Biochemical, immunological, and structural studies on a sphingolipid activator protein (SAP-1)

Sphingolipid activator protein-1 (SAP-1) is a glycoprotein found in human tissue extracts that stimulates the enzymatic hydrolysis of at least two glycosphingolipids, including GM1 ganglioside and sulfatide. The ability of purified SAP-1 to stimulate GM1 ganglioside hydrolysis by extracts of cultured fibroblasts from patients with beta-galactosidase deficiency was examined, and all patients had a pronounced deficiency (under 10% of control). Using monospecific antibodies against SAP-1, the concentration was determined in cultured fibroblasts by rocket immunoelectrophoresis. Extracts from 15 control cell lines were found to have 0.72 +/- 0.24 micrograms cross-reactive material/mg protein, while cell extracts from 8 patients with GM1 gangliosidosis involving mental retardation were found to have 1.08 +/- 0.17, which is significantly elevated. When the fibroblast extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by electroblotting, multiple bands were observed. Controls were found to have two major bands with estimated molecular weights of 9000 and 9500, and a minor band at 7800. Extracts from patients with GM1 gangliosidosis were found to have multiple bands ranging upward to 13,000. Extracts from patients with the most severe clinical types of GM1 gangliosidosis had almost exclusively high-molecular-weight forms (molecular weights above 10,000). Treatment of SAP-1 from control liver with endoglycosidase D caused a decrease in the Mr 9500 band and increased in the Mr 7800 band. When SAP-1 from GM1 gangliosidosis liver was treated sequentially with neuraminidase, beta-galactosidase, and endoglycosidase D, almost all of it was converted to the forms found in control human liver.