Glycolipid Transfer Protein Research Articles

Identification of a glycosphingolipid transfer protein GLTP1 in Arabidopsis thaliana

Arabidopsis thaliana At2g33470 encodes a glycolipid transfer protein (GLTP) that enhances the intervesicular trafficking of glycosphingolipids in vitro. GLTPs have previously been identified in animals and fungi but not in plants. Thus, At2g33470 is the first identified plant GLTP and we have designated it AtGTLP1. AtGLTP1 transferred BODIPY-glucosylceramide at a rate of 0.7 pmol x s(-1), but BODIPY-galactosylceramide and BODIPY-lactosylceramide were transferred slowly, with rates below 0.1 pmol x s(-1). AtGLTP1 did not transfer BODIPY-sphingomyelin, monogalactosyldiacylglycerol or digalactosyldiacylglycerol. The human GLTP transfers BODIPY-glucosylceramide, BODIPY-galactosylceramide and BODIPY-lactosylceramide with rates greater than 0.8 pmol.s(-1). Structural models showed that the residues that are most critical for glycosphingolipid binding in human GLTP are conserved in AtGLTP1, but some of the sugar-binding residues are unique, and this provides an explanation for the distinctly different transfer preferences of AtGLTP1 and human GLTP. The AtGLTP1 variant Arg59Lys/Asn95Leu showed low BODIPY-glucosylceramide transfer activity, indicating that Arg59 and/or Asn95 are important for the specific binding of glucosylceramide to AtGLTP1. We also show that, in A. thaliana, AtGLTP1 together with At1g21360 and At3g21260 constitute a small gene family orthologous to the mammalian GLTPs. However, At1g21360 and At3g21260 did not transfer any of the tested lipids in vitro.

Human glycolipid transfer protein (GLTP) genes: organization, transcriptional status and evolution

BackgroundGlycolipid transfer protein is the prototypical and founding member of the new GLTP superfamily distinguished by a novel conformational fold and glycolipid binding motif. The present investigation provides the first insights into the organization, transcriptional status, phylogenetic/evolutionary relationships of GLTP genes.ResultsIn human cells, single-copy GLTP genes were found in chromosomes 11 and 12. The gene at locus 11p15.1 exhibited several features of a potentially active retrogene, including a highly homologous (~94%), full-length coding sequence containing all key amino acid residues involved in glycolipid liganding. To establish the transcriptional activity of each human GLTP gene, in silico EST evaluations, RT-PCR amplifications of GLTP transcript(s), and methylation analyses of regulator CpG islands were performed using various human cells. Active transcription was found for 12q24.11 GLTP but 11p15.1 GLTP was transcriptionally silent. Heterologous expression and purification of the GLTP paralogs showed glycolipid intermembrane transfer activity only for 12q24.11 GLTP. Phylogenetic/evolutionary analyses indicated that the 5-exon/4-intron organizational pattern and encoded sequence of 12q24.11 GLTP were highly conserved in therian mammals and other vertebrates. Orthologs of the intronless GLTP gene were observed in primates but not in rodentiates, carnivorates, cetartiodactylates, or didelphimorphiates, consistent with recent evolutionary development.ConclusionThe results identify and characterize the gene responsible for GLTP expression in humans and provide the first evidence for the existence of a GLTP pseudogene, while demonstrating the rigorous approach needed to unequivocally distinguish transcriptionally-active retrogenes from silent pseudogenes. The results also rectify errors in the Ensembl database regarding the organizational structure of the actively transcribed GLTP gene in Pan troglodytes and establish the intronless GLTP as a primate-specific, processed pseudogene marker. A solid foundation has been established for future identification of hereditary defects in human GLTP genes.

Glycolipid transfer protein: Clear structure and activity, but enigmatic function

Glycosphingolipids comprize a small (typically 5–10% by weight) but vital fraction of membrane lipids in eukaryotes (Holthuis et al., 2001). They provide the plasma membrane with chemical and mechanical stabilities and take part in fundamental biological processes including differentiation, cell–cell interaction, and transmembrane signaling. For these lipids, as for most lipids in general, local metabolism and selective transport are important determinants (Sprong et al., 2001). Little is known about these processes, but it is clear that several key players in the organization and control of sphingolipid composition remain to be identified. A protein purified from bovine spleen cytosol specifically transferring glycolipids was already described more than 20 years ago ([Metz and Radin, 1980] and [Radin and Metz, 1982]). An absolute specificity was found for glycolipids containing a β-linked sugar to the hydrophobic backbone (Yamada et al., 1986). Glycolipid transfer proteins (GLTPs, EC number not assigned) are water-soluble proteins of average size of 24 kDa that have been studied extensively in vitro ([Abe and Sasaki, 1985], [Abe et al., 1982], [Brown et al., 1990], [Gammon et al., 1987] and [Metz and Radin, 1982]). Very recently, their structural and in vitro functional properties were reviewed in detail (Brown and Mattjus, 2007). These proteins have been isolated from different sources ranging from spinach chloroplasts to mammalian brain, liver and kidney ([Brown et al., 1990], [Sasaki, 1985] and [Sasaki, 1990]). GLTP orthologs in fungi and plants were found to be involved in programmed cell death ([Brodersen et al., 2002], [Mattjus et al., 2003] and [Saupe et al., 1994]) but their function in mammals is largely unknown. The activity of GLTP is preserved after expression in E. coli ([Godi et al., 2004], Lin et al., 2000 X. Lin, P. Mattjus, H.M. Pike, A.J. Windebank and R.E. Brown, Cloning and expression of glycolipid transfer protein from bovine and porcine brain, J Biol Chem 275 (2000), pp. 5104–5110. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (32)[Lin et al., 2000], [Malinina et al., 2004] and [Rao et al., 2004]) allowing for detailed structure–function studies in vitro.

Membrane Curvature Effects on Glycolipid Transfer Protein Activity

The glycolipid transfer protein (GLTP) is monomeric in aqueous solutions, and it binds weakly to membrane interfaces with or without glycolipids. GLTP is a surface-active protein and adsorbs to exert a maximal surface pressure value of 19 mN/m. The change in surface pressure following GLTP adsorption decreased linearly with initial surface pressure. The exclusion pressure for different phospholipids and sphingolipids was between 23 and 31 mN/m, being clearly highest for the negatively charged dipalmitoyl-phosphatidylserine. This can be explained by electrostatic forces when GLTP is positively charged at neutral pH (isoelectric point = 9.0) and by phosphatidylserine being negatively charged. If GLTP is injected under a palmitoyl-galactosylceramide monolayer above 30 mN/m, the presence of GLTP leads to a decrease in the surface pressure as a function of time. This suggests that GLTP is able to remove glycolipids from the monolayer without penetrating the monolayer. On the other hand, if phospholipid vesicles with or without glycolipids are also present in the subphase, no change in the surface pressure takes place. This suggests that GLTP in the presence of curved membranes is not able to transfer from or to planar membranes. We also show that transfer of fluorescently labeled galactosylceramide is faster from small highly curved palmitoyl-oleoyl-phosphatidylcholine and dipalmitoyl-phosphatidylcholine bilayer vesicles but not from palmitoyl-sphingomyelin vesicles regardless of the size.

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.

The Fungus-Specific HET Domain Mediates Programmed Cell Death in Podospora anserina

Vegetative incompatibility is a programmed cell death reaction that occurs when fungal cells of unlike genotypes fuse. Genes defining vegetative incompatibility (het genes) are highly polymorphic, and most if not all incompatibility systems include a protein partner bearing the fungus-specific domain termed the HET domain. The nonallelic het-C/het-E incompatibility system is the best-characterized incompatibility system in Podospora anserina. Cell death is triggered by interaction of specific alleles of het-C, encoding a glycolipid transfer protein, and het-E, encoding a HET domain and a WD repeat domain involved in recognition. We show here that overexpression of the isolated HET domain from het-E results in cell death. This cell death is characterized by induction of autophagy, increased vacuolization, septation, and production of lipid droplets, which are hallmarks of cell death by incompatibility. In addition, the HET domain lethality is suppressed by the same mutations as vegetative incompatibility, but not by the inactivation of het-C. These results establish the HET domain as the mediator of cell death by incompatibility and lead to a modular conception of incompatibility systems whereby recognition is ensured by the variable regions of incompatibility proteins and cell death is triggered by the HET domain.

New BODIPY lipid probes for fluorescence studies of membranes

Many fluorescent lipid probes tend to loop back to the membrane interface when attached to a lipid acyl chain rather than embedding deeply into the bilayer. To achieve maximum embedding of BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorophore into the bilayer apolar region, a series of sn-2 acyl-labeled phosphatidylcholines was synthesized bearing 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-yl (Me(4)-BODIPY-8) at the end of C(3)-, C(5)-, C(7)-, or C(9)-acyl. A strategy was used of symmetrically dispersing the methyl groups at BODIPY ring positions 1, 3, 5, and 7 to decrease fluorophore polarity. Iodide quenching of the phosphatidylcholine probes in bilayer vesicles confirmed that the Me(4)-BODIPY-8 fluorophore was embedded in the bilayer. Parallax analysis of Me(4)-BODIPY-8 fluorescence quenching by phosphatidylcholines containing iodide at different positions along the sn-2 acyl chain indicated that the penetration depth of Me(4)-BODIPY-8 into the bilayer was determined by the length of the linking acyl chain. Evaluation using monolayers showed minimal perturbation of <10 mol% probe in fluid-phase and cholesterol-enriched phosphatidylcholine. Spectral characterization in monolayers and bilayers confirmed the retention of many features of other BODIPY derivatives (i.e., absorption and emission wavelength maxima near 498 nm and approximately 506-515 nm) but also showed the absence of the 620-630 nm peak associated with BODIPY dimer fluorescence and the presence of a 570 nm emission shoulder at high Me(4)-BODIPY-8 surface concentrations. We conclude that the new probes should have versatile utility in membrane studies, especially when precise location of the reporter group is needed.

Characterization of the glycolipid transfer protein in HeLa cells

Characterization of the glycolipid transfer protein in HeLa cells

The effect of cell stress and metabolic disorders on the endogenous expression level of glycolipid transfer protein

The effect of cell stress and metabolic disorders on the endogenous expression level of glycolipid transfer protein

The Liganding of Glycolipid Transfer Protein Is Controlled by Glycolipid Acyl Structure

Glycosphingolipids (GSLs) play major roles in cellular growth and development. Mammalian glycolipid transfer proteins (GLTPs) are potential regulators of cell processes mediated by GSLs and display a unique architecture among lipid binding/transfer proteins. The GLTP fold represents a novel membrane targeting/interaction domain among peripheral proteins. Here we report crystal structures of human GLTP bound to GSLs of diverse acyl chain length, unsaturation, and sugar composition. Structural comparisons show a highly conserved anchoring of galactosyl- and lactosyl-amide headgroups by the GLTP recognition center. By contrast, acyl chain chemical structure and occupancy of the hydrophobic tunnel dictate partitioning between sphingosine-in and newly-observed sphingosine-out ligand-binding modes. The structural insights, combined with computed interaction propensity distributions, suggest a concerted sequence of events mediated by GLTP conformational changes during GSL transfer to and/or from membranes, as well as during GSL presentation and/or transfer to other proteins.

Too Long, Too Short, or Just Right: Glycosphingolipid–Protein Binding Varies with Acyl Chain Length

Glycosphingolipids (GSLs) reside in the membranes of all mammalian cells, where they play roles in both structure and signaling. They traffic between the plasma membrane—where most are found—and vesicle membranes within the cell. One of the carriers of GSLs is glycolipid transfer proteins. The interactions between these two molecules have only recently begun to be elucidated. In a new study, Lucy Malinina, Margarita Malakhova, Rhoderick Brown, Dinshaw Patel, and colleagues reveal a highly unusual binding characteristic of the protein: the sphingosine chain of the GSL either buries itself inside the protein or is left outside of it, depending on the length of the acyl chain. Every GSL has three parts: a sugar head and two long hydrocarbon chains (an 18-carbon, nitrogen-containing sphingosine chain, and an “acyl” chain whose length can vary from 16 to 26 carbons). Using x-ray crystallography, the authors recently elucidated the structure of human glycolipid transfer protein, both with and without an attached GSL, and showed that it has a novel protein fold adapted to interacting with membranes and binding with lipids. In that study, which used a GSL containing a lactose sugar and an 18-carbon monounsaturated acyl chain, they found that the sugar binds to the exterior, while the sphingosine and acyl chains lay parallel inside a hydrophobic tunnel made from an interior fold of the protein. To explore how the protein accommodated other GSLs, they varied acyl length and sugar groups and determined the structure of these protein–GSL complexes. To their surprise, they found that when the acyl chain was either longer (24 carbons) or shorter (8 or 12 carbons) than the one in their initial experiment, the sphingosine chain was not included in the tunnel, but instead jutted out away from the surface of the protein. While the effect on sphingosine is the same, the cause appears to be slightly different in the two cases. When the shorter acyl chain sits in the tunnel, it is joined by an extraneous free hydrocarbon, which denies sphingosine an entrance. The exact origin and role of this hydrocarbon is unknown, but it also occupies the tunnel in the unbound protein. In contrast, there is no extraneous hydrocarbon when the longer acyl chain is in the tunnel, but the chain curls around within, apparently blocking out sphingosine with its bulk. When the authors reverted to the 18-carbon acyl chain but introduced an additional chain-kinking double bond, once again sphingosine was excluded, suggesting that its ability to fit depends on both the length and shape of the acyl group. The tunnel itself expands and contracts with the changes in size of the chains within. Unlike the highly variable interactions of tunnel and hydrocarbon chains, the binding of sugar to the protein appears to rely mainly on a small set of invariant attractions, whether from the double sugar, lactose, or from the single sugars, galactose or glucose. In addition, in each case there are conserved hydrogen bond contacts involving an amine and carbonyl (amide linkage) in the GSL ceramide and specific amino acids of the protein, helping to position the GSL hydrocarbons for entry into the tunnel. The binding of the amide group also triggers a conformational shift in one loop of the protein at the head of the tunnel. From these observations, the authors propose a stepwise binding sequence for GSLs, in which the sugar binds first, acting as the primary determinant of GSL-protein specificity. The amide group binds next, orienting the GSL tails with the tunnel and shifting the loop to help open the tunnel for the hydrocarbons. The acyl hydrocarbon binds next. The sphingosine follows the acyl group into the tunnel in some cases, while in others, it remains outside. These results should accelerate understanding of GSL trafficking within the cell and provide insight into how these molecules play their parts in cell growth and development.

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 < POPC < DOPC, which have 0, 1, and 2 cis double bonds, respectively.

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.

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.

PRINCIPLES OF LYSOSOMAL MEMBRANE DIGESTION: Stimulation of Sphingolipid Degradation by Sphingolipid Activator Proteins and Anionic Lysosomal Lipids

Sphingolipids and glycosphingolipids are membrane components of eukaryotic cell surfaces. Their constitutive degradation takes place on the surface of intra-endosomal and intra-lysosomal membrane structures. During endocytosis, these intra-lysosomal membranes are formed and prepared for digestion by a lipid-sorting process during which their cholesterol content decreases and the concentration of the negatively charged bis(monoacylglycero)phosphate (BMP)--erroneously also called lysobisphosphatidic acid (LBPA)--increases. Glycosphingolipid degradation requires the presence of water-soluble acid exohydrolases, sphingolipid activator proteins, and anionic phospholipids like BMP. The lysosomal degradation of sphingolipids with short hydrophilic head groups requires the presence of sphingolipid activator proteins (SAPs). These are the saposins (Saps) and the GM2 activator protein. Sphingolipid activator proteins are membrane-perturbing and lipid-binding proteins with different specificities for the bound lipid and the activated enzyme-catalyzed reaction. Their inherited deficiency leads to sphingolipid- and membrane-storage diseases. Sphingolipid activator proteins not only facilitate glycolipid digestion but also act as glycolipid transfer proteins facilitating the association of lipid antigens with immunoreceptors of the CD1 family.

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.

StarD10, a START Domain Protein Overexpressed in Breast Cancer, Functions as a Phospholipid Transfer Protein

We originally identified StarD10 as a protein overexpressed in breast cancer that cooperates with the ErbB family of receptor tyrosine kinases in cellular transformation. StarD10 contains a steroidogenic acute regulatory protein (StAR/StarD1)-related lipid transfer (START) domain that is thought to mediate binding of lipids. We now provide evidence that StarD10 interacts with phosphatidylcholine (PC) and phosphatidylethanolamine (PE) by electron spin resonance measurement. Interaction with these phospholipids was verified in a fluorescence resonance energy transfer-based assay with 7-nitro-2,1,3-benzoxadiazol-4-yl-labeled lipids. Binding was not restricted to lipid analogs since StarD10 selectively extracted PC and PE from small unilamellar vesicles prepared with endogenous radiolabeled lipids from Vero monkey kidney cells. Mass spectrometry revealed that StarD10 preferentially selects lipid species containing a palmitoyl or stearoyl chain on the sn-1 and an unsaturated fatty acyl chain (18:1 or 18:2) on the sn-2 position. StarD10 was further shown to bind lipids in vivo by cross-linking of protein expressed in transfected HEK-293T cells with photoactivable phosphatidylcholine. In addition to a lipid binding function, StarD10 transferred PC and PE between membranes. Interestingly, these lipid binding and transfer specificities distinguish StarD10 from the related START domain proteins Pctp and CERT, suggesting a distinct biological function.

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-C 16. 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.

Glycolipid Transfer Protein Mediated Transfer of Glycosphingolipids between Membranes

Glycolipid Transfer Protein Mediated Transfer of Glycosphingolipids between Membranes