Mapping of the Epitope Recognized by Non‐Specific Cytotoxic Cells: Determination of the Fine Specificity Using Synthetic Peptides

Abstract

NCC recognize a conserved target cell antigen (NKTag) expressed on protozoan parasites and on transformed tumour cells. In the present study, synthetic peptides corresponding to N-terminal, C-terminal and internal NKTag(deduced) amino acid sequences were tested for binding and inhibition of NCC lysis of sensitive target cells. A 20-mer peptide equivalent to amino acids (aa) nos. 55-74 specifically inhibited NCC lysis of human EBV transformed target cells (IM-9). Inhibitory effects were nonreversible and concentration dependent, and 30 min pre-incubation produced optimum inhibition. The inhibitory 20-mer peptide was truncated into 17, 14, 10, 9 and 6-mer peptides and tested for inhibition of cytotoxicity. All produced almost complete inhibition except the 6-mer which had no activity. The NKTag sequence required for NCC binding (minimally) consisted of seven amino acids [aa nos 68-74 (ARG-ASN-LEU-THR-PHE-ILE-LEU-)]. The specificity of inhibition and the distribution of target cells expressing NKTag was determined. A 14-mer peptide composed of aa nos 61-74 inhibited lysis of HL-60, IM-9, DAUDI, YAC-1, U937 and NC-37 target cells. Flanking peptides (aa nos 35-54 and 75-94) were negative. Biotinylated aa nos 61-74 bound to NCC effector cells. The recognition requirements for aa sequence versus aa content were determined. Randomization of the aa in the cognate 9-mer obliterated the inhibitory effects. The 17-mer (cognate) synthetic peptide inhibited conjugate formation between NCC and IM-9 targets. These data demonstrate that NCC recognize a conserved antigen determinant on susceptible target cells consisting of a minimum of 7-9 amino acids in the N-terminal region of NKTag.

Comparison of select innate immune mechanisms of fish and mammals

“…Immunoprecipitation experiments using mAb5C6 (anti‐NCC antibody) resulted in the identification of a protein of 34 kDa named NCC receptor protein 1 (NCCRP‐1) [97–100]. The engagement of NCCRP‐1 by NKTag was found to be responsible for the recognition and killing of target cells by NCCs [95,96,98,101]. Further analysis, using synthetic peptides to mimic the ligand demonstrated that the ligand‐binding region of NCCRP‐1 was located between amino acids 104 and 119 [102].…”

Health and Environment in Aquaculture

“…However, the best characterization of this first step was achieved by the finding of the non-specific cytotoxic cell receptor protein-1 (NCCRP-1) by the generation and selection of a monoclonal antibody (5C6) that completely blocked catfish NCC activity (Evans et al, 1988;Jaso-Friedmann et al, 1988. This receptor showed important features: 1) the 5C6 antibody recognizes the NCCs of most studied fish and even the mammalian NK and LAK cells, demonstrating its conservation (Cuesta et al, 2005a;Evans et al, 1988;McKinney & Schmale, 1997); 2) the NCC activity is blocked by the 5C6 antibody (Evans et al, 1988;Iwanowicz et al, 2004;Jaso-Friedmann et al, 1988; 3) the NCCRP-1 is a 32-34 kDa protein found in the membrane of NCCs and binds to a 42 and 46 kDa from the tumor targets and protozoan that they kill, respectively (Evans et al, 1996;Jaso-Friedmann et al, 1997aLester et al, 1994); and 4) it is a type-III membrane protein and its activation led to tyrosine and serine phosphorylation and uses the Jak-STAT signalling pathway (Evans et al, 1999;Jaso-Friedmann et al, 1995. After binding to the target cell, mammalian NKs and fish NCCs share the same killing mechanisms including granule-dependent (release of perforin and granzymes) and granule-independent (Fas/FasL system) (Cuesta et al, 2003a;Hogan et al, 1999;Jaso-Friedmann et al, 2000;Shen et al, 2002).…”

“…A second study determined that catfish NCC were able to bind and kill 50-60% of Tetrahymena pyriformis after 10 h of coincubation (Graves et al, 1985b). Furthermore, NCC binding to xenogeneic tumor cells and Ichthyophthirius multifiliis or Tetrahymena pyriformis parasites shared the same antigen, that in the case of parasites, consist on a 46 kDa (Evans et al, 1998a(Evans et al, , 1996Graves et al, 1985a;Jaso-Friedmann et al, 1997b;Lester et al, 1994). In another study, gilthead seabream specimens were parasitized with the enteric Enteromyxum leei parasite (Cuesta et al, 2006).…”

The teleost acute-phase inflammatory response and caspase activation by a novel alarmin-like ligand

“…These substances (alarmins) are released from damaged cells, they are not microbial ligands, and they are constitutively expressed by many histological cell types throughout the animal. One member of this group (HMGB-1) [7] is secreted by macrophages (among other types of cells) and has been reported to have cytokine-like activities. HMGB-1 also produces promiscuous binding toTLR-4 and receptor for advanced glycation endproducts on epithelial and endothelial cells [8 -11].…”