Role of sphingolipids in the transport of prosaposin to the lysosomes

Prosaposin is the precursor of four lysosomal saposins that promote the deg radation of glycosphingolipids (GSLs) by acidic hydrolases, GSLs contain a hydrophobic ceramide moiety which acts as a membrane anchor, and a hydrophi lic oligosaccharide chain that faces the lumen of the Golgi apparatus and e xtracellular spaces, By using fumonisin B1, PDMP and D609? we tested the hy pothesis that sphingolipids mediate the transport of prosaposin to the lyso somes, Fumonisin BI interferes with the synthesis of ceramide, PDMP blocks the formation of glucosylceramide and D609 blocks the formation of sphingom yelin. Fumonisin B1 produced a 59-85% decrease in the density of gold parti cles in the lysosomes of CHO and NRK cells immunolabeled with anti-prosapos in antibody, and a 55% reduction in the lysosomes of CHO cells stably trans fected with an expression vector containing a human prosaposin cDNA, To exa mine whether the mannose B-phosphate receptor pathway was affected by this treatment, NRK and CHO cells treated or not with fumonisin B1 were labeled with anti-cathepsin A antibody The results showed no significant difference s in labeling of the lysosomes, suggesting that the effect of fumonisin B1 tvas specific, when fumonisin B1 and D609 were added to the media of transf ected CHO cells, a decrease in immunofluorescence with anti-prosaposin anti body was observed by confocal microscopy. PDMP did not cause any reduction in immunoreactivity indicating that sphingolmyelin appears to be involved i n this process. In conclusion, our data support the hypothesis that sphingo lipids, possibly sphingomyelin, are involved in the transport of prosaposin to the lysosomes.