In vitro mutagenesis of potential N-glycosylation sites of arylsulfatase A. Effects on glycosylation, phosphorylation, and intracellular sorting.
The correct intracellular sorting of lysosomal enzymes such as arylsulfatase A depends on the presence of mannose 6-phosphate residues on high mannose type oligosaccharides. The arylsulfatase A cDNA contains three potential N-glycosylation sites, two of which are utilized. We have mutated one or two of the N-glycosylation sites and analyzed the glycosylation, phosphorylation, and intracellular sorting of the mutant arylsulfatase A polypeptides. The results show that each of the three glycosylation sites (I, II, and III) can be glycosylated, but glycosylation at sites I and II is mutually exclusive. In mutants with one oligosaccharide side chain at positions I, II, or III all side chains can acquire mannose 6-phosphate residues irrespective of their location. This demonstrates spatial flexibility of the phosphotransferase, which specifically recognizes lysosomal enzymes and initiates the addition of mannose 6-phosphate residues on oligosaccharide side chains. However, these mutants have different intracellular sorting efficiencies and seem to use different (mannose 6-phosphate receptor-dependent and -independent) sorting pathways.
Georg-August-Universität, Abteilung Biochemie II, Göttingen, Germany.
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Gieselmann
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Schmidt
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von Figura
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D002553:Cerebroside-Sulfatase / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D006031:Glycosylation; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010766:Phosphorylation; D011233:Precipitin Tests; D014162:Transfection
D004247:DNA; D002553:Cerebroside-Sulfatase