Analysis of the glycosylation and phosphorylation of the lysosomal enzyme, beta-hexosaminidase B, by site-directed mutagenesis.
Lysosomal enzymes require a mannose 6-phosphate recognition marker, constructed on asparagine-linked oligosaccharide chains, for targeting to lysosomes. We have identified the glycosylation sites of human beta-hexosaminidase B and have determined the influence of individual oligosaccharides on the phosphorylation, lysosomal targeting, and catalytic activity of the enzyme. The five potential glycosylation sites of the hexosaminidase beta-chain were modified individually by site-directed mutagenesis, and the constructs were expressed in COS 1 cells. By this analysis, we determined that four of the five potential sites were glycosylated. Two of the four oligosaccharides were preferentially phosphorylated. The absence of these two preferentially phosphorylated oligosaccharides resulted in greatly reduced amounts of the lysosomal form of the enzyme with increased secretion into the medium. The catalytic activity of beta-hexosaminidase B was not significantly altered by the absence of individual oligosaccharides suggesting the folding and assembly of the enzyme was not disrupted.
Sonderfeld-Fresko
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Genetics and Biochemistry Branch, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892.
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D000818:Animals; D001692:Biological Transport; D002522:Chlorocebus aethiops; D004252:DNA Mutational Analysis; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006596:Hexosaminidases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008247:Lysosomes / Q000201:enzymology; D008562:Membrane Glycoproteins / Q000378:metabolism; D008970:Molecular Weight; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D013329:Structure-Activity Relationship; D014162:Transfection
D006023:Glycoproteins; D008562:Membrane Glycoproteins; C025363:mannoproteins; D006596:Hexosaminidases