Glycolipid Acquisition by Human Glycolipid Transfer Protein Dramatically Alters Intrinsic Tryptophan Fluorescence

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. ϳ40% decrease in Trp intensity and ϳ12-nm blue shift in emission wavelength maximum, involved various modes of glycolipid presentation, i.e. microinjection/dilution of lipidethanol 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 ϳ70% 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-tosubmicromolar dissociation constant that was only slightly altered by increased sugar headgroup complexity. Glycolipid transfer protein (GLTP)4 is a soluble (ϳ24-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 ␣-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 c...

Structural Determination and Tryptophan Fluorescence of Heterokaryon Incompatibility C2 Protein (HET-C2), a Fungal Glycolipid Transfer Protein (GLTP), Provide Novel Insights into Glycolipid Specificity and Membrane Interaction by the GLTP Fold

“…4C). The near-UV CD signal, originating from environmentally induced optical activity of Phe, Tyr, and Trp, is largely unaffected by incubation with PC vesicles containing glycolipid, consistent with diffraction data showing only subtle changes in tertiary folding as a consequence of glycolipid binding by GLTP (9,10,46). Nonetheless, when the vesicles lack glycolipid, a hint of tertiary change is observed in the near-UV CD signal associated with Tyr and/or Trp, prompting the further study of intrinsic Trp fluorescence in HET-C2.…”

“…6, bottom right). The latter finding was unexpected because ethanol-injected POPC has little effect on GLTP Trp emission intensity (46). The data suggested that interaction of HET-C2 with membrane alters the local environment of Trp 208 either directly or by triggering conformational changes that precede glycolipid uptake.…”

“…Lipids also were presented to HET-C2 by microinjection after dissolving in ethanol as recently detailed by Zhai et al (46). Measurements were performed under constant stirring by the addition of small aliquots (1 l) of GSL, dissolved in ethanol (0.1 mM), to protein (1 M; 2.5 ml).…”

“…Protein concentration was kept at A 295 Ͻ 0.1 to avoid inner filter effects (45,46). Fluorescence quenching experiments were performed by adding aliquots of acrylamide or KI (5 M) stocks in 10 mM sodium phosphate-saline buffer (pH 7.4) to HET-C2 (1 M; 2.5 ml) with constant stirring at 25°C.…”

“…Presentation of Lipids to HET-C2 via Ethanol MicroinjectionRecently, Zhai et al (46) showed that microinjection involving small lipid aliquots dissolved in ethanol is useful for loading the glycolipid binding site of GLTP while minimizing the accumulation of excess membrane interface in solution, thus providing a means to distinguish emission changes induced by glycolipid binding from changes produced by nonspecific partitioning to membrane interface. Fig.…”