Michaela Wendeler

Development of an assay for the intermembrane transfer of cholesterol by Niemann-Pick C2 protein.

Abstract Niemann-Pick type C disease is an inherited fatal disorder characterized by the accumulation of unesterified cholesterol and other lipids in the endosomal/lysosomal compartment. Two independent genes responsible for this neurodegenerative disorder have been identified, but the precise functions of the encoded Niemann-Pick C1 (NPC1) and C2 (NPC2) proteins are not yet known. We developed a cell-free assay for measuring intermembrane lipid transport and examined the ability of bovine NPC2 (bNPC2) for intermembrane cholesterol transfer. NPC2 specifically extracts cholesterol from phospholipid bilayers and catalyzes intermembrane transfer to acceptor vesicles in a dose- and time-dependent manner. This transfer activity is dependent on temperature, pH, ionic strength, lipid composition of the model membranes, and the ratio of donor to acceptor vesicles. In model membranes, the presence of the lysosomal anionic phospholipids bis(monooleoylglycero)phosphate and phosphatidyl inositol significantly stimulated cholesterol transfer by NPC2, whereas bis(monomyristoylglycero)phosphate, phosphatidyl serine, and phosphatidic acid had no effect. Moreover, ceramide stimulated cholesterol transfer slightly, whereas sphingomyelin reduced cholesterol transfer rates. With our assay system we identified for the first time the ability of other lysosomal proteins, most notably the GM2-activator protein, to mediate intermembrane cholesterol transfer. This assay system promises to be a valuable tool for further quantitative and mechanistic studies of protein-mediated lipid transfer.

Chemical chaperones--a new concept in drug research.

Low-molecular-weight compounds are able to stabilize the conformation of proteins that are defective in patients of inherited diseases. Unspecifically acting chemical chaperones, including osmolytes, can increase the fraction of the correctly folded variant protein encoded by the mutated gene. More recently, the concept of specifically acting chemical chaperones has been applied to two sphingolipid storage diseases, Fabrys disease (Figure 1) and Gauchers disease (Figure 2). Studies in cultured cells and, in the case of Fabrys disease also in the animal model, revealed that administration of inhibitors led to a significant increase in the activity of the variant enzymes and to a substantial improvement of therapeutic parameters.

Expression of recombinant human GM2-activator protein in insect cells: purification and characterization by mass spectrometry

The GM2-activator protein (GM2AP) is a small non-enzymatic cofactor assisting the enzyme beta-hexosaminidase A in the lysosomal degradation of ganglioside GM2. Mutations in the gene encoding this glycoprotein lead to a fatal neurological disorder, the AB variant of GM2-gangliosidoses. In this paper, we describe the overexpression of GM2AP in Sf21 cells using both the baculovirus expression vector system (BEVS) and a non-lytic, plasmid-based insect cell expression system (InsectSelect). For the BEVS, the cDNA encoding human GM2AP-preproprotein was cloned in the expression vector pAcMP3. The recombinant virus generated by cotransfection with linearized baculovirus DNA was used to infect Sf21 cells. For the non-lytic expression system, the cDNA of GM2AP was inserted into the vector pIZ/V5-His, which was used for the constitutive expression in stably transformed Sf21 cells. As it was shown by immunoblot analysis of the cell culture supernatant, in both expression systems the GM2AP precursor protein was efficiently secreted into the medium. Following expression in the BEVS, the GM2AP was purified by sequential chromatography on Ni-NTA-agarose and Con A-Sepharose, resulting in a yield of up to 9 mg purified protein from 1L of cell culture supernatant. Following expression in stably transformed insect cells, the secreted protein was first concentrated by cation-exchange and purified by metal-ion affinity chromatography, with a yield of 0.1 mg/L cell culture supernatant. The biological activity of the recombinant protein was demonstrated by its ability to stimulate the hexosaminidase A-catalyzed degradation of ganglioside GM2, and the homogeneity and glycosylation were assessed by ESI-TOF mass spectrometry. While the protein expression in the BEVS led to partly glycosylated and partly non-glycosylated protein, the stably transformed cells produced only glycosylated protein. In both expression systems, the glycosylation was found to be identical and corresponded to the structure (GlcNAc)(2)Fuc(Man)(3).

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 β‐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 cofactors 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‐β‐d‐glucopyranoside (MUGS) indicated a weakened association with the enzymes α subunit. This identifies the protein region affected by these mutations, the single short α 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.

Interaction of the GM2 activator protein with sulfated and sialylated glycosphingolipids.

Publisher Summary This chapter investigates the interaction of the G M2 activator protein with sulfated and sialylated glycosphingolipids. Data concerning the interaction of G M2 AP with gangliosides were obtained from biophysical measurements. The membrane activity of G M2 AP was measured by high-sensitivity differential scanning calorimetry (DSC) and film balance measurements, as well as by surface plasmoresonance studies. Analytical techniques such as thin-layer chromatography (TLC) overlay and fluorescence dequenching techniques provided evidence for the specific interaction between the lipid-binding protein and gangliosides. By TLC overlay it was shown that G M2 AP bound to G M1 , the precursor of G M2 in ganglioside catabolism; G M2 itself; and G M3 , the product of G M2 degradation. In vivo , the G M2 AP is required for the degradation of ganglioside G M2 and glycolipid GA2, as well as for the sufficient degradation of glycolipid S M2 a. In vitro it also acts on related GSL, such as G M1 or globotetraosylceramide. Binding and transfer studies suggested that this cofactor acts as a liftase, –it recognizes its lipid substrate, complexes it, and lifts it out of the membrane plane, thus presenting it to the water-soluble enzyme for degradation.

Pathology of Glycosphingolipid Metabolism: The Molecular and Cellular Basis of Neurodegenerative Disease

Glycosphingolipids are ubiquitous constituents of eukaryotic plasma membranes. Genetically determined deficiencies in their catabolic pathways cause the excessive intralysosomal accumulation of these lipids and give rise to a group of inherited metabolic diseases, the sphingolipidoses. The progression of these disorders often involves severe degeneration of the nervous system, and for nearly all of them, no effective treatment is available to date. Here, we discuss the physiological functions of glycosphingolipids and the topology and mechanism of their metabolism. The molecular defects associated with these storage disorders as well as their pathophysiological consequences and potential therapeutic prospects are presented. Finally, the importance of recently available animal models for the investigation of pathogenesis and the evaluation of future therapy approaches is discussed.

Recombinant Ganglioside GM2 Synthase—Expression in Insect Cells and Enzyme Assay

Publisher Summary This chapter investigates the recombinant ganglioside G M2 synthase; and describes the expression of murine β -1,4- N -acetylgalactosaminyltransferase both as a complete membrane-bound enzyme and as a soluble form in the baculovirus insect -cell expression system. A novel assay based on radiochemically labeled G M3 prepared from the tissue ganglioside, which permits the evaluation of potential UDP-GalNAc analogs is presented. Schematic protein structure of G M2 synthase showing the topology of type II transmembrane proteins is presented. For the expression of full-length G M2 synthase, the entire cDNA of G M2 synthase in the vector pCMV Blue is used as a template in the polymerase chain reaction (PCR) reaction. For expression of soluble G M2 synthase, the medium of infected cells is harvested 72 h postinfection by centrifugation (2000 rpm, 10 min) and concentrated 15-fold with a Vivaspin 4-ml concentrator with a PES membrane and 30,000 moleculor weight cut off (MWCO). This concentrated supernatant is prepared fresh for the assays and is always kept on ice. The chapter discusses the immunoblot analysis, standard glycosyltransferase assays, and kinetic analysis of recombinant G M2 synthase.