Localization of Chicken Rab22a in Cells and Its Relationship to BF or Ii Molecules and Genes
Author:
Yu Fengmei1, Raheem Muhammad Akmal23, Tan Yang1, Rahim Muhammad Ajwad4, Zha Lisha1ORCID, Zhang Jun1, Zhu Zhiwei1, Li Zhonghua1, Chen Fangfang1
Affiliation:
1. Key Laboratory of Veterinary Pathobiology and Disease Control, College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China 2. Institute of Biopharmaceutical and Health Engineering, Tsinghua Shenzhen International Graduate School, Tsinghua University, Shenzhen 518055, China 3. Tsinghua-Berkeley Shenzhen Institute, Tsinghua Shenzhen International Graduate School, Tsinghua University, Shenzhen 518055, China 4. Department of Zoology, Ghazi University Dera Ghazi Khan, Punjab 32200, Pakistan
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.
Funder
Project of Science and Technology, Anhui Province
Subject
General Veterinary,Animal Science and Zoology
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