Expression and localization of EFEMP1 wild‐type and mutant proteins
EFEMP1 is expressed in multiple ocular anterior segment cell types especially the trabecular meshwork beam cells and juxtacanilicular matrix cells (Figure 3) (van Zyl et al., 2020), two structures necessary for outflow of intraocular fluid (aqueous humor) and maintenance of normal IOP levels. EFEMP1 ocular expression is similar to Myocilin (MYOC ) also an extracellular matrix protein involved in JOAG. MYOC mutations cause protein misfolding and endoplasmic reticulum aggregation, and efforts to reduce mutation-related misfolding can result in lower IOP in animal models (Orwig et al., 2014; Jain et al., 2017).
To assess the functional effects of the JOAG-related EFEMP1variants, we hypothesized that these variants could cause protein misfolding, aggregation and intracellular retention similar to that observed for the p.Arg345Trp MLVT/DHRD variant and for JOAG-relatedMYOC mutations. To test this hypothesis we transfected COS7 cells with Gateway destination expression vectors (pCAG-V5-IRES-EGFP) (Zhang et al., 2012) for wildtype EFEMP1 cDNA, the three JOAG-relatedEFEMP1 variants, and also the MLVT/DHRD variant (p.Arg345Trp) and the POAG-related variant (p.Arg140Trp). Imaging transfected cells showed that wild type EFEMP1 was distributed throughout the cell, while significant intracellular aggregation was observed for each JOAG-related variant (Figure 4). Co-localization with an endoplasmic reticulum (ER) marker, CellLight™ ER-RFP, BacMam 2.0 (Invitrogen, Carlsbad, CA), suggests that EFEMP1 protein aggregates are formed in the vicinity of the ER. In contrast, the p.Arg345Trp variant exhibited more diffuse cellular distribution as did the POAG related variant p.Arg140Trp.
To measure intracellular protein retention, we collected an equal number of transfected cells from experiments using the wild type vector, the 3 JOAG-related vectors and the p.Arg345Trp and p.Arg140Trp vectors. Using Western blot assays (Figure 5A, Figure S1) we determined the fraction of EFEMP1 protein retained within the cell in comparison to overall protein and relative to wild type protein (Figure 5B). Compared to wild type cells intracellular EFEMP1 protein was significantly increased for all three of the JOAG-related variants (P< 0.05). JOAG-related intracellular protein was also increased compared to the MLVT/DHRD variant p.Arg345Trp and the POAG-related p.Arg140Trp.