Glycosphingolipids (GSLs) are characteristic components of the outer leaflet of eukaryotic plasma membranes. Each GSL contains a hydrophobic ceramide moiety that acts as membrane anchor and a hydrophilic, extracellular oligosaccharide chain. Ceramide itself consists of a long chain amino alcohol, D-erythro-sphingosine, which is acylated with a fatty acid. It is also a structural component of a plasma membrane phospholipid, sphingomyelin. GSLs are heterogeneous with respect to both, their carbohydrate and ceramide portion. Especially variations in the type, number and linkage of sugar residues within the oligosaccharide chain give rise to the wide range of naturally occurring GSLs. More than 300 different oligosaccharide and more than 350 ceramide structures have been characterized from natural sources. GSL structures depend on the species and can be classified into series which are characteristic for a group of evolutionary related organisms. Beside the species dependence, GSLs form cell-type specific patterns on the cell surface. In particular sialic acid containing GSLs of the ganglio-series, the gangliosides, are abundant on neuronal cells. Moreover, these patterns change with cell growth, differentiation, viral transformation, ontogenesis and oncogenesis. Together with glycoproteins and glycosaminoglycans the GSLs contribute to the glycocalix which covers the cell surface with a protecting carbohydrate wall.
At the cell surface, GSLs can interact with toxins, viruses, and bacteria. These pathogens take advantage of the close spatial neighborhood between specific carbohydrate recognition sites and the plasma membrane. GSLs can also interact with membrane bound receptors and enzymes and contribute to cell type specific adhesion processes. Various cellular functions of complex GSLs can be attributed to gangliosides. They are involved e.g. in embryogenesis, neuronal and leukocyte differentiation, cell adhesion, and signal transduction. In addition, lipophilic products of GSL metabolism such as sphingosine, ceramide and their phosphorylated derivatives, e.g. sphingosine-1-phosphate, play a significant role in signal transduction events. Finally, GSLs form a protective layer on biological membranes protecting them from inappropriate degradation and uncontrolled membrane fusion. Limited knowledge about the precise in vivo function of GSL is available today. A variety of observations indicate that they can participate in different biological events, e.g. the regulation of membrane-bound receptors and enzymes. In general, the conservation of the overall GSL structure during evolution indicate their functional importance for the living organism.

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De novo biosynthesis of GSLs takes place in the same intracellular compartments as glycoprotein biosynthesis and is coupled to intracellular vesicular transport of the growing molecules through the cisternae of the Golgi apparatus and to the plasma membrane. It starts with the formation of ceramide at the membranes of the endoplasmic reticulum (ER). The condensation of the amino acid L-serine with a fatty acyl coenzyme A, usually palmitoyl coenzyme A, to 3-ketosphinganine is catalyzed by the enzyme serine palmitoyl transferase (SPT). In the following NADPH-dependent reaction, 3-ketosphinganine is reduced to D-erythro-sphinganine by the enzyme 3-ketosphinganine reductase. Sphinganine is acylated to dihydroceramide by 6 celltype and differentiation specific sphinganine N-acyltransferases. Dihydroceramide is subsequently desaturated to ceramide in the dihydroceramide desaturase reaction. Sphingosine, the parent compound of the sphingolipids, is not an intermediate of sphingolipid biosynthesis. Instead of this it is formed during sphingolipid degradation.
Ceramide is the common precursor of GSLs and sphingomyelin. Biosynthesis of most GSLs of vertebrates requires the glucosylation of ceramide. The glucosylceramide synthase transfers a glucose residue from UDP-glucose to ceramide. Lactosylceramide, the common precursor for the five GSL series found in vertebrates, is formed by the addition of a galactose moiety from UDP-Gal to glucosylceramide catalyzed by galactosyltransferase I. Most GSL found in vertebrates share lactosylceramide as a common precursor and structural element. The sequential addition of further sugar residues including sialic acid requires the action of membrane bound glycosyltransferases in the lumen of the Golgi apparatus. Lactosylceramide and its sialylated derivatives GM3, GD3 and GT3 serve as precursors for more complex gangliosides of different series. Sequential glycosylation of these precursors is performed by less specific glycosyltransferases, which transfer a respective sugar residue to glycosyl acceptors which differ only in the number of sialic acids bound to the inner galactose. Many of the glycosyltranferases involved in GSL biosynthesis have been cloned in the recent past, although the mechanism of these enzymes is far from clear. Also several low molecular weight inhibitors of GSL biosynthesis are known and may become valuable tools for the treatment of sphingolipidoses and other diseases.
In cooperation with Rick Proia (NIH; Bethesda, USA) several mice with defects in glycolipid biosynthesis have been analyzed by us to elucidate the function of individual glycolipids.

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Not only glycosphingolipids themselves residing in the plasma membrane, but also lipophilic intermediates of sphingolipid catabolism are involved in the transmission of extracellular signals to intracellular regulatory systems. The observation that hydrolysis of sphingomyelin to ceramide can be induced by extracellular agents in various cell types led to the discovery of the so called sphingomyelin cycle. In general, ceramide appears to mediate antimitogenic effects like cell differentiation, cell cycle arrest and cell senescence. Several lines of evidence indicate ceramide to be a physiological mediator of apoptosis. The identity of the cellular targets of ceramide and other molecules downstream within the signal flow is not unambiguously known; several target proteins are currently under investigation.
A large number of events are reported to be influenced by other intermediates of sphingolipid metabolism, e.g. inhibition of protein kinase C by sphingosine and lysoGSLs lacking the amide-bonded fatty acid. Sphingosine-1-phosphate operates as mitogenic regulator in certain cell types.

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GSL degradation occurs in the acid compartments of the cells,the endosomes and the lysosomes. Parts of the plasma membrane destined for degradation are endocytosed and traffic through the endosomal compartments to reach the lysosome. The composition of sphingolipids entering the lysosomes depend on the cell type. Neuronal plasma membranes are rich in gangliosides while oligodendrocytes and Schwann cells have a higher content of galactosylceramide and sulfatide. Within the lysosome, hydrolyzing enzymes sequentially cleave off the sugar residues from the nonreducing end. Via lower glycosylated GSLs, ceramide and finally sphingosine is produced. This can leave the lysosome, reenter the biosynthetic pathway or be further degraded. For GSLs with long carbohydrate chains of more than four sugar residues the presence of an enzymatically active lysosomal hydrolase is sufficient for degradation in vivo. However, degradation of membrane bound GSLs with short oligosaccharide chains requires the cooperation of an exohydrolase and a protein cofactor, a so called sphingolipid activator protein. Several sphingolipid activator proteins are now known including the GM2 activator and the saposins SAP-A, -B, -C and -D. Inherited deficiencies of either lysosomal hydrolases or activator proteins give rise to GSL storage diseases, the sphingolipidoses. This is a group of inherited human diseases in which sphingolipids accumulate in one or more organs due to a degradation disorder. Symptoms and course of these diseases are varying widely between forms with onset in early childhood and death within the first years of life on the one hand, and chronic forms on the other hand. Their pathogenesis is poorly understood and a causal therapy is only available for the nonneuronopathic form of Gaucher's disease, a glucocerebrosidase deficiency. The genes of most proteins involved in sphingolipid degradation have recently been cloned enabling genotype-phenotype correlation and facilitating the diagnosis of sphingolipidoses. In addition, animal models of many sphingolipidoses have been created by targeted disruption of the respective genes in mice. In the future, these animals will serve as valuable tools for the investigation of the pathogenesis of these diseases and for the study of therapeutic approaches, including substrate deprivation, enzyme replacement, organ transplantation, and gene therapy.
STREICHEN: Isolation of preparative amounts of purified recombinant glycoproteins will allow to analyze structure and biophysical properties of the membrane-active sphingolipid activator proteins, exohydrolases, and glycosyltransferases.

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