Norbert Werth

Enhanced insulin sensitivity in mice lacking ganglioside GM3.

Gangliosides are sialic acid-containing glycosphingolipids that are present on all mammalian plasma membranes where they participate in recognition and signaling activities. We have established mutant mice that lack GM3 synthase (CMP-NeuAc:lactosylceramide α2,3-sialyltransferase; EC 2.4.99.-). These mutant mice were unable to synthesize GM3 ganglioside, a simple and widely distributed glycosphingolipid. The mutant mice were viable and appeared without major abnormalities but showed a heightened sensitivity to insulin. A basis for the increased insulin sensitivity in the mutant mice was found to be enhanced insulin receptor phosphorylation in skeletal muscle. Importantly, the mutant mice were protected from high-fat diet-induced insulin resistance. Our results show that GM3 ganglioside is a negative regulator of insulin signaling, making it a potential therapeutic target in type 2 diabetes.

Mice Expressing Only Monosialoganglioside GM3 Exhibit Lethal Audiogenic Seizures

Gangliosides are a family of glycosphingolipids that contain sialic acid. Although they are abundant on neuronal cell membranes, their precise functions and importance in the central nervous system (CNS) remain largely undefined. We have disrupted the gene encoding GD3 synthase (GD3S), a sialyltransferase expressed in the CNS that is responsible for the synthesis of b-series gangliosides. GD3S−/− mice, even with an absence of b-series gangliosides, appear to undergo normal development and have a normal life span. To further restrict the expression of gangliosides, the GD3S mutant mice were crossbred with mice carrying a disrupted GalNAcT gene encoding β1,4-N-acetylgalactosaminyltransferase. These double mutant mice expressed GM3 as their major ganglioside. In contrast to the single mutant mice, the double mutants displayed a sudden death phenotype and were extremely susceptible to induction of lethal seizures by sound stimulus. These results demonstrate unequivocally that gangliosides play an essential role in the proper functioning of the CNS.

Interactions of acid sphingomyelinase and lipid bilayers in the presence of the tricyclic antidepressant desipramine

The tricyclic antidepressant desipramine causes a decrease in cellular acid sphingomyelinase (A‐SMase, EC 3.1.4.12) activity when added to culture medium of human fibroblasts. This effect can be prevented by incubation of the cells with the protease inhibitor leupeptin, which suggests that desipramine induces proteolytic degradation of the lysosomal enzyme. By using surface plasmon resonance (SPR, Biacore) we were able to monitor the interactions of A‐SMase and substrate‐containing lipid bilayers immobilized on the surface of a Pioneer™ L1 sensor chip. SPR binding curves show that the enzyme hardly dissociates from the lipid surface at acidic pH values. On the other hand, a drop in binding signals (resonance units, RU) of approximately 50% occurred after injection of 20 mM desipramine. Our findings indicate that desipramine interferes with the binding of A‐SMase to the lipid bilayers and thereby displaces the enzyme from its membrane‐bound substrate. The application of control substances suggests a key role for the cationic moiety of desipramine. We hypothesize that the displacement of the glycoprotein A‐SMase from the inner membranes of late endosomes and lysosomes by desipramine renders it susceptible to proteolytic cleavage by lysosomal proteases.

Systemic inflammation in glucocerebrosidase-deficient mice with minimal glucosylceramide storage

Gaucher disease, the most common lysosomal storage disease, is caused by a deficiency of glucocerebrosidase resulting in the impairment of glucosylceramide degradation. The hallmark of the disease is the presence of the Gaucher cell, a macrophage containing much of the stored glucosylceramide found in tissues, which is believed to cause many of the clinical manifestations of the disease. We have developed adult mice carrying the Gaucher disease L444P point mutation in the glucocerebrosidase (Gba) gene and exhibiting a partial enzyme deficiency. The mutant mice demonstrate multisystem inflammation, including evidence of B cell hyperproliferation, an aspect of the disease found in some patients. However, the mutant mice do not accumulate large amounts of glucosylceramide or exhibit classic Gaucher cells in tissues.

Purified recombinant human prosaposin forms oligomers that bind procathepsin D and affect its autoactivation

Before delivery to endosomes, portions of proCD (procathepsin D) and proSAP (prosaposin) are assembled into complexes. We demonstrate that such complexes are also present in secretions of cultured cells. To study the formation and properties of the complexes, we purified proCD and proSAP from culture media of Spodoptera frugiperda cells that were infected with baculoviruses bearing the respective cDNAs. The biological activity of proCD was demonstrated by its pH-dependent autoactivation to pseudocathepsin D and that of proSAP was demonstrated by feeding to saposin-deficient cultured cells that corrected the storage of radioactive glycolipids. In gel filtration, proSAP behaved as an oligomer and proCD as a monomer. ProSAP altered the elution of proCD such that the latter was shifted into proSAP-containing fractions. ProSAP did not change the elution of mature cathepsin D. Using surface plasmon resonance and an immobilized biotinylated proCD, binding of proSAP was demonstrated under neutral and weakly acidic conditions. At pH 6.8, specific binding appeared to involve more than one binding site on a proSAP oligomer. The dissociation of the first site was characterized by a K(D1) of 5.8+/-2.9x10(-8) M(-1) (calculated for the monomer). ProSAP stimulated the autoactivation of proCD and also the activity of pseudocathepsin D. Concomitant with the activation, proSAP behaved as a substrate yielding tri- and disaposins and smaller fragments. Our results demonstrate that proSAP forms oligomers that are capable of binding proCD spontaneously and independent of the mammalian type N-glycosylation but not capable of binding mature cathepsin D. In addition to binding proSAP, proCD behaves as an autoactivable and processing enzyme and its binding partner as an activator and substrate.

Phosphatidylinositol-3,5-bisphosphate is a potent and selective inhibitor of acid sphingomyelinase

Abstract Acid sphingomyelinase (A-SMase, EC 3.1.4.12) catalyzes the lysosomal degradation of sphingomyelin to phosphorylcholine and ceramide. Inherited deficiencies of acid sphingomyelinase activity result in various clinical forms of Niemann-Pick disease, which are characterised by massive lysosomal accumulation of sphingomyelin. Sphingomyelin hydrolysis by both, acid sphingomyelinase and membrane-associated neutral sphingomyelinase, plays also an important role in cellular signaling systems regulating proliferation, apoptosis and differentiation. Here, we present a potent and selective novel inhibitor of A-SMase, L-?-phosphatidyl-D-myo-inositol-3,5-bisphosphate (PtdIns3,5P[2]), a naturally occurring substance detected in mammalian, plant and yeast cells. The inhibition constant Ki for the new A-SMase inhibitor PtdIns3,5P[2] is 0.53 M as determined in a micellar assay system with radiolabeled sphingomyelin as substrate and recombinant human A-SMase purified from insect cells. Even at concentrations of up to 50 uM, PtdIns3,5P[2] neither decreased plasma membrane associated, magnesium-dependent neutral sphingomyelinase activity, nor was it an inhibitor of the lysosomal hydrolases ?-hexosaminidase A and acid ceramidase. Other phosphoinositides tested had no or a much weaker effect on acid sphingomyelinase. Different inositol-bisphosphates were studied to elucidate structure-activity relationships for A-SMase inhibition. Our investigations provide an insight into the structural features required for selective, efficient inhibition of acid sphingomyelinase and may also be used as starting point for the development of new potent A-SMase inhibitors optimised for diverse applications.

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.