DPAGT1-CDG: Functional analysis of disease-causing pathogenic mutations and role of endoplasmic reticulum stress.

Patricia Yuste-Checa,Cristina Martín-Higueras,Celia Pérez-Cerdá,Alejandra Gámez,Belén Pérez,Ana I Vega,Celia Medrano,Magdalena Ugarte,Lourdes R Desviat,Alfred S Lewin

doi:10.1371/journal.pone.0179456

Abstract

Pathogenic mutations in DPAGT1 are manifested as two possible phenotypes: congenital disorder of glycosylation DPAGT1-CDG (also known as CDG-Ij), and limb-girdle congenital myasthenic syndrome (CMS) with tubular aggregates. UDP-N-acetylglucosamine-dolichyl-phosphate N-acetylglucosamine phosphotransferase (GPT), the protein encoded by DPAGT1, is an endoplasmic reticulum (ER)-resident protein involved in an initial step in the N-glycosylation pathway. The aim of the present study was to examine the effect of six variants in DPAGT1 detected in patients with DPAGT1-CDG, and the role of endoplasmic reticulum stress, as part of the search for therapeutic strategies to use against DPAGT1-CDG. The effect of the six mutations, i.e., c.358C>A (p.Leu120Met), c.791T>G (p.Val264Gly), c.901C>T (p.Arg301Cys), c.902G>A (p.Arg301His), c.1154T>G (p.Leu385Arg), and of the novel mutation c.329T>C (p.Phe110Ser), were examined via the analysis of DPAGT1 transcriptional profiles and GTP levels in patient-derived fibroblasts. In addition, the transient expression of different mutations was analysed in COS-7 cells. The results obtained, together with those of bioinformatic studies, revealed these mutations to affect the splicing process, the stability of GTP, or the ability of this protein to correctly localise in the ER membrane. The unfolded protein response (UPR; the response to ER stress) was found not to be active in patient-derived fibroblasts, unlike that seen in cells from patients with PMM2-CDG or DPM1-CDG. Even so, the fibroblasts of patients with DPAGT1-CDG seemed to be more sensitive to the stressor tunicamycin. The present work improves our knowledge of DPAGT1-CDG and provides bases for developing tailored splicing and folding therapies.

Highlights

Protein glycosylation refers to the co- and post-translational covalent attachment of oligosaccharide moieties to newly synthesised proteins
The results showed no induction of these genes in the DPAGT1-CDG patient-derived fibroblasts (Fig 5A), while the PMM2-CDG- and DPM1-CDG patient-derived cells showed increases in the expression of ATF4 and HSPA5 compared to the control cell line
Immunofluorescence analysis revealed GTP to be incorrectly localised in the endoplasmic reticulum (ER) in DPAGT1-CDG patient-derived fibroblasts from two patients (2 and 3)

Summary

Introduction

Protein glycosylation refers to the co- and post-translational covalent attachment of oligosaccharide moieties to newly synthesised proteins. DPAGT1 codes for UDP-Nacetylglucosamine-dolichyl-phosphate N-acetylglucosaminephosphotransferase (GPT; EC number 2.7.8.15), an enzyme involved in one of the initial steps of the N-glycosylation pathway This ER-resident transmembrane protein catalyses the transfer of N-acetylglucosamine from cytosolic UDP-N-acetylglucosamine to dolichol-phosphate, which is located in the ER membrane. DPAGT1 defects manifest as two alternative phenotypes: congenital disorder of glycosylation DPAGT1-CDG (MIM: 608093; previously known as CDG-Ij), and limb-girdle congenital myasthenic syndrome (CMS) (MIM 614750) with tubular aggregates. The former is a severe, multisystem disease, with most patients presenting moderate to severe psychomotor disability, microcephaly, hypotonia and epilepsy. This is true for mutations in other genes (e.g., GFPT1, ALG2 and ALG14) involved in the N-glycosylation pathway that result in CMS [3]

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Conclusion