BIOSYNTHESIS, PROCESSING, AND TARGETING OF SPHINGOLIPID ACTIVATOR PROTEIN (SAP) PRECURSOR IN CULTURED HUMAN FIBROBLASTS - MANNOSE 6-PHOSPHATE RECEPTOR-INDEPENDENT ENDOCYTOSIS OF SAP PRECURSOR

Sphingolipid activator proteins (SAPs) are essential cofactors for the lysosomal degradation of glycosphingolipids with short oligosaccharid e chains by acidic exohydrolases. SAP-A, -B, -C, and -D derive from pr oteolysis of a 73-kDa glycoprotein, the SAP precursor. In the present publication, we studied the intracellular transport and the endocytosi s of SAP precursor in human skin fibroblasts. Our data indicate that S AP precursor bears phosphate residues on noncomplex carbohydrate chain s linked to the SAP-C and the SAP-D domain and sulfate residues on com plex carbohydrate chains located within the SAP-A, -C, and possibly th e SAP-D domain. Treatment of fibroblasts with either bafilomycin A(1) or 3-methyladenine indicates that proteolytic cleavage of SAP precurso r begins as early as in the late endosomes. To determine whether targe ting of SAP precursor depends on mannose g-phosphate residues, we anal yzed the processing of SAP precursor in I-cell disease fibroblasts. In these cells nearly normal amounts of newly synthesized SAP-C were fou nd, although secretion of SAP precursor was enhanced 2-3-fold. Moreove r, SAP-C could be localized to lysosomal structures by indirect immuno fluorescence in normal and in I-cell disease fibroblasts. Mannose g-ph osphate was not found to interfere significantly with endocytosis of S AP precursor. Normal fibroblasts internalized SAP precursor secreted f rom I-cells nearly as efficiently as the protein secreted from normal cells. To our surprise, deglycosylated SAP precursor was taken up by m annose 6-phosphate receptor double knock out mouse fibroblasts more ef ficiently than the glycosylated protein. We propose that intracellular targeting of SAP precursor to lysosomes is only partially dependent o n mannose 6-phosphate residues, whereas its endocytosis occurs in a ca rbohydrate-independent manner.