The pH-dependent exposure of hydrophobic domains of Sap C and D paralleled their pH-dependent binding to large unilamellar vesicles composed of phosphatidylcholine, phosphatidylserine, and cholesterol. In contrast, the binding of Sap A to the vesicles was very restricted, in spite of its increased hydrophobicity at low pH. A low affinity for the vesicles was also shown by Sap B, a finding consistent with its apparent hydrophilicity both at neutral and acidic pH.At the acidic pH values needed for binding, Sap C and D powerfully destabilized the phospholipid membranes, while Sap A and B minimally affected the bilayer integrity. In the absence of the acidic phospholipid phosphatidylserine, the induced destabilization markedly decreased.Of the four saposins, only Sap C was able to promote the binding of glucosylceramidase to phosphatidylserine-containing membranes. This result is consistent with the notion that Sap C is specifically required by glucosylceramidase to exert its activity. Our finding that an acidic environment induces an increased hydrophobicity in Sap A, C, and D, making the last two saposins able to interact and perturb phospholipid membranes, suggests that this mechanism might be relevant to the mode of action of saposins in lysosomes.
Key Points Abnormal signatures in TGF-β1 signaling gene expression were identified in spleen and marrow from the Gata1low model of MF. These signatures include abnormalities in individual gene (Id2, Stat1, mTOR) in spleen and of gene pathways (Smads and BMPs) in marrow.
The function of saposin C (Sap C), a glucosylceramidase activator protein, in the enzyme stimulation by phosphatidylserine (PS) liposomes has been investigated. Using gel filtration experiments evidence was obtained for Sap C binding to PS large unilamellar vesicles (LUV) but not to glucosylceramidase. PS LUV, which by themselves are unable to tightly bind and stimulate the enzyme, acquire the capacity to also bind the enzyme after interaction with Sap C, making it express its full activity. Our results indicate that the primary step in the Sap C mode of action resides in its association with PS membranes; in turn, this association promotes the interaction between the membranes and glucosylceramidase.
Key Points Elevation of HbF in 3 patients heterozygous for distinct 2p15-p16.1 syndrome microdeletions affecting BCL11A. Identification of novel, putative regulatory elements downstream of BCL11A that govern its expression in erythroid cells.
The degradation of glucosylceramide in lysosomes is accomplished by glucosylceramidase with the assistance of, at least, another protein, saposin C (Sap C), which is generated from a large precursor together with three other similar proteins, saposins A, B, and D. In the present study, we have examined the effects of saposins on the enzymatic hydrolysis of glucosylceramide inserted in large and small phospholipid liposomes. The glucosylceramide contained in large unilamellar vesicles (LUV) was degraded by glucosylceramidase at a rate 7-8-fold lower than glucosylceramide inserted in small unilamellar vesicles (SUV). The separate addition of either Sap A or Sap C to the LUV system partially stimulated the sphingolipid degradation while saposins B and D had no effect. In the presence of both Sap A and Sap C, the rate of sphingolipid degradation was higher than the sum of the rates with the two saposins individually, indicating synergism in their actions. The stimulatory effect of the two saposins depended on the incorporation of an acidic phospholipid such as phosphatidylserine (PS) into LUV.The characteristics of glucosylceramidase activation by Sap C were different from those of Sap A. Sap C increased the rate of hydrolysis of both the artificial water soluble substrate, 4-methylumbelliferyl--D-glucopyranoside, and the lipid substrate, glucosylceramide, while Sap A only stimulated degradation of the sphingolipid. Also the binding properties of Saps A and C were markedly different. At acidic pH values, Sap C bound to PS-containing LUV and promoted the association of glucosylceramidase with the membrane. In contrast, Sap A had poor affinity for the membrane even in the presence of glucosylceramide; moreover, Sap A did not potentiate the capacity of Sap C to mediate glucosylceramidase binding.In conclusion, our results show that both Sap A and Sap C are required for maximal hydrolysis of glucosylceramide inserted in PS-containing LUV, that their effects are synergistic, and that their mode of action is different. Sap C is responsible for the membrane binding of glucosylceramidase, while Sap A stimulation is possibly related to its effect on the conformation of the enzyme. It can be envisaged that Sap A in conjunction with Sap C might have a physiological role in glucosylceramide degradation.
We have previously shown that saposin C (Sap C), a glucosylceramidase activator protein, interacts with phosphatidylserine (PS) large unilamellar vesicles (LUV), promoting the glucosylceramidase binding to the bilayer ) FEBS Lett. 336, 159-1621. In the present paper the consequences of the Sap C interaction on the lipid organization of the vesicles are reported. It was found that Sap C perturbs the PS bilayer as shown by the release of an encapsulated fluorescent dye. Three different procedures, resonance energy transfer, gel filtration and electron microscopy, indicated that the activator protein is also able to make PS liposomes fuse. The effects of Sap C on PS vesicles were observed at low but not at neutral pH. The lipid composition of the bilayer also affected the Sap C-induced destabilization; in fact, the presence of PS in mixed LUV was essential for significant leakage to occur. These results demonstrate for the first time that Sap C is a protein capable ofdestabilizing and fusing acidic phospholipid-containing membranes in a pH-dependent fashion.
The lysosomal degradation of several sphingolipids requires the presence of four small glycoproteins called saposins, generated by proteolytic processing of a common precursor, prosaposin. Saposins share several structural properties, including six similarly located cysteines forming three disulfide bridges with the same cysteine pairings. Recently it has been noted that also other proteins have the same polypeptide motif characterized by the similar location of six cysteines. These saposin-like (SAPLIP) proteins are surfactant protein B (SP-B), 'Entamoeba histolytica' pore-forming peptide, NK-lysin, acid sphingomyelinase and acyloxyacyl hydrolase. The structural homology and the conserved disulfide bridges suggest for all SAPLIPs a common fold, called 'saposin fold'. Up to now a precise fold, comprising five alpha-helices, has been established only for NK-lysin. Despite their similar structure each saposin promotes the degradation of specific sphingolipids in lysosomes, e.g. Sap B that of sulfatides and Sap C that of glucosylceramides. The different activities of the saposins must reside within the module of the alpha-helices and/or in additional specific regions of the molecule. It has been reported that saposins bind to lysosomal hydrolases and to several sphingolipids. Their structural and functional properties have been extensively reviewed and hypotheses regarding their molecular mechanisms of action have been proposed. Recent work of our group has evidenced a novel property of saposins: some of them undergo an acid-induced change in hydrophobicity that triggers their binding to phospholipid membranes. In this article we shortly review recent findings on the structure of saposins and on their interactions with lipids, with special attention to interactions with phospholipids. These findings offer a new approach for understanding the physiological role of saposins in lysosomes.
Saposins A, B, C, and D are a group of homologous glycoproteins derived from a single precursor, prosaposin, and apparently involved in the stimulation of the enzymatic degradation of sphingolipids in lysosomes. All saposins have six cysteine residues at similar positions. In the present study we have investigated the disulfide structure of saposins B and C using advanced mass spectrometric procedures. Electrospray analysis showed that deglycosylated saposins B and C are mainly present as 79- and 80-residue monomeric polypeptides, respectively. Fast atom bombardment mass analysis of peptide mixtures obtained by a combination of chemical and enzymatic cleavages demonstrated that the pairings of the three disulfide bridges present in each saposin are Cys4-Cys77, Cys7-Cys71, Cys36-Cys47 for saposin B and Cys5-Cys78, Cys8-Cys72, Cys36-Cys47 for saposin C. We have recently shown that saposin C interacts with phosphatidylserine-containing vesicles inducing destabilization of the lipid surface (Vaccaro, A. M., Tatti, M., Ciaffoni, F., Salvioli, R., Serafino, A., and Barca, A. (1994) FEBS Lett. 349, 181-186); this perturbation promotes the binding of the lysosomal enzyme glucosylceramidase to the vesicles and the reconstitution of its activity. It was presently found that the effects of saposin C on phosphatidylserine liposomes and on glucosylceramidase activity are markedly reduced when the three disulfide bonds are irreversibly disrupted. These results stress the importance of the disulfide structure for the functional properties of the saposin.
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