Cloning and analysis of non-specific cytotoxic cell receptor (NCCRP)-1 from common carp Cyprinus carpio L

Nonspecific cytotoxic cell receptor protein (NCCRP-1) provides an important function in target cell recognition and activation of cytotoxicity. NCCRP-1 has been cloned from common carp Cyprinus carpio L. from fish barbel by EST analysis. The isolated gene is composed of 945 bp with a 79 bp 5' UTR, 714 bp open reading frame and 152 bp 3' UTR. The predicted NCCRP-1 gene is composed of 237 amino acid residues and its predicted signal peptide is 19 amino acid residues in length. This gene has conservation of all the related domains characteristic to the NCCRP-1 gene in fish. Phylogenetic and genomic analyses showed that carp NCCRP-1 was similar to other fish orthologues. The expression of NCCRP-1 gene was constitutive in both lymphoid and non-lymphoid tissues. Furthermore, by semi-quantitative RT-PCR studies, we showed that NCCRP-1 gene expression is increased in anterior kidney challenged with Aeromonas hydrophila.     

The gene structure and expression of the non-specific cytotoxic cell receptor protein (NCCRP-1) in Atlantic cod (Gadus morhua L.)

The non-specific cell receptor protein (NCCRP-1) serves an important function in target cell recognition and activation of non-specific cytotoxic cells in teleosts. Atlantic cod NCCRP-1 was identified in a suppression-subtractive cDNA library and NCCRP-1 from Atlantic salmon, rainbow trout, Japanese medaka and fathead minnow was found deposited in the GenBank as EST sequences. The predicted amino acid sequences of these receptors contain the characteristic functional domains representing NCCRP-1, and phylogenetic analyses support the identification of five NCCRP-1 orthologues. Cod NCCRP-1 is shorter and has a different intron/exon organization from the common carp and channel catfish counterparts, but shows high extent of conservation in NCCRP-1 signature motives. Quantitative real-time PCR analyses showed that the gene expression of cod NCCRP-1 was higher in the lymphoid organs, head kidney (90-fold) and spleen (30-fold), compared to the organ with lowest expression. NCCRP-1 gene expression was not induced by in vitro treatment of head kidney cells with polyinosinic polycytidylic acid (poly I:C) or lipopolysaccharide (LPS), or by in vivo injections with poly I:C or formalin killed Vibrio anguillarum. These results show that the cod NCCRP-1 gene is differentially expressed in organs, and that gene expression is not induced by the tested treatments.     

The Antigen Receptor (NCCRP-1) on Catfish and Zebrafish Nonspecific Cytotoxic Cells Belongs to a New Gene Family Characterized by an F-Box-Associated Domain

The catfish nonspecific cytotoxic cell receptor protein (NCCRP-1) provides an important function in target cell recognition and activation of cytotoxicity. This report identifies and characterizes a zebrafish orthologue of the catfish NCCRP-1. The zebrafish NCCRP-1 cDNA contains an open reading frame that encodes a predicted protein of 237 amino acids with a MW of 27 kDa and a pI of 5.5. Sequence similarities comparisons show that the NCCRP-1 receptors from these two phylogenetically distant species share a high degree of identity. These results suggested that NCCRP-1 performs a crucial function in innate immunity in teleosts. Further, a zebrafish 17-mer peptide corresponding to the catfish NCCRP-1 antigen-binding domain inhibited (catfish) cytotoxicity toward conventional tumor target cells (HL-60). These data appeared to indicate that the zebrafish NCCRP-1 protein may function as an antigen recognition molecule and, as such, may participate in innate immunity in teleosts. A homology search of the zebrafish NCCRP-1 protein revealed that it shares a significant level of identity with another group of proteins belonging to an F-box subfamily. These proteins share an F-box domain in the N terminus (not present in NCCRP-1) and an extremely conserved C-terminal region that has been termed the F-box-associated domain (FBA). The FBA is currently of unknown function. A new gene family is proposed in this work, based on similarities in the FBA sequences with the catfish and zebrafish NCCRP-1 peptides. This new gene family includes several F-box domain-containing proteins and a predicted C. elegans protein. Received: 20 June 2001 / Accepted: 31 August 2001