guanosine-diphosphate-mannose and Congenital-Disorders-of-Glycosylation

GDPMan is a key substrate in glycoprotein formation. This is especially true for lower eukaryotes where, in addition to the involvement in N-glycan biosynthesis and GPI-anchor formation, GDPMan takes part in the process which is unique for yeast and fungi i.e. O-mannosylation. Several lines of evidence have been presented that the level of GDPMan affects the process occurring in the Golgi compartment i.e. the elongation of outer mannose chain of glycoproteins in Saccharomyces cerevisiae. Results from our laboratory indicate that the availability of GDPMan affects also the early steps of glycoprotein formation ascribed to the endoplasmic reticulum, i.e. assembly of the dolichol-linked oligosaccharide as well as mannosyl-phosphodolichol (MPD) formation. The biochemical basis of carbohydrate deficient glycoprotein syndrome, a severe neurological disorder related to the GDPMan deficiency, is also discussed.

Topics: Congenital Disorders of Glycosylation; Glycosylation; Guanosine Diphosphate Mannose; Humans; Saccharomyces cerevisiae

Congenital disorder of glycosylation (CDG) type Ia is a multisystem disorder that occurs due to mutations in the phosphomannomutase 2 (PMM2) gene, which encodes for an enzyme involved in the N‑glycosylation pathway. Mutated PMM2 leads to the reduced conversion of mannose‑6‑P to mannose‑1‑P, which results in low concentration levels of guanosine 5'‑diphospho‑D‑mannose (GDP‑Man), a nucleotide‑activated sugar essential for the construction of protein oligosaccharide chains. In the present study, an in vitro therapeutic approach was used, based on GDP‑Man‑loaded poly (D,L‑lactide‑co‑glycolide) (PLGA) nanoparticles (NPs), which were used to treat CDG‑Ia fibroblast cultures, thus bypassing the glycosylation pathway reaction catalysed by PMM2. To assess the degree of hypoglycosylation in vitro, the present study examined the activities of α‑mannosidase, β‑glucoronidase and β‑galactosidase in defective and normal fibroblasts. GDP‑Man (30 µg/ml GDP‑Man PLGA NPs) was incubated for 48 h with the cells and the specific activities of α‑mannosidase and β‑galactosidase were estimated at 69 and 92% compared with healthy controls. The residual activity of β‑glucoronidase increased from 6.5 to 32.5% and was significantly higher compared with that noted in the untreated CDG‑Ia fibroblasts. The glycosylation process of fibroblasts was also analysed by two‑dimensional electrophoresis. The results demonstrated that treatment caused the reappearance of several glycosylated proteins. The data in vitro showed that GDP‑Man PLGA NPs have desirable efficacy and warrant further evaluation in a preclinical validation animal model.

Topics: Cells, Cultured; Congenital Disorders of Glycosylation; Drug Carriers; Fibroblasts; Glycosylation; Guanosine Diphosphate Mannose; Humans; Nanoparticles; Phosphotransferases (Phosphomutases); Polylactic Acid-Polyglycolic Acid Copolymer

In fibroblasts from five patients with carbohydrate-deficient glycoprotein syndrome type 1, the incorporation of [2-3H] mannose into mannose phosphates, GDP-mannose, GDP-fucose, dolichol-P-mannose, lipid-linked oligosaccharides, and glycoprotein fraction was determined. We observed a 3- to 5-fold reduction of incorporation of radioactivity into mannose 1-phosphate, GDP-mannose, GDP-fucose, dolichol-P-mannose, and nascent glycoproteins. The incorporation of radioactivity into mannose 6-phosphate was normal. The formation of lipid linked oligosaccharides was only slightly affected (

Topics: Carbohydrates; Cells, Cultured; Congenital Disorders of Glycosylation; Dolichol Monophosphate Mannose; Fibroblasts; Guanosine Diphosphate Fucose; Guanosine Diphosphate Mannose; Humans; Lipopolysaccharides; Mannosephosphates; Phosphotransferases (Phosphomutases)