The Biochemical Basis of Congenital Disorders of Glycosylation

The enzyme peptide: N-glycanase (EC 3.5.1.52) is an endoglycosidase which cleaves N-linked glycans from incorrectly folded glycoproteins exported from the endoplasmic reticulum during ER-associated degradation (ERAD). Clinical mutations in this enzyme (resulting in loss or decrease in function) are responsible for the rare congenital disorder termed N-GLY1. N-GLY1 disorder was first identified by next generation sequencing in 2012, and as such there exists little information as to the cellular effects of N-glycanase deficiency. In this study, N-glycanase deficiency was examined in a well-characterised cell model (HEK-293). Cellular effects were examined following pharmacological inhibition of N-glycanase using carbobenzoxy-valyl-ananyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-fmk) and, genetic knockdown by siRNA. Attempts were also made to generate a knockout cell line using CRISPR/Cas9. Using molecular and cellular techniques, this study has confirmed that in HEK-293, pharmacological N-glycanase inhibition or siRNA knockdown does not activate ER stress, oxidative stress or change the glycoprotein profile of the cell. Both treatments were associated with a small increase in ThT fluorescence associated with protein aggregates and identified that autophagy was increased in pharmacological N-glycanase inhibition and siRNA knockdown, and that this likely represents a protective measure. Autophagy dysregulation using ATG13 KO MEF cells found a change in the redox environment of the cell. This effect was further exacerbated in both HEK293, WT MEFs and ATG13 KO MEFs by growth in glucose-free media supplemented with galactose to reduce glycolysis as an energy source. This is suggestive of mitochondrial dysregulation, although no changes in mitochondrial membrane potential or mitochondrial content were identified. Proteomics analysis of immuno-precipitated autophagosomes induced under pharmacological N-glycanase inhibition and siRNA knockdown revealed increased intensity of mitochondrial proteins in these samples indicative of mitophagy which may play a role in the cellular response to impairments of N-glycanase function