Identification and functional analysis of inhibitor of NF-κB kinase (IKK) from Scylla paramamosain: The first evidence of three IKKs in crab species and their expression profiles under biotic and abiotic stresses

Abstract

IKK (inhibitor of NF-κB kinase) is the critical regulator for NF-κB (nuclear factor-κB) pathway against pathogenic invasion in vertebrates or invertebrates. However, the IKK from crab species has not yet been identified. In the present study, three full-length cDNA sequences of IKKs from mud crab Scylla paramamosain, designated as SpIKKβ, SpIKKε1 and SpIKKε2, were firstly cloned through RT-PCR and RACE methods. This is also the first report about the identification of two IKKε genes in mud crab and even in crustaceans. The SpIKKβ cDNA was 2824 bp in length with an open reading frame (ORF) of 2382 bp, which encoded a putative protein of 793 amino acids (aa). The ORF of two SpIKKε isoforms, SpIKKε1 and SpIKKε2, were 2400 bp and 2331 bp in length encoding 799 aa and 776 aa, respectively. The crucial conserved residues and functional domains, including the kinase domains (KDs) and leucine zipper (LZ), were identified in all SpIKKs. Phylogenetic analysis suggested that SpIKKβ was classified into the IKKs class while SpIKKεs could be grouped into the IKK-related kinases class. The qRT-PCR analysis showed that three SpIKKs were constitutively expressed in all tested tissues and the highest expression levels of SpIKKβ and SpIKKεs were all in hemocyte. The gene expression profiles of SpIKKs were distinct when crabs suffered biotic and abiotic stresses including the exposures of Vibrio alginolyticus, poly (I:C), cadmium and air exposure, suggesting that the SpIKKs might play different roles in response to pathogens infections, heavy metal and air exposure. Moreover, IKKs from mud crab can significantly activate mammalian NF-κB pathway, suggesting the function of IKKs might be evolutionally well-conserved. Results of the RNAi experiments suggested that SpIKKs might regulate the immune signaling pathway when hemocytes were challenged with V. parahemolyticus or virus-analog poly (I:C). All of these results indicated that the obtained SpIKKs might be involved in stress responses against biotic or abiotic stresses, and it also highlighted their functional conservation in the innate immune system from crustaceans to mammals.