Structural and Functional Characterization of N-Glycanase-1 Pathogenic Variants

NGLY1 deficiency is a congenital disorder of deglycosylation, caused by pathogenic variants of the NGLY1 gene. It manifests as global developmental delay, hypo- or alacrima, hypotonia, and a primarily hyperkinetic movement disorder. The NGLY1 enzyme is involved in deglycosylation of misfolded N-glycosylated proteins before their proteasomal degradation, and in the activation of transcription factors that control the expression of proteasomal subunits. Here, we have characterized the pathogenic NGLY1 variants found in three Swiss NGLY deficiency patients, as well as the Arg401* variant found in several patients. Our functional and structural assessment of these variants show that they cause a profound reduction of NGLY1 activity, severely reduced expression of NGLY1 protein, and misprocessing of the transcription factor NFE2L1. Furthermore, transcription of proteasomal subunits and NGLY1 mRNA splicing are impaired by some of these variants. Our structural analysis shows that the Arg390Gln substitution results in destabilization of NGLY1 structure due to a loss of an ionic interaction network of Arg390, and potentially impairment of protein-protein interactions. Our results provide important information on the functional and structural effects of pathogenic NGLY1 variants and pave way for structure-based development of personalized treatment options.