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

Leeuwen, Tyrza van; Araman, Can; Pournara, Linda Pieper; Kampstra, Arieke S. B.; Bakkum, Thomas; Marqvorsen, Mikkel H. S.; Nascimento, Christine Rabello; Groenewold, G. J. Mirjam; Wulp, Willemijn van der; Camps, Marcel; Janssen, George M.C.; Veelen, Peter A. van; Janssen, A. M.; Florea, Bogdan I.; Overkleeft, Herman S.; Ossendorp, Ferry; Toes, René; Kasteren, Sander I. van

doi:10.1039/d1cb00009h

Cited by 6 publications

(13 citation statements)

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“…Studies in fibroblasts show that exposure to cyanate or urea can drive accumulation of intracellular homocitrullinated peptides and that these modified peptides can then be removed via proteasomal degradation (51). As seen in the context of autoimmune disease, homocitrullination of proteins can alter proteolytic cleavage and lead to MHC presentation of neoepitopes (27,38,52). However, the presence of diverse alleles that can recognize Hcit-specific peptides and the widespread presence of homocitrullinated peptides leads us to believe that this system may have a role in normal cell surveillance (10).…”

Section: Discussionmentioning

confidence: 99%

Vaccine Can Induce CD4-Mediated Responses to Homocitrullinated Peptides via Multiple HLA-Types and Confer Anti-Tumor Immunity

et al. 2022

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Homocitrullination is the post translation modification (PTM) of the amino acid lysine to homocitrulline also referred to as carbamylation. This PTM has mainly been studied in relation to autoimmune diseases including rheumatoid arthritis. Homocitrullination of lysines alters their charge which can lead to generation of neoepitopes that are differentially presented by MHC-II and induce modification-specific immune responses. Homocitrullination is often considered a process which triggers autoimmune disease by bypassing self-tolerance however, we suggest that homocitrullination may also have an alternative role in immune responses including protection against cancer. Here we demonstrate that immune responses to homocitrullinated peptides from three different proteins can be induced via multiple HLA-types. Immunization of Balb/c or HLA-transgenic DR4 and DR1 mice can induce modification-specific CD4 mediated IFNγ responses. Healthy human donors show a clear repertoire for the homocitrullinated Vimentin peptide (Vim116-135Hcit), with modification-specific and oligoclonal responses. Importantly, in vivo homocitrulline specific Vim116-135Hcit,Cyk8 371-388Hcit and Aldo 140-157Hcit responses are able to confer an anti-tumor effect in the murine B16 melanoma model. The Vim116-135Hcit anti-tumor response was dependent upon tumor expression of MHC-II suggesting the direct recognition of PTMs on tumor is an important anti-tumor mechanism. Cancer patients also have a CD4 repertoire for Vim116-135Hcit. Together these results suggest that homocitrulline-specific immune responses can be generated in healthy mice and detected in human donors through a variety of HLA-restrictions. Immunization can induce responses to Vim116-135Hcit,Aldolase 140-157Hcit and Cyk8 371-388Hcit which provide anti-tumor therapy across several HLA-types. Our results advance our understanding of homocitrulline-specific immune responses, with implications for a number of fields beyond autoimmunity, including tumor immune surveillance.

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Section: Discussionmentioning

confidence: 99%

Vaccine Can Induce CD4-Mediated Responses to Homocitrullinated Peptides via Multiple HLA-Types and Confer Anti-Tumor Immunity

et al. 2022

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“…This cell line was chosen due to its excellent imaging properties and our previous experience in using it as an in vitro model cell line for optimising bioorthogonal chemistry to study dendritic cell biology. [59] The Alexa Fluor-10.1002/anie.202207640…”

Section: Resultsmentioning

confidence: 99%

“…In contrast to cell‐free medium results, 6 – 8 and 19 – 20 exhibited successful ligation after uptake of 1 by DC2.4 dendritic cells [58] . This cell line was chosen due to its excellent imaging properties and our previous experience in using it as an in vitro model cell line for optimising bioorthogonal chemistry to study dendritic cell biology [59] . The Alexa Fluor‐based dyes 9 – 11 were unable to react with 1 in live cells.…”

Section: Resultsmentioning

confidence: 99%

Live‐Cell Imaging of Sterculic Acid—a Naturally Occurring 1,2‐Cyclopropene Fatty Acid—by Bioorthogonal Reaction with Turn‐On Tetrazine‐Fluorophore Conjugates**

et al. 2022

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In the field of lipid research, bioorthogonal chemistry has made the study of lipid uptake and processing in living systems possible, whilst minimising biological properties arising from detectable pendant groups. To allow the study of unsaturated free fatty acids in live cells, we here report the use of sterculic acid, a 1,2-cyclopropene-containing oleic acid analogue, as a bioorthogonal probe. We show that this lipid can be readily taken up by dendritic cells without toxic side effects, and that it can subsequently be visualised using an inverse electron-demand Diels-Alder reaction with quenched tetrazine-fluorophore conjugates. In addition, the lipid can be used to identify changes in protein oleoylation after immune cell activation. Finally, this reaction can be integrated into a multiplexed bioorthogonal reaction workflow by combining it with two sequential copper-catalysed Huisgen ligation reactions. This allows for the study of multiple biomolecules in the cell simultaneously by multimodal confocal imaging.

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“…We have recently reported the application of "click antigens" as tool to visualize antigen routing and processing in the context of living APCs. [14] These are recombinant antigen proteins containing bioorthogonal handles that are specifically reactive only in so-called 'click' reactions. [15] Click reactions are highly efficient bioorthogonal reactions, that enable selective conjugation of (bio)molecules labelled with a certain reactive group, without influencing the non-labelled molecules present in the cell or lysate.…”

Section: Introductionmentioning

confidence: 99%

“…We previously demonstrated that visualization of internalized click antigens could be achieve by CuAAC mediated fluorophore conjugation. [14] Incorporation of bioorthogonal amino acids introduces only minor changes in the protein structure, [14,22] while azide remains stable inside the lysosome for up to 24 hours, allowing for the imaging of antigen processing even when the antigen is degraded. [23] We hypothesized that the click antigen approach might also find application in a chemical proteomics platform.…”

Section: Introductionmentioning

confidence: 99%

Bioorthogonal Peptide Enrichment from Complex Samples Using a Rink‐Amide‐Based Catch‐and‐Release Strategy

et al. 2023

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Uptake and processing of antigens by antigen presenting cells (APCs) is a key step in the initiation of the adaptive immune response. Studying these processes is complex as the identification of low abundant exogenous antigens from complex cell extracts is difficult. Mass‐spectrometry based proteomics – the ideal analysis tool in this case – requires methods to retrieve such molecules with high efficiency and low background. Here, we present a method for the selective and sensitive enrichment of antigenic peptides from APCs using click‐antigens; antigenic proteins expressed with azidohomoalanine (Aha) in place of methionine residues. We here describe the capture of such antigens using a new covalent method namely, alkynyl functionalized PEG‐based Rink amide resin, that enables capture of click‐antigens via copper‐catalyzed azide‐alkyne [2 + 3] cycloaddition (CuAAC). The covalent nature of the thus formed linkage allows stringent washing to remove a‐specific background material, prior to retrieval peptides by acid‐mediated release. We successfully identified peptides from a tryptic digest of the full APC proteome containing femtomole amounts of Aha‐labelled antigen, making this a promising approach for clean and selective enrichment of rare bioorthogonally modified peptides from complex mixtures.

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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...

Cited by 6 publications

References 74 publications

Vaccine Can Induce CD4-Mediated Responses to Homocitrullinated Peptides via Multiple HLA-Types and Confer Anti-Tumor Immunity

Vaccine Can Induce CD4-Mediated Responses to Homocitrullinated Peptides via Multiple HLA-Types and Confer Anti-Tumor Immunity

Live‐Cell Imaging of Sterculic Acid—a Naturally Occurring 1,2‐Cyclopropene Fatty Acid—by Bioorthogonal Reaction with Turn‐On Tetrazine‐Fluorophore Conjugates**

Bioorthogonal Peptide Enrichment from Complex Samples Using a Rink‐Amide‐Based Catch‐and‐Release Strategy

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