Youngblood, H. A. | bioRxiv Science

bioRxiv2025

Monitoring mutant myocilin secretion and localization in trabecular meshwork cell cultures using a protein complementation-based luminescence assay

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 MYOC. One of the key characteristics of a pathogenic MYOC 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. HighlightsO_LIMutations in myocilin are causal for early onset open angle glaucoma C_LIO_LIIn the lab pathogenic myocilin mutations are characterized by secretion defects C_LIO_LIWe validate a complement luminescence assay for detection of myocilin localization C_LIO_LIThe luminescence-based assay is more sensitive than traditional immunoblot C_LIO_LIWe confirm secretion defects for several mutants not previously characterized C_LI TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/640780v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1647a7aorg.highwire.dtl.DTLVardef@1a4e56dorg.highwire.dtl.DTLVardef@d29165org.highwire.dtl.DTLVardef@477e4f_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Structural basis for anomalous cellular trafficking behavior of glaucoma-associated A427T mutant myocilin

Familial mutations in myocilin cause vision loss in glaucoma due to misfolding and a toxic gain of function in a senescent cell type in the anterior eye. Here we characterize the cellular behavior and structure of the myocilin (myocilin A427T) mutant, of uncertain pathogenicity. Our characterization of A427T demonstrates that even mutations that minimally perturb myocilin structure and stability can present challenges for protein quality control clearance pathways. Namely, when expressed in an inducible immortalized trabecular meshwork cell line, inhibition of the proteasome reroutes wild-type myocilin, but not myocilin A427T, from endoplasmic reticulum associated degradation to lysosomal degradation. Yet, the crystal structure of the A427T myocilin olfactomedin domain shows modest perturbations largely confined to the mutation site. The previously unappreciated range of mutant myocilin behavior correlating with variable stability and structure provides a rationale for why it is challenging to predict causal pathogenicity of a given myocilin mutation, even in the presence of clinical data for members of an affected family. Comprehending the continuum of mutant myocilin behavior in the laboratory supports emerging efforts to use genetics to assess glaucoma risk in the clinic. In addition, the study supports a therapeutic strategy aimed at enhancing autophagic clearance of mutant myocilin. Significance statementO_LIRare familial mutations cause early onset glaucoma C_LIO_LIA427T is a case of uncertain pathogenicity C_LIO_LIA427T is structurally similar to wild-type but is not efficiently degraded C_LI

biochemistry↗