PARTIAL AMINO-ACID-SEQUENCE OF A NOVEL PROTOZOAN PARASITE ANTIGEN THAT INHIBITS NONSPECIFIC CYTOTOXIC-CELL ACTIVITY

Monoclonal antibody (MoAb) 18C2, prepared against a human EBV transfor med lymphoblastic cell line (NC-37) is specific for a target cell liga nd recognized by fish NCC and by mammalian NK cells. MoAb 18C2 inhibit s the lysis of a variety of transformed murine and human cells (e.g. N C-37, YAC-1, K562, etc.). This MoAb also recognizes a determinant on t he fish protozoan parasite Tetrahymena pyriformis. In the present stud y, we used MoAb 18C2 to identify a target antigen in detergent lysates of T. pyriformis. MoAb 18C2 recognized a 46-50 kDa target antigen (NK Tag) by Western blot analysis of both crude and ammonium sulphate (AS) fractionated (25-40% saturation) T. pyriformis lysates. AS fractionat ed or purified soluble NKTag inhibited NCC mediated lysis of IM-9 targ et cells in a dose dependent fashion. AS fractionated NKTag also inhib ited NCC lysis of a variety of human and murine transformed targets (e .g. HL-60, MOLT-4, DAUDI, NC-37, U-937, YAC-1, EL-4). Inhibition was s pecific for NCC and inhibition could be removed by adsorption of AS fr actionated NKTag with MoAb 18C2 hybridoma cells. NKTag was prepared fo r amino acid sequencing by preparative SDS PAGE of whole cell detergen t (CHAPS) lysate followed by Western transfer to nitrocellulose. The M oAb 18C2 recognized NKTag was excised and submitted for microsequence analysis. Direct N-terminal analysis yielded a 12 residue sequence. Ad ditional sequences, obtained from in situ trypsin digests of the NKTag on nitrocellulose yielded four additional peptides of 10, 13, 16 and 21 residues. None of the sequences examined had significant homology t o known sequences (Swiss-Prot protein sequence database). These data i ndicate that MoAb 18C2 recognized a novel protein on T. pyriformis whi ch may be involved in target cell recognition/lysis by NCC. Further, t hese data extend our previous observation that a common target determi nant exists between higher and lower eukaryotic cells, and its express ion may provide an explanation for the susceptibility of both protozoa n parasites and transformed tumour cells to NK/NCC lysis.