Saposin A: second cerebrosidase activator protein.

Abstract

Saposin A, a heat-stable 16-kDa glycoprotein, was isolated from Gaucher disease spleen and purified to homogeneity. Chemical sequencing from its amino terminus and of peptides obtained by digestion with protease from Staphylococcus aureus strain V-8 demonstrated that saposin A is derived from proteolytic processing of domain 1 of its precursor protein, prosaposin. Processing of prosaposin (70 kDa) also generates three other previously reported saposin proteins, B, C, and D, from its second, third, and fourth domains. Similar to saposin C, saposin A stimulates the hydrolysis of 4-methylumbelliferyl ,B-glucoside and glucocerebroside by ,B-glucosylceramidase and of galactocerebroside by ,B-galactosylceramidase, mainly by increasing the maximal velocity of both reactions. Saposin A is as active as saposin C in these reactions. Saposin A has no significant effect on other sphingolipid and 4-methylumbelliferyl glycoside hydrolases tested. Saposin A has two potential glycosylation sites that appear to be glycosylated. After deglycosylation, saposin A had a subunit molecular mass of 10 kDa and was as active as native saposin A. However, reduction and alkylation abolished the activation. A three-dimensional model comparing saposins A and C reveals significant sequence homology between them, especially preservation of conserved acidic and basic residues in their middle regions. Each appears to possess a conformationally rigid hydrophobic pocket stabilized by three internal disulffide bridges, with amphipathic helical regions interrupted by helix breakers. (16). Unlike saposin B, saposin C appears to interact directly with both enzymes to stimulate activity (17). The primary structure of saposin C was determined by chemical sequencing of its amino acids (18,19) and by deducing its sequence from nucleotide sequencing of a cDNA encoding prosaposin, its precursor (20). Saposin C has been reported to be deficient in a single patient with a variant form of Gaucher disease (21).Recently, the complete nucleotide sequence of a cDNA encoding prosaposin, the precursor of saposins B and C, was elucidated (20). Prosaposin is a 511-amino acid glycoprotein, and examination of its amino acid sequence reveals four saposin-like domains, two of which are saposins B and C; these are flanked by two additional domains, which we call saposin A and saposin D. Each domain is approximately 80-amino acid residues long; has nearly identical placement of cysteine residues, glycosylation sites, and helical regions; and is flanked by proteolytic cleavage sites. Molecular models indicate that the proteins derived from each domain can fold to give rise to a conformationally rigid hydrophobic pocket held together by three disulfide bridges. Proteolytic cleavage of prosaposin at each domain boundary was predicted to give rise to four saposin proteins (20). We recently have isolated saposin D, the protein arising from domain four of prosaposin and demonstrated it to be a specific sphingomyelin phosphodiesterase (EC 3.1.4.12) activator (22). In this...

Lipids regulate the hydrolysis of membrane bound glucosylceramide by lysosomal β-glucocerebrosidase

“…Sap C was isolated from the spleen of a Gaucher disease patient, as described previously (31). The purity of the Sap C preparation was ascertained by polyacrylamide gel electrophoresis with silver staining, Western blotting, and sequencing.…”

Structural study of the oligosaccharide moieties of sphingolipid activator proteins, saposins A, C and D obtained from the spleen of a Gaucher patient

“…Recently deficiencies of saposins C and B have been demonstrated in human genetic diseases including a variant form of Gaucher disease [2] and variants of metachromatic leukodystrophy [3], respectively. From amino acid sequence analyses, human saposin A has been shown to have two glycosylation sites to which N-linked oligosaccharides are attached [4], whereas saposins B, C and D have only one such site [l].…”

Lysosomal Storage Disorders

“…Because saposins are sphingolipid activator proteins, involved in the degradation of sphingolipids, they are potential candidates for additional LSD markers. Moreover, saposins have been reported to be increased in various tissue samples from LSD patients (18,19 ). The saposin D calibration curve (Fig.…”