학술논문
In vitro treatment of congenital disorder of glycosylation type Ia using PLGA nanoparticles loaded with GDP-Man
Document Type
Academic Journal
Author
Source
International Journal of Molecular Medicine. July 2019, Vol. 44 Issue 1, p262, 11 p.
Subject
Language
English
ISSN
1107-3756
Abstract
Introduction Congenital disorder of glycosylation type la (CDG-Ia) is a rare autosomal recessive multisystem disorder with severe neurological disabilities (encephalopathy and psychomotor retardation) due to mutations in the phosphomannomutase 2 [...]
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 [alpha]-mannosidase, [beta]-glucoronidase and [beta]-galactosidase in defective and normal fibroblasts. GDP-Man (30 [micro]g/ml GDP-Man PLGA NPs) was incubated for 48 h with the cells and the specific activities of [alpha]-mannosidase and [beta]-galactosidase were estimated at 69 and 92% compared with healthy controls. The residual activity of [beta]-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.
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 [alpha]-mannosidase, [beta]-glucoronidase and [beta]-galactosidase in defective and normal fibroblasts. GDP-Man (30 [micro]g/ml GDP-Man PLGA NPs) was incubated for 48 h with the cells and the specific activities of [alpha]-mannosidase and [beta]-galactosidase were estimated at 69 and 92% compared with healthy controls. The residual activity of [beta]-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.