Processing of human acid sphingomyelinase in normal and I-cell fibroblasts.
The biosynthesis of acid sphingomyelinase in normal and I-cell disease fibroblasts was investigated by metabolic labeling with [35S]methionine and immunoprecipitation followed by polyacrylamide gel electrophoresis and fluorography. Two major polypeptides with apparent molecular masses of 75 and 72 kDa (peptide chains of 64 and 61 kDa, respectively) and a minor one with molecular mass of 57 kDa (peptide chain of 47 kDa) were found intracellularly soon after pulse labeling. The 75-kDa form is assumed to be the propropolypeptide of sphingomyelinase which is converted into the precursor form of 72 kDa. The precursor is subjected to two distinct processing events. A minor part is already cleaved in the endoplasmic reticulum-Golgi complex yielding the beta-endo-N-acetylglucosaminidase H-resistant form of 57 kDa; whereas, the major part of the precursor is processed within 4 h to a 70-kDa mature beta-endo-N-acetylglucosaminidase H-sensitive form of sphingomyelinase, which is subsequently converted into a polypeptide with molecular mass of 52 kDa within a chase of about 20 h. Both the precursor (72 kDa) as well as its early cleavage product of 57 kDa are secreted into the culture medium to a minor extent. Intracellular transport of sphingomyelinase into lysosomes depends on the phosphomannosyl specific receptor by following criteria: (i) about 80% of newly synthesized precursor was secreted in NH4Cl-treated fibroblasts as well as in I-cells, (ii) the maturation of sphingomyelinase was inhibited in normal fibroblasts exposed to NH4Cl as well as in I-cell fibroblasts, and (iii) the [32P]phosphate associated with oligosaccharides was cleavable by beta-endo-N-acetylglucosaminidase H. However, endocytosis of radiolabeled extracellular precursor by fibroblasts was not prevented by the addition of mannose 6-phosphate, whereas uptake of arylsulfatase A and beta-hexosaminidase was almost completely blocked under these conditions. This indicates that endocytosis of acid sphingomyelinase by cultured fibroblasts might be mediated by an alternative pathway.
Hurwitz
R
R
Institut für Organische Chemie und Biochemie, Bonn, Germany.
Ferlinz
K
K
Vielhaber
G
G
Moczall
H
H
Sandhoff
K
K
D000643:Ammonium Chloride / Q000494:pharmacology; D020126:Brefeldin A; D002478:Cells, Cultured; D003517:Cyclopentanes / Q000494:pharmacology; D005347:Fibroblasts / Q000201:enzymology; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D008970:Molecular Weight; D009081:Mucolipidoses / Q000201:enzymology*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012016:Reference Values; D012867:Skin / Q000201:enzymology*; D013108:Sphingomyelin Phosphodiesterase / Q000096:biosynthesis* / Q000302:isolation & purification; D013462:Sulfur Radioisotopes
D003517:Cyclopentanes; D011500:Protein Synthesis Inhibitors; D011994:Recombinant Proteins; D013462:Sulfur Radioisotopes; D000643:Ammonium Chloride; D020126:Brefeldin A; D008715:Methionine; D013108:Sphingomyelin Phosphodiesterase