Human acid ceramidase: Processing, glycosylation, and lysosomal targeting

Klaus Ferlinz, Guido Kopal, Katussevani Bernardo, Thomas Linke, Julia Bär, Bernadette Breiden, Ulrich Neumann, Florian Lang, Edward H. Schuchman, Konrad Sandhoff

Research output: Contribution to journalArticlepeer-review

98 Scopus citations


The biosynthesis of human acid ceramidase (hAC) starts with the expression of a single precursor polypeptide of ∼53-55 kDa, which is subsequently processed to the mature, heterodimeric enzyme (40 + 13 kDa) in the endosomes/lysosomes. Secretion of hAC by either fibroblasts or acid ceramidase cDNA-transfected COS cells is extraordinarily low. Both lysosomal targeting and endocytosis critically depend on a functional mannose 6-phosphate receptor as judged by the following criteria: (i) hAC-precursor secretion by NH 4Cl-treated fibroblasts and I-cell disease fibroblasts, (ii) inhibition of the formation of mature heterodimeric hAC in NH 4Cl-treated fibroblasts or in I-cell disease fibroblasts, and (iii) blocked endocytosis of hAC precursor by mannose 6-phosphate receptor-deficient fibroblasts or the addition of mannose 6-phosphate. The influence of the six individual potential N-glycosylation sites of human acid ceramidase on targeting, processing, and catalytic activity was determined by site-directed mutagenesis. Five glycosylation sites (sites 1-5 from the N terminus) are used. The elimination of sites 2, 4, and 6 has no influence on lysosomal processing or enzymatic activity of recombinant ceramidase. The removal of sites 1, 3, and 5 inhibits the formation of the heterodimeric enzyme form. None of the mutant ceramidases gave rise to an increased rate of secretion, suggesting that lysosomal targeting does not depend on one single carbohydrate chain.

Original languageEnglish
Pages (from-to)35352-35360
Number of pages9
JournalJournal of Biological Chemistry
Issue number38
StatePublished - 21 Sep 2001


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