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• W2034784128 abstract "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 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) 4The abbreviations used are: GLTP, glycolipid transfer protein; WT-GLTP, wild-type GLTP; rGLTP, recombinant GLTP; LacCer, lactosylceramide; GalCer, galactosylceramide; GlcCer, glucosylceramide; PC, phosphatidylcholine; POPC, 1-palmitoyl-2-oleyl-phosphatidylcholine; GM1, monosialoganglioside; SUV, small unilamellar vesicle; LUV, large unilamellar vesicle; GSL, glycosphingolipid; FPLC, fast protein liquid chromatography; ESI-MS, electrospray ionization mass spectrometry; SEC, size exclusion chromatography. 4The abbreviations used are: GLTP, glycolipid transfer protein; WT-GLTP, wild-type GLTP; rGLTP, recombinant GLTP; LacCer, lactosylceramide; GalCer, galactosylceramide; GlcCer, glucosylceramide; PC, phosphatidylcholine; POPC, 1-palmitoyl-2-oleyl-phosphatidylcholine; GM1, monosialoganglioside; SUV, small unilamellar vesicle; LUV, large unilamellar vesicle; GSL, glycosphingolipid; FPLC, fast protein liquid chromatography; ESI-MS, electrospray ionization mass spectrometry; SEC, size exclusion chromatography. 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 (1Viard M. Parolini I. Rawat S.S. Fecchi K. Sargiacomo M. Puri A. Blumenthal R. Glycoconj. J. 2004; 20: 213-222Crossref PubMed Scopus (35) Google Scholar, 2Schnaar R.L. Arch. Biochem. Biophys. 2004; 426: 163-172Crossref PubMed Scopus (76) Google Scholar, 3Bektas M. Spiegel S. Glycoconj. J. 2004; 20: 39-47Crossref PubMed Scopus (67) Google Scholar, 4Morales A. Colell A. Mari M. Garcia-Ruiz C. Fernandez-Checa J.C. Glycoconj. J. 2004; 20: 579-588Crossref PubMed Scopus (74) Google Scholar, 5Gouaze-Andersson V. Cabot M.C. Biochim. Biophys. Acta. 2006; 1758: 2096-2103Crossref PubMed Scopus (93) Google Scholar, 6Sabourdy F. Kedjouar B. Sorli S.C. Colié S. Milhas D. Salma Y. Levade T. Biochim. Biophys. Acta. 2008; 1781: 145-183Crossref PubMed Scopus (59) Google Scholar, 7Hannun Y.A. Obeid L.M. Nat. Rev. Mol. Cell Biol. 2008; 9: 139-150Crossref PubMed Scopus (2354) Google Scholar, 8Prinetti A. Loberto N. Chigorno V. Sonnino S. Biochim. Biophys. Acta. 2009; 1778: 184-193Crossref Scopus (112) Google Scholar). Phylogenetic/evolutionary analyses show GLTP to be highly conserved among vertebrates (9Brown R.E. Mattjus P. Biochim. Biophys. Acta. 2007; 1771: 746-760Crossref PubMed Scopus (70) Google Scholar, 10Zou X. Chung T. Lin X. Malakhova M.L. Pike H.M. Brown R.E. BMC Genomics. 2008; 9: 72Crossref PubMed Scopus (20) Google Scholar, 11West G. Viitanen L. Alm C. Mattjus P. Salminen T.A. Edqvist J. FEBS J. 2008; 275: 3421-3437Crossref PubMed Scopus (31) Google Scholar). The conformational uniqueness of the GLTP fold when compared with other lipid binding/transfer proteins (12Malinina L. Malakhova M.L. Teplov A. Brown R.E. Patel D.J. Nature. 2004; 430: 1048-1053Crossref PubMed Scopus (99) Google Scholar, 13Malinina L. Malakhova M.L. Kanak A.T. Lu M. Abagyan R. Brown R.E. Patel D.J. PLoS Biol. 2006; 4: e362Crossref PubMed Scopus (50) Google Scholar, 14Airenne T.T. Kidron H. Nymalm Y. Nylund M. West G.P. Mattjus P. Salminen T.A. J. Mol. Biol. 2006; 355: 224-236Crossref PubMed Scopus (44) Google Scholar) has resulted in GLTP being designated the prototype and founding member of the GLTP superfamily (15Murzin A.G. Brenner S.E. Hubbard T. Chothia C. J. Mol. Biol. 1995; 247: 536-540Crossref PubMed Scopus (5542) Google Scholar, 16Madera M. Vogel C. Kummerfeld S.K. Chothia C. Gough J. Nucleic Acids Res. 2004; 32: D235-D239Crossref PubMed Google Scholar). 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 (9Brown R.E. Mattjus P. Biochim. Biophys. Acta. 2007; 1771: 746-760Crossref PubMed Scopus (70) Google Scholar, 13Malinina L. Malakhova M.L. Kanak A.T. Lu M. Abagyan R. Brown R.E. Patel D.J. PLoS Biol. 2006; 4: e362Crossref PubMed Scopus (50) Google Scholar, 17Rao C.S. Chung T. Pike H.M. Brown R.E. Biophys. J. 2005; 89: 4017-4028Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 18Cho W. Stahelin R.V. Annu. Rev. Biophys. Biomol. Struct. 2005; 34: 119-151Crossref PubMed Scopus (471) Google Scholar, 19Hurley J.H. Biochim. Biophys. Acta. 2006; 1761: 805-811Crossref PubMed Scopus (124) Google Scholar, 20Cho W. Biochim. Biophys. Acta (Special Issue: Lipid-Binding Domains). 1761. 2006: 803-968Google Scholar, 21Lemmon M.A. Nat. Rev. Mol. Cell Biol. 2008; 9: 99-111Crossref PubMed Scopus (1101) Google Scholar). 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 (13Malinina L. Malakhova M.L. Kanak A.T. Lu M. Abagyan R. Brown R.E. Patel D.J. PLoS Biol. 2006; 4: e362Crossref PubMed Scopus (50) Google Scholar). Glycolipid uptake occurs via a cleft-like gating mechanism involving conformational changes to one α-helix and two interhelical loops (12Malinina L. Malakhova M.L. Teplov A. Brown R.E. Patel D.J. Nature. 2004; 430: 1048-1053Crossref PubMed Scopus (99) Google Scholar). 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 (12Malinina L. Malakhova M.L. Teplov A. Brown R.E. Patel D.J. Nature. 2004; 430: 1048-1053Crossref PubMed Scopus (99) Google Scholar, 13Malinina L. Malakhova M.L. Kanak A.T. Lu M. Abagyan R. Brown R.E. Patel D.J. PLoS Biol. 2006; 4: e362Crossref PubMed Scopus (50) Google Scholar, 14Airenne T.T. Kidron H. Nymalm Y. Nylund M. West G.P. Mattjus P. Salminen T.A. J. Mol. Biol. 2006; 355: 224-236Crossref PubMed Scopus (44) Google Scholar, 17Rao C.S. Chung T. Pike H.M. Brown R.E. Biophys. J. 2005; 89: 4017-4028Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 22Li X.-M. Malakhova M.L. Lin X. Pike H.M. Chung T. Molotkovsky J.G. Brown R.E. Biochemistry. 2004; 43: 10285-10294Crossref PubMed Scopus (29) Google Scholar, 23West G. Nylund M. Slotte J.P. Mattjus P. Biochim. Biophys. Acta. 2006; 1758: 1732-1742Crossref PubMed Scopus (26) Google Scholar), where they could help form a membrane-interaction site. Only one, Trp-96, is directly involved in glycolipid binding (12Malinina L. Malakhova M.L. Teplov A. Brown R.E. Patel D.J. Nature. 2004; 430: 1048-1053Crossref PubMed Scopus (99) Google Scholar, 13Malinina L. Malakhova M.L. Kanak A.T. Lu M. Abagyan R. Brown R.E. Patel D.J. PLoS Biol. 2006; 4: e362Crossref PubMed Scopus (50) Google Scholar, 14Airenne T.T. Kidron H. Nymalm Y. Nylund M. West G.P. Mattjus P. Salminen T.A. J. Mol. Biol. 2006; 355: 224-236Crossref PubMed Scopus (44) Google Scholar). 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. Most GalCers, LacCers, and sphingomyelins with homogeneous acyl chains were synthesized by reacylating GalSph, Lac-Sph, or lysosphingomyelin with the desired fatty acyl residue (24Smaby J.M. Momsen M. Kulkarni V.S. Brown R.E. Biochemistry. 1996; 35: 5696-5704Crossref PubMed Scopus (92) Google Scholar, 25Zhai X. Li X.-M. Momsen M.M. Brockman H.L. Brown R.E. Biophys. J. 2006; 91: 2490-2500Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar) (see supplemental information for details). Other glycolipids and all phosphoglycerides were purchased from Avanti Polar Lipids (Alabaster, AL). n-Hexyl-β-d-glucoside was obtained from Anatrace Inc. (Maumee OH). Stock phospholipid concentrations were quantitated by the Bartlett method (26Bartlett G.R. J. Biol. Chem. 1959; 234: 466-468Abstract Full Text PDF PubMed Google Scholar), and glycolipids were quantitated by gravimetric analyses. Lipid vesicles were produced by extrusion or sonication (see supplemental information). Protein Expression and Purification—Our GLTP purifications have been described previously in detail (22Li X.-M. Malakhova M.L. Lin X. Pike H.M. Chung T. Molotkovsky J.G. Brown R.E. Biochemistry. 2004; 43: 10285-10294Crossref PubMed Scopus (29) Google Scholar, 27Malakhova M.L. Malinina L. Pike H.M. Kanack A.T. Patel D.J. Brown R.E. J. Biol. Chem. 2005; 280: 26312-26320Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Briefly, the open reading frame encoding human GLTP (National Center for Biotechnology Information (NCBI) GenBank™ accession number AF209704) was subcloned into pET-30 Xa/LIC expression vector (Novagen) by ligation-independent cloning, enabling cleavage of the N-terminal His6-S tag to yield protein identical in sequence to native GLTP (12Malinina L. Malakhova M.L. Teplov A. Brown R.E. Patel D.J. Nature. 2004; 430: 1048-1053Crossref PubMed Scopus (99) Google Scholar, 27Malakhova M.L. Malinina L. Pike H.M. Kanack A.T. Patel D.J. Brown R.E. J. Biol. Chem. 2005; 280: 26312-26320Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). His6-GLTP also was prepared because structural analyses indicate that glycolipid binding might be subtly affected by the presence of the N-terminal fusion tag (13Malinina L. Malakhova M.L. Kanak A.T. Lu M. Abagyan R. Brown R.E. Patel D.J. PLoS Biol. 2006; 4: e362Crossref PubMed Scopus (50) Google Scholar). Site-directed mutants were produced by QuikChange® site-directed mutagenesis (Stratagene) and verified by sequencing. Near UV CD analysis confirmed the global folding similarity of WT-GLTP and W96F-GLTP (12Malinina L. Malakhova M.L. Teplov A. Brown R.E. Patel D.J. Nature. 2004; 430: 1048-1053Crossref PubMed Scopus (99) Google Scholar). Protein purity and concentration were determined by SDS-PAGE and bicinchoninic acid (22Li X.-M. Malakhova M.L. Lin X. Pike H.M. Chung T. Molotkovsky J.G. Brown R.E. Biochemistry. 2004; 43: 10285-10294Crossref PubMed Scopus (29) Google Scholar). Glycolipid intervesicular transfer activity of purified GLTP was monitored using established assays involving fluorophore- or radiolabeled glycolipid (see supplemental information). Fluorescence Measurements—Emission spectra were measured using a SPEX FluoroMax instrument (HORIBA Scientific, Edina, NJ). Excitation and emission band passes were 5 nm, and the cuvette holder was temperature-controlled to 25 ± 0.1 °C (Neslab RTE-111, (Thermo Fisher Scientific)). To eliminate contributions from residues other than Trp, the excitation wavelength was 295 nm. Emission spectra were recorded from 305 to 500 nm using GLTP concentrations at optical densities >0.1 to avoid inner filter effects. GSL addition (2-μl aliquots; 0.1 mm GSL stock in ethanol) to protein (2.5 ml; 1 μm) involved constant stirring. Rapid equilibration of the fluorescence emission signal (<3 min) was observed for glycolipids containing either short saturated or longer unsaturated acyl chains (supplemental Fig. S2). Binding isotherms were adjusted for the constant fractional contributions of Trp-85 and Trp-142 determined from the emission response of W96F-GLTP. Dissociation constants were determined by nonlinear curve fitting using Prism 4.0 (GraphPad Software, La Jolla, CA) (see supplemental information). Mass Spectrometry—WT-GLTP and GLTP·glycolipid complex were analyzed using an Agilent 6210 LC/MS-TOF mass spectrometer (Santa Clara, CA) by preparing (10 μm protein) in 5 mm ammonium acetate plus 5% methanol and infusing directly into the electrospray source. Spectra were collected in positive mode over an m/z range of 500–5000 using parameters optimized for complex stability, e.g. capillary, 3000 V; fragmentor, 300 V; skimmer, -60 V; octopole radio frequency, -300 V; octopole direct current, -32 V. Raw spectra data were transformed into relative molecular masses using the Agilent time-of-flight Protein Confirmation software. SHARP2 Analyses—WT-GLTP surface interaction site analysis was performed using SHARP2. The algorithm calculates parameters for overlapping patches of residues on the surface of a protein from Protein Data Bank (PDB) data (28Jones S. Thornton J.M. J. Mol. Biol. 1997; 272: 133-143Crossref PubMed Scopus (369) Google Scholar). Six parameters are assessed: solvation potential, hydrophobicity, accessible surface area, residue interface propensity, planarity, and protrusion (SHARP2) (29Jones S. Thornton J.M. J. Mol. Biol. 1997; 272: 121-132Crossref PubMed Scopus (516) Google Scholar). Patches are ranked according to combined parameter scores, enabling assessment as potential protein-protein interaction sites. Our previous x-ray crystallographic analyses showed that the 3 Trp residues of human GLTP are located in the vicinity of the glycolipid binding site (12Malinina L. Malakhova M.L. Teplov A. Brown R.E. Patel D.J. Nature. 2004; 430: 1048-1053Crossref PubMed Scopus (99) Google Scholar, 13Malinina L. Malakhova M.L. Kanak A.T. Lu M. Abagyan R. Brown R.E. Patel D.J. PLoS Biol. 2006; 4: e362Crossref PubMed Scopus (50) Google Scholar, 14Airenne T.T. Kidron H. Nymalm Y. Nylund M. West G.P. Mattjus P. Salminen T.A. J. Mol. Biol. 2006; 355: 224-236Crossref PubMed Scopus (44) Google Scholar). Trp-96 and Trp-142 are surface-localized, 14–16Å apart, and reside within a putative membrane-interaction region that encompasses the glycolipid binding site (Fig. 1, upper panel, and supplemental Fig. S1, upper panel). Trp-96 also helps bind glycolipid by providing a stacking platform for the initial ceramide-linked sugar and facilitating formation of multiple hydrogen bonds between the sugar ring hydroxyls and Asp-48, Asn-52, and Lys-55 (Figs. 1B and 6). Trp-85 resides near the protein surface but is partially buried, with its indole ring sandwiched between Pro-86 (cis configuration) and Lys-78 (supplemental Fig. S1, lower panel). The topology of the 3 Trp residues in glycolipid-free WT-GLTP is reflected by the steady-state Trp fluorescence signal, which is relatively red-shifted (λmax ∼ 347 nm) and is diminished by aqueous quenchers (22Li X.-M. Malakhova M.L. Lin X. Pike H.M. Chung T. Molotkovsky J.G. Brown R.E. Biochemistry. 2004; 43: 10285-10294Crossref PubMed Scopus (29) Google Scholar, 23West G. Nylund M. Slotte J.P. Mattjus P. Biochim. Biophys. Acta. 2006; 1758: 1732-1742Crossref PubMed Scopus (26) Google Scholar). When glycolipid-free GLTP encounters membranes containing glycolipid, dramatic changes in Trp emission occur (22Li X.-M. Malakhova M.L. Lin X. Pike H.M. Chung T. Molotkovsky J.G. Brown R.E. Biochemistry. 2004; 43: 10285-10294Crossref PubMed Scopus (29) Google Scholar, 23West G. Nylund M. Slotte J.P. Mattjus P. Biochim. Biophys. Acta. 2006; 1758: 1732-1742Crossref PubMed Scopus (26) Google Scholar), consistent with Trp involvement in both the glycolipid binding site and the membrane-interaction region of GLTP. The changes include: (i) a large decrease in intensity and (ii) a substantial blue shift (∼12 nm) in wavelength maximum (λmax). Our initial goal was to establish whether the changes in Trp emission could be used to assess the binding affinity and uptake kinetics of various glycolipids by WT-GLTP.FIGURE 6Tryptophan 96 and neighboring residues in the GLTP·N-oleoyl LacCer complex. Depictions are based on the crystal coordinates of human GLTP liganded with 18:1 LacCer (PDB 1sx6). Interaction distances between the Trp-96 indole ring and the ceramide-linked sugar of the glycolipid headgroup are indicated by lime green dashed lines. Side chains of residues that stabilize Trp-96 position in GLTP and interactions of Asp-48, Asn-52, Lys-55, and His-140 with the glycolipid polar headgroup region are indicated by turquoise dashed lines. Numerical values indicate distances in angstroms.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Glycolipid Binding to WT-GLTP Induces Dramatic Changes in Tryptophan Fluorescence—To determine whether the emission changes reflect uptake of glycolipid or protein partitioning to the membrane surface, WT-GLTP was presented with limited amounts of glycolipids, dissolved in ethanol, in titration-like fashion. The strategy of using ethanol injection was based on our previous findings showing that ethanolic solutions of glycolipids can facilitate cocrystallization of GLTP complexed with various glycolipid ligands (12Malinina L. Malakhova M.L. Teplov A. Brown R.E. Patel D.J. Nature. 2004; 430: 1048-1053Crossref PubMed Scopus (99) Google Scholar, 13Malinina L. Malakhova M.L. Kanak A.T. Lu M. Abagyan R. Brown R.E. Patel D.J. PLoS Biol. 2006; 4: e362Crossref PubMed Scopus (50) Google Scholar) and that GLTP is highly adaptable in its ability to acquire glycolipids presented in various forms (27Malakhova M.L. Malinina L. Pike H.M. Kanack A.T. Patel D.J. Brown R.E. J. Biol. Chem. 2005; 280: 26312-26320Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Fig. 2 illustrates a typical Trp emission response upon stepwise injections of LacCer ethanolic solution. The Trp emission λmax becomes progressively blue-shifted, i.e. 347–336 nm, whereas the intensity systematically decreases. Eventually, a saturation response is achieved as the λmax reaches ∼336 nm, and the total Trp emission intensity declines by ∼40%. It is important to note that each glycolipid injection corresponds to <10 mol % of total WT-GLTP and that the changes in Trp fluorescence occurred rapidly (<3–5 min) for glycolipids with short saturated or long monounsaturated acyl chains (supplemental Fig. S2), permitting fast equilibration. The emission response was similar for LacCers with oleoyl (18:1), nervonoyl (24:1), or octanoyl (8:0) acyl chains (e.g. Fig. 2A) and resulted in similar binding isotherms (Fig. 2C). In contrast, almost no changes in Trp emission were observed when POPC (Fig. 2B), l-α-dimyristoylphosphatidylcholine, 1-myristoyl,2-palmitoyl-sn-glycero 3-phosphocholine, or 1-myristoyl-2-stearoyl-sn-glycero 3-phosphocholine were presented with WT-GLTP. Formation of a WT-GLTP·glycolipid complex by GSL ethanol injection was verified by electrospray ionization mass spectrometry (ESI-MS). Fig. 3A shows spectra obtained by direct infusion of glycolipid-free GLTP plus GLTP·GalCer complex under nondenaturing conditions. Ions corresponding to monomeric, glycolipid-free rGLTP (25,248 Da) and monomeric complex (rGLTP + N-octanoyl GalCer (25,836 Da)) dominate the spectra. To further confirm that the observed changes in the Trp emission reflected glycolipid binding by WT-GLTP, alternate possibilities were evaluated. Adsorption of GLTP·glycolipid complex to the quartz cuvette walls, resulting in diminished protein solution concentration, was ruled out because applying a film of polyethyleneimine, which prevents protein adsorption to silica (30Persson D. Thorén P.E.G. Lincoln P. Nordén B. Biochemistry. 2004; 43: 11045-11055Crossref PubMed Scopus (42) Google Scholar), did not alter the outcome (data not shown). Another possibility was Trp environmental polarity change caused by nonspecific partitioning of WT-GLTP to lipid aggregates/vesicles formed in solution after ethanol injection. However, adjustment of the WT-GLTP concentration from 1 μm to either 0.5 μm or 2 μm proportionally lowered or raised the glycolipid amount needed to saturate the spectral emission changes. Also, SEC analyses using Sephacryl S-300 showed that overnight incubations of WT-GLTP, with POPC vesicles containing glycolipid, resulted in vesicles eluting in the void volume (with almost no adsorbed WT-GLTP when vesicle concentrations were kept relatively low), whereas WT-GLTP (>95%) remained soluble and eluted in the included volume (Fig. 3B). The Trp emission signal of the soluble WT-GLTP exhibited a blue-shifted λmax (∼335 nm), a finding consistent with complexation of glycolipid. Collectively, the experimental results indicated that the changes in Trp emission reflected glycolipid binding by WT-GLTP. This conclusion was further supported by experiments involving a Trp-96 point mutant (W96F), which emits via Trp-85 and Trp-142 while maintaining ∼70% or more activity (12Malinina L. Malakhova M.L. Teplov A. Brown R.E. Patel D.J. Nature. 2004; 430: 1048-1053Crossref PubMed Scopus (99) Google Scholar). As shown in Fig. 2D and supplemental Fig. S3, W96F-GLTP emits at a λmax of ∼347 nm (like glycolipid-free WT-GLTP) but with only ∼30% intensity. Stepwise ethanol microinjection of 18:1 or 8:0 GalCer with W96F-GLTP resulted in only slight changes in the Trp emission response, i.e. 2–3-nm red shift in the λmax and 4–5% intensity decrease. This finding was consistent with Trp-96 accounting for the blue shift in the λmax and the vast majority of the Trp emission intensity decrease observed when WT-GLTP acquires glycolipid. Glycolipid Structural Parameters Affecting Binding by WT-GLTP—To determine whether the changes in WT-GLTP Trp fluorescence were glycolipid-specific, the responses elicited by ceramide and sphingomyelin were investigated. As expected, removal of the glycolipid sugar headgroup (supplemental Fig. S4A) or replacement with a nonsugar headgroup (supplemental Fig. S4B) had little effect on the intrinsic Trp emission. Next, various glycolipid structural parameters were evaluated. When the sugar headgroup was changed from lactose to either glucose or galactose, the dramatic blue shift in the λmax and decrease in Trp emission intensity persisted (supplemental Fig. S5) and resulted in similar binding isotherms (Fig. 4A). Ethanol injection of ganglioside GM1, which contains five sugars including negatively charged N-acetylneuraminic acid, also elicited a λmax blue shift and decrease in Trp emission intensity, albeit reduced in magnitude and affinity when compared with glycolipids with simpler headgroups (Fig. 4A). In contrast, removal of the amide-linked acyl chain of GlcCer or LacCer elicited very modest changes, i.e. 3–6-nm λmax blue shifts and 15–25% Trp intensity decreases, but only when added in high amounts (supplemental Fig. S4, C and D). Fig. 4B shows that very weak binding occurred with hexyl-β-d-glucoside, which also can occupy the GLTP binding site (13Malinina L. Malakhova M.L. Kanak A.T. Lu M. Abagyan R. Brown R.E. Patel D.J. PLoS Biol. 2006; 4: e362Crossref PubMed Scopus (50) Google Scholar). Binding Affinity of WT-GLTP for Different Glycolipids—Table 1 shows the binding constants for the spectral responses produced by titration of WT-GLTP with various glycolipids. KD values between ∼0.6 and 2.1 μm were determined for glycolipids with one or two sugars that contained either short saturated acyl chains or long acyl chains with mono- or diunsaturation. Slow equilibration was observed when these simple glycolipids contained long saturated acyl chains (>16 carbons), thus interfering with accurate assessment of KD values. With the multiglycosylated and more soluble ganglioside GM1, fast equilibration was observed despite the predominance of stearoyl acyl chains (supplemental Fig. S2). The KD for GM1 was ∼1.7 μm. The very modest spectral changes elicited by high amounts of lyso-GlcCer and lyso-LacCer translated into KD values of ∼80 and ∼60 μm, respectively. The lack of change in Trp fluorescence signal elicited by nonglycosylated lipids (e.g. ceramide, sphingomyelin, and PC) was consistent with lack of binding.TABLE 1KD values of WT-GLTP for various glycolipids Dissociation constants were determined by nonlinear fitting analyses of glycolipid-induced changes in GLTP Trp emission intensity as detailed in the supplemental information. Values were calculated at fixed wavelength (353 nm) in the emission peak (50Halter D. Neumann S. van Dijk S.M. Wolthoorn J. de Mazière A.M. Vieira O.V. Mattjus P. Klumperman J. van Meer G. Sprong H. J. Cell Biol. 2007; 179: 101-115Crossref PubMed Scopus (229) Google Scholar). Extrusion vesicles contained 10 mol % glycolipid. Glycolipid pool size in the POPC vesicle outer leaflet and available for interaction with GLTP was estimated from previously determined transbilayer distributions (51Mattjus P. Malewicz B. Valiyaveettil J.T. Baumann W.J. Bittman R. Brown R.E. J. Biol. Chem. 2002; 277: 19476-19481Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 52Malewicz B. Valiyaveettil J.T. Jacob K. Byun H.-S. Mattjus P. Baumann W.J. Bittman R. Brown R.E. Biophys. J. 2005; 88: 2670-2680Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 53Sillence D.J. Raggers R.J. Neville D.C.A. Harvey D.J. van Meer G. J. Lipid Res. 2000; 41: 1252-1260Abstract Full Text Full Text PDF PubMed Google Scholar).KDR2μMGalCer8:0 GalCer0.60 ± 0.130.96212:0 GalCer1.60 ± 0.30" @default.
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