LAUSR

ABSTRACT Nonsense mutations are relatively frequent in the rare X-linked lysosomal α-galactosidase A (α-Gal) deficiency (Fabry disease; FD), but have been poorly investigated. Here, we evaluated the responsiveness of a wide panel (n = 14) of GLA premature termination codons (PTCs) to the RNA-based approach of drug-induced readthrough through expression of recombinant α-Gal (rGal) nonsense and missense variants. We identified four high-responders to the readthrough-inducing aminoglycoside G418 in terms of full-length protein (C56X/W209X, ≥10% of wild-type rGal) and/or activity (Q119X/W209X/Q321X, ~5-7%), resulting in normal (Q119X/Q321X) or reduced (C56X, 0.27 ± 0.11; W209X, 0.35 ± 0.1) specific activity. To provide mechanistic insights we investigated the predicted amino acid substitutions mediated by readthrough (W209C/R, C56W/R), which resulted in correct lysosomal localization and appreciable protein/activity levels for the W209C/R variants. Differently, the C56W/R variants, albeit appreciably produced and localized into lysosomes, were inactive, thus indicating detrimental effects of substitutions at this position. Noticeably, when co-expressed with the functional W209C or W209R variants, the wild-type rGal displayed a reduced specific activity (0.5 ± 0.2 and 0.6 ± 0.2, respectively) that, considering the dimeric features of the α-Gal enzyme, suggested dominant-negative effects of missense variants through their interaction with the wild-type. Overall, we provide a novel mechanism through which amino acids inserted during readthrough might impact on the functional protein output. Our findings may also have implications for the interpretation of pathological phenotypes in heterozygous FD females, and for other human disorders involving dimeric or oligomeric proteins.