Screening,Verification And Functional Characterization Of The LPS Interacting Proteins MBL、Fetuin-A In Yellow River Carp(Cyprinus Carpio)

Lipopolysaccharide（LPS）is a component of the cell wall of Gram-negative bacteria,which can directly or indirectly cause many pathophysiological processes such as fever,metabolic changes,immune dysfunction,multiple organ damage,shock and even death.In mammals,Toll-like receptor 4（TLR4）is the a pattern recognition receptor that mediates LPS responses,and the TLR4/CD14 signaling pathway is an important pathway that mediates LPS-induced inflammatory responses.However,the lack of important signaling molecules such as MD2,CD14 and TRAM in fish,and whether TLR4 recognizes LPS is still controversial.In our previous study,we found that LPS could induce TLRs expression response in Yellow River carp,among which TLR4,TLR5 and TLR21 are the most significant.It is the premise of elucidating the response mechanism of fish to LPS to find out which LPS binding proteins are in Yellow River carp and the interaction network of these proteins.Therefore,affinity chromatography,co-immunoprecipitation and LC-MS/MS were used to identify and verify the proteins that interact with bacterial LPS in the serum of Yellow River carp.And the protein complexes and protein interaction networks that mediate LPS response were preliminarily studied.The main results are as follows:1.Identification of interaction protein between LPS and serum of Yellow River carpBy using affinity chromatography and mass spectrometry,21 serum proteins of Yellow River carp interacting with LPS were identified.On this basis,through the analysis of Gene ontology（GO）annotation and KEGG Pathway,we found that the two signaling pathway related proteins MBL and Fetuin-A may interact with LPS.2.Analysis of interaction between LPS and MBL and between LPS and Fetuin-AIn order to further verify the interaction between LPS and MBL and Fetuin-A,Complete c DNA of MBL and Fetuin-A in Yellow River carp were obtained by RT-PCR and RACE.The full-length c DNA of MBL was 961 bp,with a 738 bp open reading frame encoding a 245 amino acids protein,a 136 bp5?-untranslated region,and a 87 bp 3?-untranslated region.The predicted Mw is 26.24 k Da and the p I is6.11 of the protein.The deduced amino acid sequence of MBL contained a signal peptide and a CRD domain with four conserved disulfide-bonded cysteine residues（Cys150-Cys235,Cys222-Cys243）and a conserved motif EPN-WND that affects the specificity of carbohydrate binding.Two of the transcripts of Fetuin-A gene,Fetuin-AL and Fetuin-AS,were successfully obtained.Fetuin-AL has an insert of 1606 bp,including 30 bp of the 5?-untranslated region,an opening reading frame of 1395 bp encoding a 464 amino acids protein,and 181 bp of the 3?-untranslated region.Fetuin-AS has an insert of 1115 bp,including 30 bp of the 5?-untranslated region,an opening reading frame of 903 bpencoding a 300 amino acids protein,and182 bp of the 3?-untranslated region.Compared with Fetuin-AS,Fetuin-AL added a 164 amino acid fragment,including three HRPGHGPP repeats and four AGRDPKDKTPDLRGHPEH repeats.The predicted Mw is 51.62 k Da and the p I is 6.58 of the Fetuin-AL.The predicted Mw is 33.48 k Da and the p I is 5.77 of the Fetuin-AS.The deduced amino acid sequence contains one signal peptide and two cystatin like domains.Then,the recombinant MBL（r MBL）and Fetuin-A（r Fetuin-AS）were purified from Escherichia coli BL21（DE3）,and polyclonal antibody obtained by immunizing mice with recombinant protein.Under the condition of obtaining r MBL and polyclonal antibodies,it was found that r MBL bind to LPS with a concentration-dependent manner using the method of ELISA,and Western blot results showed that r MBL can bind to 6 kinds of Gram-negative bacteria（Aeromonas hydrophila,Aeromonas Veronii,Vibrio anguillarum,Vibrio parahaemolyticus,Pseudomonas fluorescens and Escherichia coli）.And r MBL can agglutinate the above 6 pathogenic bacteria in a Ca²⁺-dependent manner.Under the condition of obtaining r Fetuin-AS and polyclonal antibodies,it was found that r Fetuin-A bind to LPS with a concentration-dependent manner using the method of ELISA,and Western blot results showed that r Fetuin-AS can also bind to 6 kinds of Gram-negative bacteria（A.hydrophila,A.Veronii,V.anguillarum,V.parahaemolyticus,P.fluorescens and E.coli）.These results show that LPS can interact with MBL and fetuin-A,which is consistent with the results of affinity chromatography and mass spectrometry.3.Regulation of LPS on MBL,fetuin-A,TLR4 and TLR5 expressionFirst,the MBL and Fetuin-A transcripts in different tissues were analyzed using the method of q RT-PCR.The results showed that MBL m RNA transcripts could be detected in all tested tissues,with the highest expression in the liver,moderately level in heart,blood,kidney,muscle,spleen,intestine,Brain,skin and gill,and relatively lower level in head kidney.Fetuin-A m RNA transcripts could be detected in the11 tested tissues,with the highest level in liver,higher level in muscle,blood,skin and heart,relatively lower level in kidney,head kidney,brain,spleen and intestine,the lowest level in gill.Furthermore,After infected by Aeromonas hydrophila and LPS in Yellow River carp,the expression levels of MBL,fetuin-A,TLR4 and TLR5 in liver,spleen and head kidney were detected,the results showed that the expression levels of these genes were up-regulated in varying degrees.4.Protein interaction network in LPS responseBy using CO-immunoprecipitation and LC-MS/MS techniques,30 proteins interacting with MBL and 18 proteins interacting with fetuin-A were identified.On this basis,through the analysis of Gene ontology（GO）annotation and KEGG Pathway,the protein interaction network of LPS were analyzed.In conclusion,in this study,the Yellow River carp was used as the experimental fish,we selected MBL and fetuin-A,which have interaction with bacterial LPS and may participate in LPS recognize,and systematically studied their molecular characteristics,tissue distribution,expression response to bacteria and immune function.The results are of great significance to reveal the role of MBL and fetuin-A in the immune defense of Yellow River carp.The results also provide an important scientific basis for the prevention and control of bacterial diseases in fish.