Phosphohexosyl components of a lysosomal enzyme are recognized by pinocytosis receptors on human fibroblasts.
Human beta-glucuronidase (beta-D-glucuronide glucuronosohydrolase, EC 3.2.1.31), like many other glycoprotein lysosomal hydrolases, is specifically taken up from the culture medium by human fibroblasts. Prior work has indicated that the enzyme exhibits charge heterogeneity and that "high-uptake" forms, i.e., those rapidly internalized by human fibroblasts, are more acidic than slowly internalized forms. Here we present two lines of evidence that the acidic group required for the high-uptake property of certain forms of the enzyme is a phosphate on, or in proximity to, a D-mannose-type carbohydrate. The first line of evidence was obtained from analysis of inhibition of enzyme pinocytosis by yeast mannans, phosphorylated sugars, and sugars. Mannans that contained phosphate were more potent inhibitors than those that did not contain phosphate. D-Mannose 6-phosphate was a more potent inhibitor than either D-mannose 1 phosphate or 2-deoxy-D-glucose 6-phosphate. D-Mannose and certain related sugars were weak pinocytosis inhibitors, while 2- and 4-epimers of mannose were noninhibitory. Competitive inhibition was demonstrated and the apparent Kis estimated for the following compounds: Saccharomyces cerevisiae mannan from mutant X2180-mnnl, 3 X 10(-6) M; mannan from wild-type S. cerebisiae, 3 X 10(-5) M; D-mannose 6-phosphate, 6 X 10(-5) M; L-fucose, 4 X 10(-2) M; and D-mannose, 6 X 10(-2) M. The second line of evidence comes from the observation that alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] treatment of human platelet beta-glucuronidase abolished its "high-uptake" activity, without diminishing its catalytic activity, and converted some forms of the heterogeneous enzyme to less acidic forms.
D000469:Alkaline Phosphatase / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D001692:Biological Transport / Q000187:drug effects; D003864:Depression, Chemical; D005347:Fibroblasts / Q000378:metabolism*; D005643:Fucose / Q000378:metabolism; D005966:Glucuronidase / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism / Q000494:pharmacology; D006600:Hexosephosphates / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology; D008358:Mannose / Q000031:analogs & derivatives / Q000378:metabolism; D008968:Molecular Conformation; D010873:Pinocytosis; D013329:Structure-Activity Relationship
D006023:Glycoproteins; D006600:Hexosephosphates; D005643:Fucose; D000469:Alkaline Phosphatase; D005966:Glucuronidase; D008358:Mannose
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