Abstract
Sphingolipid activator proteins (SAPs) are essential cofactors for the lysosomal degradation of glycosphingolipids with short oligosaccharide chains by acidic exohydrolases. SAP-A, -B, -C, and -D derive from proteolysis of a 73-kDa glycoprotein, the SAP precursor. In the present publication, we studied the intracellular transport and the endocytosis of SAP precursor in human skin fibroblasts. Our data indicate that SAP precursor bears phosphate residues on noncomplex carbohydrate chains linked to the SAP-C and the SAP-D domain and sulfate residues on complex carbohydrate chains located within the SAP-A, -C, and possibly the SAP-D domain. Treatment of fibroblasts with either bafilomycin A1 or 3-methyladenine indicates that proteolytic cleavage of SAP precursor begins as early as in the late endosomes. To determine whether targeting of SAP precursor depends on mannose 6-phosphate residues, we analyzed the processing of SAP precursor in I-cell disease fibroblasts. In these cells nearly normal amounts of newly synthesized SAP-C were found, although secretion of SAP precursor was enhanced 2-3-fold. Moreover, SAP-C could be localized to lysosomal structures by indirect immunofluorescence in normal and in I-cell disease fibroblasts. Mannose 6-phosphate was not found to interfere significantly with endocytosis of SAP precursor. Normal fibroblasts internalized SAP precursor secreted from I-cells nearly as efficiently as the protein secreted from normal cells. To our surprise, deglycosylated SAP precursor was taken up by mannose 6-phosphate receptor double knock out mouse fibroblasts more efficiently than the glycosylated protein. We propose that intracellular targeting of SAP precursor to lysosomes is only partially dependent on mannose 6-phosphate residues, whereas its endocytosis occurs in a carbohydrate-independent manner.
Highlights
Sphingolipid activator proteins (SAPs) are essential cofactors for the lysosomal degradation of glycosphingolipids with short oligosaccharide chains by acidic exohydrolases
We propose that intracellular targeting of sphingolipid activator proteins (SAPs) precursor to lysosomes is only partially dependent on mannose 6-phosphate residues, whereas its endocytosis occurs in a carbohydrate-independent manner
Increased amounts of the precursor protein could be immunoprecipitated from the media of I-cell disease (ICD) fibroblasts [11]. These results suggest that the transport of SAP precursor to lysosomes of human fibroblasts is predominantly mediated by the mannose 6-phosphate (Man-6-P)-dependent pathway
Summary
Materials—[35S]Methionine (Tran35S-labelTM, 41.77 TBq/mmol) and [32P]Na2HPO4 (3.7–37 GBq/mmol of P) were from ICN, [35S]Na2SO4 (0.9 –1.5 TBq/mg of S) was from Amersham Buchler. -Endo-N-acetylglucosaminidase H and peptide-N-glycosidase F were from New England Biolabs. Media and pretreated cell lysates were incubated with 10 l of rabbit-preimmune serum overnight at 4 °C, and the immunocomplexes were precipitated by protein A-Sepharose CL-4B (Sigma). The third aliquot was adjusted to 200 l with 50 mM sodium citrate, pH 5.5, containing 0.5% SDS and 1% -mercaptoethanol and was incubated with 50 New England Biolabs units of -endo-N-acetylglucosaminidase H (Endo H). Endocytosis—Confluent human skin fibroblasts or I-cell disease skin fibroblasts (75-cm flask) were labeled for 24 h with 0.5–1.0 mCi of [35S]methionine in 4 ml of methionine-deficient DMEM, containing 5% dialyzed fetal calf serum and 10 mM NH4Cl or 10 g/ml tunicamycin. The medium was removed, dialyzed against 1 liter of DMEM overnight, supplemented with 5 mM nonradioactive methionine and 5% fetal calf serum, and added to fresh cells in the absence or presence of various reagents as indicated, A and B. The cells were treated for 60 min at 37 °C with 3% BSA
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