Aspartylglycosaminuria (AGU) is the most common disorder of
glycoprotein degradation. AGU patients are deficient in
glycosylasparaginase (GA), which results in accumulation of
aspartylglucosamine in body fluids and tissues. Human
glycosylasparaginase was stably overexpressed in NIH-3T3 mouse fibroblasts, in which the unusual posttranslational processing and maturation of the
enzyme occurred in a high degree. The recombinant
enzyme was isolated as two
isoforms, which were both phosphorylated, and actively transported into AGU fibroblasts and lymphoblasts through
mannose-6-phosphate receptor-mediated endocytosis. The rate of uptake into fibroblasts was half-maximal when the concentration of GA in the medium was 5 x 10(-8) M. Immunofluorescence microscopy suggested compartmentalization of the recombinant
enzyme in the lysosomes. Supplementation of culture medium with either
isoform cleared AGU lymphoblasts of stored
aspartylglucosamine when
glycosylasparaginase activity in the cells reached 3-4% of that in normal lymphoblasts. A relatively small amount of recombinant GA in the culture medium was sufficient to reverse pathology in the target cells, indicating high corrective quality of the
enzyme preparations. The combined evidence indicates that
enzyme replacement therapy with the present recombinant
glycosylasparaginase might reverse pathology at least in somatic cells of AGU patients.