Role of sphingolipids in the transport of prosaposin to the lysosomes.

Prosaposin is the precursor of four lysosomal sa- posins that promote the degradation of glycosphingolipids (GSLs) by acidic hydrolases. GSLs contain a hydrophobic ceramide moiety, which acts as a membrane anchor, and a hydrophilic oligosaccharide chain that faces the lumen of the Golgi apparatus and extracellular spaces. By using fu- monisin B1, PDMP and D609, we tested the hypothesis that sphingolipids mediate the transport of prosaposin to the ly- sosomes. Fumonisin B1 interferes with the synthesis of cer- amide, PDMP blocks the formation of glucosylceramide and D609 blocks the formation of sphingomyelin. Fumoni- sin B1 produced a 59-85% decrease in the density of gold particles in the lysosomes of CHO and NRK cells immuno- labeled with anti-prosaposin antibody, and a 55% reduction in the lysosomes of CHO cells stably transfected with an ex- pression vector containing a human prosaposin cDNA. To examine whether the mannose 6-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 differences in labeling of the lysosomes, suggesting that the effect of fumo- nisin B1 was specific. When fumonisin B1 and D609 were added to the media of transfected CHO cells, a decrease in immunofluorescence with anti-prosaposin antibody was ob- served by confocal microscopy. PDMP did not cause any re- duction in immunoreactivity, indicating that sphingolmyelin appears to be involved in this process. In conclusion, our data support the hypothesis that sphingolipids, possibly sphingomyelin, are involved in the transport of prosaposin to the lysosomes. —Lefrancois, S., L. Michaud, M. Potier, S. Igdoura, and C. R. Morales. Role of sphingolipids in the transport of prosaposin to the lysosomes. J. Lipid Res. 1999. 40: 1593-1603.