Identification of a fish-specific NOD-like receptor subfamily C (NLRC) gene from common carp (Cyprinus carpio L.): Characterization, ontogeny and expression analysis in response to immune stimulation

Abstract

Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) are a large group of cytoplasmic pattern recognition receptors (PRRs), which play an important role in pathogen recognition and regulation of innate immune response. In fish, NLRs are divided into three distinct subfamilies: NLR-A resembling mammalian NODs, NLR-B resembling mammalian NALPs and fish-specific NLR-C. Presently, no data is available about the common carp NLR gene, and meanwhile the studies concerning fish NLR-C subfamily genes are relatively poor. In the present study, we cloned and characterized a novel NLRC gene (CcNLRC) from common carp. The full-length cDNA of CcNLRC was 3642 bp, with an ORF of 3078 bp encoding 1025 amino acids. CcNLRC appears to be unique to fish, consisting of a fish-specific NACHT associated (FISNA) domain, a NACHT domain, three LRR motifs and an extra B30.2 domain at C-terminus. Expression analysis revealed that CcNLRC was constitutively expressed in various healthy tissues, and during early developmental stages CcNLRC had two expression peaks (1 dpf and 24 dpf). In vivo stimulation with polyI:C and V. anguillarum showed significant up-regulation of CcNLRC expression in some immune-related tissues including liver, spleen, foregut, hindgut and skin. Additionally, in vitro study in common carp PBLs and HKLs stimulated with different ligands such as polyI:C, flagellin and PGN showed enhanced gene expression of CcNLRC. These results suggested that CcNLRC might play an important role in the innate immune defense of common carp against pathogen invasion.