title: Human lysosomal alpha-glucosidase: functional characterization of the glycosylation sites.
abstract: N-linked glycosylation is one of the important events in the post-translational modification of human lysosomal alpha-glucosidase. Phosphorylation of mannose residues ensures efficient transport of the enzyme to the lysosomes via the mannose 6-phosphate receptor. The primary structure of lysosomal alpha-glucosidase, as deduced from the cDNA sequence, indicates that there are seven potential glycosylation sites. We have eliminated these sites individually by site-directed mutagenesis and thereby demonstrated that all seven sites are glycosylated. The sites at Asn-882 and Asn-925 were found to be located in a C-terminal propeptide which is cleaved off during maturation. Evidence is presented that at least two of the oligosaccharide side chains of human lysosomal alpha-glucosidase are phosphorylated. Elimination of six of the seven sites does not disturb enzyme synthesis or function. However, removal of the second glycosylation site at Asn-233 interferes dramatically with the formation of mature enzyme. The mutant precursor is synthesized normally and assembles in the endoplasmic reticulum, but immunoelectron microscopy reveals a deficiency of alpha-glucosidase in the Golgi complex and in the more distal compartments of the lysosomal transport pathway.
authors.affiliation: Department of Cell Biology and Genetics, Erasmus University, Rotterdam, The Netherlands.
authors.forename: M M
authors.initials: MM
authors.lastname: Hermans
authors.forename: H A
authors.initials: HA
authors.lastname: Wisselaar
authors.forename: M A
authors.initials: MA
authors.lastname: Kroos
authors.forename: B A
authors.initials: BA
authors.lastname: Oostra
authors.forename: A J
authors.initials: AJ
authors.lastname: Reuser
mesh_terms: D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002451:Cell Compartmentation; D002478:Cells, Cultured; D004252:DNA Mutational Analysis; D006031:Glycosylation; D006801:Humans; D007150:Immunohistochemistry; D008247:Lysosomes / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism; D014158:Transcription, Genetic; D000520:alpha-Glucosidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D000520:alpha-Glucosidases