A congenital disorder of deglycosylation: Biochemical characterization of N-glycanase 1 deficiency in patient fibroblasts

Abstract

N-Glycanase 1, encoded by NGLY1, catalyzes the deglycosylation of misfolded N-linked glycoproteins retrotranslocated into the cytosol. We identified nine cases with mutations in NGLY1. The patients show developmental delay, seizures, peripheral neuropathy, abnormal liver function and alacrima (absence of tears). The mutations in NGLY1 resulted in the absence of N-glycanase 1 protein in patient-derived fibroblasts. Applying a recently established cellular deglycosylation-dependent Venus fluorescence assay, we found that patient fibroblasts had dramatically reduced fluorescence, indicating a pronounced reduction in N-glycanase enzymatic activity. Using this assay, we could find no evidence of other related activities. Our findings reveal that NGLY1 mutations destroy both N-glycanase 1 protein and enzymatic activity.

Keywords: N-glycanase 1 deficiency; Z-VAD; deglycosylation-dependent venus (ddVenus) fluorescence assay.

Fig. 1.

NGLY1 mRNA and protein levels are decreased in patient fibroblasts. (A) qPCR analysis of NGLY1 mRNA. Total RNA was extracted from control, patients' parents and patients’ fibroblasts followed by cDNA synthesis. Specific primer pairs (Table II) targeting human NGLY1 gene and human housekeeping gene HPRT were used in qPCR reactions to amplify the target gene (see Materials and methods). For each sample, dual replicates were run. Each error bar in the histogram represents SD of at least three independent assays. (B) Western blot analysis of N-glycanase 1. Total lysates were harvested from control, patients’ parents and patients’ fibroblasts followed by western blot analysis (top panel). Protein Coomassie blue stain serves as loading control. The lower graphs were plotted based on the quantification of western blot data. Gray intensity of N-glycanase 1 band was divided by that of the loading control to normalize the N-glycanase 1 level. The normalized N-glycanase 1 value of each cell is divided by that of the control to obtain the relative value for each individual fibroblast line. Each error bar in the histogram represents SD of 3–4 independent assays. Individual NG2D is dad of patient NG2. NG4D and NG4M are dad and mom of patient NG4, respectively. NG5D and NG5M are dad and mom of patient NG5, respectively. ND, not detected.

Fig. 2.

Cellular deglycosylation-dependent assay shows reduced N-glycanase 1 enzymatic activity in patient fibroblasts. (A) Schematic depiction of deglycosylation-dependent fluorescence assay. The ddVenus constructs containing an N-glycosylation site (NFT) only become fluorescent when they are first glycosylated in the ER and subsequently deglycosylated after retrotranlocation, where the asparagine is deamidated to aspartic acid (left panel). The fluorescence of Venus without glycosylation site (DFF) does not depend on the glycosylation and deglycosylation (right panel). (B) The patient fibroblasts were transfected with plasmids containing deglycosylation independent Venus (Venus) and deglycosylation dependent (ddVenus) by electroporation. Two days later, the cells were treated with 10 µM MG132 for 6 h. Then the cells were trypsinized and the YFP fluorescence was analyzed by flow cytometry. The cells with positive fluorescence were gated and the numbers in red color indicate the percentage. (C) The relative ddVenus/Venus median fluorescence (upper panel) and positive cell percentage (lower panel) ratios. The median fluorescence or percentage of positive cell for Venus and ddVenus was first calculated after subtract autofluorescence without transfection background. The ddVenus/Venus ratios representing the enzymatic activity of N-glycanase 1 for each line were plotted, and the error bars in the histogram represent SD of three independent assays or value range of two independent assays. This figure is available in black and white in print and in color at Glycobiology online.

Fig. 3.

Inhibition of N-glycanase 1 reduces ddVenus fluorescence. (A) siRNA knockdown of NGLY1 followed by western blot analysis. Ten nanomolar of scrambled siRNA (NC1), 10, 20 and 40 nM N-glycanase 1 siRNA Smartpool were delivered to HEK293 cells. The total lysates were harvested 24, 48 and 72 h after transfection followed by western blot analysis of NGLY1 knockdown. (B and C) siRNA knockdown of NGLY1 and ddVenus transfection followed by qPCR (B) and flow cytometry (C) analysis. Ten nanomolar of scrambled siRNA (NC1) and NGLY1 siRNA Smartpool were delivered to HEK293 cells. Forty-eight hours after siRNA knockdown, ddVenus plasmids were transfected for 24 h followed by 10 μM MG132 treatment for another 6 h. The mRNA level of NGLY1 in knockdown groups was calculated relative to that of scrambled siRNA (NC1) transfection group, which was designated as 100%. Each error bar in the histogram represents value range of two independent assays. For flow cytometry analysis, the cells with positive fluorescence were gated and the median fluorescence of gated cells was divided by that of scrambled siRNA (NC1). The relative values were plotted, and the error bars in the histogram represents value range of two independent assays. (D) After transfection, cells were treated with 30 μM Z-VAD in the presence of 5 μM MG132 for 6 h and analyzed by flow cytometry as described in Figure 2B. (E) The relative ddVenus/Venus median fluorescence ratios were plotted as described in Figure 2C. This figure is available in black and white in print and in color at Glycobiology online.

Fig. 4.

Wild type but not mutant NGLY1 restores the ddVenus fluorescence. (A) Western blot analysis of hNGLY1 overexpression by electroporation in fibroblasts. Protein Coomassie blue stain serves as loading control. (B) Patient fibroblasts were transfected with ddVenus together with the same amount of plasmids containing human wild-type NGLY1 (hNGLY1) or human NGLY1 mutant C309A. This figure is available in black and white in print and in color at Glycobiology online.

Fig. 5.

MG132 does not increase the ddVenus fluorescence accumulation in patient fibroblasts. (A) Control and patient fibroblasts were electroporated with ddVenus or Venus plasmid followed by 5 μM MG132 or DMSO treatment in the presence or absence of 30 μM Z-VAD for 6 h. After fixation and nuclei staining with Hoechst 33342, 96-well plates were imaged on high content imaging system. (B) NG1 patient fibroblasts were transfected with ddVenus and WT hNGLY1 plasmids followed by MG132 treatment in the presence or absence of Z-VAD. (C) Control and patient fibroblasts were electroporated with ddVenus followed by MG132 or DMSO treatment for shorter (upper graph) and longer (lower graph) duration. At each time point, the plates were imaged and ddVenus fluorescence intensity was quantified by Acapella software (see methods for detail). The relative ddVenus fluorescence mean value was obtained by subtracting DMSO-treated cells from the MG132-treated cells and then plotted in the graph. This figure is available in black and white in print and in color at Glycobiology online.