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.     

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.     

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.     

Human GLTP: Three distinct functions for the three tryptophans in a novel peripheral amphitropic fold

Human glycolipid transfer protein (GLTP) serves as the GLTP-fold prototype, a novel, to our knowledge, peripheral amphitropic fold and structurally unique lipid binding motif that defines the GLTP superfamily. Despite conservation of all three intrinsic Trps in vertebrate GLTPs, the Trp functional role(s) remains unclear. Herein, the issue is addressed using circular dichroism and fluorescence spectroscopy along with an atypical Trp point mutation strategy. Far-ultraviolet and near-ultraviolet circular dichroism spectroscopic analyses showed that W96F-W142Y-GLTP and W96Y-GLTP retain their native conformation and stability, whereas W85Y-W96F-GLTP is slightly altered, in agreement with relative glycolipid transfer activities of >90%, ∼85%, and ∼45%, respectively. In silico three-dimensional modeling and acrylamide quenching of Trp fluorescence supported a nativelike folding conformation. With the Trp⁹⁶-less mutants, changes in emission intensity, wavelength maximum, lifetime, and time-resolved anisotropy decay induced by phosphoglyceride membranes lacking or containing glycolipid and by excitation at different wavelengths along the absorption-spectrum red edge indicated differing functions for W142 and W85. The data suggest that W142 acts as a shallow-penetration anchor during docking with membrane interfaces, whereas the buried W85 indole helps maintain proper folding and possibly regulates membrane-induced transitioning to a glycolipid-acquiring conformation. The findings illustrate remarkable versatility for Trp, providing three distinct intramolecular functions in the novel amphitropic GLTP fold.     

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.     

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.     

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.     

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   

Functional evaluation of tryptophans in glycolipid binding and membrane interaction by HET-C2, a fungal glycolipid transfer protein

HET-C2 is a fungal glycolipid transfer protein (GLTP) that uses an evolutionarily-modified GLTP-fold to achieve more focused transfer specificity for simple neutral glycosphingolipids than mammalian GLTPs. Only one of HET-C2's two Trp residues is topologically identical to the three Trp residues of mammalian GLTP. Here, we provide the first assessment of the functional roles of HET-C2 Trp residues in glycolipid binding and membrane interaction. Point mutants HET-C2^W208F, HET-C2^W208A and HET-C2^F149Y all retained > 90% activity and 80–90% intrinsic Trp fluorescence intensity; whereas HET-C2^F149A transfer activity decreased to ~ 55% but displayed ~ 120% intrinsic Trp emission intensity. Thus, neither W208 nor F149 is absolutely essential for activity and most Trp emission intensity (~ 85–90%) originates from Trp109. This conclusion was supported by HET-C2^W109Y/F149Y which displayed ~ 8% intrinsic Trp intensity and was nearly inactive. Incubation of the HET-C2 mutants with 1-palmitoyl-2-oleoyl-phosphatidylcholine vesicles containing different monoglycosylceramides or presented by lipid ethanol-injection decreased Trp fluorescence intensity and blue-shifted the Trp λ_max by differing amounts compared to wtHET-C2. With HET-C2 mutants for Trp208, the emission intensity decreases (~ 30–40%) and λ_max blue-shifts (~ 12 nm) were more dramatic than for wtHET-C2 or F149 mutants and closely resembled human GLTP. When Trp109 was mutated, the glycolipid induced changes in HET-C2 emission intensity and λ_max blue-shift were nearly nonexistent. Our findings indicate that the HET-C2 Trp λ_max blue-shift is diagnostic for glycolipid binding; whereas the emission intensity decrease reflects higher environmental polarity encountered upon nonspecific interaction with phosphocholine headgroups comprising the membrane interface and specific interaction with the hydrated glycolipid sugar.     

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.     

A fluorescence resonance energy transfer approach for monitoring protein-mediated glycolipid transfer between vesicle membranes

A lipid transfer protein, purified from bovine brain (23.7 kDa, 208 amino acids) and specific for glycolipids, has been used to develop a fluorescence resonance energy transfer assay (anthrylvinyl-labeled lipids; energy donors and perylenoyl-labeled lipids; energy acceptors) for monitoring the transfer of lipids between membranes. Small unilamellar vesicles composed of 1 mol% anthrylvinyl-galactosylceramide, 1.5 mol% perylenoyl-triglyceride, and 97.5% 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) served as donor membranes. Acceptor membranes were 100% POPC vesicles. Addition of glycolipid transfer protein to mixtures of donor and acceptor vesicles resulted in increasing emission intensity of anthrylvinyl-galactosylceramide and decreasing emission intensity of the nontransferable perylenoyl-triglyceride as a function of time. The behavior was consistent with anthrylvinyl-galactosylceramide being transferred from donor to acceptor vesicles. The anthrylvinyl and perylenoyl energy transfer pair offers advantages over frequently used energy transfer pairs such as NBD and rhodamine. The anthrylvinyl emission overlaps effectively the perylenoyl excitation spectrum and the fluorescence parameters of the anthrylvinyl fluorophore are nearly independent of the medium polarity. The nonpolar fluorophores are localized in the hydrophobic region of the bilayer thus producing minimal disturbance of the bilayer polar region. Our results indicate that this method is suitable for assay of lipid transfer proteins including mechanistic studies of transfer protein function.     

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.     

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.     

Conformational folding and stability of the HET-C2 glycolipid transfer protein fold: does a molten globule-like state regulate activity?

The glycolipid transfer protein (GLTP) superfamily is defined by the human GLTP fold that represents a novel motif for lipid binding and transfer and for reversible interaction with membranes, i.e., peripheral amphitropic proteins. Despite limited sequence homology with human GLTP, we recently showed that HET-C2 GLTP of Podospora anserina is organized conformationally as a GLTP fold. Currently, insights into the folding stability and conformational states that regulate GLTP fold activity are almost nonexistent. To gain such insights into the disulfide-less GLTP fold, we investigated the effect of a change in pH on the fungal HET-C2 GLTP fold by taking advantage of its two tryptophans and four tyrosines (compared to three tryptophans and 10 tyrosines in human GLTP). pH-induced conformational alterations were determined by changes in (i) intrinsic tryptophan fluorescence (intensity, emission wavelength maximum, and anisotropy), (ii) circular dichroism over the near-UV and far-UV ranges, including thermal stability profiles of the derivatized molar ellipticity at 222 nm, (iii) fluorescence properties of 1-anilinonaphthalene-8-sulfonic acid, and (iv) glycolipid intermembrane transfer activity monitored by Fo?rster resonance energy transfer. Analyses of our recently determined crystallographic structure of HET-C2 (1.9 ?) allowed identification of side chain electrostatic interactions that contribute to HET-C2 GLTP fold stability and can be altered by a change in pH. Side chain interactions include numerous salt bridges and interchain cation-π interactions, but not intramolecular disulfide bridges. Histidine residues are especially important for stabilizing the local positioning of the two tryptophan residues and the conformation of adjacent chains. Induction of a low-pH-induced, molten globule-like state inhibited glycolipid intermembrane transfer by the HET-C2 GLTP fold.     

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.     

Molecular determinants for FMN-binding in Desulfovibrio gigas flavoredoxin

Flavoredoxin participates in Desulfovibrio gigas thiosulfate reduction pathway. Its 3-dimensional model was generated allowing the oxidized riboflavin-5'-phosphate (FMN) site to be predicted. Residues likely to be involved in FMN-binding were identified (N29, W35, T56, K92, H131 and F164) and mutated to alanine. Fluorescence titration with apoprotein showed that FMN is strongly bound in the wild-type protein. Comparison of K(d) values for mutants suggests that interactions with the phosphate group of FMN, contribute more to binding than the interactions with the isoalloxazine ring. The redox potential of bound FMN determined for wild-type and mutants revealed shifts to less negative values. These findings were correlated with the protein structure in order to contribute to a better understanding of the structure-function relationships in flavoredoxin.     

Enhanced selectivity for sulfatide by engineered human glycolipid transfer protein

Human glycolipid transfer protein (GLTP) fold represents a novel structural motif for lipid binding/transfer and reversible membrane translocation. GLTPs transfer glycosphingolipids (GSLs) that are key regulators of cell growth, division, surface adhesion, and neurodevelopment. Herein, we report structure-guided engineering of the lipid binding features of GLTP. New crystal structures of wild-type GLTP and two mutants (D48V and A47D‖D48V), each containing bound N-nervonoyl-sulfatide, reveal the molecular basis for selective anchoring of sulfatide (3-O-sulfo-galactosylceramide) by D48V-GLTP. Directed point mutations of "portal entrance" residues, A47 and D48, reversibly regulate sphingosine access to the hydrophobic pocket via a mechanism that could involve homodimerization. "Door-opening" conformational changes by phenylalanines within the hydrophobic pocket are revealed during lipid encapsulation by new crystal structures of bona fide apo-GLTP and GLTP complexed with N-oleoyl-glucosylceramide. The development of "engineered GLTPs" with enhanced specificity for select GSLs provides a potential new therapeutic approach for targeting GSL-mediated pathologies.     

Tryptophan fluorescence of yeast actin resolved via conserved mutations

Actin contains four tryptophan residues, W79, W86, W340, and W356, all located in subdomain 1 of the protein. Replacement of each of these residues with either tyrosine (W79Y and W356Y) or phenylalanine (W86F and W340F) generated viable proteins in the yeast Saccharomyces cerevisiae, which, when purified, allowed the analysis of the contribution of these residues to the overall tryptophan fluorescence of actin. The sum of the relative contributions of these tryptophans was found to account for the intrinsic fluorescence of wild-type actin, indicating that energy transfer between the tryptophans is not the main determinant of their quantum yield, and that these mutations induce little conformational change to the protein. This was borne out by virtually identical polymerization rates and similar myosin interactions of each of the mutants and the wild-type actin. In addition, these mutants allowed the dissection of the microenvironment of each tryptophan as actin undergoes conformational changes upon metal cation exchange and polymerization. Based on the relative tryptophan contributions determined from single mutants, a triple mutant of yeast actin (W79) was generated that showed small intrinsic fluorescence and should be useful for studies of actin interactions with actin-binding proteins.     

Investigation of the structural determinants of the intrinsic fluorescence emission of the trp repressor using single tryptophan mutants.

The fluorescence decay properties of wild-type trp repressor (TR) have been characterized by carrying out a multi-emission wavelength study of the frequency response profiles. The decay is best analyzed in terms of a single exponential decay near 0.5 ns and a distribution of lifetimes centered near 3-4 ns. By comparing the recovered decay associated spectra and lifetime values with the structure of the repressor, tentative assignments of the two decay components recovered from the analysis to the two tryptophan residues, W19 and W99, of the protein have been made. These assignments consist of linking the short, red emitting component to emission from W99 and most of the longer bluer emitting lifetime distribution to emission from W19. Next, single tryptophan mutants of the repressor in which one of each of the tryptophan residues was substituted by phenylalanine were used to confirm the preliminary assignments, inasmuch as the 0.5-ns component is clearly due to emission from tryptophan 99, and much of the decay responsible for the recovered distribution emanates from tryptophan 19. The data demonstrate, however, that the decay of the wild-type protein is not completely resolvable due both to the large number of components in the wild-type emission (at least five) as well as to the fact that three of the five lifetime components are very close in value. The fluorescence decay of the wild-type decay is well described as a combination of the components found in each of the mutants. However, whereas the linear combination analysis of the 15 data sets (5 from the wild-type and each mutant) yields a good fit for the components recovered previously for the two mutants, the amplitudes of these components in the wild-type are not recovered in the expected ratios. Because of the dominance of the blue shifted emission in the wild-type protein, it is most likely that subtle structural differences in the wild-type as compared with the mutants, rather than energy transfer from tryptophan 19 to 99, are responsible for this failure of the linear combination hypothesis.