title: Cell-free translation of human lysosomal alpha-glucosidase: evidence for reduced precursor synthesis in an adult patient with glycogenosis type II.
abstract: Early events in the biosynthesis of alpha-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 alpha-glucosidase mRNA is a 100 kDa protein. When canine microsomal membranes are added to the translation system, the nascent alpha-glucosidase precursor is cotranslationally transported across the microsomal membranes, yielding a 110 kDa glycosylated form. This protein has the same electrophoretic characteristics as the alpha-glucosidase precursor observed after in vivo labeling of control fibroblasts. Inhibition of glycosylation in vivo by tunicamycin or deglycosylation of the in vivo synthesized alpha-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 alpha-glucosidase in vitro. Northern blot analysis of the RNA, using cloned alpha-glucosidase cDNA sequences as a probe, demonstrates that in this patient the amount of the 3.4 kb alpha-glucosidase mRNA is highly reduced. The results indicate that the synthesis or stability of the mRNA is affected.
authors.affiliation: Department of Cell Biology and Genetics, Erasmus University, Rotterdam, The Netherlands.
authors.forename: G T
authors.initials: GT
authors.lastname: van der Horst
authors.forename: E H
authors.initials: EH
authors.lastname: Hoefsloot
authors.forename: M A
authors.initials: MA
authors.lastname: Kroos
authors.forename: A J
authors.initials: AJ
authors.lastname: Reuser
mesh_terms: D002474:Cell-Free System; D002478:Cells, Cultured; D005347:Fibroblasts / Q000201:enzymology; D006008:Glycogen Storage Disease / Q000235:genetics*; D006009:Glycogen Storage Disease Type II / Q000201:enzymology / Q000235:genetics*; D006801:Humans; D008247:Lysosomes / Q000201:enzymology*; D010944:Plants / Q000378:metabolism; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D012867:Skin / Q000201:enzymology; D014908:Triticum / Q000378:metabolism; D000520:alpha-Glucosidases / Q000235:genetics*
chemical_list: D012333:RNA, Messenger; D000520:alpha-Glucosidases