Cell-free translation of human lysosomal α-glucosidase: Evidence for reduced precursor synthesis in an adult patient with glycogenosis type II

Abstract

Early events in the biosynthesis of α-glucosidase (EC 3.2.1.20) were studied in a wheat-germ cell-free translation system, using control and mutant RNA. In vitro, the primary translation product of the α-glucosidase mRNA is a 100 kDa protein. When canine microsomal membranes are added to the translation system, the nascent α-glucosidase precursor is cotranslationally transported across the microsomal membranes, yielding a 110 kDa glycosylated form. This protein has the same electrophoretic characteristics as the α-glucosidase precursor observed after in vivo labeling of control fibroblasts. Inhibition of glycosylation in vivo by tunicamycin or deglycosylation of the in vivo synthesized α-glucosidase precursor by glycopeptidase F reveals a core protein similar in molecular mass to the primary translation product. Total RNA from a patient with the adult form of glycogenosis type II is not able to direct the synthesis of normal amounts of α-glucosidase in vitro. Northern blot analysis of the RNA, using cloned α-glucosidase cDNA sequences as a probe, demonstrates that in this patient the amount of the 3.4 kb α-glucosidase mRNA is highly reduced. The results indicate that the synthesis or stability of the mRNA is affected.