Human lysosomal alpha-glucosidase: functional characterization of the glycosylation sites.
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.
Hermans
M M
MM
Department of Cell Biology and Genetics, Erasmus University, Rotterdam, The Netherlands.
Wisselaar
H A
HA
Kroos
M A
MA
Oostra
B A
BA
Reuser
A J
AJ
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*
D011994:Recombinant Proteins; D000520:alpha-Glucosidases