Membrane lipids regulate ganglioside GM2 catabolism and GM2 activator protein activity

Ganglioside GM2 is the major lysosomal storage compound of Tay-Sachs disease. It also accumulates in Niemann-Pick disease types A and B with primary storage of SM and with cholesterol in type C. Reconstitution of GM2 catabolism with β-hexosaminidase A and GM2 activator protein (GM2AP) at uncharged liposomal surfaces carrying GM2 as substrate generated only a physiologically irrelevant catabolic rate, even at pH 4.2. However, incorporation of anionic phospholipids into the GM2 carrying liposomes stimulated GM2 hydrolysis more than 10-fold, while the incorporation of plasma membrane stabilizing lipids (SM and cholesterol) generated a strong inhibition of GM2 hydrolysis, even in the presence of anionic phospholipids. Mobilization of membrane lipids by GM2AP was also inhibited in the presence of cholesterol or SM, as revealed by surface plasmon resonance studies. These lipids also reduced the interliposomal transfer rate of 2-NBD-GM1 by GM2AP, as observed in assays using Förster resonance energy transfer. Our data raise major concerns about the usage of recombinant His-tagged GM2AP compared with untagged protein. The former binds more strongly to anionic GM2-carrying liposomal surfaces, increases GM2 hydrolysis, and accelerates intermembrane transfer of 2-NBD-GM1, but does not mobilize membrane lipids.     

The enzyme-binding region of human GM2-activator protein

The GM2-activator protein (GM2AP) is an essential cofactor for the lysosomal degradation of ganglioside GM2 by beta-hexosaminidase A (HexA). It mediates the interaction between the water-soluble exohydrolase and its membrane-embedded glycolipid substrate at the lipid-water interface. Functional deficiencies in this protein result in a fatal neurological storage disorder, the AB variant of GM2 gangliosidosis. In order to elucidate this cofactor's mode of action and identify the surface region of GM2AP responsible for binding to HexA, we designed several variant forms of this protein and evaluated the consequences of these mutations for lipid- and enzyme-binding properties using a variety of biophysical and functional studies. The point mutants D113K, M117V and E123K showed a drastically decreased capacity to stimulate HexA-catalysed GM2 degradation. However, surface plasmon resonance (SPR) spectroscopy showed that the binding of these variants to immobilized lipid bilayers and their ability to solubilize lipids from anionic vesicles were the same as for the wild-type protein. In addition, a fluorescence resonance energy transfer (FRET)-based assay system showed that these variants had the same capacity as wild-type GM2AP for intervesicular lipid transfer from donor to acceptor liposomes. The concentration-dependent effect of these variants on hydrolysis of the synthetic substrate 4-methylumbelliferyl-2-acetamido-2-deoxy-6-sulfo-beta-D-glucopyranoside (MUGS) indicated a weakened association with the enzyme's alpha subunit. This identifies the protein region affected by these mutations, the single short alpha helix of GM2AP, as the major determinant for the interaction with the enzyme. These results further confirm that the function of GM2AP is not restricted to a biological detergent that simply disrupts the membrane structure or lifts the substrate out of the lipid plane. In contrast, our data argue in favour of the critical importance of distinct activator-hexosaminidase interactions for GM2 degradation, and corroborate the view that the activator/lipid complex represents the true substrate for the degrading enzyme.     

Photoaffinity labelling of the human GM2-activator protein. Mechanistic insight into ganglioside GM2 degradation.

The GM2-activator protein (GM2AP) is an essential cofactor for the degradation of ganglioside GM2 by lysosomal beta-hexosaminidase A. It mediates the interaction between the water-soluble exohydrolase and its membrane-bound substrate at the lipid-water interphase. Inherited defects in the gene encoding this glycoprotein result in a fatal neurological storage disorder, the AB variant of GM2-gangliosidosis. To elucidate the mode of action of this glycoprotein cofactor, we synthesized the two photoaffinity labels [14C]C3-TPD-GM2 and [14C]C7-TPD-GM2. Incubation of GM2AP with these substrate analogues and subsequent irradiation led to covalent labelling of the protein. After separation of tryptic peptides by reverse-phase HPLC, the labelled peptide fractions were analysed by MALDI-TOF and sequenced by ESI-Q-TOF mass spectrometry. Both labels were found to be specifically photoincorporated into a part of the surface loop comprising residues V153-L163, a stretch of amino acids that was previously identified as the most flexible region in the crystal structure of the activator. Our results provide strong evidence that this loop constitutes the part of the activator protein that directly interacts with the ganglioside substrate, suggesting that the hydrophobicity and the great structural mobility of this element are crucial for the extraction of the membrane-embedded glycolipid, its stabilization inside the spacious cavity and its guidance to the enzyme's active site. This study demonstrates that the approach of photoaffinity labelling in conjunction with accurate mass measurements can provide insight into substrate binding interactions that complements structural information.     

Study of Interaction of GM2 Activator Protein with GM2 Using Circular Dichroism and Fluorescence Spectroscopy

GM2 activator protein (GM2AP) is a cofactor for stimulating the enzymatic hydrolysis of the glycolipid GM2 by -hexosaminidase A to produce GM3. We have examined the conformation of GM2AP before and after its interaction with GM2, GM3, and GA2 using circular dichroism and fluorescence spectroscopy techniques. In the presence of GM2, a blue shift of the fluorescence emission maximum and a strong decrease of molar ellipticity values in circular dichroism spectra were observed only at pH 4.5 and at GM2/GM2AP molar ratio higher than 10:1 (up to 50:1). These results suggest that GM2AP assumed a more organized -helical conformation with the tryptophan residues moving from the polar medium toward the hydrophobic environment of the protein. The conformation of GM2AP in the presence of the downstream reaction product, GM3, or a less favorable substrate, GA2, clearly differed from that in the presence of GM2. The relationships between spectroscopic changes and enzymatic activity, herein discussed, strongly suggest that the specific conformation exhibited by GM2AP in the presence of GM2 is functional to serve as an activator for the enzymatic hydrolysis of GM2.     

Membrane-spanning lipids for an uncompromised monitoring of membrane fusion and intermembrane lipid transfer

A Förster resonance energy transfer-based fusion and transfer assay was developed to study, in model membranes, protein-mediated membrane fusion and intermembrane lipid transfer of fluorescent sphingolipid analogs. For this assay, it became necessary to apply labeled reporter molecules that are resistant to spontaneous as well as protein-mediated intermembrane transfer. The novelty of this assay is the use of nonextractable fluorescent membrane-spanning bipolar lipids. Starting from the tetraether lipid caldarchaeol, we synthesized fluorescent analogs with fluorophores at both polar ends. In addition, we synthesized radioactive glycosylated caldarchaeols. These labeled lipids were shown to stretch through bilayer membranes rather than to loop within a single lipid layer of liposomes. More important, the membrane-spanning lipids (MSLs) in contrast to phosphoglycerides proved to be nonextractable by proteins. We could show that the GM2 activator protein (GM2AP) is promiscuous with respect to glycero- and sphingolipid transfer. Saposin (Sap) B also transferred sphingolipids albeit with kinetics different from GM2AP. In addition, we could unambiguously show that the recombinant activator protein Sap C x His₆ induced membrane fusion rather than intermembrane lipid transfer. These findings showed that these novel MSLs, in contrast with fluorescent phosphoglycerolipids, are well suited for an uncompromised monitoring of membrane fusion and intermembrane lipid transfer.     

Complexing of glycolipids and their transfer between membranes by the activator protein for degradation of lysosomal ganglioside GM2

The lysosomal degradation of ganglioside GM2 by hexosaminidase A depends on the presence of the specific activator protein which mediates the interaction between micellar or membrane-bound ganglioside and water-soluble hydrolase. The mechanism and the glycolipid specificity of this activator were studied in more detail. 1. It could be shown with three different techniques (isoelectric focusing, centrifugation and electrophoresis) that the activator protein extracts glycolipid monomers from micelles or liposomes to give water-soluble complexes with a stoichiometry of 1 mol of glycolipid/mol of activator protein. Liposome-bound ganglioside GM2 is considerably more stable against extraction and degradation than micellar ganglioside. 2. In the absence of enzyme the activator acts in vitro as glycolipid transfer protein, transporting glycolipids from donor to acceptor membranes. 3. The activator protein is rather specific for ganglioside GM2. Other glycolipids (GM3 GM1, GD1a and GA2) form less stable complexes with the activator and are transferred at a slower rate (except for ganglioside GM1) than ganglioside GM2.     

Synthesis of a fluorescent ganglioside GM1 derivative and screening of a synthetic peptide library for GM1 binding sequence motifs

A ganglioside GM1 probe bearing a dark-red fluorescent dye at the sphingosine moiety of the molecule was prepared by a convenient one-pot synthesis. The labeled GM1 permitted the detection of the natural ganglioside GM1 ligand Escherichia coli heat-labile enterotoxin subunit B (EtxB) in picomole quantities on a solid support. When an epitope mapping of several ganglioside binding proteins and protein fragments was performed by screening a cellulose membrane-bound synthetic library of 64 16mer peptides with the new probe, several peptides displaying ganglioside GM1 affinity could be identified. We consider the labeled glycolipid described herein a versatile tool for manifold biochemical investigations.     

Properties of a specific glycolipid transfer protein from bovine brain

A transfer protein specific for glycolipids has been isolated from bovine brain. As judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, the protein is 68% pure and has a molecular weight of 20 000. Three different assays were employed to study the protein's specificity and glycolipid binding properties. The protein transferred several different neutral glycosphingolipids and ganglioside GM1 equally well, but failed to accelerate phosphatidylcholine or sphingomyelin intervesicular movement. The protein's ability to interact with glycolipids was strongly influenced by the physical properties of the matrix phospholipid in which the glycolipids reside. Both the phase state of the phospholipid matrix and bilayer curvature affected glycolipid intervesicular transfer rates. Protein binding to phospholipid vesicles containing either tritium-labeled or pyrene-labeled glucosylceramide could not be demonstrated by density gradient centrifugation or fluorescence energy transfer measurements, respectively. A specific association of the transfer protein for pyrene-labeled glucosylceramide was found when the fluorescence emission of the pyrene excimer-to-monomer ratio was measured suggesting that a portion of the fluorescent glycolipid was being sequestered from the phospholipid vesicles and was binding to the freely soluble protein.     

Substrate specificity of GM2 and GD3 synthase of Golgi vesicles derived from rat liver

Several GM3 derivatives have been synthesized. Among them were lyso-GM3 derivatives and GM3 analogues with modifications in the sialic acid moiety. They were used as glycolipid acceptors in assays for GM2 and GD3 synthase of rat liver Golgi. Analysis of the resulting enzyme activities and of the reaction products revealed different substrate specificities for GM2 and GD3 synthase although the normal glycolipid acceptor for both transferases is ganglioside GM3. Specificity of GD3 synthase is strongly determined by the substrate's negative charge and the acyl residue in amide bond to the amino group of neuraminic acid, while GM2 synthase reacts quite indifferently to these changes in the sialic moiety of the substrate. Both enzymes seem to be sensitive to the spatial extension at the neuraminic acid's carboxylic group.     

Crystal structure analysis of phosphatidylcholine-GM2-activator product complexes: evidence for hydrolase activity

GM2-activator protein (GM2AP) is a lysosomal lipid transfer protein with important biological roles in ganglioside catabolism, phospholipid metabolism, and T-cell activation. Previous studies of crystal structures of GM2AP complexed with the physiological ligand GM2 and platelet activating factor (PAF) have shown binding at two specific locations within the spacious apolar pocket and an ordering effect of endogenous resident lipids. To investigate the structural basis of phospholipid binding further, GM2AP was cocrystallized with phosphatidylcholine (PC), known to interact with GM2AP. Analysis of three crystal forms revealed binding of single chain lipids and fatty acids only and surprisingly not intact PC. The regions of best defined electron density are consistent with the presence of lyso-PC and oleic acid, which constitute deacylation products of PC. Their acyl tails are in stacking contact with shorter, less well-defined stretches of electron density that may represent resident fatty acids. The GM2AP associated hydrolytic activity that generates lyso-PC was further confirmed by mass spectrometry and enzymatic assays. In addition, we report the structures of (i) mutant Y137S, assessing the role of Tyr137 in lipid transfer via the hydrophobic cleft, and (ii) apo-mouse GM2AP, revealing a hydrophobic pocket with a constricted opening. Our structural results provide new insights into the biological functions of GM2AP. The combined effect of hydrolytic and lipid transfer properties has profound implications in cellular signaling.     

How helminth lipid-binding proteins offload their ligands to membranes: differential mechanisms of fatty acid transfer by the ABA-1 polyprotein allergen and Ov-FAR-1 proteins of nematodes and Sj-FABPc of schistosomes

Three different classes of small lipid-binding protein (LBP) are found in helminth parasites. Although of similar size, the ABA-1A1 (also designated As-NPA-A1) and Ov-FAR-1 (formerly known as Ov20) proteins of nematodes are mainly alpha-helical and have no known structural counterparts in mammals, whereas Sj-FABPc of schistosomes is predicted to form a beta-barrel structure similar to the mammalian family of intracellular fatty acid binding proteins. The parasites that produce these proteins are unable to synthesize their own complex lipids and, instead, rely entirely upon their hosts for supply. As a first step in elucidating whether these helminth proteins are involved in the acquisition of host lipid, the process by which these LBPs deliver their ligands to acceptor membranes was examined, by comparing the rates and mechanisms of ligand transfer from the proteins to artificial phospholipid vesicles using a fluorescence resonance energy transfer assay. All three proteins bound the fluorescent fatty acid 2-(9-anthroyloxy)palmitic acid (2AP) similarly, but there were clear differences in the rates and mechanisms of fatty acid transfer. Sj-FABPc displayed a collisional mechanism; 2AP transfer rates increased with acceptor membrane concentration, were modulated by acceptor membrane charge, and were not diminished in the presence of increasing salt concentrations. In contrast, transfer of ligand from Ov-FAR-1 and ABA-1A1 involved an aqueous diffusion step; transfer rates from these proteins were not modulated by acceptor membrane concentration or charge but did decrease with the ionic strength of the buffer. Despite these differences, all of the proteins interacted directly with membranes, as determined using a cytochrome c competition assay, although Sj-FABPc interacted to a greater extent than did Ov-FAR-1 or rABA-1A1. Together, these results suggest that Sj-FABPc is most likely to be involved in the intracellular targeted transport and metabolism of fatty acids, whereas Ov-FAR-1 and ABA-1A1 may behave in a manner analogous to that of extracellular LBPs such as serum albumin and plasma retinol binding protein.     

Complete localization of disulfide bonds in GM2 activator protein.

Lysosomal degradation of ganglioside GM2 by hexosaminidase A requires the presence of a small, non-enzymatic cofactor, the GM2-activator protein (GM2AP). Lack of functional protein leads to the AB variant of GM2-gangliosidosis, a fatal lysosomal storage disease. Although its possible mode of action and functional domains have been discussed frequently in the past, no structural information about GM2AP is available so far. Here, we determine the complete disulfide bond pattern of the protein. Two of the four disulfide bonds present in the protein were open to classical determination by enzymatic cleavage and mass spectrometry. The direct localization of the remaining two bonds was impeded by the close vicinity of cysteines 136 and 138. We determined the arrangement of these disulfide bonds by MALDI-PSD analysis of disulfide linked peptides and by partial reduction, cyanylation and fragmentation in basic solution, as described recently (Wu F, Watson JT, 1997, Protein Sci 6:391-398).     

Site specific point mutation changes specificity: A molecular modeling study by free energy simulations and enzyme kinetics of the thermodynamics in ribonuclease T1 substrate interactions

We have theoretically and experimentally studied the binding of two different ligands to wild-type ribonuclease T₁ (RNT1) and to a mutant of RNT1 with Glu-46 replaced by Gln. The binding of the natural substrate 3′-GMP has been compared with the binding of a fluorescent probe, 2-aminopurine 3′-monophosphate (2AP), and relative free energies of binding of these ligands to the mutant and the wild-type (wt) enzyme have been calculated by free energy perturbation methods. The free energy perturbations predict that the mutant RNT1-Gln-46 binds 2AP better than 3′GMP, in agreement with experiments on dinucleotides. Four free energy perturbations, forming a closed loop, have been performed to allow the detection of systematic errors in the simulation procedure. Because of the larger number of atoms involved, it was necessary to use a much longer simulation time for the change in the protein, i.e., the perturbation from Glu to Gln, than in the perturbation from 3′-GMP to 2AP. Finally the structure of the binding site is analyzed for understanding differences in catalytic speed and binding strength. © 1993 Wiley-Liss, Inc.     

Role of endosomal membrane lipids and NPC2 in cholesterol transfer and membrane fusion

We examined the effect of Niemann-Pick disease type 2 (NPC2) protein and some late endosomal lipids [sphingomyelin, ceramide and bis(monoacylglycero)phosphate (BMP)] on cholesterol transfer and membrane fusion. Of all lipid-binding proteins tested, only NPC2 transferred cholesterol at a substantial rate, with no transfer of ceramide, GM3, galactosylceramide, sulfatide, phosphatidylethanolamine, or phosphatidylserine. Cholesterol transfer was greatly stimulated by BMP, little by ceramide, and strongly inhibited by sphingomyelin. Cholesterol and ceramide were also significantly transferred in the absence of protein. This spontaneous transfer of cholesterol was greatly enhanced by ceramide, slightly by BMP, and strongly inhibited by sphingomyelin. In our transfer assay, biotinylated donor liposomes were separated from fluorescent acceptor liposomes by streptavidin-coated magnetic beads. Thus, the loss of fluorescence indicated membrane fusion. Ceramide induced spontaneous fusion of lipid vesicles even at very low concentrations, while BMP and sphingomyelin did so at about 20 mol% and 10 mol% concentrations, respectively. In addition to transfer of cholesterol, NPC2 induced membrane fusion, although less than saposin-C. In this process, BMP and ceramide had a strong and mild stimulating effect, and sphingomyelin an inhibiting effect, respectively. Note that the effects of the lipids on cholesterol transfer mediated by NPC2 were similar to their effect on membrane fusion induced by NPC2 and saposin-C.     

A mutation in the gene of a glycolipid-binding protein (GM2 activator) that causes GM2-gangliosidosis variant AB

GM2-gangliosidoses are neurological disorders caused by a genetic deficiency of either the β-hexosaminidase A or the GM2 activator, a glycolipid binding protein. In a patient with an immunologically proven GM2 activator protein deficiency, A T⁴¹² → C transition (counted from A of the initiation codon) was found in the coding sequence, which results in the substitution of Arg for the normal Cys¹⁰⁷ in the mature GM2 activator protein. The remainder of the coding sequence remained entirely normal.     

GLTP Mediated Non-Vesicular GM1 Transport between Native Membranes

Lipid transfer proteins (LTPs) are emerging as key players in lipid homeostasis by mediating non-vesicular transport steps between two membrane surfaces. Little is known about the driving force that governs the direction of transport in cells. Using the soluble LTP glycolipid transfer protein (GLTP), we examined GM1 (monosialotetrahexosyl-ganglioside) transfer to native membrane surfaces. With artificial GM1 donor liposomes, GLTP can be used to increase glycolipid levels over natural levels in either side of the membrane leaflet, i.e., external or cytosolic. In a system with native donor- and acceptor-membranes, we find that GLTP balances highly variable GM1 concentrations in a population of membranes from one cell type, and in addition, transfers lipids between membranes from different cell types. Glycolipid transport is highly efficient, independent of cofactors, solely driven by the chemical potential of GM1 and not discriminating between the extra- and intracellular membrane leaflet. We conclude that GLTP mediated non-vesicular lipid trafficking between native membranes is driven by simple thermodynamic principles and that for intracellular transport less than 1 µM GLTP would be required in the cytosol. Furthermore, the data demonstrates the suitability of GLTP as a tool for artificially increasing glycolipid levels in cellular membranes.     

The trafficking of prosaposin (SGP-1) and GM2AP to the lysosomes of TM4 Sertoli cells is mediated by sortilin and monomeric adaptor proteins

Prosaposin (SGP-1) and GM2 activator protein (GM2AP) are soluble sphingolipid activator proteins (SAPs) that are targeted to the lysosomal compartment of Sertoli cells to aid hydrolases in the breakdown of glycosphingolipids. To reach the lysosome, most soluble proteins must interact with the mannose 6-phosphate receptor (MPR). To be sorted from the Golgi, the MPR must bind to the Golgi associated, gamma-adaptin homologous, ARF binding proteins (GGAs), a group of monomeric adaptor proteins responsible for the recruitment of clathrin. It is well established, however, that the lysosomes of I-cell disease (ICD) patients have near normal levels of several lysosomal proteins, including prosaposin and GM2AP. ICD results from a mutation in the phosphotransferase that adds mannose 6-phosphate to hydrolases. Thus, prosaposin and GM2AP can traffic to lysosomes in a MPR independent manner. Previous work has demonstrated that an interaction with sphingomyelin in the Golgi membrane is necessary for the targeting of prosaposin by an unknown receptor. Using a TM4 Sertoli cell line, we tested the hypothesis that prosaposin and GM2AP are targeted to the lysosomal compartment via the sortilin receptor, which has been recently shown to have a GGA binding motif. Interestingly, dominant-negative GGAs, unable to bind clathrin to shuttle from the Golgi, prevented the trafficking of prosaposin and GM2AP to lysosomes. A dominant negative construct of sortilin lacking the GGA binding domain retained prosaposin and GM2AP in the Golgi. In conclusion, our results showed that the trafficking of prosaposin and GM2AP to the lysosome is dependent on sortilin.     

Detection of protein mediated glycosphingolipid clustering by the use of resonance energy transfer between fluorescent labelled lipids. A method established by applying the system ganglioside GM1 and cholera toxin B subunit.

Glycosphingolipids labelled in the ceramide moiety with 3-(p-(6-phenyl)-1,3,5-hexatrienyl)phenylpropionic acid (DPH) or 6-(4-nitrobenz-2-oxa-1,3-diazole-7-yl)aminohexanoic acid (NBD) were incorporated into small unilamellar lecithin liposomes. They were used in resonance energy transfer (RET) experiments between the donor fluorophore DPH and the acceptor NBD to study glycosphingolipid distribution. In pure lecithin liposomes the fluorescent derivatives of GM1, GA1, galactosylceramide and sulfatide behaved almost identically and Ca2+ ions (5 microM or 150 mM) did not influence their transfer efficiencies. But cholera toxin B subunit (CTB) specifically clustered GM1 and enhanced the transfer efficiency. This RET-based method facilitated determination of binding specificity, complex stoichiometry (CTB/GM1 = 1:5), halftime of complex formation (5 s), cooperativity in binding and had a maximal sensitivity at a liposome dotation rate of just 0.25 mol%. In contrast to this, anisotrophy of the fluorophores and the excimer to monomer ratio of pyrene-GM1 were not affected by CTB. This demonstrates the advantage of the presented technique in detection of protein mediated glycosphingolipid clustering.