title: Expression and routeing of human lysosomal alpha-glucosidase in transiently transfected mammalian cells.
abstract: Previously isolated lysosomal alpha-glucosidase cDNA clones were ligated to full-length constructs for expression in vitro and in mammalian cells. One of these constructs (pSHAG1) did not code for functional enzyme, due to an arginine residue instead of a tryptophan residue at amino acid position 402. The mutation does not affect the rate of enzyme synthesis, but interferes with post-translational modification and intracellular transport of the acid alpha-glucosidase precursor. Using immunocytochemistry it is demonstrated that the mutant precursor traverses the endoplasmic reticulum and the Golgi complex, but does not reach the lysosomes. Pulse-chase experiments suggest premature degradation. The Trp-402-containing enzyme (encoded by construct pSHAG2) is processed properly, and has catalytic activity. A fraction of the enzyme is localized at the plasma membrane. It is hypothesized that membrane association of the acid alpha-glucosidase precursor, as demonstrated by Triton X-114 phase separation, is responsible for transport to this location. Transiently expressed acid alpha-glucosidase also enters the secretory pathway, since a catalytically active precursor is found in the culture medium. This precursor has the appropriate characteristics for use in enzyme replacement therapy. Efficient uptake via the mannose 6-phosphate receptor results in degradation of lysosomal glycogen in cultured fibroblasts and muscle cells from patients with glycogenosis type II.
authors: MGC-Department of Cell Biology and Genetics, Erasmus University, Rotterdam, The Netherlands.
authors: L H
authors: LH
authors: Hoefsloot
authors: R
authors: R
authors: Willemsen
authors: M A
authors: MA
authors: Kroos
authors: M
authors: M
authors: Hoogeveen-Westerveld
authors: M M
authors: MM
authors: Hermans
authors: A T
authors: AT
authors: Van der Ploeg
authors: B A
authors: BA
authors: Oostra
authors: A J
authors: AJ
authors: Reuser
mesh_terms: D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology* / Q000648:ultrastructure; D010957:Plasmids; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D000520:alpha-Glucosidases / Q000235:genetics* / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D004247:DNA; D000520:alpha-Glucosidases