Phospholipid vesicle fusion induced by saposin C

Saposin C is a small Trp-free, multifunctional glycoprotein that enhances the hydrolytic activity of acid beta-glucosidase in lysosomes. Saposin C's functions have been shown to include neuritogenic/neuroprotection effects and membrane fusion induction. Here, the mechanism and kinetics of saposin C's fusogenic activity were evaluated by fluorescence spectroscopic methods including dequenching, fluorescence resonance energy transfer, and stopped-flow analyses. Trp or dansyl groups were introduced as fluorescence reporters into selected sites of saposin C to serve as topological probes for protein-protein and protein-membrane interactions. Saposin C induction of liposomal vesicle enlargement was dependent upon anionic phospholipids and acidic pH. The initial fusion burst was completed in the timeframe of a few seconds to minutes and was dependent upon the unsaturated anionic phospholipid content. Two events were associated with saposin C-membrane interaction: membrane insertion of the saposin C terminal helices and reorientation of its central helical region. The latter conformational change likely exposed a binding site for saposins anchored on vesicles. Addition of selected saposin C peptides prior to intact saposin C in reaction mixtures abolished the liposomal fusion. These results indicated that saposin-membrane and saposin-saposin interactions are needed for the fusion process.     

Cathepsin-mediated regulation of autophagy in saposin C deficiency

Saposin C deficiency, a rare variant form of Gaucher disease, is due to mutations in the prosaposin gene (PSAP) affecting saposin C expression and/or function. We previously reported that saposin C mutations affecting one cysteine residue result in autophagy dysfunction. We further demonstrated that the accumulation of autophagosomes, observed in saposin C-deficient fibroblasts, is due to an impairment of autolysosome degradation, partially caused by the reduced amount and enzymatic activity of CTSB (cathepsin B) and CTSD (cathepsin D). The restoration of both proteases in pathological fibroblasts results in almost completely recovery of autophagic flux and lysosome homeostasis.     

Autophagy in Gaucher disease due to saposin C deficiency

Gaucher disease, due to a deficit of glucosylceramidase or, rarely, of its activator saposin C, is characterized by accumulation of glucosylceramide in the lysosomes of monocyte/macrophage lineage. In our study we demonstrate that saposin C deficiency due to mutations involving a cysteine residue results in increased autophagy. Autophagy was monitored by LC3 analysis and confirmed by electron microscopy; we observed a correlation among saposin C mutation, Gaucher phenotype and increased autophagy.     

Solution structure of human saposin C in a detergent environment

Saposin C is a lysosomal, membrane-binding protein that acts as an activator for the hydrolysis of glucosylceramide by the enzyme glucocerebrosidase. We used high-resolution NMR to determine the three-dimensional solution structure of saposin C in the presence of the detergent sodium dodecyl sulfate (SDS). This structure provides the first representation of membrane bound saposin C at the atomic level. In the presence of SDS, the protein adopts an open conformation with an exposed hydrophobic pocket. In contrast, the previously reported NMR structure of saposin C in the absence of SDS is compact and contains a hydrophobic core that is not exposed to the solvent. NMR data indicate that the SDS molecules interact with the hydrophobic pocket. The structure of saposin C in the presence of SDS is very similar to a monomer in the saposin B homodimer structure. Their comparison reveals possible similarity in the type of protein/lipid interaction as well as structural components differentiating their quaternary structures and functional specificity.     

Role of lysine residues in membrane anchoring of saposin C

Molecular dynamics (MD) simulations of the N-terminal region of saposin C, containing amino acid residues 4-20 (saposin C4-20), were performed over 2.5 ns in 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) monolayers. The simulations revealed several strong specific interactions of lysine 13 (Lys13) and lysine 17 (Lys17) in saposin C4-20 with the anionic phospholipids, which are required for membrane anchoring of the peptide. Membrane anchoring of saposin C4-20 facilitates saposin C-induced liposomal membrane fusion. Substitutions of Lys13 or Lys17 with alanine or glutamic acid led to a substantial loss of saposin C's fusogenicity. However, arginine replacement of Lys13 or Lys17 caused a partial loss of saposin C's fusogenic activity. The membrane anchoring of saposin C was altered in the presence of 0.4 M sodium chloride. Differential salt effects on Lys-mutant saposin Cs were observed using Trp fluorescence analysis. Low salt concentration had a more significant impact on Lys-mutant saposin C with a negatively charged amino acid residue replacement than those mutants with a positively charged or neutral residue replacement. These results indicate that positively charged amino acids at positions 13 and 17 are required for the fusogenic function of saposin C. In addition, the side-chain structure of lysine is crucial to the precise membrane anchoring which is necessary for the total fusion activity of saposin C. The MD simulations and vesicle size measurements of lysine-mutant saposins confirm the importance of the two lysine residues in saposin C4-20 for saposin C-induced fusion of negatively charged phospholipid membranes.     

Altered autophagy in the mice with a deficiency of saposin A and saposin B

Combined saposin A and saposin B deficiency (AB^−/−) was created in mice by knock-in of point mutations into the saposin A and B domains of the Psap (encoding prosaposin) locus. PSAP is the precursor of saposin A, saposin B and two other members, saposin C and saposin D. Those four saposins have multiple functions including their roles as glycosphingolipid activator proteins in a lysosomal glycosphingolipid degradation pathway. Saposin A participates in the removal of galactose from galactosylceramide and galactosylsphingosine by enhancing β-galactosylceramidase activity. Saposin B has lipid binding properties and is involved in glycosphingolipid metabolism by presenting the substrates to specific enzymes for degradation, i.e., sulfatide to ARSA/arylsulfatase A, lactosylceramide to GALC/GM-1-β-galactosylceramidase, and globotriaosylceramide to GLA/α-galactosidase. Galactosylceramide and sulfatide are myelin glycosphingolipids involved in carbohydrate interaction between synapses. The AB^−/− mice develop accumulation of multiple glycosphingolipids in various organs. Sulfatide and galactosylsphingosine, a deacylated form of galactosylceramide, are the major substrates accumulated in the CNS of AB^−/− mice. The latter is a toxic metabolite to oligodendrocytes and results in demyelination and cell death.     

Conformational and amino acid residue requirements for the saposin C neuritogenic effect.

Prosaposin is the precursor of four activator proteins, termed saposins A, B, C, and D, that are required for much of glycosphingolipid hydrolysis. The intact precursor also has neurite outgrowth activity ex vivo and in vivo that is localized to amino acid residues 22-31 of saposin C. Across species, this saposin C region has a high degree of identity and similarity with amino acids in the analogous region of saposin A. Wild-type and mutant saposins C and A from human and mouse were expressed in E. coli. Pure proteins, synthetic peptide analogues, conformation-specific antibodies, and CD spectroscopy were used to evaluate the basis of the ex vivo neuritogenic effect. Wild-type saposin A had no neuritogenic activity whereas reduced and alkylated saposin A did. Introduction of the conserved saposin A Tyr 30 (Y30) into saposin C at the analogous position 31, a conserved Ala(A)/Gly(G)31, diminished neuritogenic activity by 50-60%. Nondenatured saposin A with an introduced A30 acquired substantial neuritogenic activity. Polyclonal antibodies directed against the NH2-terminus of saposin C cross-reacted well with reduced and alkylated saposins C and A, wild-type saposin C, and saposin A [Y30A], poorly with saposin C [A31Y], and not at all with wild-type saposin A. CD spectra of wild-type and mutant saposins C and A, the corresponding neuritogenic region of saposin C, and the analogous region of saposin A showed that more "saposin C-like" molecules had neuritogenic properties. Those with more "saposin A-like" spectra did not. These studies show that the neuritogenic activity of saposin C requires specific placement of amino acids, and that Y30 of saposin A significantly alters local conformation in this critical region and suppresses neuritogenic activity.     

Phospholipid membrane interactions of saposin C: in situ atomic force microscopic study

Saposin C (Sap C) is a small glycoprotein required for hydrolysis of glucosylceramidase in lysosomes. The full activity of glucosylceramidase requires the presence of both Sap C and acidic phospholipids. Interaction between Sap C and acidic phospholipid-containing membranes, a crucial step for enzyme activation, is not fully understood. In this study, the dynamic process of Sap C interaction with acidic phospholipid-containing membranes was investigated in aqueous buffer using atomic force microscopy. Sap C induced two types of membrane restructuring: formation of patch-like structural domains and the occurrence of membrane destabilization. The former caused thickness increase whereas the latter caused thickness reduction in the gel-phase membrane bilayer, possibly as a result of lipid loss or an interdigitating process. Patch-like domain formation was independent of acidic phospholipids, whereas membrane destabilization is dependent on the presence and concentration of acidic phospholipids. Sap C effects on membrane restructuring were further studied using synthetic peptides. Synthetic peptides corresponding to the amphipathic alpha-helical domains 1 (designated "H1 peptide") and 2 (H2 peptide) of Sap C were used. Our results indicated that H2 contributed to domain formation but not to membrane destabilization, whereas H1 induced neither type of membrane restructuring. However, H1 was able to mimic Sap C's destabilization effect in conjunction with H2, but only when H1 was present first and H2 was added afterwards. This study provides an approach to investigate the structure-function aspects of Sap C interaction with phospholipid membranes, with insights into the mechanism(s) of Sap C-membrane interaction.     

Solution structure of human saposin C: pH-dependent interaction with phospholipid vesicles

Saposin C binds to membranes to activate lipid degradation in lysosomes. To get insights into saposin C's function, we have determined its three-dimensional structure by NMR and investigated its interaction with phospholipid vesicles. Saposin C adopts the saposin-fold common to other members of the family. In contrast, the electrostatic surface revealed by the NMR structure is remarkably different. We suggest that charge distribution in the protein surface can modulate membrane interaction leading to the functional diversity of this family. We find that the binding of saposin C to phospholipid vesicles is a pH-controlled reversible process. The pH dependence of this interaction is sigmoidal, with an apparent pK(a) for binding close to 5.3. The pK(a) values of many solvent-exposed Glu residues are anomalously high and close to the binding pK(a). Our NMR data are consistent with the absence of a conformational change prior to membrane binding. All this information suggests that the negatively charged electrostatic surface of saposin C needs to be partially neutralized to trigger membrane binding. We have studied the membrane-binding behavior of a mutant of saposin C designed to decrease the negative charge of the electrostatic surface. The results support our conclusion on the importance of protein surface neutralization in binding. Since saposin C is a lysosomal protein and pH gradients occur in lysosomes, we propose that lipid degradation in the lysosome could be switched on and off by saposin C's reversible binding to membranes.     

A mutation within the saposin D domain in a Gaucher disease patient with normal glucocerebrosidase activity

Only two Gaucher disease (GD) patients bearing mutations in the prosaposin gene (PSAP), and not in the glucocerebrosidase gene (GBA), have been reported. In both cases, one mutant allele remained unidentified. We report here the identification of the second mutation in one of these patients, being the first complete genotype described so far in a SAP-C-deficient GD patient. This mutation, p.Q430X, is the first one reported in the saposin D domain and probably produces a null allele by nonsense mediated mRNA decay.     

A peptide with alternating lysines can act as a highly specific Z-DNA binding domain

Many nucleic acid binding proteins use short peptide sequences to provide specificity in recognizing their targets, which may be either a specific sequence or a conformation. Peptides containing alternating lysine have been shown to bind to poly(dG–d5meC) in the Z conformation, and stabilize the higher energy form [H. Takeuchi, N. Hanamura, H. Hayasaka and I. Harada (1991) FEBS Lett., 279, 253–255 and H. Takeuchi, N. Hanamura and I. Harada (1994) J. Mol. Biol., 236, 610–617.]. Here we report the construction of a Z-DNA specific binding protein, with the peptide KGKGKGK as a functional domain and a leucine zipper as a dimerization domain. The resultant protein, KGZIP, induces the Z conformation in poly(dG–d5meC) and binds to Z-DNA stabilized by bromination with high affinity and specificity. The binding of KGZIP is sufficient to convert poly(dG–d5meC) from the B to the Z form, as shown by circular dichroism. The sequence KGKGKGK is found in many proteins, although no functional role has been established. KGZIP also has potential for engineering other Z-DNA specific proteins for future studies of Z-DNA in vitro and in vivo.     

Fusogenic activity of SIV (simian immunodeficiency virus) peptides located in the GP32 NH2 terminal domain

Peptides of 12, 16 and 24 amino acids length corresponding to the NH2 terminal sequence of SIV gp32 were synthesized. Fluorescence energy transfer studies have shown that those peptides can induce lipid mixing of SUV (Small Unilamellar Vesicles) of various compositions at pH 7.4 and 37 degrees C. LUV (Large Unilamellar Vesicles) were shown to undergo fusion, provided they contained PE in their lipid composition. This work is an attempt to determine how the fusogenic activity depends on the structure of the peptide inserted into a lipidic environment. The peptides secondary structure and orientation in the lipid bilayer were determined using Fourier Transform infrared spectroscopy (FTIR). They adopt mainly a beta-sheet conformation in the absence of lipids. After interaction with DOPC SUV, the beta-sheet is partly converted into alpha-helix oriented obliquely with respect to the membrane interface. We bring here evidence that this oblique orientation is a prerequisite to the fusion process.     

Investigating the interaction of saposin C with POPS and POPC phospholipids: a solid-state NMR spectroscopic study

The interaction of Saposin C (Sap C) with negatively charged phospholipids such as phosphatidylserine (PS) is essential for its biological function. In this study, Sap C (initially protonated in a weak acid) was inserted into multilamellar vesicles (MLVs) consisting of either 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-L-serine] (negatively charged, POPS) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (neutrally charged, POPC). The MLVs were then investigated using solid-state NMR spectroscopy under neutral pH (7.0) conditions. The (2)H and (31)P solid-state NMR spectroscopic data of Sap C-POPS and Sap C-POPC MLVs (prepared under the same conditions) were compared using the (2)H order parameter profiles of the POPC-d(31) or POPS-d(31) acyl chains as well as the (31)P chemical shift anisotropy width and (31)P T(1) relaxation times of the phospholipids headgroups. All those solid-state NMR spectroscopic approaches indicate that protonated Sap C disturbs the POPS bilayers and not the POPC lipid bilayers. These observations suggest for the first time that protonated Sap C inserts into PS bilayers and forms a stable complex with the lipids even after resuspension under neutral buffer conditions. Additionally, (31)P solid-state NMR spectroscopic studies of mechanically oriented phospholipids on glass plates were conducted and perturbation effect of Sap C on both POPS and POPC bilayers was compared. Unlike POPC bilayers, the data indicates that protonated Sap C (initially protonated in a weak acid) was unable to produce well-oriented POPS bilayers on glass plates at neutral pH. Conversely, unprotonated Sap C (initially dissolved in a neutral buffer) did not interact significantly with POPS phospholipids allowing them to produce well-oriented bilayers at neutral pH.     

Phospholipid membrane restructuring induced by saposin C: a topographic study using atomic force microscopy

The enzymatic activity of glucosylceramidase depends on the presence of saposin C (Sap C) and acidic phospholipid-containing membranes. In order to delineate the mechanism underlying Sap C stimulation of the enzyme activity, it is important to understand how Sap C interacts with phospholipid membranes. We studied the dynamic process of Sap C interaction with planar phospholipid membranes, in real time, using atomic force microscopy (AFM). The phospholipid membrane underwent restructuring upon addition of Sap C. The topographic characteristics of the membrane restructuring include the appearance of patch-like new features, initially emerged at the edge of phospholipid membranes and extended laterally with time. Changes in the image contrast of the phospholipid membrane observed after the Sap C addition indicate that a new phase of lipid-protein structure has formed during membrane restructuring. The process of membrane restructuring is dynamic, commencing shortly after Sap C addition, and continuing throughout the duration of AFM imaging (about 30 min, sometimes over 1 h). This study demonstrated the potential of AFM real-time imaging in studying protein-membrane interactions.     

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 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.     

Fusogenic activity of EFF-1 is regulated via dynamic localization in fusing somatic cells of C. elegans

BACKGROUND: Many animal tissues form via fusion of cells. Yet in all instances of developmental cell fusion, the mechanism underlying fusion of plasma membranes remains poorly understood. EFF-1 is required for most somatic cell fusions in C. elegans, and misexpressed EFF-1 alters the normal pattern of fusing hypodermal cells. However, the autonomous activity of EFF-1, the rules governing its specificity, and the mechanism of its action have not been examined. RESULTS: We show that EFF-1 acts as a cellular fusogen, capable of inducing fusion of virtually any somatic cells in C. elegans, yet targeted precisely to fusion-fated contacts during normal development. Misexpression of EFF-1 in early embryos causes fusion among groups of cells composed entirely of nonfusion-fated members. Measurements of cytoplasm diffusion in induced fusion events show that ectopic EFF-1 expression produces fusion pores similar to those in normal fusion events. GFP-labeled EFF-1 is specifically targeted to fusion-competent cell contacts via reciprocal localization to the touching membranes of EFF-1-expressing cells. EFF-1 function is also governed by intercellular barriers that prohibit cell fusion between distinct tissues. Analysis of mutant versions of EFF-1 indicates a novel mode of fusogenicity, employing neither a phospholipase active site nor hydrophobic fusion-peptide acting solely in pore formation. CONCLUSIONS: EFF-1 can confer potent fusogenic activity to nonfusing cell types. However, it is normally targeted only to fusion-fated cell borders via mutual interaction between EFF-1-expressing cells and relocalization to the plasma membrane. Because EFF-1 appears evolutionarily unique to nematodes, multiple mechanisms may have evolved for controlled plasma-membrane fusion in development.     

Evidence for essential lysines in heparin cofactor II

Covalent modification with pyridoxal 5'-phosphate was used to study the function of lysyl residues in heparin cofactor II, a heparin-dependent plasma protease inhibitor. Reduction of the Schiff base with sodium borohydride resulted in modification of 3-4 lysyl residues of heparin cofactor II at high concentrations of pyridoxal 5'-phosphate, one of which was protected in the presence of heparin. The antithrombin activity of modified heparin cofactor II was enhanced compared to the native protein. However, the heparin cofactor activity for thrombin inhibition was reduced significantly or completely eliminated in the modified protease inhibitor depending on the extent of phosphopyridoxylation. In contrast to native heparin cofactor II, the modified protease inhibitor did not bind to a heparin-agarose column. The results suggest that lysyl residues are essential for heparin cofactor activity during thrombin inhibition.     

Adiponectin multimerization is dependent on conserved lysines in the collagenous domain: evidence for regulation of multimerization by alterations in posttranslational modifications

Adiponectin is a secreted, multimeric protein with insulin-sensitizing, antiatherogenic, and antiinflammatory properties. Serum adiponectin consists of trimer, hexamer, and larger high-molecular-weight (HMW) multimers, and these HMW multimers appear to be the more bioactive forms. Multimer composition of adiponectin appears to be regulated; however, the molecular mechanisms involved are unknown. We hypothesize that regulation of adiponectin multimerization and secretion occurs via changes in posttranslational modifications (PTMs). Although a structural role for intertrimer disulfide bonds in the formation of hexamers and HMW multimers is established, the role of other PTMs is unknown. PTMs identified in murine and bovine adiponectin include hydroxylation of multiple conserved proline and lysine residues and glycosylation of hydroxylysines. By mass spectrometry, we confirmed the presence of these PTMs in human adiponectin and identified three additional hydroxylations on Pro71, Pro76, and Pro95. We also investigated the role of the five modified lysines in multimer formation and secretion of recombinant human adiponectin expressed in mammalian cell lines. Mutation of modified lysines in the collagenous domain prevented formation of HMW multimers, whereas a pharmacological inhibitor of prolyl- and lysyl-hydroxylases, 2,2'-dipyridyl, inhibited formation of hexamers and HMW multimers. Bacterially expressed human adiponectin displayed a complete lack of differentially modified isoforms and failed to form bona fide trimers and larger multimers. Finally, glucose-induced increases in HMW multimer production from human adipose explants correlated with changes in the two-dimensional electrophoresis profile of adiponectin isoforms. Collectively, these data suggest that adiponectin multimer composition is affected by changes in PTM in response to physiological factors.