NLRC3L Inflammasome Related Genes In The Spotted Sea Bass(Lateolabrax Maculatus):Molecular Structure And Expression Profiling Analysis

OneInflammasome is a kind of multi-protein complex in the cell,which can mediate the activation of cysteine aspartate specific protease-1(Caspase-1)to promote the maturation and secretion of pro-inflammatory cytokines interleukin-1β(IL-1β)and IL-18;at the same time,activated Caspase-1 can trigger cell pyrolysis mechanism.The inflammasome is mainly composed of three parts: recognition receptor,apoptosis-associated speck-like protein containing CARD(ASC)and effector molecule pro-caspase-1.The inflammasomes found in current research include NLRP1,NLRP3,NLRC4(IPAF),NLRP6,NLRP7,NLRP12,AIM2 and IFI16.When stimulated,the innate immune system activates and stimulates the inflammasome through the pattern-recognition receptor(PRR)on the cell surface,which in turn triggers the downstream immune response.In this study,the receptor NLRC3 L,ASC and two Caspase-1 genes of Lateolabrax maculatus were identified and characterized.The NLRC3 L,ASC and two Caspase-1 gene sequences of spotted sea bass were obtained by RACE-PCR technology.The c DNA sequence of NLRC3 L is 3755 bp in length,of which the 5’-untranslated region(UTR)is 108 bp,3’-UTR is 320 bp,and the open reading frame(ORF)is 3327 bp,encoding 1108 amino acids.The molecular weight(MW)of the protein is 125 k Da,and the theoretical isoelectric point(PI)is 5.71.Domain analysis showed that the seabass NLRC3 L has a FISNA domain(fish-specific NACHTrelated domain),a NACHT domain(nucleotide binding oligomerization domain),four LRR domains(leucine-rich repeat domains)and a B30.2(PRY / SPRY)domain.The full length of ASC c DNA sequence is 827 bp,5’-UTR is 59 bp,3’-UTR is 150 bp,ORF is 618 bp,which encodes 205 amino acids.The MW of the protein is 23 k Da,and the PI is 5.86.The domain analysis showed that the spotted sea bass ASC has a PYD domain(pyrin domain)and a CARD domain(caspase recruitment domain).There are two Caspase-1 genes in spotted sea bass,named Caspase1-1 and Caspase1-2.Their c DNA sequence lengths are2230 and 2209 bp,respectively,in which 5’-UTR is 518 and 326 bp,3’-UTR is 716 bp,and ORF is 1185 and 972 bp,encoding 394 and 323 amino acids,respectively.The MW of the protein is 44 and 37 k Da,and the PI is 5.71 and 5.70.Domain analysis showed that Caspase1-1 has a CARD domain and a CASc domain;Caspase1-2 has a CASc domain,and they all contain the conserved active site QACRG.Real-time fluorescent quantitative PCR detection results showed that NLRC3 L,ASC and two Caspase-1 genes were constitutively expressed in the gill,intestine,thymus,brain,skin,liver,head kidney and spleen of spotted sea bass.And these four genes were found to be highly expressed in the gill,followed by the head kidney,spleen and muscle tissues,while the expression levels in the skin,intestine,liver and brain are relatively weak.This indicates that NLRC3 L,ASC and Caspase-1 may interact in the form of inflammasomes when resisting the invasion of exogenous pathogens.LPS(lipopolysaccharide),Poly(I:C)and Edwardsiella tarda(E.tarda)have effects on the m RNA expression of NLRC3 L,ASC and Caspase1-1 in spotted sea bass.In the initial stage of LPS stimulation,the expression of the three genes was upregulated and began to down-regulate after 24 hours.After E.tarda infection,the m RNAs of NLRC3 L,ASC and Caspase1-1 were up-regulated in all tissues.Compared with bacterial infection,Poly(I:C)stimulation results are not obvious.At the initial stage of stimulation,only NLRC3 L was up-regulated in the spleen;at the later stage of stimulation,ASC and Caspase1-1 were down-regulated in the tested tissues.TwoCaspases are a family of cysteine proteases in the cytoplasm.Their main function is to regulate apoptosis and mediate inflammation,which play an important role in maintaining homeostasis and innate immune defense.So far,at least 14 Caspase family members have been found in mammals.Among them,Caspase-8 is a kind of initial Caspase,which fulfils a vital role in apoptosis caused by external and internal pathways.The structure and function of Caspase-8 have been extensively studied in mammals,but the characterization of this initial Caspase is still very few in fish.In this study,the full-length Caspase-8 gene of spotted seabass was cloned.The tissue expression pattern and the changes in the expression of spotted sea bass Caspase-8 in the head kidney、spleen and gill after stimulation by LPS,Poly(I:C)and E.tarda were analyzed by RT-PCR.The study provided a basis for exploring the Caspase-8 gene of spotted sea bass in the prevention and treatment of fish infectious diseases.