{"doi":"10.1016/j.jbc.2021.101197","title":"Rational design of a hydrolysis-resistant mycobacterial phosphoglycolipid antigen presented by CD1c to T cells","abstract":"Whereas proteolytic cleavage is crucial for peptide presentation by classical major histocompatibility complex (MHC) proteins to T cells, glycolipids presented by CD1 molecules are typically presented in an unmodified form. However, the mycobacterial lipid antigen mannosyl-β1-phosphomycoketide (MPM) may be processed through hydrolysis in antigen presenting cells, forming mannose and phosphomycoketide (PM). To further test the hypothesis that some lipid antigens are processed, and to generate antigens that lead to defined epitopes for future tuberculosis vaccines or diagnostic tests, we aimed to create hydrolysis-resistant MPM variants that retain their antigenicity. Here, we designed and tested three different, versatile synthetic strategies to chemically stabilize MPM analogs. Crystallographic studies of CD1c complexes with these three new MPM analogs showed anchoring of the lipid tail and phosphate group that is highly comparable to nature-identical MPM, with considerable conformational flexibility for the mannose head group. MPM-3, a difluoromethylene-modified version of MPM that is resistant to hydrolysis, showed altered recognition by cells, but not by CD1c proteins, supporting the cellular antigen processing hypothesis. Furthermore, the synthetic analogs elicited T cell responses that were cross-reactive with nature-identical MPM, fulfilling important requirements for future clinical use. Whereas proteolytic cleavage is crucial for peptide presentation by classical major histocompatibility complex (MHC) proteins to T cells, glycolipids presented by CD1 molecules are typically presented in an unmodified form. However, the mycobacterial lipid antigen mannosyl-β1-phosphomycoketide (MPM) may be processed through hydrolysis in antigen presenting cells, forming mannose and phosphomycoketide (PM). To further test the hypothesis that some lipid antigens are processed, and to generate antigens that lead to defined epitopes for future tuberculosis vaccines or diagnostic tests, we aimed to create hydrolysis-resistant MPM variants that retain their antigenicity. Here, we designed and tested three different, versatile synthetic strategies to chemically stabilize MPM analogs. Crystallographic studies of CD1c complexes with these three new MPM analogs showed anchoring of the lipid tail and phosphate group that is highly comparable to nature-identical MPM, with considerable conformational flexibility for the mannose head group. MPM-3, a difluoromethylene-modified version of MPM that is resistant to hydrolysis, showed altered recognition by cells, but not by CD1c proteins, supporting the cellular antigen processing hypothesis. Furthermore, the synthetic analogs elicited T cell responses that were cross-reactive with nature-identical MPM, fulfilling important requirements for future clinical use. The ability of T cells to respond to peptides presented by major histocompatibility complex (MHC) proteins is more widely known than their ability to respond to nonclassical MHC class I-like CD1 proteins. Whereas mice only express CD1d, humans express a functionally diverse family of CD1 antigen presenting molecules, namely CD1a, CD1b, CD1c, and CD1d (1Moody D.B. Zajonc D.M. Wilson I.A. Anatomy of CD1-lipid antigen complexes.Nat. Rev. Immunol. 2005; 5: 387-399Crossref PubMed Scopus (149) Google Scholar). Unlike MHC genes, CD1 genes are essentially monomorphic, so all individuals express the same CD1 genes. The nonpolymorphic nature of CD1 proteins likely enables them to present identical antigens in all individuals, which makes ligands of CD1 proteins attractive candidates for subunit vaccine and diagnostics development. Several lipid antigens that are presented by CD1 and recognized by T cells are found in the cell wall of Mycobacterium tuberculosis. Here we focus on mycoketides, a class of mycobacterial lipids presented by CD1c. CD1c proteins are constitutively expressed on the surface of marginal zone and mantle zone B cells in spleen and lymp","journal":"Journal of Biological Chemistry","year":2021,"id":191265,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":13,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9546,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2021-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":756317,"name":"Laura Marino","orcid":"0000-0001-6325-5738","position":1,"is_corresponding":false},{"id":756318,"name":"Thinh-Phat Cao","orcid":"0000-0001-9387-5460","position":2,"is_corresponding":false},{"id":314930,"name":"Tan‐Yun Cheng","orcid":"0000-0002-5178-6985","position":3,"is_corresponding":false},{"id":756319,"name":"Dennis Dam","orcid":"0000-0002-2510-9913","position":4,"is_corresponding":false},{"id":312631,"name":"Adam Shahine","orcid":"0000-0001-7717-4437","position":5,"is_corresponding":false},{"id":493562,"name":"Martin D. Witte","orcid":"0000-0003-4660-2974","position":6,"is_corresponding":false},{"id":618533,"name":"Dmitri V. Filippov","orcid":"0000-0002-6978-7425","position":7,"is_corresponding":false},{"id":252291,"name":"Sara Suliman","orcid":"0000-0002-5154-576X","position":8,"is_corresponding":false},{"id":756320,"name":"Gijsbert A. van der Marel","orcid":"0000-0001-8917-3458","position":9,"is_corresponding":false},{"id":312635,"name":"D. Branch Moody","orcid":"0000-0003-2306-3058","position":10,"is_corresponding":false},{"id":493563,"name":"Adriaan J. Minnaard","orcid":"0000-0002-5966-1300","position":11,"is_corresponding":false},{"id":256186,"name":"Jamie Rossjohn","orcid":"0000-0002-2020-7522","position":12,"is_corresponding":false},{"id":756321,"name":"Jeroen D. C. Codée","orcid":"0000-0003-3531-2138","position":13,"is_corresponding":false},{"id":252300,"name":"Ildiko Van Rhijn","orcid":"0000-0002-1446-5701","position":14,"is_corresponding":false},{"id":308665,"name":"Josephine F. Reijneveld","orcid":"0000-0001-8695-964X","position":0,"is_corresponding":true}],"reference_count":54,"raw_metadata":null,"created_at":"2026-07-18T23:49:31.121780Z","pmid":"34536421","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}