Screening And Preliminary Identification Of Interaction Protein Between Toxocara Canis Mucin 1 And Mouse Macrophage

Toxocariasis is a serious zoonosis caused by the Toxocara canis（T.canis）parasitizing in humans and various animals.Human and animal infections of toxocariasis are usually food-borne infections,which is caused by accidently ingestion of infectious eggs of T.canis or the undercooked meat,or the infectious larvae in internal organs.The infective larvae of T.canis can only develop into adults in small intestine of the final host.Instead of developing into adults,the infective larvae migrate to other tissues and organs in human or other paratenic hosts therefore causes toxocariasis.The clinical types of this disease can be divided into Visceral larva migrans（VLM）,Ocular larve migrans（OLM）,Covert toxocariasis（CT）and Neurological toxocariasis（NT）and so on.Mucin（MUC）is a kind of high molecular weight glycoprotein comprising mucopolysaccharide,mainly exists in epithelial tissues and possesses PTS structure.PTS structure contains abundant serine（P）,threonine（T）and proline（S）residues which are in highly glycosylation and rich in asparagine at the N term and cysteine at the C term.MUC has diverse biological functions in the process of cell adhesion,differentiation,immune response,tumor formation,and cell signaling and is mainly divided into membrane-bound mucin and secretory mucin.A transmembrane domain was located at the carboxyl end of membrane-bound mucin,which can ligate itself to plasma membrane（such as MUC-1,MUC-3,MUC-4,MUC-16,MUC-17）in order to facilitate cell to cell/protein to protein incorporation and signal transduction and protein stability.Secretory mucin（such as MUC-2,MUC-5AC,MUC-5B）can be packaged into secretion goes into vesicles to participate in intracellular signal transduction.In mammalian tissues,mucin provides viscous and physical protection for the epithelial surface,and carries a large amount of glycan for cell transport and adhesion.Mucin-1 is an important member of the mucin family,which plays a role in cell identification,cell protection and viscosity maintain.Tc-MUC-1 is an excretory-secretory protein（ESP）produced by T.canis larvae in the paratenic host,and mainly participates in body immunity and plays an important role in host immune evasion.There are few studies on Tc-MUC-1 at home and abroad at the moment.In this study,we cloned T.canis mucin 1 gene（Tc-muc-1）,screened the Tc-MUC-1proteins interacted with mouse macrophage by His pull-down and mass spectrometry,and confirmed the interplayed proteins with Co-immunoprecipitation technology.The main experimental results are summarized as follows:1.Molecular cloning and sequence analysis of Tc-muc-1 geneTotal RNA of T.canis larva was used as template,the coding sequence（CDS）of Tc-muc-1 gene was cloned and sequence analyzed.The results showed that the CDS is in length of 531bp,coding 176 animo acids in total.Functional domain analysis showed Tc-MUC-1 comprising a Mucin domain which consists of eleven STSSSSA repeat sequences and two Sh KT domains which contain 36 animo acids.GO analysis indicated it has the binding ability of protein and metal ions.Multiple sequence alignment revealed that Tc-MUC-1 and other 4 mucins（Tc-MUC-2–Tc-MUC-5）all possess 2Sh KT domains,phylogenetic tree analysis showed Tc-MUC-1 has a close evolution relation with Pristionchus pacificus and Caenorhabditis elegans,but distant from Ancylostoma caninum and Strongyloides ratti.2.Construction and expression of prokaryotic expression vector Tc-MUC-1The Tc-muc-1 gene CDS was cloned and the Tc-muc-1/p Cold TF prokaryotic expression vector was constructed.The sequencing result showed the sequence length inserted into p Cold TF was 546 bp.Transformed the recombination plasmid into Escherichia coli Transetta（DE3）and the positive colonies expressed soluble recombinant protein after IPTG induction,which is 75 k Da.Optimize the conditions of induction expression,the highest expression can be obtained as IPTG was added to a final concentration of 0.6 m M when cell OD₆₀₀reaches 1.0,and incubation at 15?C for24 h.Purification of recombinant protein using Ni-NTA showed that when using 250m M imidazole for protein elution will get the best result and high concentration of recombinant protein.3.Screen of interaction proteins between Tc-MUC-1 and mouse macrophagesUsing His pull-down technology,the induced recombinant protein Tc-MUC-1 was used as the bait protein for pull-down for in vitro fishing mouse macrophages（RAW264.7）total proteins which interacted with Tc-MUC-1.After identification by mass spectrometry,219 candidate Tc-MUC-1 interaction proteins were obtained.According to GO functional annotation analysis of the identified interaction proteins,it was found that Tc-MUC-1 interaction proteins mainly exist in extracellular bodies,vesicles,extracellular regions and other cellular components,which participate in various biological processes such as protein process in ribosomes,phagosomes and endoplasmic reticulum,synthesis and metabolism of organic nitrogen compounds,and cytoamide metabolism.KEGG function annotation analysis found Tc-MUC-1interaction proteins have potential molecular function in RNA binding,heterocyclic compound binding and organic cyclic compound binding.Interacting protein network construction showed that Tc-MUC-1 mediated interaction proteins mainly participate in biological processes such as intracellular carbon metabolism,amino acid biosynthesis and endoplasmic reticulum protein processing.4.Verification of interaction protein between Tc-MUC-1 and mouse macrophagesCo-Immunoprecipitation（Co-IP）was used to verify the interplay between proteins,and according to mass spectrometry identification and bioinformatics analysis,6candidate Tc-MUC-1 interaction proteins were selected as research subject,which are PRDX1,PFN1,CFL1,AKR1B3,FABP5 and ARHGDIA.First,Tc-muc-1 gene and CDS of mouse Prdx1,pfn1,Cfl1,Akr1b3,Fabp5,Arhgdia gene were cloned,and constructed eukaryotic expression vector Tc-muc-1/p CDNA3.1-Flag,pfn1/p CMV-HA,Akr1b3/p CMV-HA,Cfl1/p CMV-HA,Prdx1/p CMV-HA,Fabp5/p CMV-HA and Arhgdia/p CMV-HA.The expressed protein was collected and was performed Co-IP after Tc-muc-1/p CDNA3.1-Flag plasmid was transfected with each of the following plasmid,pfn1/p CMV-HA,Akr1b3/p CMV-HA,Cfl1/p CMV-HA,Prdx1/p CMV-HA,Fabp5/p CMV-HA and Arhgdia/p CMV-HA,separately,in HEK293T cells.The results showed Tc-MUC-1 was sufficient to bind CFL1 and FABP5,but couldn’t bind PRDX1,PFN1,AKR1B3 and ARHGDIA.This indicated that Tc-MUC-1 can interact with CFL1and FABP5 proteins in mouse macrophages.It is speculated that Tc-MUC-1 plays an important role in inducing host immune response when T.canis infects the hosts.