Localization of Chicken Rab22a in Cells and Its Relationship to BF or Ii Molecules and Genes

SIMPLE SUMMARY: Rab22a is an important molecule in endocytosomal transport, while BF and cIi areantigen-presenting presenting molecules in poultry. The relationship between Chicken Rab22a (cRab22a) and BF/cIi is unclear. In this study, the intracellular localization of cRab22a and its key domains and amino acids were determined by gene mutation and immunofluorescence methods, then the relationship between cRab22a and BF/cIi genes and molecules was studied by RT-PCR and Co-IP methods. The results showed that the Rab22a amino acid sequences of different species are highly conserved. Positions Ser41 and Tyr74 in Switch region are key amino acids for intracellular localization. Both low expression and high expression of cRab22a genes will affect the expression of BF gene, while only low expression will affect the expression of cIi gene. The cRab22a molecule does not directly interact with the BF/cIi molecules. This study clarified the relationship between cRab22a and BF or cIi, and provided an experimental basis for further research on the intracellular transport of BF or cIi and related immune response. ABSTRACT: Rab22a is an important small GTPase protein the molecule that is involved in intracellular transportation and regulation of proteins. It also plays an important role in antigens uptake, transportation, regulation of endosome morphology, and also regulates the transport of antigens to MHC (Major Histocompatibility Complex) molecules. To investigate the role of Rab22a, the intracellular co-localization of chicken Rab22a (cRab22a) molecule and its relationship to BF and chicken invariant chain (cIi) molecules was studied. A 3D protein structure of Rab22a was constructed by using informatics tools (DNASTAR 4.0 and DNAMAN). Based on the model, the corresponding recombinant eukaryotic plasmids were constructed by point mutations in the protein’s structural domains. HEK 293T cells were co-transfected with plasmids pEGFP-C1-cIi to observe the intracellular co-localization. Secondly, the DC2.4 Mouse Dendritic Cell and Murine RAW 264.7 cells were transfected with recombinant plasmids of pmCherry-cRab22a and pmCherry-mRab22a respectively. Subsequently, the intracellular localization of cRab22a in early and late endosomes was observed with specific antibodies against EEA1 and LAMP1 respectively. For gene expression-based studies, the cRab22a gene was down-regulated and up-regulated in HD11 cells, following the detection of transcription levels of the BFa (MHCIa) and cIi genes by real-time quantitative PCR (RT-qPCR). The interactions of the cRab22a gene with BFa and cIi were detected by co-immunoprecipitation (Co-IP) and Western blot. The results showed that the protein structures of chicken and mouse Rab22a were highly homologous (95.4%), and both localize to the early and late endosomes. Ser41 and Tyr74 are key amino acids in the Switch regions of Rab22a which maintain its intracellular localization. The down-regulation of cRab22a gene expression significantly reduced (p < 0.01) the transcription of BFa (MHCIa) and cIi in HD11 cells. However, when the expression of the cRab22a gene was increased 55 times as compared to control cells, the expression of the BFa (MHCIa) gene was increased 1.7 times compared to the control cells (p < 0.01), while the expression of the cIi gene did not significantly differ from control (p > 0.05). Western blot results showed that cRab22a could not directly bind to BFa and cIi. So, cRab22a can regulate BFa and cIi protein molecules indirectly. It is concluded that cRab22a was localized with cIi in the endosome. The Switch regions of cRab22a are the key domains that affect intracellular localization and colocalization of the cIi molecule.