{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,9,10]],"date-time":"2026-09-10T06:05:39Z","timestamp":1789020339897,"version":"build-2803163510"},"posted":{"date-parts":[[2026,4,2]]},"group-title":"Biophysics","reference-count":54,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,4,2]],"date-time":"2026-04-02T00:00:00Z","timestamp":1775088000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"award":["R35-GM147039"],"award-info":[{"award-number":["R35-GM147039"]}],"id":[{"id":"https:\/\/ror.org\/04q48ey07","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000001","name":"U.S. National Science Foundation","doi-asserted-by":"crossref","award":["DGE-2139841"],"award-info":[{"award-number":["DGE-2139841"]}],"id":[{"id":"10.13039\/100000001","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,4,1]]},"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-terminal 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-glycans that comprise more than 50% of its molecular weight remain largely unknown. To address this gap, we leveraged mass spectrometry (MS)-based glycoproteomics and molecular dynamics (MD) to explore the structural role of site-specific O-glycans on C-terminal lacritin. In doing do, we identified distinct glycosylation profiles between monomeric and multimeric lacritin, particularly at glycosites located near crosslinking residues (Lys101 and Lys104) that modulate multimer formation. Building on our glycoproteomics data, we performed MD simulations on monomer and multimer glycoforms and revealed that O-glycans participate in intra-glycan-protein interactions, thereby affecting the conformational flexibility of lacritin and the spatial arrangement of Lys101 and Lys104. Finally, we quantified the solvent-accessible surface area (SASA) of Lys101 and Lys104, highlighting that proximal O-glycosylation is predicted to affect the propensity of these residues to participate in crosslinking. Taken together, these findings underscore a central role for lacritin O-glycans in affecting structural topology with implications for its downstream biological activity.<\/jats:p>","DOI":"10.64898\/2026.03.30.715376","type":"posted-content","created":{"date-parts":[[2026,4,3]],"date-time":"2026-04-03T00:00:11Z","timestamp":1775174411000},"source":"Crossref","is-referenced-by-count":1,"title":["Site-specific O-glycans influence lacritin structure and multimerization in tears"],"prefix":"10.64898","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 CT, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ryan J.","family":"Chen","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Yale University, New Haven CT, 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