{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,4,11]],"date-time":"2026-04-11T19:44:09Z","timestamp":1775936649583,"version":"3.50.1"},"posted":{"date-parts":[[2025,3,3]]},"group-title":"Biochemistry","reference-count":31,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,3,3]],"date-time":"2025-03-03T00:00:00Z","timestamp":1740960000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,3,3]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Approximately 2-4% of adult onset and 10% of juvenile onset cases of primary open angle glaucoma can be attributed to non-synonymous coding mutations in\n                  <jats:italic>MYOC<\/jats:italic>\n                  . One of the key characteristics of a pathogenic\n                  <jats:italic>MYOC<\/jats:italic>\n                  mutant is the inability of the resulting protein to be secreted from trabecular meshwork cells. Instead, pathogenic myocilin variants accumulate in the endoplasmic reticulum. Typically, localization of MYOC mutants is compared to wild-type myocilin in cellular secretion assays that use immunoblot to detect myocilin in extracellular media, alongside intracellular soluble and insoluble (aggregated) fractions. Here, we implement a new method that utilizes a complement-based luminescence method in which an 11-residue HiBiT tag is appended to myocilin and complements a truncated nanoluciferase. The method allows for highly sensitive luminescence detection and does not require immunoblot. We tested non-synonymous coding variants T377R, D384G, D395ins, C433Y, T455K, and L486F, in an established immortalized trabecular meshwork cell line. Secretion was tested in 96-well plate format, revealing poor secretion for these mutants compared to wild-type myocilin. For assays conducted in 6-well plates, myocilin mutants were accumulated in intracellular fractions. HiBiT luminescence signals correlated well with immunofluorescence as well as immunoblot but is more sensitive than the latter. Overall, our study demonstrates that complement-based detection of mutant myocilin using luminescence allows for facile and sensitive detection of myocilin localization and has confirmed secretion defects for seven variants.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Highlights<\/jats:title>\n                  <jats:list list-type=\"bullet\">\n                    <jats:list-item>\n                      <jats:p>Mutations in myocilin are causal for early onset open angle glaucoma<\/jats:p>\n                    <\/jats:list-item>\n                    <jats:list-item>\n                      <jats:p>In the lab pathogenic myocilin mutations are characterized by secretion defects<\/jats:p>\n                    <\/jats:list-item>\n                    <jats:list-item>\n                      <jats:p>We validate a complement luminescence assay for detection of myocilin localization<\/jats:p>\n                    <\/jats:list-item>\n                    <jats:list-item>\n                      <jats:p>The luminescence-based assay is more sensitive than traditional immunoblot<\/jats:p>\n                    <\/jats:list-item>\n                    <jats:list-item>\n                      <jats:p>We confirm secretion defects for several mutants not previously characterized<\/jats:p>\n                    <\/jats:list-item>\n                  <\/jats:list>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>TOC Graphic<\/jats:title>\n                  <jats:fig id=\"ufig1\" position=\"float\" fig-type=\"figure\" orientation=\"portrait\">\n                    <jats:graphic xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"640780v1_ufig1\" position=\"float\" orientation=\"portrait\"\/>\n                  <\/jats:fig>\n                <\/jats:sec>","DOI":"10.1101\/2025.02.28.640780","type":"posted-content","created":{"date-parts":[[2025,3,3]],"date-time":"2025-03-03T22:45:12Z","timestamp":1741041912000},"source":"Crossref","is-referenced-by-count":0,"title":["Monitoring mutant myocilin secretion and localization in trabecular meshwork cell cultures using a protein complementation-based luminescence assay"],"prefix":"10.64898","author":[{"given":"Hannah A.","family":"Youngblood","sequence":"first","affiliation":[{"name":"School of Chemistry & Biochemistry, Georgia Institute of Technology"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ethan F.","family":"Harris","sequence":"additional","affiliation":[{"name":"School of Chemistry & Biochemistry, Georgia Institute of Technology"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kaylee P.","family":"Lankford","sequence":"additional","affiliation":[{"name":"Department of Ophthalmology, University of Texas Southwestern Medical 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Technology"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2026041111592961000_2025.02.28.640780v1.1","first-page":"e11686","article-title":"Epidemiology of glaucoma: the past, present, and predictions for the future","volume":"12","year":"2020","journal-title":"Cureus"},{"key":"2026041111592961000_2025.02.28.640780v1.2","doi-asserted-by":"publisher","DOI":"10.1016\/j.gene.2013.02.054"},{"key":"2026041111592961000_2025.02.28.640780v1.3","doi-asserted-by":"publisher","DOI":"10.1002\/humu.24482"},{"key":"2026041111592961000_2025.02.28.640780v1.4","first-page":"1944","article-title":"A novel MYOC heterozygous mutation identified in a Chinese Uygur pedigree with primary open-angle glaucoma","volume":"18","year":"2012","journal-title":"Mol 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