Human acid ceramidase - Processing, glycosylation, and lysosomal targeting

The biosynthesis of human acid ceramidase (hAC) starts with the expression of a single precursor polypeptide of similar to 53-55 kDa, which is subsequ ently processed to the mature, heterodimeric enzyme (40 + 13 kDa) in the en dosomes/lysosomes. Secretion of hAC by either fibroblasts or acid ceramidas e cDNA-transfected COS cells is extraordinarily low. Both lysosomal targeti ng and endocytosis critically depend on a functional mannose 6-phosphate re ceptor as judged by the following criteria: (i) hAC-precursor secretion by NH4Cl-treated fibroblasts and I-cell disease fibroblasts, (ii) inhibition o f the formation of mature heterodimeric hAC in NH4Cl-treated fibroblasts or in I-cell disease fibroblasts, and (iii) blocked endocytosis of hAC precur sor by mannose 6-phosphate receptor-deficient fibroblasts or the addition o f mannose 6-phosphate. The influence of the six individual potential N-glyc osylation sites of human acid ceramidase on targeting, processing, and cata lytic activity was determined by site-directed mutagenesis. Five glycosylat ion sites (sites 1-5 from the N terminus) are used. The elimination of site s 2, 4, and 6 has no influence on lysosomal processing or enzymatic activit y 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 targ eting does not depend on one single carbohydrate chain.