Structural and membrane‐binding properties of saposin D

Saposin D is generated together with three similar proteins, saposins A, B and C, from a common precursor,called prosaposin, in acidic organelles such as late endosomes and lysosomes. Although saposin D has beenreported to stimulate the enzymatic hydrolysis of sphingomyelin and ceramide, its physiological role has not yetbeen clearly established. In the present study we examined structural and membrane-binding properties ofsaposin D. At acidic pH, saposin D showed a great affinity for phospholipid membranes containing an anionicphospholipid such as phosphatidylserine or phosphatidic acid. The binding of saposin D caused destabilization ofthe lipid surface and, conversely, the association with the membrane markedly affected the fluorescenceproperties of saposin D. The presence of phosphatidylserine-containing vesicles greatly enhanced the intrinsictyrosine fluorescence of saposin D, which contains tyrosines but not tryptophan residues.The structural properties of saposin D were investigated in detail using advanced MS analysis. It was foundthat the main form of saposin D consists of 80 amino acid residues and that the six cysteine residues are linked inthe following order: Cys5–Cys78, Cys8–Cys72 and Cys36–Cys47. The disulfide pattern of saposin D isidentical with that previously established for two other saposins, B and C, which also exhibit a strong affinity forlipids. The common disulfide structure probably has an important role in the interaction of these proteins withmembranes.The analysis of the sugar moiety of saposin D revealed that the single N-glycosylation site present in themolecule is mainly modified by high-mannose-type structures varying from two to six hexose residues.Deglycosylation had no effect on the interaction of saposin D with phospholipid membranes, indicating that theglycosylation site is not related to the lipid-binding site.The association of saposin D with membranes was highly dependent on the composition of the bilayer. Neitherceramide nor sphingomyelin, sphingolipids whose hydrolysis is favoured by saposin D, promoted its binding,while the presence of an acidic phospholipid such as phosphatidylserine or phosphatidic acid greatly favoured theinteraction of saposin D with vesicles at low pH. These results suggest that, in the acidic organelles wheresaposins are localized, anionic phospholipids may be determinants of the saposin D topology and, conversely,saposin D may affect the lipid organization of anionic phospholipid-containing membranes.Keywords: disulfide bridges; mass spectrometry, membrane interaction; phospholipids; saposin D.Saposin D is released together with three other similar proteins,saposins A, B and C, from a common precursor calledprosaposin [1–5]. This precursor is proteolytically processedinto mature saposins in late endosomes and lysosomes [6,7].Genetic defects of saposins have provided insights into theirrole in the degradation of sphingolipids. When all saposins aremissing, as seen in a patient carrying a mutation in theprosaposin initiation codon, accumulation of several sphingo-lipids such as ceramide, glucosylceramide, lactosylceramideand ganglioside GM3 has been observed [8,9]. A mutant mouseline in which the prosaposin gene was inactivated exhibited aclinical, pathological and biochemical phenotype closelyresembling that of the human disease [10]. Saposins B and Cappear to be involved in the catabolism of specific sphingo-lipids, as indicated by the storage of sulfatide caused by aselective deficiency of saposin B in an atypical form ofmetachromatic leukodystrophy and the storage of glucosylcer-amide caused by a selective deficiency of saposin C in a variantform of Gaucher’s disease [11–13]. An isolated deficiency insaposin A or D has not been reported so far and thus their actualphysiological functions are still not known. In vitro, saposin Ais able to activate the degradation of glucosylceramide [14,15]and galactosylceramide [14], and saposin D that of sphingo-myelin and ceramide [16,17]. The addition of saposin D to theculture medium of fibroblasts from patients with prosaposindeficiency leads to a decrease in the accumulated ceramide[18], and thus the function of this saposin seems to be related toceramide degradation rather than sphingomyelin degradation.As saposins are involved in the catabolism of membranecomponents such as sphingolipids, a knowledge of theirinteractions with lipid bilayers is of critical importance to ourunderstanding of their physiological behaviour. We haverecently found that at least two saposins, C and D, are able tobind to phospholipid large unilamellar vesicles (LUVs) at lowEur. J. Biochem. 263, 486–494 (1999) q FEBS 1999