AGAL misprocessing-induced ER stress and the unfolded protein response: lysosomal storage-independent mechanism of Fabry disease pathogenesis?
Classic Fabry disease (FD) is caused by GLA mutations that result in enzymatic deficiency of alpha-galactosidase A (AGAL), lysosomal storage of globotriaosylceramide, and a resulting multisystemic disease.  In non-classic later-onset FD, patients have some preserved AGAL activity and a milder disease course, though female carriers may also be affected.  While FD pathogenesis has been mostly attributed to catalytic deficiency of mutated AGAL, lysosomal storage and impairment of lysosomal functions, other pathogenic factors may be important, especially in non-classic later-onset FD.  Clinical findings in affected males revealed a milder clinical course with ~15% residual AGAL activity and borderline plasma lyso-Gb3Cer levels. Kidney biopsies did not show lysosomal storage. Laboratory investigations documented intracellular retention of mutated AGAL with resulting endoplasmic reticulum (ER) stress and the unfolded protein response (UPR), which were alleviated with BRD4780, a small molecule clearing misfolded proteins from the early secretory compartment. We observed similar findings of ER stress and UPR with several other classic and non-classic FD missense AGAL variants. We identified defective proteostasis of mutated AGAL resulting in chronic ER stress and UPR of AGAL expressing cells (hereafter referred to as AGALopathy) as an important contributor to FD pathogenesis. These findings provide insight into non-classic later-onset FD and may better explain clinical manifestations with implications for pathogenesis, clinical characterization and treatment of all FD forms. Overall design: We characterized the genetic, clinical, biochemical, molecular, cellular and organ pathology correlates of the p.L394P AGAL variant that was identified in six individuals with end-stage kidney disease by the Czech national screening program for FD and by further screening in an additional 24 family members. Among other material we used HEK cells stably producing either wt-AGAL_FLAG or mut-AGAL_FLAG tagged. Preparation of cell lines stably expressing AGAL_FLAG in HEK cells: HEK 293 cells were maintained in Dulbecco's Modified Eagle Medium : Nutrient Mixture F-12 (DMEM/F-12) high glucose medium supplemented with 10% (vol/vol) fetal calf serum (Gibco), 100 U/ml penicillin G/streptomycin (Sigma, Prague, Czech Republic). HEK293 cells were transfected with 2.5 µg of plasmid DNA using  Lipofectamine 3000TM (Invitrogen, Paisley, UK). Three days post transfection, cells were trypsinized, diluted and cultured in selective medium containing 0.5 mg/ml G418 (Invitrogen-Gibco, Paisley, UK). For each selected clone, the presence and correct sequence of GLA_FLAG was confirmed by Sanger sequencing; the amount of GLA_FLAG transcript was determined by real-time PCR, and expression levels of AGAL_FLAG tagged proteins were assesed by Western blot with mouse monoclonal anti-FLAG antibody (F1804, SIGMA-Aldrich, Prague, Czech Republic). Clones demonstrating similar GLA_FLAG transcript and AGAL_FLAG protein amounts were selected for further analyses.