The Localization Of Chicken Rab22a In Cells And Its Relationship With MHC-related Molecules

The processing and presentation of antigens by major histocompatibility complex(MHC)molecules is an important process for initiating immune response.The simplest molecular structure of avian MHC is to study the ’model’ of MHC molecules,which mainly includes MHC class I and MHC class II and their partner invariant chain(Ii).Rab22 a is an important small GTPases protein located in endosome pathway and plays an important role in antigen presenting molecules(MHC Ⅰ and CD1a)transport and regulation,antigen uptake and maintenance of endosome morphology.At present,the structure,localization characteristics and relationship with MHC-related molecules of avian Rab22 a are still unclear.In this study,chicken Rab22a(chicken Rab22 a,c Rab22a)and MHC Iα and MHCⅡβ and Ii were used as the research objects,and the following three aspects were mainly studied.First,the localization of c Rab22 a in cells was clarified.Firstly,the Rab22 a gene sequences of chicken and mouse were amplified by PCR and inserted into pm Cherry-C1 to construct the corresponding eukaryotic recombinant plasmids.Secondly,the constructed recombinant plasmids were transfected into mouse dendritic cell line DC2.4 and mouse macrophage line RAW264.7,respectively.Finally,the intracellular localization of c Rab22 a was observed by immunofluorescence with antibodies against early and late endocytic marker proteins EEA1 and LAMP1,respectively.The results showed that the recombinant plasmid pm Cherry-C1-c Rab22a/m Rab22 a was successfully constructed.The recombinant plasmids containing c Rab22 a and m Rab22 a could be co-localized with EEA1 and LAMP1 in DC2.4 and RAW264.7 cells,indicating that Rab22 a in chickens and mice had similar localization characteristics and could be localized in the early and late endosomes of cells.Second,the protein structure of c Rab22 a was analyzed and the key domains and amino acids affecting its intracellular localization were identified..Firstly,the protein structure of c Rab22 a and m Rab22 a was compared and analyzed by biological software,and the protein3 D model of c Rab22 a was constructed by inputting SWISS-MODEL.Secondly,the key domain of c Rab22a(such as corner region)and amino acids were mutated and inserted into pm Cherry-C1 vector to construct eukaryotic recombinant plasmids containing mutants.Then these plasmids were transfected into 293 T cells to observe the intracellular localization.Finally,c Rab22a(RFP/c Rab22a)containing red fluorescence and its mutant recombinant plasmids were co-transfected into 293 T cells with Ii(RFP/c Ii)containing green fluorescence for intracellular localization.The results showed that the structure of chicken and mouse Rab22 a was highly homologous(95.4%).Nine mutants(four domain mutants and five key amino acid mutants)were successfully constructed.The mutant Switch Ⅰ domain,Switch Ⅱ domain,Ser41 of Switch I and Tyr74 of Switch II cannot be localized in the endosome but in the whole cell.Comparative analysis findings,these two amino acids are highly conserved among species,indicating that the key amino acids affecting Rab22 a intracellular localization and co-localization with Ii are the 41 st and 74 th amino acids in the Switch structure.Third,the interaction between chicken Rab22 a and MHC molecules was clarified at the gene and protein levels.Firstly,pm Cherry-C1-c Rab22 a was transfected into HD11,DT40 and DF-1 cells to overexpress c Rab22 a gene.RT-PCR was used to detect the gene transcription levels of c Rab22 a,Mhc Iα,Mhc IIβ and Ii.Secondly,si RNA was transfected into HD11 for c Rab22 a gene silencing,and RT-PCR was used to detect the expression of related genes.Finally,the interaction between c Rab22 a and MHC Iα and Ii molecules was detected by immunoprecipitation.The results showed that the expression levels of c Rab22 a in the three cells were increased by 55,4.2 and 88 folds,the Mhc Iα and Mhc IIβ genes were significantly up-regulated by 50%-70%(P<0.01),but had no effect on the transcription level of Ii gene(P>0.05)in HD11 cells.The transcription levels of Mhc IIβand Ii were not affected in DT40 cells(P>0.05),but the Mhc Iα gene was significantly down-regulated by 50%(P<0.01).Compared with the control group,si RNA could down-regulate the expression levels of c Rab22 a in HD11 and DT40 cells to 35% and 67%,respectively and significantly down-regulate the transcription levels of Mhc Iα,Mhc IIβand Ii in HD11 cells to 53.7%,64.3% and 37.0%(P<0.01).Immunoprecipitation results showed that c Rab22 a could not bind to MHC Iα and Ii.It is suggested that the effect of c Rab22 a on MHC molecules is indirect rather than direct.In summary,c Rab22 a in chicken and mouse has similar structure and can be located in early and late endosomes,which is determined by the Ser41 and Tyr74 amino acids of Switch.Overexpression and silenced of c Rab22 a gene affect the transcription level of Mhc-related gene in immune cells,and this effect is indirect rather than direct.These provide new data for further study of the relationship between Rab22a and MHC.