Recombinant glycosylasparaginase and in vitro correction of aspartylglycosaminuria.

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.