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 ␣2,3-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.

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 ␤1,4-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.Gangliosides are glycosphingolipids that contain sialic acid (reviewed in Ref. 1). They are found on the external leaflet of the plasma membrane on eucaryotic cells and are most abundant in the central nervous system (CNS) 1 where they represent the major sialoglycoconjugate. Because of their dramatic changes in expression during neuronal development and differentiation (3-6), as well as their prominence in the mature CNS, gangliosides have long been assumed to have fundamental roles in CNS development and function.In the ganglioside biosynthetic pathway (1) (see Fig. 2A), lactosylceramide serves as the core structure. The first ganglioside synthesized, GM3, 2 is produced by the transfer of an ␣2,3-linked sialic acid residue to lactosylceramide. Subsequently, GM3 can be modified by the action of ␤1,4-N-acetylgalactosaminyltransferase (GalNAcT, EC 2.4.1.92) to produce GM2 and other complex gangliosides. Alternatively, GM3 can be modified by the action of GD3 synthase (CMP-sialic acid: GM3 ␣-2,8-sialyltransferase, EC 2.4.99.8) to produce the disialoganglioside GD3, which diverts the pathway to the synthesis of b-and c-series gangliosides. Gene targeting in mice has been a particularly fertile approach for uncovering the functions of gangliosides in the CNS. Disruption of the GalNAcT gene (7) blocks the synthesis of complex gangliosides and results in the expression of only the simple gangliosides GM3 and GD3. Surprisingly, these mutant mice are viable, with a normal life span and a CNS that is largely intact both morphologically and functionally (8, 9). These mice do, however, exhibit an agerelated dysmyelination process that is associated with axonal degeneration (10). The mechanism for dysmyelination may be the absence of neuronal ganglioside ligands for myelin-associated glycoprotein (MAG) resulting in myelin instability. Ultimately, motor defects are observed in aged, 12-month-old GalNAcTϪ/Ϫ mice, suggesting a role for complex gangliosides in long-term CNS maintenance ...

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 ¢bro-blasts. This e¡ect 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 Pioneer1 1 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 ¢ndings 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. ß 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

Gangliosides, which are sialylated glycosphingolipids, are the major class of glycoconjugates on neurons and carry the majority of the sialic acid within the central nervous system (CNS). To determine the role of ganglioside synthesis within the CNS, mice carrying null mutations in two critical ganglioside-specific glycosyltransferase genes, Siat9 (encoding GM3 synthase) and Galgt1 (encoding GM2 synthase), were generated. These double-null mice were unable to synthesize gangliosides of the ganglio-series of glycosphingolipids, which are the major ganglioside class in the CNS. Soon after weaning, viable mice developed a severe neurodegenerative disease that resulted in death. Histopathological examination revealed striking vacuolar pathology in the white matter regions of the CNS with axonal degeneration and perturbed axon-glia interactions. These results indicate that ganglioside synthesis is essential for the development of a stable CNS, possibly by means of the promotion of interactions between axon and glia.glycosphingolipid ͉ neurodegeneration ͉ glycosyltransferase

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.