Bioorthogonal protein labelling enables the study of antigen processing of citrullinated and carbamylated auto-antigens

Abstract

Proteolysis is fundamental to many biological processes. In the immune system, it underpins the activation of the adaptive immune response: degradation of antigenic material into short peptides and presentation thereof on major histocompatibility complexes, leads to activation of T-cells. This initiates the adaptive immune response against many pathogens. Studying proteolysis is difficult, as the oft-used polypeptide reporters are susceptible to proteolytic sequestration themselves. Here we present a new approach that allows the imaging of antigen proteolysis throughout the processing pathway in an unbiased manner. By incorporating bioorthogonal functionalities into the protein in place of methionines, antigens can be followed during degradation, whilst leaving reactive sidechains open to templated and non-templated post-translational modifications, such as citrullination and carbamylation. Using this approach, we followed and imaged the post-uptake fate of the commonly used antigen ovalbumin, as well as the post-translationally citrullinated and/or carbamylated auto-antigen vinculin in rheumatoid arthritis, revealing differences in antigen processing and presentation.

Fig. 1. Bioorthogonal antigens closely approximate the biophysical behavior of wild type antigens. (A) Expression of antigenic proteins in the E. coli auxotrophic strain B834 allows replacement of methionines with azidohomoalanine (Aha) or homopropargylglycine (Hpg). These labels allow the in cell imaging of degradation whilst at the same time allowing the study of the antigens by T-cell activation; (B) native-PAGE analysis shows similar migration behavior of wt-Ova, Aha-Ova and Hpg-Ova, whilst showing altered migration of fluorophore modified Ova-analogues; (C) circular dichroism of bioorthogonal and fluorophore Ova-analogues; (D) activation of OT-I cells by Ova-variants as measured by IL-2 production; (E) activation of OT-II cells by these same antigens. Asterisks refer to given P values, * p < 0.05, ** p < 0.005, **** p < 0.00005. Group mean values were analyzed by two-way analysis of variance with Tukey post hoc significant difference test using GraphPad Prism 6.0. Data are represented as means ± SD; (F) tracking of degradation of antigen inside D1 dendritic cells.

Fig. 2. Imaging processing of bioorthogonal and fluorophore-modified Ova in DC 2.4-APCs via confocal microscopy. DCs were incubated for 2 h (pulse) with Aha or A647-Ova. Cells were fixed with 0.5% PFA and processed for immunofluorescence with LAMP-1 as a lysosomal marker (green in merged images). The nucleus was stained with DAPI (blue in merged images). Aha-Ova was stained using ccHc with A647-alkyne fluorophore (grey in single channel, red in merged images). (A) Upper panel: imaging of Aha-Ova, lower panel: imaging of A647-Ova (B) screenshot from Movie S1 (ESI†). Scale bar is 10 μm (white bar, left corner).

Fig. 3. Activation of anti-DERAA T-cells by bioorthogonal vinculin derivatives. PBMC derived dendritic cells, isolated from HLA-DQ8+ donors were pulsed with peptides or Vin variants were cocultured with the Jurkat cells and used for stimulation. Activation of Vin-DERAA-specific Jurkat cells was measured with eCFP expression within the cells. The eCFP expression is dependent on NFκB and is therefore a marker of TCR activation and costimulation, the percentage of live eCFP-positive cells is plotted in bars. HSV2 (Herpes Simplex Virus 2 peptide) and Tetanus toxoid protein are negative controls. VCL-DERAA peptide and PMA-ionomycin are controls. Error bars show the SD. Data is representative of two experiments with biological triplicates.

Fig. 4. Effect of post-translational modifications on vinculin degradation. Incubation of vinculin variants with isolated lysosomes (A) or CatS (B) show decreased proteolysis of wt and Aha-Vin upon carbamylation. Vin peptides recovered after CatS digestion (n = 3 per variant) were mapped against the protein sequence. Peptide sequences were marked green, when found in at least 2/3 replicates.

Fig. 5. FACS read-out for T-cell activation with titration of wt- and Cit-VCL-DERAA peptide. Percentage of eCFP positive cells was read out as a measure for T-cell activation (n = 3).

Fig. 6. Molecular docking of wt- and Cit-VCL-DERAA in HLA-DQA1*03:01/DQB1*03:02 (HLA-DQ8, PDB: 6DFX). Arg residues in wt-VCL-DERAA (A) are exposed extracellularly whereas for Cit-VCL-DERAA (B) citrullinated arginines are buried (C, blue arrows) and Glu residue becomes exposed extracellularly (C, red arrow).