and required both Ca2+and Mg2+for maximal activity. Purine and pyrimidine nucleotides were competitive inhibitors of the catalytic reaction. Inhibition increased with the addition of increasing negative charge of the phosphate side chain and was also dependent on contributions from the nucleoside. NK3.3 ectoATPase activity was inhibited by reaction with the affinity label [p-(fluorosulfonyl)benzoyl]-5’-adenosine (5’-FSBA), which is shown to modify the enzyme a t or near the ATP-binding domain. Photoaffinity labeling of intact NK3.3 cells with [ ~ ~ - ~ ~ P ] - 8 - a z i d odemonstrated ATP an ATP-binding protein of 68-80 kDa unique to NK3.3 cells. A positive correlation was observed between the ability of thevarious nucleotides to block photoincorporation into the 68-80-kDa protein and their ability to inhibit ectoATPase activity. NK3.3 cells which were made ectoATPase-deficient by reaction with 5’-FSBA demonstrated that this enzyme does not have a major role in the protection of this cytolytic effector cell from the possible lytic effects of extracellular ATP.
Adenine nucleotides are released into the extracellular space
in millimolar concentrations from a variety of cells in response to activating stimuli and also as a result of tissue damage or cell death (Haslam & Cusak, 1981; Holmsen, 1985). Extracellular adenine nucleotides cannot cross the cell membrane, but rather mediate their physiologic effects through purinergic receptors (Luthje, 1989; Burnstock, 1990). The nucleotides thus act as signaling molecules to influence physiological processes, cell membrane integrity, and a variety of intracellular biochemical reactions (Ikehara et al., 1981). For example, extracellular ATP stimulates in vivo DNA synthesis in bone marrow and thymocytes but inhibits DNA synthesis in spleen, lymph nodes, and peripheral blood lymphocytes (Ikehara et al., 1981). Also, ATP can trigger histamine secretion from mast cells (Diamant & Kruger, 1967;Cockcroft & Gomperts, 1979a,b) and the secretion of specific granules from neutrophils and monocytes (Dubyak et al., 1988; Cockcroft & Stutchfield, 1980). In addition, ATP has been shown to cause both a cytostatic and a cytotoxic effect on some tumor cells (Rapaport, 1990) and to inhibit macrophage~~
This work was supported in part by grants from seed grant funds from the Texas Tech University Health Sciences Center (K.E.D. and C.A.P.), the Amarillo Area Foundation (C.A.P.), and the Department of Veterans Affairs (J.M.T. and Stephen E. Wright). A preliminary version of this work has been presented at the Eighth Natural Killer Cell Workshop and the First Meeting of The Society for Natural Immunity, Oct 4-6, 1992, and at the Tenth Texas Immunology Conference, Richardson, TX, Nov 13-1 5, 1992. ‘Author to whom correspondence should be addressed at the Department of Internal Medicine, Texas Tech University Health Sciences Center, 1400 Wallace Blvd., Amarillo, TX 79106. t Department of Veterans Affairs Medical Center. Department of Internal Medicine, Texas Tech University Health Sciences Center Regional Academic Center at Amarillo. 1 Department of Biochemistry and Molecular Biology, Texas Tech University Health Sciences Center. * Texas A&M University. # Present address: Department of Internal Medicine, Texas Tech University Health Sciences Center, Amarillo, TX 79106.
mediated (Blanchard et al., 1991) and natural killer (NK)’ cell-mediated (Henriksson, 1983; Schmidt et al., 1984) cytotoxicities. Alternatively, extracellular nucleotides can be degraded to the nucleoside by extracellular, cell-surfacelocated, nucleotide-metabolizing enzymes (ectonucleotidases) and be transported into the cell (Luthje, 1989; Che et al., 1992). The nucleotides can then be salvaged and enter into the regulation of de novo purine and pyrimidine biosynthesis or general cell metabolism. Ectonucleotidases have been described on the cell surface of hepatocytes (Lin, 1990), renal cells (Sabolic et al., 1992), and lymphoid and erythroid cells (Ikehara et al., 198 1). These cell types are in contact with the lumen of blood vessels where high local concentrations of extracellularnucleotides may exist. It has been postulated that ectonucleotidases, and more specifically ectoATPases, may provide protection for certain cell populations against the lytic effects of extracellular ATP (Fillipini et al., 1990). However, the role of these enzymes in a cell-mediated event has not been directly demonstrated. In the immune system, ectoATPases are associated with B-cells (Kragballe & Ellegaard, 1978; Barankiewicz et al., 1989a,b; Barankiewicz & Cohen, 1990), macrophages (Steinberg et al., 1990), and CTL (Fillipini et al., 1990). NK cells, like CTL and macrophages, are cytotoxic effector cells in the immune system. NK cells, unlike CTL, can spontaneously kill certain susceptible tumor target cells in a major histocompatibility complex unrestricted manner and function in the natural resistance against cancer and a variety of infections. Abbreviations: NK, natural killer;DMF, N,N-dimethylformamide; 5’-FSBA, [p-(fluorosulfonyl)benzoyl]-5’-adenosine; 8-azidoATP,8-azidoadenosine 5’-triphosphate; AMPPNP, 5’-adenylyl imidodiphosphate; NEM, N-ethylmaleimide; CTL, cytotoxic T-lymphocytes; FBS, fetal bovine serum; BSA, bovine serum albumin; Pi, inorganic phosphate; HEPES, N-(2-hydroxyethyl)piperazine-N‘-2-ethanesulfonicacid; DTT, dithiothreitol; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis;kDa, kilodalton(s);Is0,concentrationofan inhibitorwhich gives half the maximal velocity.
0006-2960/93/0432-6515$04.00/0 0 1993 American Chemical Society
6516 Biochemistry, Vol. 32, No. 26, 1993 In this paper, evidence is presented that the human natural killer cell line NK3.3 possesses an enzymic activity capable of hydrolyzing extracellular ATP that fits the criteria to be classified as an ectoATPase, and possible roles of this enzyme in natural killer cell function are discussed.
MATERIALS AND METHODS Materials. Nucleotides were purchased as the sodium salts from Sigma Chemical Co. (St. Louis, MO). Activated charcoal, HEPES, glucose, BSA (fraction V), and N E M were purchased from Sigma. [ T - ~ ~ P I A T (triethylammonium P salt in 50% aqueous ethanol) was purchased from Amersham (Arlington Heights, IL). [~r-~~P]-8-AzidoATP was purchased from ICN (Irvine, CA). Sodium [SICr]chromatewas purchased from NEN/Dupont Research Products (Boston, MA). RPMI 1640, penicillin/streptomycin, and L-glutamine were purchased from GIBCO BRL (Gaithersburg, MD). Lymphocult-T, a source of cellular growth factors, was purchased from Biotest Diagnostics (Denville, NJ). Rehatuin fetal bovine serum was purchased from Intergen Co. (Purchase, NY). All other chemicals were of reagent-grade purity. Cell Culture and Maintenance. The NK3.3 cell line was a generous gift from Dr. Jackie Kornbluth, University of Arkansas, Little Rock, AR. This cell line was derived from an allogeneic mixed lymphocyte culture between normal human donors and is interleukin 2-dependent (Kornbluth et al., 1982; Leiden et al., 1988). These cells were maintained in RPMI 1640 with 100 units/mL penicillin/streptomycin, 2 mM L-glutamine, 15% FBS, and 15% Lymphocult-T at a concentration of 1 X lo6 cells/mL at 37 OC in a humidified 5% C02/95% air atmosphere. To ensure that these cells function optimally under the above culture conditions, natural cytotoxicity was routinely determined against the NK-sensitive target cell line K562 (an erythromyeloid leukemia cell line purchased from American Type Culture Collection, Rockville, MD) using a SICr-releaseassay (Brunner et al., 1976). Only cell populations exhibiting an acceptable cytotoxic activity of 50-90 lytic units (Pross et al., 198 1) were used for experiments. NK3.3 cells were assayed for ectoATPase activity 24 h after feeding. The human T-cell leukemia line Jurkat (B2.7 clone) has a T-helper surface phenotype and was obtained from Dr. Seth Lederman, Columbia University, College of Surgeons and Physicians. Jurkat cells were grown and maintained in RPMI 1640 containing 10% FBS. Jurkat cells do not possess ectoATPase activity (Barankiewicz & Cohen, 1990). Raji, a B-cell lymphoma deficient in ectoATPase activity (Barankiewicz & Cohen, 1990), was purchased from ATCC and grown in RPMI 1640 containing 10% FBS. Jurkat and Raji were assayed as negative controls. Assay of ATPase Activity. ATPase activity was measured using [ Y - ~ ~ P ] A as T Psubstrate and counting the amount of [32P]Pireleased in supernatants after precipitation of nucleotides with activated charcoal (Reddy et al., 1978). The reaction buffer consisted of 10 mM HEPES (pH 7.4) containing 135 mM NaC1, 5 mM KCl, 2 mM CaC12, 2 mM MgC12,lO mM glucose, and 1%BSA. The final concentration of ATP was 0.3 mM and contained 0.3 pCi of [y-32P]ATP. The final assay volume was 200 p L and contained 1 X lo4 cells. Alternatively, the reaction was carried out in RPMI 1640 medium, pH 7.4, without the addition of BSA. Concentrations of divalent metal ions, ligands, and nucleotides were varied accordingly as described in the text. The assay mixture was incubated at 37 OC for 20 min and the reaction stopped by the addition of 0.5 mL of cold 20% (w/v) activated
Dombrowski et al. charcoal in 1.0 M HCl. The assay tubes were incubated on ice for 10 min and centrifuged at 10000gfor 10 min to pellet the charcoal. Aliquots (0.2 mL) of the supernatant containing the released [32P]Pi were transferred to scintillation vials containing 2.5 mL of Biofluor scintillation fluid (Amersham), and the radioactivity was determined using a Beckman LS 5801 scintillation counter. All assays were performed in triplicate and reported as the mean f standard deviation. Before addition to the assay, cells were centrifuged and resuspended either in a 10 mM HEPES buffer, pH 7.4, containing 135 mM NaCl and 5 mM KC1 or in RPMI 1640. Theviability of NK3.3 cells used for the assay of ectoATPase activity was routinely >98% by trypan blue exclusion, and the spontaneous SICrrelease was 90%) activity was observed when either assay buffer was used (Table and spontaneous W r release from Wr-labeled NK3.3 cells 1)* (90% after incubation with 5’-FSBA or 2.5% D M F alone (control) as assayed by trypan blue exclusion, and spontaneous S*Crrelease was 80% of the ectoATPase activity, and
