Partial amino acid sequence of a novel protozoan parasite antigen that inhibits non-specific cytotoxic cell activity.
Monoclonal antibody (MoAb) 18C2, prepared against a human EBV transformed lymphoblastic cell line (NC-37) is specific for a target cell ligand recognized by fish NCC and by mammalian NK cells. MoAb 18C2 inhibits the lysis of a variety of transformed murine and human cells (e.g. NC-37, YAC-1, K562, etc.). This MoAb also recognizes a determinant on the fish protozoan parasite Tetrahymena pyriformis. In the present study, we used MoAb 18C2 to identify a target antigen in detergent lysates of T. pyriformis. MoAb 18C2 recognized a 46-50 kDa target antigen (NKTag) by Western blot analysis of both crude and ammonium sulphate (AS) fractionated (25-40% saturation) T. pyriformis lysates. AS fractionated or purified soluble NKTag inhibited NCC mediated lysis of IM-9 target cells in a dose dependent fashion. AS fractionated NKTag also inhibited 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 specific for NCC and inhibition could be removed by adsorption of AS fractionated NKTag with MoAb 18C2 hybridoma cells. NKTag was prepared for amino acid sequencing by preparative SDS PAGE of whole cell detergent (CHAPS) lysate followed by Western transfer to nitrocellulose. The MoAb 18C2 recognized NKTag was excised and submitted for microsequence analysis. Direct N-terminal analysis yielded a 12 residue sequence. Additional 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 to known sequences (Swiss-Prot protein sequence database). These data indicate that MoAb 18C2 recognized a novel protein on T. pyriformis which may be involved in target cell recognition/lysis by NCC. Further, these data extend our previous observation that a common target determinant exists between higher and lower eukaryotic cells, and its expression may provide an explanation for the susceptibility of both protozoan parasites and transformed tumour cells to NK/NCC lysis.
Leary
J H
JH
Department of Medical Microbiology, College of Veterinary Medicine, University of Georgia, Athens 30602.
Evans
D L
DL
Jaso-Friedmann
L
L
D000595:Amino Acid Sequence; D000818:Animals; D000953:Antigens, Protozoan / Q000276:immunology* / Q000302:isolation & purification; D015153:Blotting, Western; D003601:Cytotoxicity Tests, Immunologic; D000939:Epitopes / Q000276:immunology; D005260:Female; D006801:Humans; D007059:Ictaluridae / Q000276:immunology*; D007694:Killer Cells, Natural / Q000276:immunology*; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D010802:Phylogeny; D013769:Tetrahymena pyriformis / Q000276:immunology*; D014407:Tumor Cells, Cultured / Q000276:immunology
D000953:Antigens, Protozoan; D000939:Epitopes