Probing for preferential interactions among sphingolipids in bilayer vesicles using the glycolipid transfer protein.

We have investigated the intervesicular transfer of galactosylceramide between unilamellar bilayer vesicles composed of differing sphingomyelin and phosphatidylcholine molar ratios. To monitor glycolipid transfer from donor to acceptor vesicles, we used a fluorescence resonance energy transfer assay involving anthrylvinyl-labeled galactosylceramide (AV-GalCer) and perylenoyl-labeled triglyceride. The transfer was mediated by glycolipid transfer protein (GLTP), purified from bovine brain and specific for glycolipids. The initial transfer rate and the total accessible pool of glycolipid in the donor vesicles were both measured. An increase in the sphingomyelin content of 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) vesicles decreased the transfer rate in a nonlinear fashion. Decreased transfer rates were clearly evident at sphingomyelin mole fractions of 0.22 or higher. The pool of AV-GalCer available for GLTP-mediated transfer also was smaller in vesicles containing high sphingomyelin content. In contrast, AV-GalCer was more readily transferred from vesicles composed of POPC and different disaturated phosphatidylcholines. Our results show that GLTP acts as a sensitive probe for detecting interactions of glycosphingolipids with neighboring lipids and that the lateral mixing of glycolipids is probably affected by the matrix lipid composition. The compositionally driven changes in lipid interactions, sensed by GLTP, occur in membranes that are either macroscopically fluid-phase or gel/fluid-phase mixtures. Gaining insights into how changes in membrane sphingolipid composition alter accessibility to soluble proteins with affinity for membrane glycolipids is likely to help increase our understanding of how sphingolipid-enriched microdomains (i.e., "rafts" and caveolae) are formed and maintained in cells.     

Protein mediated glycolipid transfer is inhibited FROM sphingomyelin membranes but enhanced TO sphingomyelin containing raft like membranes

The mammalian glycolipid transfer protein, GLTP, catalyzes the transfer in vitro of glycolipids between membranes. In this study we have examined on one hand the effect of the variations in the donor vesicle composition and on the other hand the effects of variations in the acceptor vesicle composition on the GLTP-catalyzed transfer kinetics of galactosylceramide between bilayer vesicles. For this purpose a resonance energy transfer assay was used, the energy donor being anthrylvinyl-galactosylceramide and the energy acceptor DiO-C16. First, we show that the transfer of anthrylvinyl-galactosylceramide from palmitoyl-oleoyl-phosphatidylcholine donor vesicles was faster than from dipalmitoyl-phosphatidylcholine vesicles, and that there is no transfer from palmitoyl-sphingomyelin vesicles regardless of the cholesterol amount. In this setup the acceptor vesicles were always 100% palmitoyl-oleoyl-phosphatidylcholine. We also showed that the transfer in general is faster from small highly curved vesicles compared to that from larger vesicles. Secondly, by varying the acceptor vesicle composition we showed that the transfer is faster to mixtures of sphingomyelin and cholesterol compared to mixtures of phosphatidylcholines and cholesterol. Based on these experiments we conclude that the GLTP mediated transfer of anthrylvinyl-galactosylceramide is sensitive to the matrix lipid composition and membrane bending. We postulate that a tightly packed membrane environment is most effective in preventing GLTP from accessing its substrates, and cholesterol is not required to protect the glycosphingolipid in the membrane from being transferred by GLTP. On the other hand GLTP can more easily transfer glycolipids to 'lipid raft' like membranes, suggesting that the protein could be involved in raft assembly.     

Protein mediated glycolipid transfer is inhibited FROM sphingomyelin membranes but enhanced TO sphingomyelin containing raft like membranes

The mammalian glycolipid transfer protein, GLTP, catalyzes the transfer in vitro of glycolipids between membranes. In this study we have examined on one hand the effect of the variations in the donor vesicle composition and on the other hand the effects of variations in the acceptor vesicle composition on the GLTP-catalyzed transfer kinetics of galactosylceramide between bilayer vesicles. For this purpose a resonance energy transfer assay was used, the energy donor being anthrylvinyl-galactosylceramide and the energy acceptor DiO-C₁₆. First, we show that the transfer of anthrylvinyl-galactosylceramide from palmitoyl-oleoyl-phosphatidylcholine donor vesicles was faster than from dipalmitoyl-phosphatidylcholine vesicles, and that there is no transfer from palmitoyl-sphingomyelin vesicles regardless of the cholesterol amount. In this setup the acceptor vesicles were always 100% palmitoyl-oleoyl-phosphatidylcholine. We also showed that the transfer in general is faster from small highly curved vesicles compared to that from larger vesicles. Secondly, by varying the acceptor vesicle composition we showed that the transfer is faster to mixtures of sphingomyelin and cholesterol compared to mixtures of phosphatidylcholines and cholesterol. Based on these experiments we conclude that the GLTP mediated transfer of anthrylvinyl-galactosylceramide is sensitive to the matrix lipid composition and membrane bending. We postulate that a tightly packed membrane environment is most effective in preventing GLTP from accessing its substrates, and cholesterol is not required to protect the glycosphingolipid in the membrane from being transferred by GLTP. On the other hand GLTP can more easily transfer glycolipids to ‘lipid raft’ like membranes, suggesting that the protein could be involved in raft assembly.     

GLTP Mediated Non-Vesicular GM1 Transport between Native Membranes

Lipid transfer proteins (LTPs) are emerging as key players in lipid homeostasis by mediating non-vesicular transport steps between two membrane surfaces. Little is known about the driving force that governs the direction of transport in cells. Using the soluble LTP glycolipid transfer protein (GLTP), we examined GM1 (monosialotetrahexosyl-ganglioside) transfer to native membrane surfaces. With artificial GM1 donor liposomes, GLTP can be used to increase glycolipid levels over natural levels in either side of the membrane leaflet, i.e., external or cytosolic. In a system with native donor- and acceptor-membranes, we find that GLTP balances highly variable GM1 concentrations in a population of membranes from one cell type, and in addition, transfers lipids between membranes from different cell types. Glycolipid transport is highly efficient, independent of cofactors, solely driven by the chemical potential of GM1 and not discriminating between the extra- and intracellular membrane leaflet. We conclude that GLTP mediated non-vesicular lipid trafficking between native membranes is driven by simple thermodynamic principles and that for intracellular transport less than 1 µM GLTP would be required in the cytosol. Furthermore, the data demonstrates the suitability of GLTP as a tool for artificially increasing glycolipid levels in cellular membranes.     

Human glycolipid transfer protein--intracellular localization and effects on the sphingolipid synthesis

Glycolipid transfer proteins (GLTPs) are small proteins that specifically transfer glycolipids from one bilayer membrane to another in vitro. However, the precise biological function is still unknown. In this study the intracellular distribution of GLTP was determined. We have used several independent methods, including differential and discontinuous density gradient centrifugation, plasma membrane permeabilization and confocal microscopy imaging, and we demonstrate that GLTP has a cytosolic location. The GLTP is not located in the Golgi apparatus, endoplasmic reticulum, nucleus, lysosomes, mitochondria or peroxisomes in HeLa cells. We have also used a fluorescence resonance energy transfer assay to detect transfer of fluorescently labeled BODIPY-glucosylceramide in the cytosolic fraction from both wild-type and GLTP-overexpressing HeLa cells. Furthermore, we have studied de novo sphingolipid changes in cells overexpressing GLTP using sphinganine metabolic labeling. The results show a significant increase in the synthesis of glucosylceramide (GlcCer) and a decrease in the sphingomyelin (SM) synthesis. However, no changes were detected in the de novo sphingolipid synthesis in GLTP-knockdown cells compared to control cells. We propose that GLTP is not likely involved in the de novo synthesis of glycosphingolipids, but could rather have a role as a glycolipid sensor for the cellular levels of glucosylceramide.     

Glycolipid transfer proteins

Glycolipid transfer proteins (GLTPs) are small (24 kDa), soluble, ubiquitous proteins characterized by their ability to accelerate the intermembrane transfer of glycolipids in vitro. GLTP specificity encompasses both sphingoid- and glycerol-based glycolipids, but with a strict requirement that the initial sugar residue be beta-linked to the hydrophobic lipid backbone. The 3D architecture of GLTP reveals liganded structures with unique lipid-binding modes. The biochemical properties of GLTP action at the membrane surface have been studied rather comprehensively, but the biological role of GLTP remains enigmatic. What is clear is that GLTP differs distinctly from other known glycolipid-binding proteins, such as nonspecific lipid transfer proteins, lysosomal sphingolipid activator proteins, lectins, lung surfactant proteins as well as other lipid-binding/transfer proteins. Based on the unique conformational architecture that targets GLTP to membranes and enables glycolipid binding, GLTP is now considered the prototypical and founding member of a new protein superfamily in eukaryotes.     

Human glycolipid transfer protein: probing conformation using fluorescence spectroscopy

Glycolipid transfer protein (GLTP) is a soluble 24 kDa protein that selectively accelerates the intermembrane transfer of glycolipids in vitro. Little is known about the GLTP structure and dynamics. Here, we report the cloning of human GLTP and characterize the environment of the three tryptophans (Trps) of the protein using fluorescence spectroscopy. Excitation at 295 nm yielded an emission maximum (lambda(max)) near 347 nm, indicating a relatively polar average environment for emitting Trps. Quenching with acrylamide at physiological ionic strength or with potassium iodide resulted in linear Stern-Volmer plots, suggesting accessibility of emitting Trps to soluble quenchers. Insights into reversible conformational changes accompanying changes in GLTP activity were provided by addition and rapid dilution of urea while monitoring changes in Trp or 1-anilinonaphthalene-8-sulfonic acid fluorescence. Incubation of GLTP with glycolipid liposomes caused a blue shift in the Trp emission maximum but diminished the fluorescence intensity. The blue-shifted emission maximum, centered near 335 nm, persisted after separation of glycolipid liposomes from GLTP, consistent with formation of a GLTP-glycolipid complex at a glycolipid-liganding site containing Trp. The results provide the first insights into human GLTP structural dynamics by fluorescence spectroscopy, including global conformational changes that accompany GLTP folding into an active conformational state as well as more subtle conformational changes that play a role in GLTP-mediated transfer of glycolipids between membranes, and establish a foundation for future studies of membrane rafts using GLTP.     

Alternation in the Glycolipid Transfer Protein Expression Causes Changes in the Cellular Lipidome

The glycolipid transfer protein (GLTP) catalyzes the binding and transport of glycolipids, but not phospholipids or neutral lipids. With its all-alpha helical fold, it is the founding member for a new superfamily, however its biological role still remains unclear. We have analyzed changes in the HeLa cell lipidome in response to down- and up-regulation of GLTP expression. We used metabolic labeling and thin layer chromatography analysis, complemented with a lipidomics mass spectroscopic approach. HeLa cells were treated with GLTP siRNA or were transiently overexpressing the GLTP gene. We identified eight different lipid classes that changed as a result of the GLTP down- or up-regulation treatments; glucosylceramide, lactosylceramide, globotriaosylceramide, ceramide, sphingomyelin, cholesterol-esters, diacylglycerol and phosphatidylserine. We discovered that the amount of globotriaosylceramide (Gb₃) was extensively lowered after down-regulation of GLTP. Further, an up-regulation of GLTP caused a substantial increase in both the Gb₃ and glucosylceramide levels compared to the controls. Total galactosylceramide levels remained unchanged. Both lactosylceramide and ceramide showed small changes, an increase with increasing GLTP and a decrease in the HeLa cell GLTP knockdowns. The cholesterol-esters and diacylglycerol masses increased in cells that had upregulated GLTP protein levels, wheras down-regulation did not affect their amounts. For the glycerophospholipids, phosphatidylserine was the only species that was lower in GLTP overexpressing cells. Phosphatidylethanolamine, phosphatidylglyerol and phosphatidylinositol remained unaltered. A total of 142 lipid species were profiled and quantified using shotgun lipidomics analyses. This work provides for the first time insights into how alternations in the levels of a protein that binds and transfers glycolipids affects the cellular lipid metabolism. We discuss the observed changes in the lipidome and how these relate to GLTP. We suggest, that GLTP not only could be a significant player in cellular sphingolipid metabolism, but also could have a much broader role in the overall lipid metabolism.     

GLTP-fold interaction with planar phosphatidylcholine surfaces is synergistically stimulated by phosphatidic acid and phosphatidylethanolamine

Among amphitropic proteins, human glycolipid transfer protein (GLTP) forms a structurally-unique fold that translocates on/off membranes to specifically transfer glycolipids. Phosphatidylcholine (PC) bilayers with curvature-induced packing stress stimulate much faster glycolipid intervesicular transfer than nonstressed PC bilayers raising questions about planar cytosol-facing biomembranes being viable sites for GLTP interaction. Herein, GLTP-mediated desorption kinetics of fluorescent glycolipid (tetramethyl-boron dipyrromethene (BODIPY)-label) from lipid monolayers are assessed using a novel microfluidics-based surface balance that monitors lipid lateral packing while simultaneously acquiring surface fluorescence data. At biomembrane-like packing (30–35 mN/m), GLTP uptake of BODIPY-glycolipid from POPC monolayers was nearly nonexistent but could be induced by reducing surface pressure to mirror packing in curvature-stressed bilayers. In contrast, 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) matrices supported robust BODIPY-glycolipid uptake by GLTP at both high and low surface pressures. Unexpectedly, negatively-charged cytosol-facing lipids, i.e., phosphatidic acid and phosphatidylserine, also supported BODIPY-glycolipid uptake by GLTP at high surface pressure. Remarkably, including both 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (5 mol%) and POPE (15 mol%) in POPC synergistically activated GLTP at high surface pressure. Our study shows that matrix lipid headgroup composition, rather than molecular packing per se, is a key regulator of GLTP-fold function while demonstrating the novel capabilities of the microfluidics-based film balance for investigating protein-membrane interfacial interactions.     

Point mutational analysis of the liganding site in human glycolipid transfer protein. Functionality of the complex

Mammalian glycolipid transfer proteins (GLTPs) facilitate the selective transfer of glycolipids between lipid vesicles in vitro. Recent structural determinations of the apo- and glycolipid-liganded forms of human GLTP have provided the first insights into the molecular architecture of the protein and its glycolipid binding site (Malinina, L., Malakhova, M. L., Brown, R. E., and Patel, D. J. (2004) Nature 430, 1048-1053). In the present study, we have evaluated the functional consequences of point mutation of the glycolipid liganding site of human GLTP within the context of a carrier-based mechanism of glycolipid intermembrane transfer. Different approaches were developed to rapidly and efficiently assess the uptake and release of glycolipid by GLTP. They included the use of glass-immobilized, glycolipid films to load GLTP with glycolipid and separation of GLTP/glycolipid complexes from vesicles containing glycolipid (galactosylceramide or lactosylceramide) or from monosialoganglioside dispersions by employing nickel-nitrilotriacetic acid-based affinity or gel filtration strategies. Point mutants of the sugar headgroup recognition center (Trp-96, Asp-48, Asn-52) and of the ceramide-accommodating hydrophobic tunnel (Phe-148, Phe-183, Leu-136) were analyzed for their ability to acquire and release glycolipid ligand. Two manifestations of point mutation within the liganding site were apparent: (i) impaired formation of the GLTP/glycolipid complex; (ii) impaired acquisition and release of bound glycolipid by GLTP. The results are consistent with a carrier-based mode of GLTP action to accomplish the intermembrane transfer of glycolipid. Also noteworthy was the inefficient release of glycolipid by wtGLTP into phosphatidylcholine acceptor vesicles, raising the possibility of a function other than intermembrane glycolipid transfer in vivo.     

The glycolipid transfer protein interacts with the vesicle-associated membrane protein-associated protein VAP-A

The glycolipid transfer protein (GLTP) is a cytoplasmic protein with an ability to bind glycolipids and catalyze their in vitro transfer. In this study, we have found a FFAT-like motif in GLTP. The FFAT (two phenylalanines in an acidic tract) motif in lipid-binding proteins has previously been shown to interact with the VAPs (vesicle-associated membrane protein-associated proteins) in the endoplasmic reticulum. Here we used glutathione S-transferase pull-down experiments to confirm that GLTP and VAP-A interact. By displacing different amino acids in the motif we clearly show that the interaction is dependent on the FFAT-like motif in GLTP. The potential role of GLTP in the endoplasmic reticulum association is discussed.     

Glycolipid transfer protein interaction with bilayer vesicles: modulation by changing lipid composition

Glycosphingolipids (GSLs) are important constituents of lipid rafts and caveolae, are essential for the normal development of cells, and are adhesion sites for various infectious agents. One strategy for modulating GSL composition in lipid rafts is to selectively transfer GSL to or from these putative membrane microdomains. Glycolipid transfer protein (GLTP) catalyzes selective intermembrane transfer of GSLs. To enable effective use of GLTP as a tool to modify the glycolipid content of membranes, it is imperative to understand how the membrane regulates GLTP action. In this study, GLTP partitioning to membranes was analyzed by monitoring the fluorescence resonance energy transfer from tryptophans and tyrosines of GLTP to N-(5-dimethyl-aminonaphthalene-1-sulfonyl)-1,2-dihexadecanoyl-sn-glycero-3-phospho-ethanolamine present in bilayer vesicles. GLTP partitioned to POPC vesicles even when no GSL was present. GLTP interaction with model membranes was nonpenetrating, as assessed by protein-induced changes in lipid monolayer surface pressure, and nonperturbing in that neither membrane fluidity nor order were affected, as monitored by anisotropy of 1,6-diphenyl-1,3,5-hexatriene and 6-dodecanoyl-N,N-dimethyl-2-naphthylamine, even though the tryptophan anisotropy of GLTP increased in the presence of vesicles. Ionic strength, vesicle packing, and vesicle lipid composition affected GLTP partitioning to the membrane and led to the following conclusion: Conditions that increase the ratio of bound/unbound GLTP do not guarantee increased transfer activity, but conditions that decrease the ratio of bound/unbound GLTP always diminish transfer. A model of GLTP interaction with the membrane, based on the partitioning equilibrium data and consistent with the kinetics of GSL transfer, is presented and solved mathematically.     

Glycolipid Acquisition by Human Glycolipid Transfer Protein Dramatically Alters Intrinsic Tryptophan Fluorescence: INSIGHTS INTO GLYCOLIPID BINDING AFFINITY*S?

Glycolipid transfer proteins (GLTPs) are small, soluble proteins that selectively accelerate the intermembrane transfer of glycolipids. The GLTP fold is conformationally unique among lipid binding/transfer proteins and serves as the prototype and founding member of the new GLTP superfamily. In the present study, changes in human GLTP tryptophan fluorescence, induced by membrane vesicles containing glycolipid, are shown to reflect glycolipid binding when vesicle concentrations are low. Characterization of the glycolipid-induced “signature response,” i.e. ∼40% decrease in Trp intensity and ∼12-nm blue shift in emission wavelength maximum, involved various modes of glycolipid presentation, i.e. microinjection/dilution of lipid-ethanol solutions or phosphatidylcholine vesicles, prepared by sonication or extrusion and containing embedded glycolipids. High resolution x-ray structures of apo- and holo-GLTP indicate that major conformational alterations are not responsible for the glycolipid-induced GLTP signature response. Instead, glycolipid binding alters the local environment of Trp-96, which accounts for ∼70% of total emission intensity of three Trp residues in GLTP and provides a stacking platform that aids formation of a hydrogen bond network with the ceramide-linked sugar of the glycolipid headgroup. The changes in Trp signal were used to quantitatively assess human GLTP binding affinity for various lipids including glycolipids containing different sugar headgroups and homogenous acyl chains. The presence of the glycolipid acyl chain and at least one sugar were essential for achieving a low-to-submicromolar dissociation constant that was only slightly altered by increased sugar headgroup complexity.Glycolipid transfer protein (GLTP)⁴ is a soluble (∼24-kDa) protein that selectively transfers glycosphingolipids (GSLs) between membranes. GSLs play key roles in cell recognition, adhesion, differentiation, proliferation, and programmed death in normal and disease states (1–8). Phylogenetic/evolutionary analyses show GLTP to be highly conserved among vertebrates (9–11). The conformational uniqueness of the GLTP fold when compared with other lipid binding/transfer proteins (12–14) has resulted in GLTP being designated the prototype and founding member of the GLTP superfamily (15, 16). GLTP employs a novel two-layer “sandwich motif,” dominated by α-helices and achieved without intramolecular disulfide bridges, to accommodate glycolipid within a single lipid binding site and to form a membrane-interaction domain that differs from other known membrane targeting/translocation domains, i.e. C1, C2, PH, PX, and FYVE (9, 13, 17–21). The glycolipid binding site of GLTP consists of a sugar headgroup recognition center that anchors the ceramide-linked sugar to the protein surface via multiple hydrogen bonds and a hydrophobic tunnel that accommodates the hydrocarbon chains of ceramide. The crystal structures of glycolipid-free GLTP and of GLTP complexed with a half-dozen glycolipids differing in sugar headgroup and/or lipid acyl composition reveal the basis for specific recognition and adaptive accommodation of various GSLs. A conserved, concerted sequence of events, initiated by anchoring of the GSL headgroup to the sugar headgroup recognition center, seems to facilitate entry and exit of the lipid chains in the membrane-associated state (13). Glycolipid uptake occurs via a cleft-like gating mechanism involving conformational changes to one α-helix and two interhelical loops (12). The selectivity of GLTP for glycolipids makes this protein a prime candidate for molecular manipulation of GSL-enriched microdomains in membranes as well as a potential vehicle for selectively delivering glycolipids to cells. However, the binding affinity of various glycolipids for GLTP and the time frame of GSL uptake by GLTP remain unclear. In the present study, these issues are investigated using fluorescence approaches.GLTP is intrinsically fluorescent by virtue of having 3 Trp and 10 Tyr residues among its 209 amino acids. All 3 Trp residues reside on or near the surface of GLTP (12–14, 17, 22, 23), where they could help form a membrane-interaction site. Only one, Trp-96, is directly involved in glycolipid binding (12–14). Given the likely roles in membrane interaction and GSL binding, our goal was to define the relative contributions of the Trp fluorescence changes caused by membrane interaction versus glycolipid binding. A signature Trp emission response, indicative of GSL binding by WT-GLTP, has been identified and characterized using select GLTP point mutants and different modes of glycolipid presentation, i.e. ethanol injection of pure GSLs and titration with membrane vesicles (LUVs and SUVs) containing GSLs as minor components. The signature Trp emission response has been used to comprehensively assess the glycolipid binding affinity of the novel GLTP fold for the first time, focusing on the impact of compositional variation of the sugar headgroup and nonpolar acyl chain moieties of the glycolipid.     

Membrane interaction and activity of the glycolipid transfer protein

In this study we have addressed the ability of the glycolipid transfer protein (GLTP) to transfer anthrylvinyl-galactosylceramide at different pH and sodium chloride concentrations, and the ability of three different mutants to transfer the fluorescently labeled galactosylceramide between donor and acceptor model membranes. We constructed single tryptophan mutants with site-directed mutagenesis where two of the three tryptophan (W) of wild-type human GLTP were substituted with phenylalanine (F) and named W85 GLTP (W96F and W142F), W96 GLTP (W85F and W142F) and W142 GLTP (W85F and W96F) accordingly. Wild-type GLTP and W96 GLTP were both able to transfer anthrylvinyl-galactosylceramide, but the two variants W85 GLTP and W142 GLTP did not show any glycolipid transfer activity, indicating that the tryptophan in position 96 is crucial for transfer activity. Tryptophan fluorescence emission showed a blue shift of the maximal emission wavelength upon interaction of glycolipid containing vesicle with wild-type GLTP and W96 GLTP, while no blue shift was recorded for the protein variants W85 GLTP and W142 GLTP. The quantum yield of tryptophan emission was highest for the W96 GLTP protein whereas W85 GLTP, W142 GLTP and wild-type GLTP showed a lower and almost similar quantum yield. The lifetime and anisotropy decay of the different tryptophan mutants also changed upon binding to vesicles containing galactosylceramide. Again wild-type GLTP and W96 GLTP showed similar behavior in the presence of vesicles containing glycolipids. Taken together, our data show that the W96 is involved not only in the activity of the protein but also in the interaction between the protein and glycolipid containing membranes.     

Membrane interaction and activity of the glycolipid transfer protein

In this study we have addressed the ability of the glycolipid transfer protein (GLTP) to transfer anthrylvinyl-galactosylceramide at different pH and sodium chloride concentrations, and the ability of three different mutants to transfer the fluorescently labeled galactosylceramide between donor and acceptor model membranes. We constructed single tryptophan mutants with site-directed mutagenesis where two of the three tryptophan (W) of wild-type human GLTP were substituted with phenylalanine (F) and named W85 GLTP (W96F and W142F), W96 GLTP (W85F and W142F) and W142 GLTP (W85F and W96F) accordingly. Wild-type GLTP and W96 GLTP were both able to transfer anthrylvinyl-galactosylceramide, but the two variants W85 GLTP and W142 GLTP did not show any glycolipid transfer activity, indicating that the tryptophan in position 96 is crucial for transfer activity. Tryptophan fluorescence emission showed a blue shift of the maximal emission wavelength upon interaction of glycolipid containing vesicle with wild-type GLTP and W96 GLTP, while no blue shift was recorded for the protein variants W85 GLTP and W142 GLTP. The quantum yield of tryptophan emission was highest for the W96 GLTP protein whereas W85 GLTP, W142 GLTP and wild-type GLTP showed a lower and almost similar quantum yield. The lifetime and anisotropy decay of the different tryptophan mutants also changed upon binding to vesicles containing galactosylceramide. Again wild-type GLTP and W96 GLTP showed similar behavior in the presence of vesicles containing glycolipids. Taken together, our data show that the W96 is involved not only in the activity of the protein but also in the interaction between the protein and glycolipid containing membranes.     

Molecular features of phospholipids that affect glycolipid transfer protein-mediated galactosylceramide transfer between vesicles

The glycolipid transfer protein (GLTP)-mediated movement of galactosylceramide from model membrane donor vesicles to acceptor vesicles is sensitive to the membrane environment surrounding the glycolipid. GLTP can catalyze the transfer of a fluorescently labeled GSL, anthrylvinyl-galactosylceramide (AV-GalCer), from vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and dipalmitoylphosphatidylcholine matrices, but not from vesicles prepared from N-palmitoylsphingomyelin, regardless of the cholesterol content of the vesicles. In this study, we have examined the structural features of sphingomyelin (SM) that are responsible for its inhibition of the rate of GLTP-catalyzed transfer of AV-GalCer. The rate of glycolipid transfer was enhanced when the N-palmitoyl chain of SM was replaced with an N-oleoyl chain. Analogs of N-palmitoyl-SM in which the 4,5-double bond of the long-chain base is reduced or the 3-hydroxy group is removed did not inhibit GLTP-catalyzed transfer of AV-GalCer. When the donor vesicles were prepared with phosphatidylcholines or ether-linked phosphatidylcholine analogs, the transfer rates of AV-GalCer increased with increasing degree of unsaturation. The rate of AV-GalCer transfer was strongly dependent on the unsaturation degree of the acyl and/or alkyl chains. For ester-linked PCs, the transfer rate increased in the order DPPC相似文献   

Human glycolipid transfer protein (GLTP) expression modulates cell shape

Glycolipid transfer protein (GLTP) accelerates glycosphingolipid (GSL) intermembrane transfer via a unique lipid transfer/binding fold (GLTP-fold) that defines the GLTP superfamily and is the prototype for GLTP-like domains in larger proteins, i.e. phosphoinositol 4-phosphate adaptor protein-2 (FAPP2). Although GLTP-folds are known to play roles in the nonvesicular intracellular trafficking of glycolipids, their ability to alter cell phenotype remains unexplored. In the present study, overexpression of human glycolipid transfer protein (GLTP) was found to dramatically alter cell phenotype, with cells becoming round between 24 and 48 h after transfection. By 48 h post transfection, ～70% conversion to the markedly round shape was evident in HeLa and HEK-293 cells, but not in A549 cells. In contrast, overexpression of W96A-GLTP, a liganding-site point mutant with abrogated ability to transfer glycolipid, did not alter cell shape. The round adherent cells exhibited diminished motility in wound healing assays and an inability to endocytose cholera toxin but remained viable and showed little increase in apoptosis as assessed by poly(ADP-ribose) polymerase cleavage. A round cell phenotype also was induced by overexpression of FAPP2, which binds/transfers glycolipid via its C-terminal GLTP-like fold, but not by a plant GLTP ortholog (ACD11), which is incapable of glycolipid binding/transfer. Screening for human protein partners of GLTP by yeast two hybrid screening and by immuno-pulldown analyses revealed regulation of the GLTP-induced cell rounding response by interaction with δ-catenin. Remarkably, while δ-catenin overexpression alone induced dendritic outgrowths, coexpression of GLTP along with δ-catenin accelerated transition to the rounded phenotype. The findings represent the first known phenotypic changes triggered by GLTP overexpression and regulated by direct interaction with a p120-catenin protein family member.     

Structural evidence for adaptive ligand binding of glycolipid transfer protein

Glycolipids participate in many important cellular processes and they are bound and transferred with high specificity by glycolipid transfer protein (GLTP). We have solved three different X-ray structures of bovine GLTP at 1.4 angstroms, 1.6 angstroms and 1.8 angstroms resolution, all with a bound fatty acid or glycolipid. The 1.4 angstroms structure resembles the recently characterized apo-form of the human GLTP but the other two structures represent an intermediate conformation of the apo-GLTPs and the human lactosylceramide-bound GLTP structure. These novel structures give insight into the mechanism of lipid binding and how GLTP may conformationally adapt to different lipids. Furthermore, based on the structural comparison of the GLTP structures and the three-dimensional models of the related Podospora anserina HET-C2 and Arabidopsis thaliana accelerated cell death protein, ACD11, we give structural explanations for their specific lipid binding properties.     

Molecular features of phospholipids that affect glycolipid transfer protein-mediated galactosylceramide transfer between vesicles

The glycolipid transfer protein (GLTP)-mediated movement of galactosylceramide from model membrane donor vesicles to acceptor vesicles is sensitive to the membrane environment surrounding the glycolipid. GLTP can catalyze the transfer of a fluorescently labeled GSL, anthrylvinyl-galactosylceramide (AV-GalCer), from vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and dipalmitoylphosphatidylcholine matrices, but not from vesicles prepared from N-palmitoylsphingomyelin, regardless of the cholesterol content of the vesicles. In this study, we have examined the structural features of sphingomyelin (SM) that are responsible for its inhibition of the rate of GLTP-catalyzed transfer of AV-GalCer. The rate of glycolipid transfer was enhanced when the N-palmitoyl chain of SM was replaced with an N-oleoyl chain. Analogs of N-palmitoyl-SM in which the 4,5-double bond of the long-chain base is reduced or the 3-hydroxy group is removed did not inhibit GLTP-catalyzed transfer of AV-GalCer. When the donor vesicles were prepared with phosphatidylcholines or ether-linked phosphatidylcholine analogs, the transfer rates of AV-GalCer increased with increasing degree of unsaturation. The rate of AV-GalCer transfer was strongly dependent on the unsaturation degree of the acyl and/or alkyl chains. For ester-linked PCs, the transfer rate increased in the order DPPC < POPC < DOPC, which have 0, 1, and 2 cis double bonds, respectively.     

Glycolipid Transfer Protein Expression Is Affected by Glycosphingolipid Synthesis

Members of the glycolipid transfer protein superfamily (GLTP) are found from animals and fungi to plants and red micro-alga. Eukaryotes that encode the glucosylceramide synthase responsible for the synthesis of glucosylceramide, the precursor for most glycosphingolipids, also produce GLTPs. Cells that does not synthesize glucosylceramide neither express GLTPs. Based on this genetic relationship there must be a strong correlation between the synthesis of glucosylceramide and GLTPs. To regulate the levels of glycolipids we have used inhibitors of intracellular trafficking, glycosphingolipid synthesis and degradation, and small interfering RNA to down-regulate the activity of glucosylceramide synthase activity. We found that GLTP expression, both at the mRNA and protein levels, is elevated in cells that accumulate glucosylceramide. Monensin and brefeldin A block intracellular vesicular transport mechanisms. Brefeldin A treatment leads to accumulation of newly synthesized glucosylceramide, galactosylceramide and lactosylceramide in a fused endoplasmic reticulum-Golgi complex. On the other hand, inhibiting glycosphingolipid degradation with conduritol-B-epoxide, that generates glucosylceramide accumulation in the lysosomes, did not affect the levels of GLTP. However, glycosphingolipid synthesis inhibitors like PDMP, NB-DNJ and myriocin, all decreased glucosylceramide and GLTP below normal levels. We also found that an 80% loss of glucosylceramide due to glucosylceramide synthase knockdown resulted in a significant reduction in the expression of GLTP. We show here that interfering with membrane trafficking events and simple neutral glycosphingolipid synthesis will affect the expression of GLTP. We postulate that a change in the glucosylceramide balance causes a response in the GLTP expression, and put forward that GLTP might play a role in lipid directing and sensing of glucosylceramide at the ER-Golgi interface.