Biomedical subjects

Xiaoyang Qi

Publications and source records attributed to Xiaoyang Qi.

15 recordsLinked to original sources

PubMed2006

Spontaneously forming ellipsoidal phospholipid unilamellar vesicles and their interactions with helical domains of saposin C.

We have observed a bimodal distribution of ellipsoidal unilamellar vesicles (ULVs) in a phospholipid mixture composed of dioleoyl phosphatidylserine (DOPS) and dipalmitoyl and dihexanoyl phosphatidylcholine, DPPC and DHPC, respectively. Dynamic light scattering and transmission electron microscopy data indicate a bimodal size distribution of these nanoparticles with hydrodynamic radii of approximately 200 and >500 nm, while small-angle neutron scattering data were fit using a model of coexisting monodisperse morphologies, namely, oblate and triaxial ellipsoidal vesicles. Unlike DOPS ULV formed by sonication, which can fuse days after being formed, these ULVs are stable over a period of 12 months at 4 degrees C. We also report on the structure of these ULVs associated with the two helical peptide domains (H1 and H2) of a glucosylprotein, namely, Saposin C, to gain some insight into protein-membrane interactions.

Animals↗

PubMed2005

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.

Binding Sites↗

PubMed2005

Structural changes in a binary mixed phospholipid bilayer of DOPG and DOPS upon saposin C interaction at acidic pH utilizing 31P and 2H solid-state NMR spectroscopy.

Saposin C (Sap C) is known to stimulate the catalytic activity of the lysosomal enzyme glucosylceramidase (GCase) that facilitates the hydrolysis of glucosylceramide to ceramide and glucose. Both Sap C and acidic phospholipids are required for full activity of GCase. In order to better understand this interaction, mixed bilayer samples prepared from dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylserine (DOPS) (5:3 ratio) and Sap C were investigated using (2)H and (31)P solid-state NMR spectroscopy at temperatures ranging from 25 to 50 degrees C at pH 4.7. The Sap C concentrations used to carry out these experiments were 0 mol%, 1 mol% and 3 mol% with respect to the phospholipids. The molecular order parameters (S(CD)) were calculated from the dePaked (2)H solid-state NMR spectra of Distearoyl-d70-phosphatidylglycerol (DSPG-d70) incorporated with DOPG and DOPS binary mixed bilayers. The S(CD) profiles indicate that the addition of Sap C to the negatively charged phospholipids is concentration dependent. S(CD) profiles of 1 mol% of the Sap C protein show only a very slight decrease in the acyl chain order. However, the S(CD) profiles of the 3 mol% of Sap C protein indicate that the interaction is predominantly increasing the disorder in the first half of the acyl chain near the head group (C1-C8) indicating that the amino and the carboxyl termini of Sap C are not inserting deep into the DOPG and DOPS mixed bilayers. The (31)P solid-state NMR spectra show that the chemical shift anisotropy (CSA) for both phospholipids decrease and the spectral broadening increases upon addition of Sap C to the mixed bilayers. The data indicate that Sap C interacts similarly with the head groups of both acidic phospholipids and that Sap C has no preference to DOPS over DOPG. Moreover, our solid-state NMR spectroscopic data agree with the structural model previously proposed in the literature [X. Qi, G.A. Grabowski, Differential membrane interactions of saposins A and C. Implication for the functional specificity, J. Biol. Chem. 276 (2001) 27010-27017] [1].

Acids↗

PubMed2005

Saposin C-LBPA interaction in late-endosomes/lysosomes.

Acidic phospholipids and saposins associations are involved in the degradation process of glycosphingolipids/sphingolipids in late endosomes/lysosomes. In this report, we showed the colocalization of saposin C and lysobisphosphatidic acid (LBPA) in human fibroblasts by using cytoimmunofluorescence analysis. This colocalization pattern was not seen with other saposins. Large numbers of saposins A, B, and D illustrated the staining patterns that differ from LBPA. In addition, ingested anti-LBPA antibody altered the location of saposin C in human wild-type fibroblasts. In vitro assays demonstrated that saposin C at nM concentrations induced membrane fusion of LBPA containing phospholipid vesicles. Under the same condition, other saposins had no fusion induction on these vesicles. These results suggested a specific interaction between saposin C and LBPA. Total saposin-deficient fibroblasts showed a massive accumulation of multivesicular bodies (MVBs) by electron microscopic analysis. No significant increase of MVBs was found in saposins A and B deficient cells. Interestingly, the accumulated MVBs were significantly reduced by loading saposin C alone into the total saposin-deficient cells. Therefore, we propose that saposin C-LBPA interaction plays a role in the regulation of MVB formation in cells.

Animals↗

PubMed2005

Mapping and analysis of the lytic and fusogenic domains of surfactant protein B.

Surfactant protein B (SP-B) is a hydrophobic, 79 amino acid peptide that regulates the structure and function of surfactant phospholipid membranes in the airspaces of the lung. Addition of SP-B to liposomes composed of DPPC/PG (7:3) leads to membrane binding, destabilization, and fusion, ultimately resulting in rearrangement of membrane structure. The goal of this study was to map the fusogenic and lytic domains of SP-B and assess the effects of altered fusion and lysis on surface activity. Synthetic peptides were generated to predicted helices and/or interhelical loops of SP-B and tested for fusion, lytic, and surface activities. The N-terminal half of SP-B (residues 1-37), which includes the nonhelical N-terminal amino acids in addition to helices 1 and 2, promoted rapid liposome fusion whereas shorter peptides were significantly less effective. The requirements for optimal surface tension reduction were similar to those for fusion; in contrast, helix 1 (residues 7-22) alone was sufficient for liposome lysis. The C-terminal half of SP-B (residues 43-79), which includes helices 3, 4, and 5, exhibited significantly lower levels of fusogenic, lytic, and surface tension reducing activities compared to the N-terminal region. These results indicate that SP-B fusion, lytic and surface activities map predominantly to the N-terminal half of SP-B. Amino acid substitutions in synthetic peptides corresponding to the N-terminal half of SP-B indicated that, in general, decreased fusion or lytic activities were associated with altered surface tension reducing properties of the peptide. However, the presence of fusion and lytic activities alone could not account for the surface tension reducing property of SP-B. We propose a model in which association of helix 1 with lipids leads to membrane permeabilization but not aggregation; helix 2 mediates membrane cross-linking (aggregation), which, in turn, facilitates lipid mixing, membrane fusion, and interfacial adsorption/surface tension reduction.

Amino Acid Sequence↗

PubMed2005

Saposin C: neuronal effect and CNS delivery by liposomes.

Saposin C is one of four small lipid-binding proteins that derive from a single precursor protein, named prosaposin (PSAP). PSAP has several neuronal effects, including neurite outgrowth stimulation, neuron preservation, and nerve regeneration enhancement. A minimal domain required for PSAP's neurotrophic function is located in the amino-terminal half of saposin C. Genetic defects of the PSAP gene in humans and mice lead to a complex lysosomal storage disease. The skin fibroblasts from PSAP- and saposin C-deficient patients have a massive accumulation of multivesicular bodies (MVBs). Incorporation of exogenous saposin C-containing liposomes into the cultured PSAP-/- cells reduced the accumulated MVBs to normal levels. Internalized saposin C was localized to late endosomes and lysosomes. MVBs are crucial for maintaining the cellular homeostasis required for neuronal development and growth. PSAP-/- mice have a short life span (30 days) and central nervous system (CNS) neuronal degeneration. Similar to PSAP-/- fibroblasts, excessive MVBs accumulated in CNS neurons and brain tissues of PSAP-null mice. Cultured cortical and hippocampal neurons from PSAP-/- mice had poor survival and displayed a neurite degenerative pattern. Delivery of saposin C ex vivo into cultured neurons and in vivo into brain neuronal cells in mice across the blood-brain barrier was accomplished with intravenously administered dioleoylphosphatidylserine (DOPS) liposomes. These studies may yield a new therapeutic approach for neuron protection, preservation, and regeneration.

Animals↗

PubMed2004

Fusogenic domain and lysines in saposin C.

Saposin C, a sphingolipid activator protein with fusogenic activity, interacts specifically with the membrane containing negatively charged, unsaturated phospholipids. The kinetics and mechanism of saposin C-induced membrane fusion were previously investigated using acidic phospholipid liposomes. A hypothetic clip-on model for such a fusion process was illustrated by the ionic binding between saposin C and lipids, as well as the inter-saposin C hydrophobic interaction. Here, we report the location of the fusogenic domain in a linear sequence at the amino-terminal half of saposin C. This domain consisted of the first and second helical sequences. Selected positively charged lysines in the fusogenic domain were mutated to study the roles of basic residues in the saposin C-induced vesicle fusion. Based on the results, Lys13 and Lys17 were critical for the fusogenic activity, but had no effect on the enzymatic activation of acid beta-glucosidase (GCase). These results clearly indicate the segregation of the fusion and activation function into two different regions of saposin C. Interestingly, all the Lys mutant saposin Cs anchored on the acidic phospholipid membrane. Our data suggest that saposin C's fusogenic and activation functions have different requirements for the orientation and insertion manners of helical peptides in membranes.

Amino Acid Substitution↗

PubMed2004

Direct AFM observation of saposin C-induced membrane domains in lipid bilayers: from simple to complex lipid mixtures.

Saposin C (Sap C) is a small glycoprotein required by glucosylceramidase (GCase) for hydrolysis of glucosylceramide to ceramide and glucose in lysosomes. The molecular mechanism underlying Sap C stimulation of the enzyme activation is not fully understood. Here, atomic force microscopy (AFM) has been used to study Sap C-membrane interactions under physiological conditions. First, to establish how Sap C-membrane interactions affect membrane structure, lipid bilayers containing zwitterionic and anionic phospholipids were used. It was observed that Sap C induced two types of membrane restructuring effects, i.e., the formation of patch-like domains and membrane destabilization. Bilayers underwent extensive structural reorganization. To validate the biological importance of the membrane restructuring effects, interaction of Sap C with lipid bilayers composed of cholesterol, sphingomyelin, and zwitterionic and anionic phospholipids were studied. Although similar membrane restructuring effects were observed, Sap C-membrane interactions, in this case, were remarkably modulated and their effects were restricted to a limited area. As a result, nanometer-sized domains were formed. The establishment of a model membrane system will allow us to further study the dynamics, structure and mechanism of the Sap C-associated membrane domains and to examine the important role that these domains may play in enzyme activation.

Complex Mixtures↗

PubMed2003

Editing of CD1d-bound lipid antigens by endosomal lipid transfer proteins.

It is now established that CD1 molecules present lipid antigens to T cells, although it is not clear how the exchange of lipids between membrane compartments and the CD1 binding groove is assisted. We report that mice deficient in prosaposin, the precursor to a family of endosomal lipid transfer proteins (LTP), exhibit specific defects in CD1d-mediated antigen presentation and lack Valpha14 NKT cells. In vitro, saposins extracted monomeric lipids from membranes and from CD1, thereby promoting the loading as well as the editing of lipids on CD1. Transient complexes between CD1, lipid, and LTP suggested a "tug-of-war" model in which lipid exchange between CD1 and LTP is on the basis of their respective affinities for lipids. LTPs constitute a previously unknown link between lipid metabolism and immunity and are likely to exert a profound influence on the repertoire of self, tumor, and microbial lipid antigens.

Animals↗

PubMed2003

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.

Fluorescence Resonance Energy Transfer↗

PubMed2003

Saposin C is required for normal resistance of acid beta-glucosidase to proteolytic degradation.

Saposins (A, B, C, and D) are small sphingolipid activator proteins that are derived by proteolytic processing of a common precursor, prosaposin. In the lysosomal sphingolipid degradation pathway, acid beta-glucosidase (GCase) requires saposin C for optimal in vitro and in vivo hydrolysis of glucocerebroside. The deficiency of prosaposin/saposins (PS-/-) in humans and mice leads to a decrease of GCase activity in selected tissues. Concordant decreases (>50%) of GCase protein and in vitro activity were detected in extracts of cultured fibroblasts and hepatocytes from PS-/- mice and human prosaposin-deficient fibroblasts. GCase RNA in the PS-/- cells was at wild-type levels. Compared with that in wild-type cells (t(1/2) >24 h), the GCase protein in the PS-/- cells had a faster disappearance rate (t(1/2) approximately 1 h in mouse and approximately 8 h in human) as determined by metabolic labeling and immunoprecipitation with anti-GCase antibodies. Treatment of PS-/- cells with leupeptin, an inhibitor of cysteine proteases, led to significant increases (approximately 2-fold) in GCase protein and in vitro activity. Loading saposin C to human PS-/- fibroblasts resulted in an enhancement of GCase protein and in vitro activity. Saposin D loading had no effect. These data indicate that saposin C is required for GCase resistance to proteolytic degradation in the cell. Thus, diminished in vivo GCase activity would be greater than expected only from the lack of GCase activation by saposin C. These results indicate a new property for saposin C, an anti-proteolytic protective function toward GCase.

Animals↗

PubMed2003

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.

Glycoproteins↗

PubMed2002

Ex vivo localization of the mouse saposin C activation region for acid beta-glucosidase.

Saposin C is a biological activator of acid beta-glucosidase (GCase), the lysosomal hydrolase with activity towards glucosylceramide (GC). In addition, saposin C possesses a functional domain that determines the in vitro and ex vivo neuritogenic effects of prosaposin, the precursor of saposins A, B, C, and D. The domains for enzymatic activation and neuritogenic function segregate in vitro, respectively, to the carboxyl- and amino-terminal halves of human and mouse saposin C. A chimeric mouse saposin C(1-8)B(8-28)C(30-80) was created to obliterate the neuritogenic region by substituting amino acids 9-29 of saposin C with amino acids 8-28 of saposin B. This saposin showed normal in vitro enzymatic activation effects toward GCase, but no neuritogenic activity. An altered prosaposin was made to contain the chimeric saposin C region. Expression of this altered or wild-type prosaposin was driven by the PGK-1 promoter as a transgene in prosaposin knock-out mice. In cultured fibroblasts from such mice, expressed saposins localized to the lysosomal compartments. Metabolic lipid labeling using L-[3-(14)C]serine showed retention or clearance of GC in prosaposin deficient or transgene reconstituted cells, respectively. In addition, sulfatide catabolism, that requires saposin B and arylsulfatase, was also normalized in prosaposin KO cells reconstituted with the transgenes. These data show that the transgenic prosaposins were expressed and processed to functional saposins in fibroblasts. These results also show that the enzymatic activation domain is located at carboxyl-terminal half of saposin C and functions only in the context of the general saposin structure.

Amino Acid Sequence↗

PubMed2002

Prosaposin: threshold rescue and analysis of the "neuritogenic" region in transgenic mice.

Prosaposin is the precursor of four glycoprotein activators (saposins) for lysosomal hydrolases. Intact prosaposin also has lipid transfer properties in vitro as well as neuritogenic effects ex vivo and in vivo. Such "neuritogenic" effects of saposin C were evaluated in vivo using transgenic mice with prosaposin cDNAs having normal (PS-N) or mutated neuritogenic region. The mutant prosaposin cDNA (PS-CBC) encoded a chimeric saposin C that contained the non-neuritogenic sequence of saposin B, but retained acid beta-glucosidase (GCase) activation effects. When driven by the PGK (3-phosphoglycerate kinase) promoter, transgene expression was highest in the cerebrum for any of the transgenes (range from 15% to 42% of wild-type). Low levels were in visceral tissues. Prosaposin knock-out (PS-/-) mice expressing N or CBC transgenes, even at low levels, had delayed onset of neurologic signs and neuropathology, and significant lengthening of life span (from 1.7- to 7-fold) with age dependent partial correction of GlcCer and LacCer accumulation in the brain. Neuropathologic progression and neuronal glycosphingolipid storage were related directly to the transgene expression levels in the brain. Purkinje cell loss was age dependent. Gross brain and neuronal organizations were indistinguishable in PS-/- mice with or without the various transgenes, albeit the phenotype appeared later in the mice with transgenes. These studies show the degree of neuropathologic manifestations in each transgenic line depended on expression level rather than on the nature of the transgene. These studies also show in vivo localization of the GCase activation region to the carboxy terminal half of saposin C and the lack of a significant gross trophic effect of saposin C on CNS organization in vivo.

Animals↗

PubMedDate not supplied

Computational models for the helix tilt angle.

The concept of hydrophobic imbalance and that of hydrophobic and hydrophilic centers are used along with side chain models in the computation of helix orientation and tilt angle in or near a membrane. Rotamer statistics are used to infer typical side chain positions and chain length for each amino acid, and the results are used in fast computation of helix orientation. Sliding windows are used to compute local tilt angles on long alpha-helices that defy idealized modeling and generate tilt angle profiles. Seven different procedures based on different formulas and hydrophobicity scales are used for comparison. These procedures generated very similar tilt angle profiles. These profiles provide insights into helix deformation, membrane destabilization, and similarity and differences between membrane proteins.

Algorithms↗