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Molecular characterization of a protozoan parasite target antigen recognized by nonspecific cytotoxic cells
L Jaso-Friedmann1, J H Leary, J Warren
1College of Veterinary Medicine, University of Georgia, Athens 30602, USA.
Abstract:
The target cell antigen(s) on tumor cells and on protozoan parasites recognized by NK and nonspecific cytotoxic cells (NCC) has not yet been specifically identified. NCC may be the teleost equivalent of NK cells and IL-2-activated NK cells. A ligand recognized by NCC has been identified. It is expressed on both protozoan parasites and mammalian tumor target cells. In the present study, a protozoan parasite antigen (NK target antigen/NKTag/p46) was purified from Tetrahymena pyriformis and the entire amino acid sequence was deduced from cDNA. Soluble and purified NKTag inhibited NCC lysis of human and mouse transformed target cells. Homology comparisons using Swissprot database revealed that NKTag is a novel protein. Molecular weight computation of the deduced sequence demonstrated that NKTag is a 48.17-kDa protein containing 422 amino acids with relatively high percentages of tyrosine and serine residues. Expression of NKTag on various mammalian tumor target cells, normal tissue, and T. pyriformis was determined using anti-multiple antigenic peptide (MAP) monoclonal antibody (mab) 22A12 [generated against an N-terminal 20-mer (aa 61-80) of p46]. This mab bound to tissue-cultured and tumor cells (YAC-1, IM-9, NC-37, MOLT-4, and U937) with low levels of binding to fish, mouse, and equine cells. Studies were also done to determine if purified and iodinated NKTag bound specifically to NCC. Binding was saturable and specific. These data provide evidence that NCC recognize a target cell ligand which is found on both protozoan and tumor cells. This may provide an explanation as to how NCC (including activated NK cells) recognize a vast array of targets in the absence of haplotype recognition and in spite of a diverse species of origin.
Insights
Researchers identified a novel antigen, NKTag, present on both protozoan parasites and tumor cells. This discovery explains how nonspecific cytotoxic cells (NCC) recognize diverse targets without haplotype matching.
Area of Science:
- Immunology
- Parasitology
- Cell Biology
Background:
- Natural Killer (NK) cells and nonspecific cytotoxic cells (NCC) target various cells, but the specific antigens involved remain unidentified.
- NCC may represent the fish equivalent of mammalian NK cells and IL-2-activated NK cells.
- A shared ligand recognized by NCC has been observed on both protozoan parasites and mammalian tumor cells.
Purpose of the Study:
- To purify and characterize the NK target antigen (NKTag/p46) from Tetrahymena pyriformis.
- To determine if NKTag is the ligand recognized by NCC on tumor cells and parasites.
- To investigate the expression of NKTag on different cell types and its binding specificity to NCC.
Main Methods:
- Purification of NKTag from Tetrahymena pyriformis and cDNA sequencing to deduce its amino acid sequence.
- Inhibition assays using purified NKTag to assess its effect on NCC-mediated cytotoxicity.
- Monoclonal antibody generation and binding studies to determine NKTag expression on various cell lines and tissues.
- Radioligand binding assays to evaluate the specific binding of NKTag to NCC.
Main Results:
- NKTag was purified, and its full amino acid sequence was determined, revealing it as a novel 48.17-kDa protein.
- Soluble NKTag significantly inhibited NCC lysis of mammalian tumor target cells.
- Monoclonal antibody 22A12 confirmed NKTag expression on several human tumor cell lines and limited expression on fish, mouse, and equine cells.
- Purified NKTag demonstrated saturable and specific binding to NCC.
Conclusions:
- NCC recognize a shared target cell ligand, NKTag, which is expressed on both protozoan parasites and mammalian tumor cells.
- NKTag provides a molecular basis for the broad target recognition by NCC, independent of haplotype.
- This finding offers a potential explanation for how NCC identify diverse targets across species and in the absence of specific immune recognition mechanisms.