The carbohydrate moiety of the activator protein for glucosylceramide β-glucosidase

Sano, Akira; Radin, Norman S.

doi:10.1016/0006-291x(88)90267-7

Cited by 18 publications

(6 citation statements)

References 24 publications

(14 reference statements)

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“…The carbohydrate moiety does not appear to be critical for the in vitro activity of saposins. In fact, deglycosylation of saposins A, B and C does not impair their ability to activate galactosylceramidase, sulfatide sulfatase and glucosylceramidase, respectively [14,30,31]. In the present study, we found that removal of the sugar moiety did not even influence the association of saposin D with phospholipid membranes, suggesting that the glycosylation site is not related to the region interacting with the lipid bilayer.…”

Section: Discussionmentioning

confidence: 45%

Structural and membrane‐binding properties of saposin D

Tatti

Salvioli

Ciaffoni

et al. 1999

European Journal of Biochemistry

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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 been reported to stimulate the enzymatic hydrolysis of sphingomyelin and ceramide, its physiological role has not yet been clearly established. In the present study we examined structural and membrane-binding properties of saposin D. At acidic pH, saposin D showed a great affinity for phospholipid membranes containing an anionic phospholipid such as phosphatidylserine or phosphatidic acid. The binding of saposin D caused destabilization of the lipid surface and, conversely, the association with the membrane markedly affected the fluorescence properties of saposin D. The presence of phosphatidylserine-containing vesicles greatly enhanced the intrinsic tyrosine 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 found that the main form of saposin D consists of 80 amino acid residues and that the six cysteine residues are linked in the following order: Cys5±Cys78, Cys8±Cys72 and Cys36±Cys47. The disulfide pattern of saposin D is identical with that previously established for two other saposins, B and C, which also exhibit a strong affinity for lipids. The common disulfide structure probably has an important role in the interaction of these proteins with membranes.The analysis of the sugar moiety of saposin D revealed that the single N-glycosylation site present in the molecule 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 the glycosylation 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. Neither ceramide 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 the interaction of saposin D with vesicles at low pH. These results suggest that, in the acidic organelles where saposins 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 called prosaposin [1±5]. This precursor is proteolytically processed into mature saposins in late endosomes and lysosomes [6,7]. Genetic defects of saposins have provided insights into their role in the degradation of sphingolipids. When all saposins are missing, as seen in a patient carrying a mutation in the prosaposin initi...

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Section: Discussionmentioning

confidence: 45%

Structural and membrane‐binding properties of saposin D

Tatti

Salvioli

Ciaffoni

et al. 1999

European Journal of Biochemistry

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“…Each of saposins B, C, and D has one such sequence, whereas saposin A has two. Nonglycosylated saposins retain their respective activation effects using in vitro assays (12)(13)(14)(15).…”

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confidence: 99%

Differential Membrane Interactions of Saposins A and C

Grabowski

2001

Journal of Biological Chemistry

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Saposins are small, heat-stable glycoprotein activators of lysosomal glycosphingolipid hydrolases that derive from a single precursor, prosaposin, by proteolytic cleavage. Three of these saposins (B, C, and D) share common structural features including a lack of tryptophan, a single glycosylation sequence, the presence of three conserved disulfide bonds, and a common multiamphipathic helical bundle motif. Saposin A contains an additional glycosylation site and a single tryptophan. The oligosaccharides on saposins are not required for in vitro activation functions. Saposins A and C were produced in Escherichia coli to contain single tryptophans at various locations to serve as intrinsic fluorescence reporters, i.e. as topological probes, for interaction with phospholipid membranes. Maximum emission shifts, aqueous and solid quenching, and resonance energy transfer were quantified by fluorescence spectroscopy. Amphipathic helices at the amino-and carboxyl termini of saposins A and C were shown to insert into the lipid bilayer to about five carbon bond lengths. In comparison, the middle region of saposins A or C were either embedded in the bilayer or solvent-exposed, respectively. Conformational changes of saposin C induced by phosphatidylserine interaction suggested the reorientation of functional helical domains. Differential interaction models are proposed for the membrane-bound saposins A and C. By site-directed mutagenesis of saposin A and C, their membrane topological structures were correlated with their activation effects on acid ␤-glucosidase. These findings show that proper orientation of the middle segment of saposin C to the outside of the membrane surface is critical for its specific and multivalent interaction with acid ␤-glucosidase. Such membrane interactions and orientations of the saposins determine the proximity of their activation and/or binding sites to lysosomal hydrolases or lipoid substrates.Saposins, a family of small (ϳ80 amino acids) heat-stable glycoproteins, are essential for the in vivo hydrolytic activity of several lysosomal enzymes in the catabolic pathway of glycosphingolipids (1-3). Four members of saposins, A, B, C, and D, are proteolytically derived from a single precursor protein, termed prosaposin (4 -8). The primary sequences of saposins are highly homologous with ϳ60% amino acid similarity. In addition, each of the saposins has six conserved cysteines that form three intradomain disulfide bridges whose placements are identical (9). In a native state, five consensus N-glycosylation sequences in prosaposin are occupied by oligosaccharide chains (10 -12). Each of saposins B, C, and D has one such sequence, whereas saposin A has two. Nonglycosylated saposins retain their respective activation effects using in vitro assays (12-15).A multiple ␣-helical bundle motif, characterized by a threeconserved-disulfide structure and several amphipathic peptides, is found in the saposins and also in saposin-like proteins and domains, i.e. NK-lysin, surfactant-associated protein B (SP-B), 1 a...

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“…After removal of its signal peptide, prosaposin is glycosylated at five glycosylation sites [two in saposin A, one each in saposins B, C, and D (1,3,5,23)]. Prosaposin is proteolyzed to generate saposins A, B, C, and D, with cleavage occurring at or near basic dipeptides at their polypeptide boundaries (1,2,24).…”

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confidence: 99%