Rescue of sarcoglycan mutations by inhibition of endoplasmic reticulum quality control is associated with minimal structural modifications

Abstract

Sarcoglycanopathies (SGP) are a group of autosomal recessive muscle disorders caused by primary mutations in one of the four sarcoglycan genes. The sarcoglycans (α-, β-, γ-, and δ-sarcoglycan) form a tetrameric complex at the muscle membrane that is part of the dystrophin-glycoprotein complex and plays an essential role for membrane integrity during muscle contractions. We previously showed that the most frequent missense mutation in α-sarcoglycan (p.R77C) leads to the absence of the protein at the cell membrane due to its blockade by the endoplasmic reticulum (ER) quality control. Moreover, we demonstrated that inhibition of the ER α-mannosidase I activity using kifunensine could rescue the mutant protein localization at the cell membrane. Here, we investigate 25 additional disease-causing missense mutations in the sarcoglycan genes with respect to intracellular fate and localization rescue of the mutated proteins by kifunensine. Our studies demonstrate that, similarly to p.R77C, 22 of 25 of the selected mutations lead to defective intracellular trafficking of the SGs proteins. Six of these were saved from ER retention upon kifunensine treatment. The trafficking of SGs mutants rescued by kifunensine was associated with mutations that have moderate structural impact on the protein.

Partial IFN-γR2 deficiency is due to protein misfolding and can be rescued by inhibitors of glycosylation

“…25 Kifunensine was the only inhibitor tested that complemented all patients' cells following stimulation with IFN-g. NB-DNJ and castanospermine also rescued P3 cells. The precise mechanism of action of these modifiers of glycosylation is unclear, but they affect glucosidases I and II, ER-mannosidase I, Golgi mannosidase I, and other enzymes, 25,[34][35][36][37] possibly explaining their different impacts in our experiments. In addition, the mutations may have subtly different structural impacts, in terms of folding and glycosylation of IFN-gR2.…”

“…It has been shown that the use of modifiers of N-glycosylation (eg, kifunensine) can improve the maturation of misfolded proteins. 25,[34][35][36][37] We therefore treated the transfected HEK293T cells with kifunensine before western blotting. This treatment impaired the processing of the WT and all mutant proteins, which were also sensitive to Endo H treatment ( Figure 5A).…”

Unveiling the degradative route of the V247M α-sarcoglycan mutant responsible for LGMD-2D

“…Most of sarcoglycanopathy cases are in fact associated with missense mutations that can lead to a misfolded protein. However, many disease-causing missense mutations are without functional consequences (12,17), nonetheless, the mutant is intercepted by the quality control system and delivered to the proteasome. The premature degradation precludes the possibility for the mutated, but probably functional protein, to assemble with its partners, exit the ER, and move to the plasma membrane.…”

“…Sarcoglycanopathy clinical phenotype is very heterogeneous for age of onset, rate of progression and severity, often correlated to the level of residual sarcoglycans. Most of the LGMD-2D cases, due to defects of the α-sarcoglycan coding gene, are associated with missense mutations that could lead to a misfolded protein that becomes an ERAD substrate (11,16,17). Loss or strong reduction of α-sarcoglycan usually causes the absence or reduction of the other subunits, thus depriving muscle fibers of a critical complex (18) and identifying ERAD action as the key pathogenetic mechanism of LGMD-2D (12,16).…”

“…Even though many disease-causing missense mutations do not have functional consequences, because of the quality control rigid rules, these mutants are prematurely degraded. However, treatments interfering with either ERAD or proteasome attenuated the degradation of misfolded but ‘functional’ sarcoglycans and allowed a significant fraction of the surviving protein to reach the cell surface (11,16,17). …”