Phosphorylation of lysosomal enzymes in fibroblasts. Marked deficiency of N-acetylglucosamine-1-phosphotransferase in fibroblasts of patients with mucolipidosis III.
N-Acetylglucosamine-1-phosphotransferase activity was assayed in human skin fibroblasts using [beta-32P]UDP-N-acetylglucosamine as donor and dephosphorylated beta-N-acetyl-D-hexosaminidase as acceptor. An optimal transfer rate of N-acetylglucosamine 1-phosphate required CDP-choline and ADP in order to inhibit the breakdown of [beta-32P]UDP-N-acetylglucosamine and a combination of leupeptin and iodoacetamide to protect the transferase. The transferase required Mg2 or Mn2. Using doubly labelled UDP-N-acetylglucosamine, simultaneous transfer of N-acetyl-[6-3H]glucosamine and [32P]phosphate to endogenous acceptors was demonstrated. Membranes prepared from fibroblasts from patients with mucolipidosis III were defective in transfer of N-acetylglucosamine 1-phosphate. A residual transferase activity of less than 10% of controls was detectable in fibroblast membranes of eight patients with mucolipidosis III. In membranes from fibroblasts from patients with mucolipidosis II,N-acetylglucosamine-1-phosphotransferase activity was not detectable. Our results indicate that the primary defect in mucolipidoses II and III is a deficiency in N-acetylglucosamine-1-phosphotransferase, the residual activity being higher in mucolipidosis III than in mucolipidosis II.
D002462:Cell Membrane / Q000201:enzymology; D004288:Dolichol Phosphates / Q000172:deficiency / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D005964:Glucosyltransferases / Q000172:deficiency* / Q000378:metabolism; D006801:Humans; D008247:Lysosomes / Q000201:enzymology*; D009081:Mucolipidoses / Q000201:enzymology*; D010766:Phosphorylation; D017855:Transferases (Other Substituted Phosphate Groups)
D004288:Dolichol Phosphates; D005964:Glucosyltransferases; D017855:Transferases (Other Substituted Phosphate Groups); C023222:UDPacetylglucosamine-dolichyl-phosphate acetylglucosamine-1-phosphate transferase