Congenital disorder of glycosylation syndrome type Ia (CDG Ia) is an autosomal recessive multisystem disorder with neurological involvement due to mutations into gene sequence that codifies phosphomannomutase 2 (PMM2), enzyme involved in N-glycosylation pathway. Mutated PMM2 leads to a missed conversion of mannose-6P to mannose 1P which results in low concentrations of GDP-MAN in cytosol and little branched oligosaccharide chains. Previous studies have been demonstrating a reduced activity of several lysosomal enzymes such as α-fucosidase, β-hexosaminidase, β-glucuronidase and α–mannosidase in leukocytes and sera of CDG-Ia patients.
We propose an in vitro treatment of CDG fibroblasts using PLGA nanoparticles loaded with guanosine diphosphate D-mannose (GDP-Man), a nucleotide–activated sugar essential to building olygosaccharide chains bypassing glycosylation pathway reaction catalized by PMM2. PLGA-NPS loaded with the commercial guanosine 5'-diphospho-D-mannose sodium salt were prepared by W/O/W (double emulsion) solvent evaporation method. Human fibroblast controls and CDG 1a fibroblasts were obtained from skin biopsies. Specific activities of β-galactosidase, α-mannosidase, β-glucuronidase were measured using 4-methyl-umbelliferyl β-D-galactopyranoside, 4-methyl-umbelliferyl α-d-Mannopyranoside, 4-methyl-umbelliferyl β-d-glucuronide substrates.
In order to determine in vitro hypoglycosylation correction we assayed specific activity of different lysosomal enzymes including β-galactosidase, α-mannosidase and β-glucoronidase from CDG 1a patient’s fibroblast cultures. Three fibroblast cultures derived from three CDG patients, called F1, F2 and F3, were incubated with PLGA nanoparticles loaded with 1.5 ug of GDP-Man, corresponding to 2,3 nmol of GDP-Man for three or six hours at 37°C.
The specific activity of lysosomal enzymes like α-mannosidase and β-glucoronidase were assayed at three different times after treatment: 24 hours, 48 hours and 72 hours. As shown in Figure 1, α-mannosidase attains levels of activity close to fibroblast control activity after 48 hours post-incubation in F1, F2 and F3, demonstrating the efficiency of the proposed treatment. To date there is no therapy for CDG-Ia patients, but PLGA nanoparticles loaded with GDP-Man might provide a suitable treatment to bypass PMM2 deficiency and improve protein glycosylation due to their high biodegradability and toxicity profile.
Keywords: Congenital disorder of glycosylation, poly(lactic-co-glycolic acid)
nanoparticles, Guanosine diphosphate and Mannose, phosphomannomutase 2, lysosomal enzyme, specific activity.
