Mus musculus Vti1b Binding to Invariant Chain (Ii) and Its Effect on MHC-associated Molecules
CHEN Fang-Fang1#,*, XU Wan-Yi1,2,*, ZHANG Jun1, ZHOU Xu-Qing1, LI Shuai1, LIU Xue-Lan1, LI Jin-Chun1
1 Anhui Agricultural University, College of Veterinary Medicine/Anhui Provincial Key Laboratory of Zoonoses, Hefei 230036, China; 2 Shandong Agricultural University, College of Veterinary Medicine, Taian 271017, China
Abstract：The invariant chain (Ii), a key immunoregulatory molecule in antigen processing and presentation, plays a critical role in regulating the assembly and transport of major histocompatibility complex (MHC) class Ⅰ/Ⅱ molecules, endosomal sorting, and antigen peptide loading. Vesicle transport through interaction with t-SNAREs 1B (Vti1b), a crucial regulatory factor in endosomal membrane fusion, participates in antigen processing and presentation pathways by mediating endosome-lysosome fusion and autophagosome maturation; however, the relationship between Ii and Vti1b remains unclear. This study focused on Vti1b and Ii of Mus musculus aiming to clarify their subcellular localization and protein-protein interaction, and to investigate the effects of Vti1b under conditions of low and high intracellular expression on the expression levels of Ii and its related MHC molecules. First, Vti1b molecules from different mouse cell types and growth phases of M. musculus were cloned, followed by amino acid sequence alignment; Subsequently, Vti1b and the P31/P41 isoforms of the Ii gene were separately cloned into eukaryotic expression vectors. Laser confocal microscopy was employed to analyze the subcellular colocalization of Vti1b and Ii in Raw264.7 cells. Meanwhile, co-immunoprecipitation (Co-IP), Pull-down and Western blot assays were used to investigate their protein-protein interactions in eukaryotic cells. Finally, through siRNA-mediated gene silencing and eukaryotic recombinant plasmid transfection for Vti1b overexpression, qPCR was used to detect the transcriptional level changes of Vti1b in Raw264.7 cells, as well as the transcription levels of Ii and MHC-associated genes under low and overexpression states of this gene. Results showed that the amino acid sequences of Vti1b exhibited only 1~2 amino acid variations across different mouse cell types and growth phases. Vti1b and Ii proteins were found to interact and colocalize in late endosomes of Raw264.7 cells. When Vti1b gene transcription was significantly downregulated to 42% of the normal control level (P<0.05), the transcription levels of Ii, MHC Ⅰα, and MHC Ⅱβ genes were significantly reduced to 45% (P<0.01), 48% (P<0.01), and 47% (P<0.05) of the control group, respectively. The Vti1b gene was overexpressed by 40.88-fold, and the transcriptional level of MHCⅡβ gene was significantly upregulated, reaching 154% of that in the control group (P<0.01). In contrast, the transcriptional levels of Ii and MHCⅠα genes showed no significant difference compared with the control group. The transcriptional level of Vti1b was correlated with those of Ii and its MHC-associated molecule genes, particularly showing that reduced Vti1b expression led to downregulation of Ii and MHC-related gene transcription. This results clarified the co-localization and protein interaction between Vti1b and Ii in late endosomes, confirmed that the Vti1b gene exerts a regulatory effect on the expression of Ii and MHC-related genes. This study provides an important basis for elucidating their cell biological functions and immunological mechanisms.
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