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Recognizing the inciting event in the production of anti-citrullinated protein antibodies (ACPAs) may shed light on the origin of RA. Here, we demonstrate that ACPAs originate from germline-encoded antibodies targeting a distinct but structurally similar modification, called carbamylation, which is pathogenic and environmentally driven. The transition from anti-carbamylated protein (anti-CarP) antibodies to ACPAs results from somatic hypermutations, indicating that the change in reactivity is acquired via antigen-driven affinity maturation. During this process, a single germline anti-CarP antibody transitions from anti-CarP to double positive (anti-CarP\/ACPA) to ACPA according to the pattern and number of somatic hypermutations, explaining their coexistence and diverse specificity in RA. Artificial intelligence-based structural modeling revealed that an ACPA and its germline precursor exhibit distinct structural and biophysical properties, and pointed to heavy-chain tryptophan 48 (H-W48) as a critical residue in the differential recognition of citrullinated vs. carbamylated proteins. Indeed, a single methionine substitution in H-W48 changes the antibody specificity from ACPA to anti-CarP. These data indicate that the existence of germline-encoded anti-CarP antibodies is most likely the first event in the production of ACPAs during the early stages of RA development.<\/jats:p>","DOI":"10.1101\/2025.03.22.644346","type":"posted-content","created":{"date-parts":[[2025,3,26]],"date-time":"2025-03-26T03:24:49Z","timestamp":1742959489000},"source":"Crossref","is-referenced-by-count":1,"title":["Anti-citrullinated protein antibodies arise during affinity maturation of germline antibodies to carbamylated proteins in rheumatoid arthritis"],"prefix":"10.64898","author":[{"given":"Marta","family":"Escarra-Senmarti","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael","family":"Chungyoun","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dylan","family":"Ferris","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6380-2324","authenticated-orcid":false,"given":"Jeffrey J.","family":"Gray","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3415-0704","authenticated-orcid":false,"given":"Felipe","family":"Andrade","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2025032707401762000_2025.03.22.644346v1.1","doi-asserted-by":"publisher","DOI":"10.1111\/joim.12980"},{"key":"2025032707401762000_2025.03.22.644346v1.2","doi-asserted-by":"crossref","unstructured":"Trejo-Zambrano, M.I. , Gomez-Banuelos, E. & Andrade, F . Redox-Mediated Carbamylation As a Hapten Model Applied to the Origin of Antibodies to Modified Proteins in Rheumatoid Arthritis. 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