Molecular Identification And Preliminary Functional Study Of TLR3 Signaling Pathway In Northeast Chinese Lamprey(Lethenteron Morii)

The innate immune system is the first line of defense for vertebrates against pathogens.Pathogens that invade vertebrates are frst identifed by the innate immune system through various pattern recognition receptors(PRRs).Pathogens recognized by PRRs are called pathogen-associated molecular patterns(PAMP),such as bacterial lipopolysaccharide and viral double-stranded RNA.PRRs recognize the pathogen and activate relevant immune cells through downstream signaling pathways,inducing immune cells to participate in inflammation and various immune responses.Toll-like receptors(TLR)is one of the important pattern recognition receptors in vertebrates.According to the type of Toll-like receptors,it can recognize bacterial lipopolysaccharides,viral double-stranded RNA,bacterial flagella and other pathogen-associated molecular patterns.After Toll-like receptor recognizes the corresponding pathogen,it signals and activates important transcription factors such as NF-kB and IRF by binding to downstream adaptor such as MyD88 and TRIF,thereby inducing the production of inflammatory cytokines and interferons and producing innate immune response.Northeast Chinese lamprey(Lethenteron morii)belongs to Cyclostomata and is one of the most primitive vertebrates.In order to study the origin and evolution of Toll-like receptors and their mediated signaling pathways in the innate immune system,we identified Northeast Chinese lamprey TLR signaling pathway related molecules and performed preliminary functional studies.In this study,polymerase chain reaction(PCR)technology was used to clone the cds sequence of LmTLR3 and its adaptor molecule Lm TRIF from Northeast Chinese lamprey Toll-like receptor family.The LmTLR3 cds sequence and Lm TRIF cds sequence were identified and analyzed by bioinformatics method.Preliminary tissue expression analysis of LmTLR3 and Lm TRIF were performed by real-time quantitative PCR.The eukaryotic expression vector of LmTLR3 and its related domains and Lm TRIF were constructed.The expression localization of LmTLR3 and Lm TRIF in Northeast Chinese lamprey was explored by immunofluorescence assay.In order to study the immune response function involved in LmTLR3,The activity of Northeast Chinese lamprey NF-kB(Lm NF-kB)promoter reporter gene was verified using dual luciferase reporter gene assay,and the relationship between LmTLR3 and its adaptor molecule Lm TRIF and Lm NF-kB were explored.In addition,as a comparison,the relationship between members of Lm TLR1 subfamily and its adaptor molecule Lm MyD88 and Lm NF-kB was explored.The research results obtained are as follows: 1.Cloning,identification,tissue expression and subcellular localization of LmTLR3The LmTLR3 cds sequence of Northeast Chinese lamprey was 2670 bp in length and encoded 889 aa.The LmTLR3 amino acid sequence included a signal peptide,13 LRR domains,a transmembrane domain and a TIR domain.Multiple sequence alignment results showed that the LmTLR3 TIR domain contained three conserved regions.The phylogenetic tree showed that mammalian TLR3 and reptile TLR3 were clustered into one branch,and then they were clustered with the teleost fish TLR3,and finally with the LmTLR3.LmTLR3 was between vertebrates and invertebrates.Northeast Chinese lamprey TRIF cds sequence was 1242 bp in length,encoding 413 aa,and had a relative molecular mass of 46.99 k D.Preliminary real-time quantitative PCR results showed that the relative expression of LmTLR3 in the skin was highest,while the relative expression of liver,brain,intestine,and gill was higher.The results of cellular immunofluorescence showed that LmTLR3 could be expressed in HEK293 T cells,and LmTLR3 was located in the cytoplasm and mainly distributed around the nucleus.LmTLR3 LRR truncated protein were widely distributed in cytoplasm.2.Cloning,identification and subcellular localization of Lm TRIFThe Lm TRIF amino acid sequence contained a TIR domain,without the N-terminal domain of a typical TRIF.The phylogenetic tree showed that Mammalian TRIF was clustered with birds and reptiles,and then they were clustered with the teleost fish TRIF,and finally with Lm TRIF into a large branch.Lm TRIF had the highest homology with the teleost fish TRIF.Preliminary real-time quantitative PCR results showed that the relative expression of Lm TRIF in the gill was highest,while the relative expression of intestine,brain,and skin was higher.The results of cellular immunofluorescence showed that Lm TRIF could be expressed in HEK293 T cells and EPC cells,and it was located in the cytoplasm.Lm TRIF in EPC cells distributed in an aggregated form.3.Verification of Lm NF-kB promoter reportor gene activityLm NF-kB promoter sequence contained CREB,Oct-1,GATA-1,AP-1,NF-kappa B,IRF-1,Sp-1,c-Jun,c-Fos and other transcription factor binding sites,and contained TATA box transcription core elements.Cp G islands with a CG content greater than 50% and a length of 196 bp were found at the sequence of 1207-1402 bp.The dual luciferase reporter system was used to detect Lm NF-kB promoter reporter gene in HEK293 T and EPC cells had significantly more activity than the control group.And after LPS stimulated HEK293 T cells,Lm NF-kB promoter reporter gene was significantly more active than Lm NF-kB promoter reporter gene that was not stimulated by LPS.4.Relationship between LmTLR3 and Lm TRIF and Lm NF-kBThe results of the dual luciferase reporter assay showed that transient transfection of Lm TRIF could significantly activate Lm NF-kB promoter activity.Under the premise of overexpression of Lm TRIF,after co-transfection with LmTLR3,with the increase of the amount of LmTLR3,the downstream Lm NF-kB activity gradually decreased,but there was no significant difference compared with the control group without LmTLR3.5.Identification and subcellular localization of Lm MyD88Northeast Chinese lamprey MyD88 gene cds sequence was previously cloned by our laboratory,and its encoded amino acid sequence contained the typical domains of the MyD88 protein: the N-terminus death domain and the C-terminus TIR domain,and contained three conserved sites of the death domain and three conserved regions of the TIR domain.Multiple sequence alignment results showed that the similarity of MyD88 amino acid sequence between Northeast Chinese lamprey and other species was 47%–54%,of which the similarity of the DD domain was 42%–51%,and that of the TIR domain was 61%–66%.The phylogenetic tree showed that the MyD88 of Mammalia,Aves,and Reptilia were clustered on a branch,Osteichthyes was clustered on a branch,and lampreys and other vertebrates were clustered on a big branch.Meanwhile,MyD88 had a high homology between lampreys and Osteichthyes.The phylogenetic tree of MyD88 was in line with the evolutionary position of species,and MyD88 of Northeast Chinese lamprey was between vertebrates and invertebrates.The results of cellular immunofluorescence confrmed the location of Northeast Chinese lamprey MyD88 in cells.Under confocal microscopy,Northeast Chinese lamprey MyD88 was clustered in HEK 293 T cells and expressed in the nucleus and cytoplasm.However,although Northeast Chinese lamprey MyD88 overlapped with markers of mitochondria and endoplasmic reticulum,it had no specifc co-localization with these markers.6.Relationship between Lm TLR1 subfamily members and Lm MyD88 and Lm NF-kBThe results of the dual luciferase reporter assay showed that overexpression of Lm MyD88 could significantly promote Lm NF-kB promoter reporter gene activity.The Northeast Chinese lamprey LRR domain of Lm TLR 2d significantly promoted the activity of the Lm NF-kB promoter,while the Lm TLR 2d and Lm TLR 2d TIR domain did not significantly affect the activity of the Lm NF-kB promoter.Lm TLR 2d,Lm TLR 2d LRR domain,Lm TLR 2d TIR domain,and Lm MyD88 co-transfection group significantly promoted the activity of Lm NF-kB promoter compared with their respective transfection groups.Moreover,these co-transfection groups had a certain promotion effect on the activity of the Lm NF-kB promoter compared with the Lm MyD88 alone transfection group.Transfection of Lm TLR 14 d,Lm TLR 14 d TIR domain,and Lm TLR 14 d LRRCT domain alone did not significantly affect the activity of Lm NF-kB promoter.However,when they were co-transfected with Lm MyD88,they significantly promoted the activity of the Lm NF-kB promoter compared with their respective transfection groups.In addition,the Lm TLR 14 d and Lm TLR 14 d LRRCT domain co-transfected with Lm MyD88 significantly promoted the activity of the Lm NF-kB promoter compared with the Lm MyD88 alone transfected group.To sum up,in this study,LmTLR3 and its adaptor molecule Lm TRIF were identified for the first time.Among them,LmTLR3 had conserved domains and sites.Lm TRIF lacked the N-terminal classical domain and had certain sequence specificity.Studies have shown that LmTLR3 and its adaptor molecule Lm TRIF could be expressed in cells and were localized in the cytoplasm.This study verified the activity of Lm NF-kB promoter reporter gene activity,and found that Lm TRIF could promote Lm NF-kB activity.In addition,it was identified that Lm MyD88,another important adaptor molecule of the TLR signaling pathway in Northeast Chinese lamprey,had conserved domains and functional sites,it could be expressed in cells in a morphology similar to other vertebrates MyD88,and could promote the downstream immune transcription factor NF-kB activity.This study also found that Lm TLR1 family members Lm TLR 2d,Lm TLR 14 d and Lm MyD88 may cooperate to activate Lm NF-kB.In this study,the characteristics and interrelationships of molecules related to the LmTLR3 signaling pathway in Northeast Chinese lamprey were explored,as a comparison,was conducted on whether Lm TLR1 can activate Lm NF-kB through the Lm MyD88 adaptor molecule,which laid a foundation for the study of the innate immune system of primitive vertebrates.