Abstract In humans, N-glycanase 1 (NGLY1; Peptide: N-glycanase, PNGase) is responsible for the deglycosylation of misfolded glycoproteins. Pathogenic mutations in NGLY1 cause a clinical condition known as congenital disorder of deglycosylation (NGLY1-CDDG), a rare autosomal recessive disease first reported in 2012. Although NGLY1-CDDG was diagnosed through whole-exome or whole-genome sequencing and by evaluating the expression levels of NGLY1, the clinical relevance of a detected mutation in NGLY1 needs to be further confirmed. In this study, an in vitro enzymatic assay system was established to evaluate the thermal stability and substrate specificity of NGLY1, as well as the optimum reaction conditions for its activity. A panel of all mutations at the amino acid site R328 in NGLY1 was subjected to this assay. The results revealed that R328A, R328D, R328E, R328F, R328G, R328I, R328P, R328V, R328W, and R328Y were dysfunctional mutations (10/19); NGLY1 mutations with R328H and R328T exhibited similar activity as wild-type NGLY1 (2/19); and NGLY1 mutations with R328C, R328K, R328L, R328M, R328N, R328Q, and R328S showed decreased activity (7/19) compared to wild-type NGLY1. In addition, the effect of potential regulatory compounds, including N-acetyl-L-cysteine and dithiothreitol, on NGLY1 was examined. This in vitro assay may serve as a standard protocol to facilitate rapid diagnosis of all mutations on NGLY1 and a practical screening method for drugs and compounds with potential therapeutic value for NGLY1-CDDG patients.
