Study On The Mechanism Of Rab Molecules Involved In The Transport Of Ii And Active Fragments In Cells

The invariant chain?Ii?,also known as CD74,has the immunological functions of assisting the major histocompatibility complex?MHC?to resist protopeptide transport,promote B cell maturation and act as a receptor of cytokines.Ii is composed of cytosolic region?Cyt?,transmembrane region?Tra?and endoplasmic reticulum region,in which the reticulum region is composed of CLIP?class II-associated invariant chain peptide?and trimer region?Trim?.At the same time,Rab like protein?Rab?is a small GTP enzyme related to Ras.In antigen-presenting cells,Rab plays an important role in regulating the formation,transport and fusion of cell vesicles.It has been found that the antigen peptide linked by Ii or its active fragment can target the antigen to the endocytosis,which facilitates the transport of antigen.In this study,the transport of Ii and its active fragments in cells was studied.It mainly includes the following three aspects:Firstly,it is clear that Rab5a molecule is involved in the transport of Ii in the early cell endocytosis,which is related to the CLIP region of Ii.Firstly,the Rab5a gene was cloned with self-designed primers,and the recombinant expression vectors containing chicken c Ii,c Ii mutants(Ii _D81-87aa and Ii _D91-99aa)were constructed.Secondly,the protein structure of Rab5a in chicken?Gallus gallus?and mouse?Mus musculus?was compared by software.Finally,Rab5a and Ii were transfected into DC2.4 cells respectively.The localization and co-localization of Rab5a,c Ii,c Ii _D81-87aa and c Ii _D91-99aa in eukaryotic cells were observed by immunofluorescence and laser confocal.The interaction between Rab5a and Ii was detected by pull-down method and western-blot.The results showed that the Rab5a gene was 648 bp,which was consistent with the expected size.The molecular structure analysis showed that Rab5a of chicken was highly similar to that of mouse,and the homology was as high as 95.4%.The results of homologous modeling analysis showed that Rab5a of chicken and mouse contained 5?helices and 6?helices,indicating that Rab5a of mammal and poultry were homologous.Laser confocal observation showed that Rab5a and Ii were located in the early endocytosis of chicken cells,while the mutant Ii _D81-87aa and Ii _D91-99aacould not be located in the early endocytosis.The results of pull-down and western-blot showed that Rab5a could bind to Ii.Secondly,Rab7b is clearly involved in the transport of Ii in the late endocytosis.Firstly,Rab7b gene of chicken and mouse was amplified by self-designed primers,and amino acid homology was compared.Meanwhile,prokaryotic and eukaryotic recombinant plasmids containing Rab7b Q67L and Rab7b7b Q67L were constructed.Secondly,the constructed recombinant plasmids,pmcherry-C1-c Rab7b,pmcherry-C1-c Rab7b Q67L and pmcherry-C1-c Ii,were transfected into Raw264.7 cells and co-located with FITC labeled late endocytosis protein 1?LAMP1?.The co-location of Rab7b,Rab7b Q67L and Ii in late endocytosis was observed by laser confocal microscopy.Finally,the binding of Rab7b and Rab7b Q67L to Ii was detected by pull down method and western-blot.The results showed that the Rab7b gene was the same size as expected,with an open reading frame of 624 bp,encoding 208 amino acids.The results of homology comparison showed that the homology of Rab7b protein structure in chicken and mouse was 74%;The prokaryotic and eukaryotic expression plasmids of Rab7b and its mutant Rab7b Q67L were constructed.The results of laser confocal analysis showed that Rab7b and Ii could co-locate in the late endocytosis,but Rab7b Q67L could not.The results of pull-down and western-blot showed that Rab7b and Rab7b Q67L in chicken could combine with Ii.Thirdly,it is clear that the Ii active fragments carrying different antigenic peptides are co-located with MHC I and Ii molecules in the endocytosomes.According to the mouse Ii gene sequence designed in NCBI and the gene sequence designed in its active region,primers were used to amplify the Ii_?Cyt/Tra?,Ii_{?Cyt/Tra/Clip?}and the control region Ii_?Trim?.The recombinant eukaryotes containing the three fragments of Ii and T cell epitope(OVA_257-264)or B cell epitope(OVA_323-339)were constructed by overlapping extension PCR.The constructed recombinant plasmids were co-transfected with MHC I or MHC II to 293T cells respectively.The results showed that Ii_?Cyt/Tra?/OVA_257-264/Ii_?Cyt/Tra?/OVA_323-339,Ii_{?Cyt/Tra/Clip?}/OVA_257-264/Ii_{?Cyt/Tra/Clip?}/OVA_323-339 could be co-located in the endocytosomes of MHC I and MHC II,but the control group Ii_?Trim?/OVA_257-264/Ii_?Trim?/OVA_323-339 could not.This indicated that the antigen peptide with Ii as carrier could participate in antigen cross presentation in cells.Further confocal results showed that Ii_?Cyt/Tra?/OVA_257-264/Ii_?Cyt/Tra?/OVA_323-339,Ii_{?Cyt/Tra/Clip?}/OVA_257-264/Ii_{?Cyt/Tra/Clip?}/OVA_323-339 could be co-located in Rab5a in the endocytosis,which indicated that Rab5a was involved in the transport of antigen peptide in the active region of Ii.To sum up,Rab5a and Rab7b are involved in the transport of Ii in cells.The carrier with Ii as the active fragment can carry different antigens and target into endocytosomes,which is related to the transport of Rab molecules.This will provide a theoretical basis for further study of the transport of Ii and its active fragments in cells and the application of Ii carrier.