The role of glycosylation and phosphorylation in the expression of active human beta-glucuronidase.
Phosphorylation of mannose residues on N-linked oligosaccharide side chains of lysosomal enzymes targets them to lysosomes. We used site-directed mutagenesis to observe the effect of eliminating selective glycosylation sites from human beta-glucuronidase on enzyme sorting. Expression studies allowed us to determine which of four potential sites were glycosylated, preferentially phosphorylated, and required for catalytic activity. All four sites of the human enzyme were glycosylated, whereas in the mouse and rat enzymes, only three of four sites are used. Sites 2 and 3 were preferentially phosphorylated. Elimination of sites 2 and 3 in combination markedly decreased sorting to lysosomes and increased enzyme secretion. Each of the four glycosylation sites could be eliminated individually without drastic reduction in catalytic activity. Activity was progressively lost as combinations of two, three, and four sites were eliminated. Wild-type enzyme produced in the presence of tunicamycin was also inactive, indicating that glycosylation is required for realization of enzyme activity. However, active enzyme could be deglycosylated with only minimal loss of activity. Mutant enzyme completely lacking glycosylation did not form tetramers. Partial restoration of tetramerization was achieved by the co-expression of normal rat enzyme, provided that the normal rat enzyme supplied at least two subunits to the tetramer.
Shipley
J M
JM
Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, Missouri 63104.
Grubb
J H
JH
Sly
W S
WS
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D005966:Glucuronidase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D008247:Lysosomes / Q000201:enzymology; D046911:Macromolecular Substances; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D014162:Transfection
D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D008715:Methionine; D005966:Glucuronidase