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.