A two-hybrid antibody micropattern assay reveals specific in cis interactions of MHC I heavy chains at the cell surface

Abstract

We demonstrate a two-hybrid assay based on antibody micropatterns to study protein-protein interactions at the cell surface of major histocompatibility complex class I (MHC I) proteins. Anti-tag and conformation-specific antibodies are used for individual capture of specific forms of MHC I proteins that allow for location- and conformation-specific analysis by fluorescence microscopy. The assay is used to study the in cis interactions of MHC I proteins at the cell surface under controlled conditions and to define the involved protein conformations. Our results show that homotypic in cis interactions occur exclusively between MHC I free heavy chains, and we identify the dissociation of the light chain from the MHC I protein complex as a condition for MHC I in cis interactions. The functional role of these MHC I protein-protein interactions at the cell surface needs further investigation. We propose future technical developments of our two-hybrid assay for further analysis of MHC I protein-protein interactions.

Keywords: cell biology; endocytosis; immunology; in cis interactions; inflammation; major histocompatibility complex molecules; membrane proteins; protein micropatterns.

Figure 1.. Specific capture of cell surface K^b on antibody micropatterns.

(A) Schematic presentation of the capture assay. Cells transduced with K^b (red) fused to GFP (green) are incubated on the Y3 antibody micropatterns (anti K^b; magenta). Upon specific antibody-antigen interaction, K^b-GFP is captured on its extracellular epitope by the Y3 antibody pattern elements (see enlargement). (B) Printed antibodies are target-specific. Control experiments demonstrate that K^b-GFP is only captured by the anti-K^b antibody Y3 and not by an antibody specific for D^b (27-11-13S). (C) Schematic displaying the different antibody epitopes on the K^b molecule. The Y3 epitope reacts specifically with residues of the α₂ helix of K^b-GFP whereas the anti-HA antibody recognizes the additional HA-tag that was N-terminally fused to K^b-GFP. (D) Surface K^b-GFP can be directly captured by the anti-K^b antibody Y3 or by the anti-HA antibody against the N-terminally tagged HA-K^b-GFP. Cells were transduced with K^b-GFP or HA-K^b-GFP and tested for specificity on Y3 or anti-HA antibody micropatterns. Y3 successfully captures both constructs, whereas HA only recognizes the HA-tagged molecules. Scale bar: 25 µm.

Figure 2.. Antibody micropatterns determine stability of the captured K^b population.

(A) Cells expressing HA-K^b-GFP were captured on Y3 or anti-HA antibody micropatterns and incubated at 25 or 37°C to allow for the dissociation of β₂m. To identify the nature of the captured K^b-GFP population (green channel), specific fluorescent peptide SIINFEKL (SL8^TAMRA; red channel) was added to the samples. Based on their ability to bind peptide, one can distinguish between the peptide-receptive K^bHC/β₂m dimer and the K^b free heavy chains, which are incapable to bind peptide. (B) For further characterization of the captured HA-K^b-GFP on Y3 or anti-HA antibody micropatterns, immunostaining experiments were performed. Immunostaining of captured HA-K^b-GFP molecules with the anti-β₂m antibody (BBM.1^Atto542) reveals dissociation of β₂m from anti-HA antibody micropatterns at 37°C (column 3). Addition of the specific ligand peptide SIINFEKL (SL8) during 37°C incubation prevents β₂m dissociation (column 4). Scale bars: 25 µm.

Figure 3.. Antibody micropatterns reveal conformation-dependent in cis interaction of captured K^b-GFP.

(A) For the two-hybrid assay, cells were co-transduced with two K^b constructs: K^b with an N terminal HA tag (HA-K^b) and K^b-GFP (GFP fused to the cytoplasmic tail). (B) Cells were incubated on anti-HA or Y3 antibody micropatterns at different temperatures. Recruitment of K^b-GFP (green channel) to the anti-HA antibody micropatterns occurs specifically at 37°C and can be inhibited by addition of the SIINFEKL (SL8) peptide (column 3 and 4). The single chain mutant, scK^b-GFP (which has β₂m covalently linked to the K^b heavy chain) is also not recruited to the antibody micropatterns (column 5). From top to bottom: Antibody micropatterns, K^b-GFP, phase contrast, and schematic representation. Scale bar: 25 µm. (C) For quantification of co-capture, the mean fluorescence intensities of K^b-GFP of the total cell and the areas of pattern elements were determined. The redistribution of K^b-GFP leads to increased fluorescence intensity levels in the areas of the pattern elements and is represented as an increase of the ratio of the fluorescence intensity of the pattern elements over the fluorescence intensity of the entire cell (see Materials and methods). The plot shows the mean (red) ± SEM and the distribution of the calculated ratios from individual cells (black symbols; n (cells) ≥ 14) of ≥ 2 independent experiments (****: Significant difference, p<0.0001, two-tailed t-test).

Figure 3—figure supplement 1.. Staining E3-HA-K^b with K4-peptide.

The stable cell line STF1/E3 HA-K^b was electroporated (*) with K^b-GFP and incubated overnight on anti-HA micropatterns and either left at 25°C or shifted to 37°C to allow for co-capture as described. Cells were then stained with the K4^Atto633 peptide, which binds specifically to the E3 tag of the E3-HA-K^b construct, which was captured by its HA-tag on the anti-HA micropatterns. Scale bar: 25 µm.

Figure 3—figure supplement 2.. Binding SIINFEKL^TAMRA to co-captured K^b-.

Co-transduced cells with E3-HA-K^b and K^b-GFP were incubated overnight on anti-HA micropatterns and either left at 25°C or shifted to 37°C to allow for co-capture as described. Cells were then incubated with SIINFEKL^TAMRA (SL8^TAMRA) peptide. Peptide binding was reduced in co-captured K^b at 37°C (columns 3 and 4), indicating that only free heavy chains interact in cis. Duplicates are shown. Scale bar: 25 µm.

Figure 4.. In cis interactions of K^b-GFP and HA-K^b are peptide-dependent and generally occur in cells at 37°C.

For co-immunoprecipitation, the same co-transduced cells from the previous experiment were used (STF1/HA-K^b and K^b-GFP). Cells were incubated at 25°C overnight to increase K^b cell surface levels and then shifted to 37°C to allow for the dissociation of β₂m from the K^b heavy chain. The SIINFEKL (SL8) peptide was added as control to inhibit β₂m dissociation (lanes 2 and 4). Cells were then lysed and successfully immunoprecipitated with an anti-HA antibody (bottom row). Immunoisolates and lysate control samples were analysed by western blotting by sequential staining with an anti-GFP antibody (top row) and an anti-HA antibody (bottom row). The K^b-GFP construct was specifically co-immunoprecipitated in the absence of peptide, similar to the result on antibody micropatterns (lane 1).

Figure 4—figure supplement 1.. Co-immunoprecipitation of cell surface K^b molecules with K4-peptide.

For co-immunoprecipitation of cell surface proteins, stable STF1 cells co-transfected with E3-HA-K^b and K^b-GFP were used. Cells were incubated at 25°C overnight to enrich K^b cell surface levels and incubated in presence or absence of SIINFEKL (SL8) as indicated. For cell surface labeling, the biotinylated K4 peptide (K4^biotin; binds to the extracellular E3 tag of E3-HA-K^b) was added and cells were shifted to 37°C to allow for β₂m dissociation as described. Cells were lysed and co-immunoprecipitated with neutravidin agarose binding to the biotinylated E3-HA-K^b cell surface population. The immunoisolates were then treated with EndoF1 as indicated to distinguish the cell surface population (EndoF1 resistant, top bands) from the intracellular population of isolated K^b molecules (EndoF1 sensitive K^b molecules, see asterisk). (A) Sequential Western blot analysis with an anti-GFP antibody (top row) and anti-HA antibody (bottom row) demonstrates that only free heavy chains co-precipitate (lane 1) with E3-HA-K^b. (B) Quantification of (A): The ratio of co-precipitated K^b-GFP to total protein amounts was quantified.

Author response image 1.. Comparison of cell surface expression of HA-K^b in STF1 and STF1+TAP2 cells by flow cytometry.

STF1 cells were transduced with HA-K^b and stained with anti-HA or 25-D1.16 and anti-mouse IgG conjugated to Alexa Fluor 488 and subjected to flow cytometry. (A) Surface intensities of HA-K^b of both cell lines are represented as bar charts. TAP-deficient STF1 cells are represented in black and TAP2-proficient cells (STF1+TAP2) are represented in grey. Cells were incubated with (+) and without (-) 10 µM of the high affinity peptide SL8 and stained with the indicated antibodies. (B) The increase in surface signal after peptide addition in (A) for both antibodies is displayed as ratio. (n= 3; standard deviations as indicated).