A NOVEL LIGAND IN LYMPHOCYTE-MEDIATED CYTOTOXICITY - EXPRESSION OF THE BETA-SUBUNIT OF H-CELL LINES( TRANSPORTING ATP SYNTHASE ON THE SURFACE OF TUMOR)

Extracellular adenosine triphosphate (eATP) has been suggested to play a role in lymphocyte-induced tumor destruction. We now provide eviden ce that a protein responsible for ATP synthesis in mitochondria may al so play a physiologic role in major histocompatibility complex-indepen dent, lymphocyte-mediated cytotoxicity. A 51.5-kD protein (p51.5) bear ing structural and immunologic characteristics of the beta subunit of H+ transporting ATP synthase (E.C. 3.6.1.34, beta-H+ ATPase, published molecular mass of 51.6 kD) was detected on the plasma membrane of thr ee different human tumor cell lines studied. NH2-terminal amino acid s equence analysis of purified p51.5 from K562 tumor cells revealed 100% homology of 16 residues identified in the first 21 positions to the k nown sequence of human mitochondrial beta-H+ ATPase. Antibody directed against a 21-mer peptide in the ATP binding region of beta-H+ ATPase (anti-beta) reacted with only one band on Western blots of whole tumor extracts and tumor membrane extracts suggesting that the antiserum re acts with a single species of protein. Anti-beta reacted with the cell membranes of tumor cells as determined by fluorescence-activated flow cytometry and immunoprecipitated a 51.5-kD protein from surface-label ed neoplastic cells (but not human erythrocytes and lymphocytes). Puri fied p51.5 bound to human lymphocytes and inhibited natural killer (NK ) cell-mediated cytotoxicity. Furthermore, anti-beta treatment of the K562 and A549 tumor cell lines inhibited NK (by >95%) and interleukin 2-activated killer (LAK) cell (by 75%) cytotoxicity, respectively. Sol uble p51.5 upon binding to lymphocytes retained its reactivity to anti -beta suggesting that the ATP binding domain and the lymphocyte-recept or binding domain reside in distinct regions of the ligand. These resu lts suggest that beta-H+ ATPase or a nearly identical molecule is an i mportant ligand in the effector phase (rather than the recognition pha se) of a cytolytic pathway used by naive NK and LAK cells.