Identification of Roles for Peptide: N-Glycanase and Endo-β-N-Acetylglucosaminidase (Engase1p) during Protein N-Glycosylation in Human HepG2 Cells

BACKGROUND: During mammalian protein N-glycosylation, 20% of all dolichol-linked oligosaccharides (LLO) appear as free oligosaccharides (fOS) bearing the di-N-acetylchitobiose (fOSGN2), or a single N-acetylglucosamine (fOSGN), moiety at their reducing termini. After sequential trimming by cytosolic endo beta-N-acetylglucosaminidase (ENGase) and Man2c1 mannosidase, cytosolic fOS are transported into lysosomes. Why mammalian cells generate such large quantities of fOS remains unexplored, but fOSGN2 could be liberated from LLO by oligosaccharyltransferase, or from glycoproteins by NGLY1-encoded Peptide-N-Glycanase (PNGase). Also, in addition to converting fOSGN2 to fOSGN, the ENGASE-encoded cytosolic ENGase of poorly defined function could potentially deglycosylate glycoproteins. Here, the roles of Ngly1p and Engase1p during fOS metabolism were investigated in HepG2 cells. METHODS/PRINCIPAL FINDINGS: During metabolic radiolabeling and chase incubations, RNAi-mediated Engase1p down regulation delays fOSGN2-to-fOSGN conversion, and it is shown that Engase1p and Man2c1p are necessary for efficient clearance of cytosolic fOS into lysosomes. Saccharomyces cerevisiae does not possess ENGase activity and expression of human Engase1p in the png1Delta deletion mutant, in which fOS are reduced by over 98%, partially restored fOS generation. In metabolically radiolabeled HepG2 cells evidence was obtained for a small but significant Engase1p-mediated generation of fOS in 1 h chase but not 30 min pulse incubations. Ngly1p down regulation revealed an Ngly1p-independent fOSGN2 pool comprising mainly Man(8)GlcNAc(2), corresponding to approximately 70% of total fOS, and an Ngly1p-dependent fOSGN2 pool enriched in Glc(1)Man(9)GlcNAc(2) and Man(9)GlcNAc(2) that corresponds to approximately 30% of total fOS. CONCLUSIONS/SIGNIFICANCE: As the generation of the bulk of fOS is unaffected by co-down regulation of Ngly1p and Engase1p, alternative quantitatively important mechanisms must underlie the liberation of these fOS from either LLO or glycoproteins during protein N-glycosylation. The fully mannosylated structures that occur in the Ngly1p-dependent fOSGN2 pool indicate an ERAD process that does not require N-glycan trimming

GDP-mannose: GlcNAc₂-PP-dolichol mannosyltransferase deficiency (CDG Ik): 5 new patients and 7 novel mutations

International audienceBackground: In type I Congenital Disorders of Glycosylation (CDG I), proteins necessary for the biosynthesis of the lipid-linked oligosaccharide (LLO) required for protein N-glycosylation are defective. A deficiency in GDP-mannose: GlcNAc-PP-dolichol mannosyltransferase (MT-1) causes CDG Ik (OMIM 608540), and only five patients, with severe multisystemic clinical presentations, have been described with this disease. Objective: To characterise genetic, biochemical and clinical data in 5 new CDG Ik cases and compare these findings with those of the 5 previously described patients. Methods: LLO biosynthesis was examined in skin biopsy fibroblasts, mannosyltransferases were assayed in microsomes prepared from these cells, and -encoding MT-1 was sequenced at both the DNA and cDNA levels. Clinical data for the 5 new patients were collated. Results: Cells from 5 patients with non-typed CDG I revealed accumulations of GlcNAc-PP-dolichol, the second intermediate in the biosynthesis of LLO. Assay of MT-1, 2 and 3, the first three mannosyltransferases required for extension of this intermediate demonstrated only MT-1 to be deficient. DNA sequencing of ALG1 revealed 9 different mutations, 7 of which have not been previously reported. Clinical presentations are severe with CNS involvement and ocular disturbances being prevalent. Conclusions: CDG Ik presents at the severe end of the CDG I clinical spectrum, and in our experience, along with CDG Ib (MPI deficiency: OMIM 602579), is the second most frequently diagnosed CDG I after CDG Ia (PMM2 deficiency: OMIM 601785). Accordingly, we propose that CDG Ik should be given more consideration when diagnostic strategies are prioritised based on apparent CDG I subtype frequencies

From splitting GLUT1 deficiency syndromes to overlapping phenotypes.

International audienceIntroduction Glucose transporter type 1 deficiency syndrome (GLUT1DS) is a rare genetic disorder due to mutations or deletions in SLC2A1, resulting in impaired glucose uptake through the blood brain barrier. The classic phenotype includes pharmacoresistant epilepsy, intellectual deficiency, microcephaly and complex movement disorders, with hypoglycorrhachia, but milder phenotypes have been described (carbohydrate-responsive phenotype, dystonia and ataxia without epilepsy, paroxysmal exertion-induced dystonia). The aim of our study was to provide a comprehensive overview of GLUT1DS in a French cohort. Methods 265 patients were referred to the French national laboratory for molecular screening between July 2006 and January 2012. Mutations in SLC2A1 were detected in 58 patients, with detailed clinical data available in 24, including clinical features with a focus on their epileptic pattern and electroencephalographic findings, biochemical findings and neuroimaging findings. Results 53 point mutations and 5 deletions in SLC2A1 were identified. Most patients (87.5%) exhibited classic phenotype with intellectual deficiency (41.7%), epilepsy (75%) or movement disorder (29%) as initial symptoms at a medium age of 7.5 months, but diagnostic was delayed in most cases (median age at diagnostic 8 years 5 months). Sensitivity to fasting or exertion in combination with those 3 main symptoms were the main differences between mutated and negative patients (p < 0.001). Patients with myoclonic seizures (52%) evolved with more severe intellectual deficiency and movement disorders compared with those with Early Onset Absence Epilepsy (38%). Three patients evolved from a classic phenotype during early childhood to a movement disorder predominant phenotype at a late childhood/adulthood. Conclusions Our data confirm that the classic phenotype is the most frequent in GLUT1DS. Myoclonic seizures are a distinctive feature of severe forms. However a great variability among patients and overlapping through life from milder classic phenotype to paroxysmal-prominent- movement-disorder phenotype are possible, thus making it difficult to identify definite genotype–phenotype correlations