Suppression of the 'uncovering' of mannose-6-phosphate residues in lysosomal enzymes in the presence of NH4Cl.
The uncovering ratio of phosphate groups in lysosomal enzymes is defined as the percentage of phosphomonoester groups in the oligosaccharide side chains based on the sum of phosphomonoester and phosphodiester groups. Using a new procedure for the specific and complete hydrolysis of uncovered phosphomonoester groups in denatured immunoprecipitates of human cathepsin D, we show that the uncovering ratio varies between different forms of the enzyme and may be used as an indicator of the maturation of its carbohydrate side chains. The uncovering ratio in the total (cellular and secreted) cathepsin D from U937 promonocytes is greater than 95%. It is only slightly decreased in cells incubated in the presence of 1 alpha,25-dihydroxycholecalciferol, in which the rate of synthesis of cathepsin D is several times higher than in the control cells. In U937 cells and also in fibroblasts, the uncovering is nearly complete in intermediate and mature forms of the intracellular cathepsin D but less extensive in the intracellular and secreted precursor. In both cell types, incubation with 10 mM NH4Cl results in a decrease in the uncovering ratio of total cathepsin D. However, the activity of the uncovering enzyme, N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase, as determined with UDP-N-acetylglucosamine is not affected with up to 60 mM NH4Cl. Our results suggest that NH4Cl, in addition to its known effects on the acidic-pH-dependent functions of lysosomal compartments and of mannose-6-phosphate receptors, impairs the processing or transport of lysosomal enzyme precursors at, or proximally to, the site of the uncovering of their mannose-6-phosphate residues.
Isidoro
C
C
Dipartimento di Medicina ed Oncologia Sperimentale, Università di Torino, Italy.
Radons
J
J
Baccino
F M
FM
Hasilik
A
A
D000643:Ammonium Chloride / Q000494:pharmacology*; D002117:Calcitriol / Q000494:pharmacology; D002402:Cathepsin D / Q000378:metabolism*; D002460:Cell Line; D005347:Fibroblasts / Q000201:enzymology; D006600:Hexosephosphates / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D008247:Lysosomes / Q000201:enzymology*; D008360:Mannosephosphates / Q000378:metabolism*; D009000:Monocytes / Q000201:enzymology; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D011498:Protein Precursors / Q000378:metabolism
D006600:Hexosephosphates; D008360:Mannosephosphates; D011498:Protein Precursors; D000643:Ammonium Chloride; C027693:mannose-6-phosphate; D010727:Phosphoric Diester Hydrolases; C026645:N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase; D002402:Cathepsin D; D002117:Calcitriol