A congenital disorder of deglycosylation: biochemical characterization of N‐glycanase 1 deficiency in patient fibroblasts (607.3)

Abstract

N‐glycanase 1, encoded by NGLY1, catalyzes the deglycosylation of misfolded N‐linked glycoproteins. Using whole‐genome and ‐exome sequencing, we identified 6 cases with mutations in NGLY1. The patients show developmental delay, seizures, peripheral neuropathy, abnormal liver function, and absent tears. The mutations in NGLY1 resulted in drastic reduction of N‐glycanase 1 protein in patient‐derived fibroblasts. We probed N‐glycanase 1 enzymatic activity firstly by measuring the enzyme released free oligosaccharides (fOS). The patient fibroblasts produced 2‐3 folds less fOS and showed an altered size distribution. Applying a recently established cellular deglycosylation dependent Venus fluorescence assay, we found that patient fibroblasts had dramatically reduced fluorescence indicating pronounced enzymatic activity reduction in N‐glycanase 1. Since deglycosylation of misfolded glycoproteins precedes proteasomal degradation, we speculated that N‐glycanase 1 deficiency might also accumulate misfolded glycoproteins, leading to ER stress. We observed 5‐10 fold increase of transfected ERAD substrates accumulation in the cytoplasm. However, under physiological conditions, we did not observe abnormalities of the three branches of UPR signal transduction (IRE1, PERK, and ATF6) in patient fibroblasts, indicating that the N‐glycanase 1 deficiency itself is not sufficient to cause ER stress.Grant Funding Source: Supported by the Bertrand Might Research Fund