ACCUMULATION OF SPHINGOLIPIDS IN SAP-PRECURSOR (PROSAPOSIN)-DEFICIENTFIBROBLASTS OCCURS AS INTRALYSOSOMAL MEMBRANE STRUCTURES AND CAN BE COMPLETELY REVERSED BY TREATMENT WITH HUMAN SAP-PRECURSOR

The degradation of glycosphingolipids takes place in lysosomes iby act ion of specific exohydrolases, with the assistance of sphingolipid act ivator proteins (SAPs), Four of the SAPs, SAP-A to -D (also called sap osins A to D), are synthesized from a single protein, the SAP-precurso r (prosaposin). Deficiency in this precursor protein, a rape inherited disease in humans, results in tile storage of sphingolipids with shor t oligosaccharide head groups within the patients' tissues, and electr on microscopy revealed the accumulation of large multivesicular storag e organelles. In this study we analyze the multivesicular storage orga nelles in cultivated fibroblasts from these patients. The results supp ort our hypothesis that endocytosis of plasma membrane-derived lipids occurs via small intraendosomal and intralysosomal vesicles and membra ne structures that are then digested within the lysosomes (Sanehoff, K ., T. Kolter, Trends in Cell Biol, 6, 98-103 (1996). First, we show th at the storage compartment consists of late endosomes and lysosomes by immunogold labeling for marker proteins of these organelles, The tran sport of endocytosed bovine serum albumin-colloidal gold or cationized ferritin into the compartment occurs with tile timing expected for tr ansport to late endocytic organeiles. Second, complementation of the m edium of the SAP-precursor-deficient fibroblasts with only nanomolar c oncentrations of purified SAP-precursor nearly completely reversed the aberrant accumulation of multivesicular structures, thereby abolishin g most of the intralysosomal membrane structures, Analysis of the sphi ngolipid pattern of the cells after metabolic labeling with [C-14]seri ne reveals that the cells' ability to degrade glycosphingolipids is co mpletely restored by feeding of SAP-precursor at the same concentratio ns. This is tile first demonstration in vivo that endocytosed SAP-prec ursor is processed into functional active SAPs A,- B,- C, and D and th at the degradation of the vesicular structures within the lysosomes de pends on tile presence of the SAPs. Moreover, these studies suggest th at a therapy program based on feeding purified SAP-precursor mag be va luable in treating the entire family of diseases which result from the loss of one or more of the SAPs.