Scallop NEMO interacts with IKK1 and regulates host innate immunity against PAMPs

Abstract

The IκB kinase (IKK) complex, which is essential for the activation of the transcription factor NF-κB, is composed of IKK1 (IKKα), IKK2 (IKKβ), and the key regulatory protein NF-κB essential modulator (NEMO). However, there are few studies on the function of NEMO in invertebrates. In this study, we obtained the complete coding sequence of the NEMO gene of the Zhikong scallop Chlamys farreri and studied its response to pathogen-related molecular pattern (PAMP) stimulation and its interaction with the IKK1 protein. Sequence analysis revealed that Chlamys farreri NEMO (CfNEMO) encodes 778 amino acids, and the encoded protein contains a typical NEMO domain, two coiled coils, a putative leucine zipper motif, and a C-terminal zinc finger domain. Phylogenetic relationship analysis showed that CfNEMO belongs to the NEMO family of mollusks in invertebrates. Real-time quantitative PCR showed that CfNEMO transcripts were expressed in all tissues tested in scallops, with the highest expression in the gills. Furthermore, the expression level of CfNEMO in the gills was significantly induced after lipopolysaccharide, peptidoglycan, and polyinosinic–polycytidylic acid challenge. Finally, the results of co-immunoprecipitation showed that CfNEMO can interact with IKK1 protein (IKKα/IKKβ family member). And by constructing truncated mutants, we found that the interaction between the two proteins is mediated by the NEMO domain of CfNEMO and the SDD (scaffold dimerization domain) domain of IKK1. In conclusion, our results lay the foundation for in-depth understanding of the innate immune function of invertebrate NEMO and the formation of the IKK complex. Our findings provide valuable information on disease control and management in scallop farming.