Synthesis and processing of sphingolipid activator protein-2 (SAP-2) in cultured human fibroblasts.
Sphingolipid activator proteins (SAP) are relatively small molecular weight proteins that stimulate the enzymatic hydrolysis of sphingolipids in the presence of specific lysosomal hydrolases. SAP-2 has previously been demonstrated to activate the hydrolysis of glucosylceramide, galactosylceramide, and, possibly, sphingomyelin. Using monospecific rabbit antibodies against human spleen SAP-2, the synthesis and processing of SAP-2 were studied in cultured human fibroblasts. When [35S]methionine was presented in the medium to control human cells for 4 h, five major areas of radiolabeling were found. These had apparent molecular weights of 73,000, 68,000, 50,000, 12,000, and 9,000. Further studies indicated that the major extracellular product in normal cells given NH4Cl along with the [35S]methionine and in medium from cultures from patients with I cell disease had an apparent molecular weight of 73,000. The Mr = 68,000 and 73,000 species can be converted to a species with an apparent molecular weight of 50,000 by the action of endoglycosidase F. After labeling cells for 1 h followed by a 1-h chase, the Mr = 12,000 and 9,000 species appear. Treatment of the immunoprecipitated mixture with endoglycosidase F resulted in conversion of these species to one band with an apparent molecular weight of 7,600. These studies indicate that this relatively low molecular weight protein is rapidly synthesized from a relatively large molecular weight highly glycosylated precursor.
D002478:Cells, Cultured; D005347:Fibroblasts / Q000378:metabolism; D006023:Glycoproteins; D006801:Humans; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D008970:Molecular Weight; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification; D049231:Saposins; D012867:Skin / Q000378:metabolism; D049128:Sphingolipid Activator Proteins; D013462:Sulfur Radioisotopes
D006023:Glycoproteins; C483345:PSAP protein, human; D011506:Proteins; D049231:Saposins; D049128:Sphingolipid Activator Proteins; D013462:Sulfur Radioisotopes; D008715:Methionine