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

Abstract

Proteins are considered to be amphitropic when translocation to/from biomembranes: i ) is required for function; ii ) occurs transiently and reversibly from the cytoplasm; and iii ) involves interaction with the lipid bilayer ( 1-3 ). Glycolipid transfer proteins (GLTPs) comprise a structurallyunique amphitropic protein superfamily defi ned by their ability to reversibly interact with membranes to achieve selective transport of glycosphingolipids ( 4 ). Human GLTP resides in the cytosol ( 5 ), but is capable of delivering glucosylceramide to the cytosolic face of the plasma membrane ( 6, 7 ) and contains a nonclassical diphenylalanine-in-anacidic-tract targeting sequence to the endoplasmic reticulum ( 8 ). GLTPs occur widely among eukaryotes ( 4, 9 ) and GLTP-like domains serve as key functional regions in larger human proteins, i.e., FAPP2 ( 10, 11 ). The GLTPfold is dominated by ␣ -helices, lacks intramolecular disulfi des, and uses a two-layer "sandwich" motif to form a single glycolipid binding site consisting of a surface-localized sugar headgroup recognition center and an expandable hydrophobic pocket for accommodation of the ceramide hydrocarbon chains of the glycolipid ( 12-17 ). During glycolipid transfer, GLTP interacts with membranes transiently and in a minimally perturbing manner ( 18, 19 ) via a membrane interaction domain formed by a cluster of Trp, Tyr, Lys, and nonpolar residues on the GLTP surface Abstract Among amphitropic proteins, human glycolipid transfer protein (GLTP) forms a structurally-unique fold that translocates on/off membranes to specifi cally transfer glycolipids. Phosphatidylcholine (PC) bilayers with curvature-induced packing stress stimulate much faster glycolipid intervesicular transfer than nonstressed PC bilayers raising questions about planar cytosol-facing biomembranes being viable sites for GLTP interaction. Herein, GLTP-mediated desorption kinetics of fl uorescent glycolipid (tetramethyl-boron dipyrromethene (BODIPY)-label) from lipid monolayers are assessed using a novel microfl uidics-based surface balance that monitors lipid lateral packing while simultaneously acquiring surface fl uorescence data. At biomembrane-like packing (30-35 mN/m), GLTP uptake of BODIPY-glycolipid from POPC monolayers was nearly nonexistent but could be induced by reducing surface pressure to mirror packing in cur vature-stressed bilayers. In contrast, 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) matrices supported robust BODIPY-glycolipid uptake by GLTP at both high and low surface pressures. Unexpectedly, negativelycharged cytosol-facing lipids, i.e., phosphatidic acid and phosphatidylserine, also supported BODIPY-glycolipid uptake by GLTP at high surface pressure. Remarkably, including both 1-palmitoyl-2-oleoyl-sn -glycero-3-phosphate (5 mol%) and POPE (15 mol%) in POPC synergistically activated GLTP at high surface pressure. Our study shows that matrix lipid headgroup composition, rather than molecular packing per se, is a key regulator of GLTP-fold function while demonstrating th...

Sphingolipid transfer proteins defined by the GLTP-fold

“…All three Trp residues are conserved in vertebrate GLTPs (Zou et al 2008), but the only Trp universally conserved in more distantly related GLTPs is the Trp associated with the glycolipid-binding site. The emission signal of Trp is red-shifted in wild-type GLTP devoid of glycolipid (Li et al 2004; West et al 2006; Zhai et al 2009). The 347–348 nm emission wavelength maximum (λ max ) is consistent with exposure of the emitting Trp residues to the aqueous environment (Fig.…”

“…Zhai et al (2013) recently made use of an innovative, custom-built, microfluidics-based surface balance, developed by Howard Brockman and co-workers (Brown & Brockman, 2007; Hoang et al 2006; Momsen et al 2005), that simultaneously measures surface pressure and fluorescence via a Wilhelmy nichrome wire and a laser-coupled fiber optic cable/diode array platform. GLTP-mediated desorption kinetics of fluorescent glycolipid (tetramethyl-boron dipyrromethene (BODIPY)-label) from lipid monolayers was investigated when lipid packing was systematically controlled to reproduce conditions found in either nonstressed bilayers or high-curvature vesicles, a topic of renewed and timely interest (Brown, 2012; Chong et al 2014; Huang & Ramamurthi, 2010).…”

“…Prior to the study by Zhai et al (2013), the only membrane packing feature known to markedly stimulate glycolipid uptake by GLTP was high positive curvature stress in PC bilayers (Brown et al 1985; Nylund et al 2007; Rao et al 2004, 2005). This finding led to questions about whether cell membrane interaction sites need to be highly curved to be viable interaction sites for GLTP.…”

Synthesis of a novel photoactivatable glucosylceramide cross-linker

“…XLA and XLB were cross-linked in DU145 microsomes using GLTP to deliver and insert the cross-linker into the microsomal membranes, as GLTP has been previously used to insert and extract GSLs in model membranes (26,31), and our studies show GLTP enhancement of microsomal GSL synthesis. Recombinant GLTP was cross-linked by both XLA and XLB in DU145 microsomes, but crosslinking of microsomal proteins was not enhanced by GLTP delivery.…”

Investigation of the biophysical properties of a fluorescently modified ceramide-1-phosphate

“…It was previously shown by Zhai et al that the human glycolipid transfer protein, with structural similarity to CPTP, was successful in transfer of BODIPY-GalCer (Zhai et al, 2013) in a similar manner to GalCer. If CPTP is able to transport TopFluor-C1P from the TGN to the plasma membrane, this may contribute to the restoration of some of the TopFluor-C1P in the plasma membrane following photobleaching.…”