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PLoS One
2013 Jan 01;84:e60903. doi: 10.1371/journal.pone.0060903.
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Comparison of the intracellular trafficking itinerary of ctla-4 orthologues.
Kaur S
,
Qureshi OS
,
Sansom DM
.
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CTLA-4 is an essential inhibitor of T cell immune responses. At steady state, most CTLA-4 resides in intracellular compartments due to constitutive internalisation mediated via a tyrosine based endocytic motif (YVKM) within the cytoplasmic domain. This domain is highly conserved in mammals suggesting strong selective pressure. In contrast, the C-terminal domain varies considerably in non-mammals such as fish, xenopus and birds. We compared the ability of the C-terminus of these species to direct the trafficking of CTLA-4 with human CTLA-4. Using a chimeric approach, endocytosis was found to be conserved between human, xenopus and chicken CTLA-4 but was reduced substantially in trout CTLA-4, which lacks the conserved YXXM motif. Nevertheless, we identified an alternative YXXF motif in trout CTLA-4 that permitted limited endocytosis. Post-internalisation, CTLA-4 was either recycled or targeted for degradation. Human and chicken CTLA-4, which contain a YVKM motif, showed efficient recycling compared to xenopus CTLA-4 which contains a less efficient YEKM motif. Specific mutation of this motif in human CTLA-4 reduced receptor recycling. These findings suggest evolutionary development in the endocytic and recycling potential of CTLA-4, which may facilitate more refined functions of CTLA-4 within the mammalian immune system.
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23565286
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Figure 2. Cellular localisation of CTLA-4 chimeras.A. CHO cells expressing CTLA-4 chimeras were incubated with unlabeled anti-CTLA-4 Ab at 37°C for 1 hour, cooled to 4°C and surface CTLA-4 stained red with anti-mouse Alexa 555. Cells were subsequently fixed, permeabilised and stained with Alexa488 anti-mouse IgG (green) and imaged by confocal microscopy. B. The ratio of plasma membrane to internalised CTLA-4 fluorescence (PM/I) was calculated by outlining cells in ImageJ. C. CHO cells expressing human CTLA-4 were labeled with anti-CTLA-4 PE at 37°C for 30 minutes followed by labeling surface CTLA-4 on ice (4°C) with Alexa647 anti-mouse IgG. Cells were analysed by flow cytometry and data are plotted as cycling CTLA-4 (37°C label) vs surface CTLA-4 (4°C label). D. CHO cells expressing the CTLA-4 chimeras were labeled as described in C and analysed by flow cytometry. Dotted line provides a standard gradient for reference purposes.
Figure 3. Endocytosis rates of CTLA-4 chimeras.A. CHO cells expressing CTLA-4 chimeras were labeled at 4°C with anti-CTLA-4 to label surface CTLA-4. Cells were then warmed to 37°C to allow endocytosis for the times indicated. Cells were then placed on ice and any remaining surface CTLA-4 detected with Alexa647 anti-mouse IgG. The 647 signal was plotted against time as the fraction remaining compared to 4°C. B. CHO cells expressing CTLA-4 chimeras were labeled as in A but in medium supplemented with sucrose (0.45 M) to prevent endocytosis. C. CHO cells expressing the chimeric CTLA-4 constructs were incubated with a transferrin (Tf) Alexa633 conjugate (Invitrogen) and anti-CTLA-4 PE at 37°C for 45 minutes. Cells were subsequently fixed and analysed by confocal microscopy. The red arrows indicate co-localisation. D. Rate of endocytosis of VGNF mutant was performed as described in A. E. Transferrin uptake of VGNF mutant was performed as described in C and analysed by confocal microscopy.
Figure 4. Degradation of CTLA-4 chimeras.A. CHO cells expressing CTLA-4 chimeras were incubated in medium or medium supplemented with cycloheximide (CHX) at 37°C for 3 hours. Cells were fixed, permeabilised and stained for total CTLA-4 with an unlabeled anti-CTLA-4 Ab followed by Alexa488 anti-mouse IgG (green) and nuclei counterstained using DAPI (blue). Cells were analysed by confocal microscopy. B Total CTLA-4 was quantified by outlining cells in ImageJ and MFI plotted (left column). For flow cytometric quantitation (right hand column) cells were stained for total CTLA-4 using anti-CTLA-4 PE after 3 hours of CHX or NH4Cl treatment at 37°C and MFI plotted as a percentage of initial fluorescence. C. CHO cells expressing CTLA-4 chimeras were transfected with CD63-GFP. Cells were incubated in medium supplemented with NH4Cl at 37°C for 3 hours. Cells were fixed, permeabilised, and stained with an unlabeled anti-CTLA-4 Ab and Alexa565 anti-human IgG (red) to stain total CTLA-4 protein and analysed by confocal microscopy. The red arrows indicate co-localisation.
Figure 5. Recycling rates of CTLA-4 chimeras.A. CHO cells expressing WT human CTLA-4 were labeled with mouse anti-CTLA-4 PE at 37°C to detect cycling CTLA-4, washed and any recycling CTLA-4-PE antibody detected by addition of Alexa647 anti-mouse IgG at either 4°C or 37°C. Representative FACS plots are shown for PE-label (cycling CTLA-4) vs Alexa647 label (re-cycling CTLA-4) at the indicated time points. B. Recycling rates are plotted for the chimeras as normalised to the 4°C control.
Figure 6. Recycling efficiency is regulated by the YVKM motif.A. CHO cells expressing WT human CTLA-4 YVKM or YEKM motif were labeled at 4°C with anti-CTLA-4 to label surface CTLA-4. Cells were warmed to 37°C for the time indicated. Cells were then placed on ice and the remaining surface CTLA-4 detected with Alexa647 anti-mouse IgG and plotted over time. B. CHO cells expressing WT human CTLA-4 YVKM or a point mutant YEKM motif were labeled with mouse anti-CTLA-4 PE at 37°C to detect cycling CTLA-4. Recycling protein was detected with Alexa647 anti-mouse IgG at 37°C for the indicated time points. Recycling rates are plotted for the CTLA-4 variants normalised to the 4°C control. C. Representative data comparing human and xenopus CTLA-4 recycling after 30 minutes is shown.
Figure 1. Generation and localisation of CTLA-4 chimeras.
A. C-terminal sequence alignments of selected mammalian CTLA-4 based on sequence data from Ensembl and in ref 12. B. Diagram of human CTLA-4 chimeras containing the extracellular and transmembrane domain of human CTLA-4 and the C-terminus of species shown. C-terminal amino acid sequence alignments of human, chicken, xenopus and trout CTLA-4 are shown below, based on alignments using Clustal W. C. CHO cells expressing CTLA-4 chimeras were incubated with WGA-tetramethylrhodamine at 4°C for 45minutes. Cells were subsequently fixed, permeabilised, and stained with an unlabeled anti-CTLA-4 Ab followed by Alexa488 anti-human IgG (green) to stain total CTLA-4 protein. Cells were analysed by confocal microscopy. D Relative expression of surface (4°C) and total CTLA-4 for each chimera as determined by flow cytometry.
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