Wild-type and missense mutants of retinoschisin co-assemble resulting in either intracellular retention or incorrect assembly of the functionally active octamer

Abstract

The X-linked disease retinoschisis is caused by mutations in the RS1 gene encoding retinoschisin, most commonly missense mutations leading to a lack of secretion of functional protein. One potential approach to treat this disease would be the introduction of the wild-type protein by gene therapy in affected individuals. Retinoschisin normally forms homo-octamers, so co-expression of the wild-type protein with the mutant could result in their co-assembly. In the present study, we show that retinoschisin assembles into an octamer before transport from the endoplasmic reticulum and that co-assembly of wild-type and mutant protein can occur when they are co-expressed in the same cell. This co-assembly results in the retention of some, but not all, expressed wild-type retinoschisin. Moreover, when the wild-type protein is expressed with a missense mutant that is normally secreted, co-assembly occurs resulting in the secretion of a heterogeneous mixture of oligomers. Missense mutations of retinoschisin which cause intracellular retention also lead to an unfolded protein response. However, this is not sufficient to decrease cell viability suggesting that the pathology of the disease is not likely to be linked to programmed cell death.

Molecular Mechanisms Leading to Null-Protein Product from Retinoschisin (RS1) Signal-Sequence Mutants in X-Linked Retinoschisis (XLRS) Disease

“…The extent to which ER stress modulates the XLRS clinical phenotype is not clear. A recent study [Gleghorn et al, 2010] demonstrated that UPR in cells expressing pCys110Tyr mutant RS1 may actually promote cell viability. Apoptosis of photoreceptor cells would have drastic consequences for vision.…”

Recycling of peroxiredoxin IV provides a novel pathway for disulphide formation in the endoplasmic reticulum

“…For overexpression, cells were seeded to 60% confluence in 15 cm cell culture dishes and transfected with 12 μg plasmid DNA encoding the appropriate V5‐tagged PDI‐family member (construct creation described elsewhere (Jessop et al , 2009b)). Transfection was performed using polyethylenimine exactly as established previously (Gleghorn et al , 2010), with volumes increased three‐fold to reflect increased quantity of DNA. Cells were harvested for SP‐cell preparation at 48 h post‐transfection.…”

X-linked juvenile retinoschisis: Clinical diagnosis, genetic analysis, and molecular mechanisms

“…Accordingly, wild-type retinoschisin expressed in these cells by gene delivery could interact with the endogenously expressed mutant retinoschisin diminishing the therapeutic effect of gene therapy. This has been studied by analyzing protein expression, cellular localization and secretion of wild-type retinoschisin co-expressed with various disease-linked missense mutants in culture cells (Dyka and Molday, 2007; Gleghorn et al, 2010). These studies indicate that wild-type retinoschisin undergoes protein folding, subunit assembly, and secretion largely independent of the misfolded mutants.…”