Related Experiment Videos
NCCRP-1: a novel receptor protein sequenced from teleost nonspecific cytotoxic cells
L Jaso-Friedmann1, J H Leary, D L Evans
1Department of Medical Microbiology, College of Veterinary Medicine, University of Georgia, Athens 30602, USA.
Abstract:
The recognition structure responsible for binding to conventional antigen on target cells has not previously been described for nonspecific cytotoxic cells (NCC) or for mammalian natural killer (NK) cells. Although several biochemical pathways may be available for initiation of the lytic cycle in NCC, evidence presented indicates that initial contact with a tumor cell or protozoan parasite is facilitated by recognition of a target antigen by a membrane protein of Mr 34,000 on NCC (NCC receptor protein, NCCRP-1). Binding to NCCRP-1 by monoclonal antibody 5C6, by target cell antigen or by cognate synthetic peptide initiates a signalling response leading to increased cytotoxicity. In the present study, three 20-mer microsequences were obtained from tryptic digests of purified NCCRP-1. Degenerate primers were synthesized (based on each peptide sequence) and were used for RT-PCR with mRNA purified from homogeneous NCC populations. An NCCRP-1 specific cDNA sequence was used to synthesize nondegenerate primers. These primers were used in a 5'/3' RACE PCR to obtain the entire NCCRP-1 specific cDNA. A deduced aa sequence consisted of 235 aa with a derived molecular weight of 30,628 Da. NCCRP-1 is proline rich (9%), has two glycosylation sites and 18% of all amino acids are potential phosphorylation sites (serine, threonine, tyrosine). The identity of the protein was confirmed by finding the previously microsequenced peptides in the derived sequence. Homology searches revealed that NCCRP-1 is a novel protein. Northern blot analysis of mRNA content from teleost NCC, B-cells and T-cells revealed only one band in NCC preparations. Functional studies demonstrated a decrease in membrane NCCRP-1 expression and inhibition of NCC cytotoxicity following treatment with NCCRP-1 anti-sense oligonucleotides. Treatment of NCC with sense oligonucleotides had no inhibitory effects on cytotoxicity. An algorithm predicting the membrane conformation of NCCRP-1 suggests one extracellular proline-rich domain, a transmembrane portion of 15 18 aa and a cytoplasmic tail composed of a high frequency of phosphorylation sites. Current studies suggest that NCC and NCCRP-1 may participate in innate resistance functions in teleost fish.
Insights
Researchers identified a novel membrane protein, NCCRP-1, on nonspecific cytotoxic cells (NCC). This protein is crucial for target cell recognition and initiating cytotoxic responses, suggesting a role in innate immunity in fish.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Nonspecific cytotoxic cells (NCC) and mammalian natural killer (NK) cells lack a described recognition structure for target cell binding.
- Initial contact between NCC and target cells (tumor cells, protozoa) is thought to involve a membrane protein, NCC receptor protein-1 (NCCRP-1).
Purpose of the Study:
- To characterize the NCCRP-1 protein involved in NCC-mediated cytotoxicity.
- To elucidate the molecular identity and function of NCCRP-1 in teleost fish.
Main Methods:
- Purification and microsequencing of NCCRP-1.
- RT-PCR and 5'/3' RACE PCR for NCCRP-1 cDNA synthesis.
- Northern blot analysis and anti-sense oligonucleotide treatment for functional studies.
Main Results:
- The NCCRP-1 cDNA sequence was obtained, encoding a 235 amino acid protein (30,628 Da).
- NCCRP-1 is proline-rich, has glycosylation and phosphorylation sites, and is a novel protein.
- NCCRP-1 expression is specific to NCC, and its down-regulation inhibits NCC cytotoxicity.
Conclusions:
- NCCRP-1 is a novel membrane protein essential for NCC recognition and cytotoxic function.
- NCCRP-1 likely plays a significant role in the innate immune resistance of teleost fish.