title: Enzymatic identification of mannose 6-phosphate on the recognition marker for receptor-mediated pinocytosis of beta-glucuronidase by human fibroblasts.
abstract: Human beta-glucuronidase (beta-D-glucuronide glucuronosohydrolase, EC 3.2.1.31), like many other glycoprotein lysosomal hydrolases, is subject to receptor-mediated endocytosis by fibroblasts. Prior work demonstrated charge heterogeneity in beta-glucuronidase and showed that high-uptake forms are more acidic than slowly internalized forms. Considerable indirect evidence implicated mannose 6-phosphate as an essential part of the recognition marker on high-uptake enzyme forms. Here we report the purification of beta-glucuronidase from human spleen and demonstrate enzymatically that mannose 6-phosphate is released on acid hydrolysis of pure enzyme varies directly with its susceptibility to pinocytosis by fibroblasts. Enzyme forms resolved by CM-Sephadex chromatography differed over an 18-fold range in uptake rate and in mannose 6-phosphate content. The most acidic forms had 4.4 mol of mannose 6-phosphate per mol of enzyme. The mannose 6-phosphate was released from the enzyme by treatment with endoglycosidase H with concomitant loss of susceptibility to adsorptive endocytosis. Thus, these studies provide direct evidence that mannose 6-phosphate is present on high-uptake enzyme forms, that it is present in the recognition marker for uptake, and that it is present on oligosaccharide that is released by endoglycosidase H.
mesh_terms: D001693:Biological Transport, Active; D005347:Fibroblasts / Q000378:metabolism; D005966:Glucuronidase / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism; D006600:Hexosephosphates / Q000502:physiology*; D006801:Humans; D008360:Mannosephosphates / Q000502:physiology*; D010873:Pinocytosis; D011955:Receptors, Drug / Q000378:metabolism*; D013154:Spleen / Q000201:enzymology
chemical_list: D006023:Glycoproteins; D006600:Hexosephosphates; D008360:Mannosephosphates; D011955:Receptors, Drug; D006026:Glycoside Hydrolases; D005966:Glucuronidase