An Investigation Into The Innate Immunity Regulated By Mex3A And Mex3B In Grass Carp

Mex3A and Mex3 B are two important members of the Mex3 family.Previous studies showed that they play important roles in cell polarization,asymmetric division and apoptosis.Previously,we found that fish Mex3 A and Mex3 B may participate in innate immune responses,but their functions and related mechanisms have not yet been reported.Fish is lower aquatic vertebrate and mainly relies on innate immunity to fight against various pathogenic microorganisms.To better understand the functions of Mex3 A and Mex3 B in grass carp,CiMex3 A and CiMex3 B were first cloned and identified in this study using homologous cloning methond.CiMex3A(Gene number: MW368974),which is 1521 bp in length encoding a deduced polypeptide of 506 amino acids.To investigate what role CiMex3 A may play in innate immunity,the profiles of CiMex3 A expression were analyzed following the stimulations with LPS,Z-DNA and poly(I:C).The results showed that CiMex3 A expression is up-regulated after stimulation with LPS,Z-DNA or poly(I:C),especially with intracellular poly(I:C)in CIK cells,suggesting that CiMex3 A may be involved in innate immune response in grass carp.In addition,the over-expression of CiMex3 A reduces the expression of IFN1,ISG15 as well as pro-inflammatory factors IL8 and TNFα.CiMex3 A also reduces the phosphorylation level of IRF3 after stimulation with poly(I:C),but the mechanism by which CiMex3 A acts as a negative regulator still remains obscure.The potential target of CiMex3 A was screened using immunoprecipitation assay,the results demonstrated that CiMex3 A can interact with Ci RIG-I.We also found that both CiMex3 A and Ci RIG-I are located in the endoplasmic reticulum but rarely in the lysosome under stimulation with poly(I:C).Except for the endoplasmic reticulum,CiMex3 A is hardly located in the mitochondria or endosome.However,Ci RIG-I is mainly located in the early endosome and can be transferred to the late endosome following stimulation with poly(I:C).Besides,Ci RIGI can also be located in the mitochondrion.Previous studies revealed that ubiquitination is an important activation form of RIG-I.CiMex3 A is known as E3 ubiquitin ligase,it is necessary to explore whether CiMex3 A can ubiquitinate Ci RIG-I.Our results showed that CiMex3 A significantly promotes the level of Ci RIG-I ubiquitination and induces the level of Ci RIG-I degradation upon treatment with poly(I:C).The molecular structure of CiMex3 A indicated that CiMex3 A mainly contains KH domain and RING domain.We found that CiMex3 A is dependent on its RING domain to promote the ubiquitination and degradation of Ci RIG-I,although the CiMex3 A mutant(deletion in RING domain)still interacts with Ci RIG-I,suggesting that the RING domain is essential for CiMex3 A to exert its function.The results described as above revealed that CiMex3 A promotes Ci RIG-I ubiquitination and leads to degradation of Ci RIG-I.Therefore,CiMex3 A is considered as a negative regulator of innate immunity in grass carp.CiMex3B(Gene number: MT276802.1)is 1578 bp in length encoding 525 amino acids.Just like CiMex3 A,CiMex3 B also belongs to the Mex3 family.It is interesting to figure out whether CiMex3 B also plays a pivotal role in innate immunity.We conducted an over-expression experiment of CiMex3 B in CIK cells.The results showed that CiMex3 B up-regulates the expressions of IFN1,ISG15,MX2,as well as inflammatory cytokines IL6,IL8 and TNFα following stimulation with poly(I:C),indicating that CiMex3 B is a positive regulator to participate in the innate immunity response.We further found that Ci TLR3 and Ci TRIF are important substrates of CiMex3 B.Many previous studies revealed that TLR3 is located in the endosome to perform its functions.Our results showed that both CiMex3 B and Ci TLR3 can be located in the late endosome,mitochondria and endoplasmic reticulum after poly(I:C)stimulation,but rarely located in the lysosomes,suggesting that the subcellular localizations of CiMex3 B and Ci TLR3 in cells are dynamically transferred with their functional change.Previous studies confirmed that CiMex3 B serves as a positive regulator in innate immunity.Additionally,we found the phosphorylation level of IRF3 is induced and IFN1 expression is up-regulated when the cells were co-transfected with CiMex3 B and Ci TLR3.Therefore,CiMex3 B can cooperate with Ci TLR3 to positively regulate innate immune response.In consideration of the activated form of TLR3,we investigated the effect of CiMex3 B on the ubiquitination of Ci TLR3.The results showed that CiMex3 B promotes the K63-linked ubiquitination of Ci TLR3.Our study also found that the subcellular localizations of different CiMex3 B mutants are various in CIK cells.The mutant of CiMex3B(1-220)is located in the cytoplasm,which is in consistent with that of wild-type Mex3 B.The mutant of CiMex3B(221-525)is located in the cytoplasm and the nucleus as well.CiMex3 B mutant(221-525)still can interact with Ci TLR3,but fail to promote K63-linked ubiquitination of Ci TLR3.Therefore,CiMex3 B positively regulates innate immunity by promoting K63-linked ubiquitination of Ci TLR3.Given that both CiMex3 A and CiMex3 B are involved in innate immunity,it is necessary to investigate the functional correlation between CiMex3 A and CiMex3 B.We found that both CiMex3 A and CiMex3 B share the highest level of similarity with that of D.rerio.We also found that both CiMex3 A and CiMex3 B contain similar KH and RING domains.Furthermore,co-localization analysis showed that CiMex3 A and CiMex3 B are co-located in CIK cells.The expressions of CiMex3 A and CiMex3 B are also up-regulated following stimulation with GCRV.Taken together,CiMex3 A and CiMex3 B share similar molecular structure as well as subcellular localization pattern,and both of them participate in innate immune response;however,they are involved in different signaling pathways.CiMex3 A negatively regulates innate immune pathways by promoting the ubiquitination and degradation of Ci RIG-I;CiMex3 B positively regulates the innate immune response by promoting K63-linked ubiquitination of Ci TLR3.Further investigation is still needed to determine the functional differentiation and biological significance of CiMex3 A and CiMex3 B in grass carp.