{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,1]],"date-time":"2026-10-01T12:08:03Z","timestamp":1790856483421,"version":"4.1.0"},"reference-count":54,"publisher":"Wiley","issue":"11","license":[{"start":{"date-parts":[[2026,10,1]],"date-time":"2026-10-01T00:00:00Z","timestamp":1790812800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"},{"start":{"date-parts":[[2026,10,1]],"date-time":"2026-10-01T00:00:00Z","timestamp":1790812800000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"funder":[{"DOI":"10.13039\/100000057","name":"National Institute of General Medical Sciences","doi-asserted-by":"publisher","award":["R35\u2010GM147039"],"award-info":[{"award-number":["R35\u2010GM147039"]}],"id":[{"id":"10.13039\/100000057","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000001","name":"National Science Foundation","doi-asserted-by":"publisher","award":["DGE\u20102139841"],"award-info":[{"award-number":["DGE\u20102139841"]}],"id":[{"id":"10.13039\/100000001","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Protein Science"],"published-print":{"date-parts":[[2026,11]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Lacritin is an abundantly expressed glycoprotein in tear fluid and plays key roles in immune response, tear secretion, and bacterial killing. These biological functions are tightly regulated through several biochemical mechanisms including multimerization, proteolysis, and alternative splicing, especially within its C\u2010terminal domain. Given its critical role at the ocular surface, lacritin is currently under investigation as a diagnostic biomarker and therapeutic candidate for dry eye disease (DED). However, despite over three decades since its initial discovery, the functional significance of the O\u2010glycans that comprise more than 50% of its molecular weight remains largely unknown. To address this gap, we leveraged mass spectrometry (MS)\u2010based glycoproteomics, AlphaFold 3.0, and molecular dynamics (MD) to explore the structural role of site\u2010specific O\u2010glycans on C\u2010terminal lacritin. In doing so, we identified distinct glycosylation profiles between monomeric and multimeric lacritin, particularly at glycosites located near crosslinking residues (Lys101 and Lys104) that modulate multimer formation. Based on our glycoproteomics data, we performed MD simulations on monomer and multimer glycoforms and revealed that O\u2010glycans may participate in intramolecular glycan\u2013protein interactions that influence its structure and the spatial arrangement of Lys101 and Lys104. Differences in the solvent accessible surface area (SASA) and root mean squared fluctuation (RMSF) of these residues further suggested that proximal O\u2010glycosylation could affect their ability to participate in crosslinking. To test these predictions, we performed in vitro crosslinking assays and demonstrated reduced TGM2\u2010mediated multimerization of glycosylated versus unmodified lacritin. Finally, we show that recombinant glycoforms bearing O\u2010glycan patterns similar to endogenous lacritin multimers are enriched after TGM2 crosslinking. Taken together, these findings underscore a central role for lacritin O\u2010glycans in affecting structural topology and multimerization with implications for its downstream biological activity.<\/jats:p>","DOI":"10.1002\/pro.70812","type":"journal-article","created":{"date-parts":[[2026,10,1]],"date-time":"2026-10-01T11:42:54Z","timestamp":1790854974000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Site\u2010specific O\u2010glycans influence lacritin structure and multimerization in tears"],"prefix":"10.1002","volume":"35","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-4692-0451","authenticated-orcid":false,"given":"Vincent","family":"Chang","sequence":"first","affiliation":[{"name":"Department of Chemistry Yale University  New Haven Connecticut USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ryan J.","family":"Chen","sequence":"additional","affiliation":[{"name":"Department of Chemistry Yale University  New Haven Connecticut USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Isaac","family":"Lian","sequence":"additional","affiliation":[{"name":"Department of Chemistry Yale University  New Haven Connecticut USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Madilynn","family":"Hamilton","sequence":"additional","affiliation":[{"name":"Department of Chemistry Yale University  New Haven Connecticut USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4561-9838","authenticated-orcid":false,"given":"Keira E.","family":"Mahoney","sequence":"additional","affiliation":[{"name":"Department of Chemistry Yale University  New Haven Connecticut USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jeff","family":"Romano","sequence":"additional","affiliation":[{"name":"Department of Cell Biology University of Virginia  Charlottesville Virginia USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5311-2650","authenticated-orcid":false,"given":"Gordon","family":"Laurie","sequence":"additional","affiliation":[{"name":"Department of Cell Biology University of Virginia  Charlottesville Virginia USA"},{"name":"Department of Ophthalmology University of Virginia  Charlottesville Virginia USA"},{"name":"Department of Biomedical Engineering University of Virginia  Charlottesville Virginia USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2382-5067","authenticated-orcid":false,"given":"Stacy A.","family":"Malaker","sequence":"additional","affiliation":[{"name":"Department of Chemistry Yale University  New Haven Connecticut 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